M. S. De Mitri

HCV-associated liver cancer without cirrhosis

Chronic infection with hepatitis C virus (HCV) is regarded as a risk factor for hepatocellular cancer, mostly in patients with liver cirrhosis. We looked for HCV genomes in the livers of patients with hepatocellular cancer who did not have cirrhosis to see whether HCV was directly oncogenic. Cancerous and non-cancerous liver tissue, and serum samples from 19 patients negative for hepatitis B surface antigen were analysed by polymerase chain reaction for the presence of HCV genome, HCV replication, HCV genotyping, and HBV genome. 13 of 19 patients were HCV RNA-positive in cancerous and non-cancerous liver tissue; 8 of 17 tested were anti-HCV positive. Among the 13 HCV RNA-positive patients, 11 had genotype 1b and 2 had genotype 2a. 7 of 13 serum samples were HCV RNA positive. 7 of 19 patients were HBV DNA positive in cancerous and non-cancerous liver tissue, 5 of them anti-HBc positive. 4 patients were both HCV RNA and HBV DNA positive and 3 were both HCV RNA and HBV DNA negative. Our results provide evidence for the association of HCV, mostly genotype 1b, with hepatocellular cancer without the intermediate step of cirrhosis.

Distribution of three major hepatitis C virus genotypes in Italy. A multicentre study of 49 5 patients with chronic hepatitis C

SUMMARY. Different genotypes of hepatitis C virus (HCV) have been shown to have distinct geographical distribution and to associate with variable clinical features. To evaluate their role in chronic hepatitis in Italian patients, we studied 495 consecutive cases with chronic hepatitis C seen in nine sentinel centres homo geneously distributed over Italy. HCV genotyping was carried out using a dot‐blot hybridization assay with genotype‐specific probes. Four hundred and eleven patients were viraemic and could be evaluated: 57% were found to be infected with HCV‐1,31% with HCV‐2, 8% with HCV‐3. 1% showed mixed infection and 3% were ascribed to HCV‐2b or HCV‐4 by direct sequencing. Geographical distribution showed discrete territorial variations. A history of drug addiction was commoner in patients infected with HCV‐3. There were no signifi cant differences in activity of liver disease among different HCV genotypes but the response to interferon therapy was reduced in patients infected with HCV‐1 compared to HCV‐2 or HCV‐3.

Virological analysis, genotypes and mutational patterns of the HBV precore/core gene in HBV/HCV-related hepatocellular carcinoma.

Summary.  We investigated the replicative profile of hepatitis B (HBV) and hepatitis C (HCV) viruses and the mutational pattern of the HBV precore/core (pre‐C/C) domain in hepatocellular carcinoma (HCC). Thirty‐eight consecutive patients with HCC were included in the study – 18 of them with HBV/HCV co‐infection and 20 with HBV single infection. Twenty‐three additional patients with co‐infection, without HCC were recruited as the control group. Replication activity was evaluated by detecting and quantitating both HBV and HCV genomes. The HBV pre‐C/C region, encompassing the pregenome encapsidation signal involved in viral replication, was analysed by direct sequencing. HBV viraemia levels were significantly lower (P = 0.04) in patients with co‐infection in comparison with single‐infected HCC, whereas two different HBV viraemia profiles were detected in co‐infection with or without circulating HCV. HBV genotype D was prevalent in the three groups and HCV genotype 1b was found to be the infecting strain in all patients. Lower variability in the pre‐C/C region was found in co‐infection in comparison with HBV single infection (P = 0.0004). A synonymous T1936C mutation was found in all co‐infected HCC cases not related to the presence or absence of circulating HCV, and a hypermutated pre‐C strain, characterized by the same mutational pattern, was identified in three HCC cases. The mutational pattern of the pre‐C/C region was closely related to HBV replication efficiency, and specific HBV mutations selectively associated with HCV co‐infection could be linked with accelerated HBV/HCV‐related disease progression.

A novel stop codon mutation within the hepatitis B surface gene is detected in the liver but not in the peripheral blood mononuclear cells of HIV-infected individuals with occult HBV infection

Summary.  To characterize occult HBV infection (OHB) in different compartments of HIV+ individuals. This retrospective study involved 38 consecutive HIV+ patients; 24 HBsAg negative (HBV−) and 14 HBsAg positive (HBV+). OHB was assessed in serum samples, liver tissue (LT) and peripheral blood mononuclear cells (PBMC) by genomic amplification of the partial S, X and precore/core regions. HBV genomic analysis was inferred by direct sequencing of PCR products. The intracellular HBV‐DNA was measured by a quantitative real‐time PCR. HBV+ patients were used as a control for HBV replication and genomic profile. In HBV− patients, HBV‐DNA was undetectable in all serum samples, while it was found positive in 7/24 (29%) LT in which genotype D prevailed (57%). HBV‐DNA was found in 6/7 (86%) PBMC of occult‐positive and none of occult‐negative LT. Significantly lower HBV‐DNA load was present in both compartments in OHB+ with respect to the HBV+ group (LT: P = 0.002; PBMC: P = 0.026). In the occult‐positive cases, HBV replication was significantly higher in LT than in PBMC (P = 0.028). A hyper‐mutated S gene in PBMC and a nucleotide mutation at position C695 in LT that produces a translational stop codon at amino acid 181 of the HBs gene characterized OHB. In this group of HIV+ persons, OHB is frequent and exhibits lower replication levels than chronic HBV in the different compartments examined. HBV‐DNA detection in PBMC may offer a useful tool to identify OHB in serum‐negative cases. The novel HBs gene stop codon found in LT could be responsible for reduced production leading to undetectability of HBsAg.

