Interleukin 28B polymorphisms as predictors of sustained virological response in chronic hepatitis C: systematic review and meta-analysis.

Polymorphism of interleukin 28B gene represents a powerful outcome predictor for interferon-based regimens in hepatitis C virus infection. However, some studies report conflicting results. The predictive value of interleukin 28B genotype over the outcome interferon-α/ribavirin treatment was thoroughly evaluated and compared with virological predictors of response. Literature revision was performed on PubMed. Pooled odds ratios (ORs) were calculated by fixed- or random-effects models. Heterogeneity and publication bias were also assessed. Sixty-two eligible papers including 20 290 patients were retrieved. Both polymorphisms (rs12979860 and rs8099917) were strongly associated with response (OR=4.09 and 4.00, respectively), however, the association was weaker for subjects infected with viral genotypes 2 and 3 (OR=1.52 and 1.49, respectively). Compared with interleukin 28B genotype, the association with response was lower for baseline viremia (OR=2.15) and higher for rapid virological response (OR=13.86). These results provide a critical evaluation of interleukin 28B genotype as a pharmacogenetic predictor in hepatitis C patients.

Role of viral and host factors in HCV persistence: which lesson for therapeutic and preventive strategies?

Several lines of evidence support the view that hepatitis C virus is not directly cytopathic for infected host cells and that the immune response plays a central role in the pathogenesis of liver damage. Innate and adaptive immune responses are induced in most individuals infected with hepatitis C virus but are insufficient to eliminate the virus. The mechanisms responsible for this failure are largely unknown but the kinetics of hepatitis C virus replication relative to the priming of the adaptive responses may exert a profound influence on the balance between virus and host. Immediately after hepatitis C virus infection, the virus replicates efficiently, inducing the production of type I interferons. However, the rapid increase in viral replication seems to be ignored by the adaptive immune response, and after a short interval from exposure, viral load can reach levels comparable to those of patients with established persistent infection. The CD8-mediated response shows functional defects, with impaired production of interferon-gamma, low perforin content, decreased capacity of expansion and lysis of target cells. Late appearance and functional defects of T cells in hepatitis C virus infection might be the result of the rapid increase of the viral load that could create the conditions for exhaustion of the adaptive response or reflect an insufficient function of the innate immune response. This possibility is suggested by in vitro studies showing that hepatitis C virus gene products can interfere with the anti-viral activity of type I interferons and natural killer cells as well as with the maturation of dendritic cells. While T-cell defects are reversed in a minority of infected individuals who succeed in controlling the infection, the T-cell impairment becomes progressively more profound as infection progresses to chronicity. In this situation, therapeutic restoration of adaptive responses may represent a rational strategy to obtain resolution of infection and to complement available therapies. The peculiar kinetics of hepatitis C virus replication and T-cell induction soon after infection may have important implications also for the design of protective vaccines since memory responses may not be able to precede the early peak of viral replication. Therefore, vaccines against hepatitis C virus may be unable to prevent infection but may rather be effective in facilitating a self-limited evolution of infection.

Prolonged treatment with imatinib mesylate in patients with advanced chronic myeloid leukemia causes a reduction of bcr/abl mRNA levels independent of cytogenetic response.

Bcr/abl mRNA levels were monitored in 13 patients with chronic myeloid leukemia receiving imatinib mesylate over a period of 78 weeks. During treatment median bcr/abl mRNA levels progressively declined from 77.2 normalized dose (nD) at baseline to 11.28 nD after 13 weeks ( P<0.05) and to 1.28 nD after 78 weeks ( P<0.05). After 13 weeks, bcr/abl mRNA levels were significantly lower in cytogenetic responders compared to nonresponders ( P<0.05), but subsequent decrease in the transcript levels caused the loss of any correlation to the cytogenetic status. These results suggest that bcr/abl mRNA levels may reflect cytogenetic response only during the early phases of imatinib therapy.

Prevalence and genomic variability of transfusion transmitted virus in Italian cryptogenic chronic liver disease and healthy blood donors.