New infection with heterotypic hepatitis C virus in a patient with long-term hepatitis C virus eradication

BACKGROUND Experimental hepatitis C virus infection in chimpanzees has shown that natural hepatitis C virus infection does not induce protective immunity and reinfection can occur in seroconverted animals. AIM To study the clinical, virological and histological outcome of a new infection sustained by a different hepatitis C virus strain after a primary infection with eradication of the original virus. PATIENTS AND METHODS A young Italian man with chronic hepatitis C virus type 4 hepatitis was treated with Interferon therapy and achieved a sustained biochemical and virological response. After long follow-up, an asymptomatic flare-up of alanine transaminase occurred. This alanine transaminase increase was associated with serum hepatitis C virus RNA positivity and a low viral load, and the infecting hepatitis C virus genotype was type 3. The clinical and virological course of this new infection is described. RESULTS AND CONCLUSIONS This report shows that there is no protective immunity against hepatitis C virus type 3 after infection by hepatitis C virus type 4 strain.

Low replication and variability of HBV pre-core in concomitant infection with hepatitis B and hepatitis C viruses

Summary.In an attempt to define the virological profile of HBV in HCV co-infection, we analysed the viral load, the infecting genotype, and the mutational pattern of the HBV pre-core region (pre-C), which is involved in viral encapsidation and DNA replication. Eighty-six patients were studied: 32 with serological HBV/HCV-1b co-infection (group BC), 32 infected by HBV alone (group B), and 22 by HCV-1b alone (group C). Sequence analysis of the HBV pre-S and pre-C regions identified genotypes and mutational patterns. The HBV viral load was significantly lower in group BC than in group B (p < 0.001), and the distribution of HBV pre-C mutations showed a higher prevalence of wild type in concomitant infection than in the control group (p < 0.006). The predominant HBV infecting strain was genotype D in both the BC (96%) and B (87%) groups. No difference was observed in HCV viremia levels between the two groups, whereas in HBV/HCV infection, the low levels of circulating HBV were closely associated with the low degree of variability of pre-C domain (p = 0.005). In conclusion, in HBV/HCV infection, the virological pattern was characterised by the dominance of HCV associated with lower HBV replication capacity and decreased emergence of HBV pre-C variants.

Genetic variability of hepatitis C virus in HBV/HCV co-infection and HCV single-infection

Summary.To describe the virological profile of HCV in HBV/HCV co-infection, we investigated the variability of HVR-1 and NS5A domains, which may be involved in viral persistence and replication efficiency. We studied 95 patients: 37 with serological markers of HBV/HCV co-infection, 33 with single HBV and 25 with single HCV infection. HVR-1 complexity and NS5A gene variability were respectively explored by means of PCR-SSCP and direct sequencing. Serum HBV genomes were detected in all coinfected patients: 19 also had circulating HCV particles (group BC-I), whereas HCV were undetectable in the other 18 (group BC-II). Group BC-I was characterised by a significantly lower HBV replication capacity, that reflects the replicative dominance of HCV, although the dominant virus had the same degree of variability as the HCV in single infection. HBV viral load was higher in group BC-II, but not significantly different from that observed in the single infection. Our data indicate an alternation in replicative dominance in co-infection: HBV can suppress HCV replication to undetectable levels, whereas HCV may reduce but does not abrogate the replication capacity of HBV. Furthermore, in the cases of HCV dominance, circulating HBV genomes did not have a significant effect on the viral heterogeneity of HCV.

Potential Molecular Mechanisms of Viral Liver Carcinogenesis

Hepatocellular carcinoma is one of the most common malignant tumors worldwide. The geographic prevalence of HCC is largely variable and this reflects the different distribution of possible etiological factors. Liver cirrhosis is the major determinant of neoplastic transformation (1). The strong association between chronic HBV and HCV infection and developement of HCC is widely demonstrated by epidemiological studies (2,3) but the exact mechanism by which malignant trasformation occurs remains still unclear. HBV might exert a direct role in liver carcinogenesis by a combination of different non exclusive effects as DNA integration (4) and transactivating activity of HBX and truncated pre-S2/S proteins (5,6). Concerning HCV related liver oncogenesis, it is still obscure whether HCV might exert, in addition to inducing cirrhosis, a direct effect in malignant transformation. The evidence that HCV persists and replicate in HCC developing in absence of cirrhosis (7) may be a basis for further molecular studies on the potential oncogenic effect of HCV.