BACKGROUND: Infection with transfusion transmitted virus, a new member of the Parvoviridae family, has been found in patients both with chronic and fulminant post-transfusion cryptogenic hepatitis. AIM: To evaluate the prevalence and clinical impact of transfusion transmitted virus infection in Italy. PATIENTS AND METHODS: Studies were carried out on 256 patients and control subjects from three centres from Northern, Central and Southern Italy (92 nonA-nonC chronic hepatitis, 10 acute non fulminant cryptogenic hepatitis, 41 hepatitis C virus-related chronic hepatitis and 113 blood donors). Serum transfusion transmitted virus was detected by nested polymerase chain reaction using two overlapping sets of primers. RESULTS: A total of 52 of the 92 patients (54.3%) with chronic cryptogenic liver disease and 17 of the 41 hepatitis C virus chronic hepatitis patients (41.4%) were transfusion transmitted virus-DNA positive. Transfusion transmitted virus co-infection in hepatitis C virus patients was not associated with either a higher severity of liver histology or higher alanine transaminase levels or signs of cholestasis, transfusion transmitted virus was found in 48 out of 113 (42.4%) blood donors. In the majority of samples, transfusion transmitted virus DNA was detected with only one of the two sets of primers used. Genotyping and phylogenetic analysis performed on 21 randomly selected viral isolates showed the presence of both type 1 and type 2 transfusion transmitted virus and allowed identification of two isolates with high homology to genotype 6, described, so far, mostly in Japan. CONCLUSIONS: Transfusion transmitted virus type 1 and 2 infection is common among blood donors and patients with liver disease in Italy. The pathogenic potential of transfusion transmitted virus type 1 and 2 in nonA-nonC hepatitis patients is unlikely but further studies are needed to evaluate the epidemiological and clinical impact of other transfusion transmitted virus subtypes.

Quantitative analysis of hepatitis B virus DNA by real-lime amplification.

Diagnostic assays allowing the quantification of hepatitis B virus (HBV) DNA over a wide range of concentrations are important for monitoring patients during antiviral therapy. The aim of this study was to develop a new real-time method for HBV DNA quantification. Primers and probe were selected in a highly conserved region of the HBV S gene, and a plasmid containing the pre-S/S region was used as a standard. Linear quantification of the standard was obtained between 10 and 10(9) copies/reaction, with high correlation between ayw and adw genomes (P<0.001). HBV DNA was detected in serial dilutions of a high-titer serum sample with linear results until 2.4 x 10(3) copies/ml. One hundred eight serum samples positive for hepatitis B surface antigen were tested in both the real-time assay and the Digene Hybrid Capture assay (Digene, USA). HBV DNA could be detected by both assays in 70 samples, with significant correlation of results (P<0.001). Results for 38 samples were below the sensitivity limit of the Digene assay, but they could be quantified by the real time polymerase chain reaction assay. These results show that real-time polymerase chain reaction allows sensitive, rapid and linear quantification of HBV DNA in serum.

Hepatitis C virus (HCV) RNA determination after two weeks of induction interferon treatment is an accurate predictor of nonresponse: comparison of two treatment schedules.

The aim of this study was to analyze of HCV kinetics during interferon treatment administered daily or three times weekly. Seventy-seven naive patients were randomized to two treatment courses starting with four weeks of high-dose interferon administered daily or three times weekly. Twenty-two patients (28.6%) achieved end-of-treatment response and nine (11.7%, four of whom received daily induction) sustained response. The initial decline of viral load was sharper in patients receiving daily induction, but the rates of early RNA clearance were independent of treatment schedule, being higher in patients with genotype non-1. Detectable HCV RNA during treatment predicted nonresponse more significantly than high pretreatment viral load or genotype 1. HCV RNA at week 2 was the best predictor (100% sensitivity in patients receiving daily induction). In conclusion, daily induction increased the HCV decline slope, but not the rate of virological response. HCV RNA at week 2 reliably identified nonresponders.

Influence of maternal CD4 levels on the predictive value of virus load over mother-to-child transmission of human immunodeficiency virus type 1 (HIV-1). Study Group for Vertical Transmission.

Forty-four anti-HIV seropositive pregnant women were enrolled in a study of maternal factors related to mother-to-infant human immunodeficiency virus type 1 (HIV-1) transmission. HIV-1 infection was documented in 11 of 45 infants (24.4%). Obstetric factors, maternal CD4 counts, and disease stage were not related to the risk of transmission. HIV-1 RNA levels at delivery were significantly higher in mothers who transmitted the infection (P = .024). A strong relationship between viral load and risk of transmission was observed in women with stage A1 (P= .006), but not in those with stages A2-A3. These results suggest that vertical transmission of HIV-1 is multifactorial and that viral load plays a major role in mothers with early-stage HIV-1 infection.

Prospective study of mother-to-infant transmission of hepatitis C virus (HCV) infection. Study Group for Vertical Transmission.

Seventy-five women with anti-hepatitis C virus (HCV) antibody were enrolled prospectively during pregnancy or at delivery for study of mother-to-child transmission of HCV. Twenty-three women were coinfected with the human immunodeficiency virus (HIV). Seventy babies were monitored for at least 6 months. HCV infection was diagnosed in six infants (8.6%), four of whom were born to anti-HIV-positive mothers. HCV RNA was first detected between 2 and 6 months, and the genotypes of infected babies matched those of their mothers (type 1: n = 4; type 3: n = 2). Identical master sequences of the hypervariable region (HVR1) were detected in a mother-infant pair. In three babies coinfected with HCV and HIV, anti-HCV disappeared between 2 and 7 months, being persistently negative in two cases monitored for 11 and 26 months. Transmitting mothers did not differ significantly from those who did not transmit the infection with anti-HIV, HCV genotypes, and viral load at delivery, but had lower rate of reactivity to C100 by the recombinant immunoblot assay (RIBA) (P < .01). This prospective study confirms transmission of HCV from anti-HIV-negative mothers (4.4% in this series). Absence of anti-C100 antibodies at delivery is apparently related to increased risk of vertical transmission. Seronegative HCV infection can be observed in children coinfected with HIV.

Virological response to interferon treatment in hepatitis C virus carriers with normal aminotransferase levels and chronic hepatitis.

Hepatitis C virus (HCV) carriers with normal aminotransferase levels often show histological chronic hepatitis. This study was undertaken to determine the effect of interferon (IFN) in such patients. Nineteen HCV carriers with normal aminotransferase activities and chronic hepatitis were randomized to receive IFN-alpha2b (3 million units 3 times weekly for 12 months) or no treatment. Therapy was monitored by qualitative and quantitative determination of viral RNA. Patients who did not clear HCV RNA after 6 months discontinued therapy. In all, 9 patients constituted the control group, while 10 patients were treated. Five of these patients, still viremic after 6 months, stopped IFN. The remaining 5 patients, who cleared the viral RNA within 6 months, completed the 12-month course. Three of these patients relapsed off treatment, and 2 were still free of viremia 12 months after stopping therapy. A transient flare-up of aminotransferase activities was detected in 2 patients during treatment and in 3 patients after. None of the 9 control patients cleared the viral RNA during follow-up. A variable degree of sequence heterogeneity was detected in the hypervariable region before therapy, and IFN treatment decreased sequence diversity in all patients. These results indicate that IFN therapy can be effective in chronic HCV carriers with normal aminotransferase activities, inducing short-term virological response in 3 of 10 patients and sustained response in 2. The effects of treatment on viral load and quasispecies complexity were similar to those reported previously in patients with increased aminotransferase activities.

Effects of interferon treatment on the antiviral T-cell response in hepatitis C virus genotype 1b- and genotype 2c-infected patients.

The viral genotype may influence the response to interferon (IFN) treatment in chronic hepatitis C virus (HCV) infection. To characterize potential mechanisms responsible for this effect, we assessed whether IFN modulation of HCV-specific T-cell responses differs in patients infected by different genotypes. The T-cell response to HCV core protein was sequentially analyzed before and during IFN treatment in two groups of patients chronically infected with HCV genotype 1b (eight patients) or 2c (eight patients). Overlapping 20 mer peptides corresponding to the amino acid sequence of the prevalent viral population identified in the serum of each patient were used for the analysis of the T-cell proliferative response to avoid possible problems caused by amino acid differences between infecting virus and HCV proteins used in vitro. Recombinant HCV core antigen was used in parallel. The level of viremia was monitored by competitive polymerase chain reaction (PCR). The T-cell response to HCV peptides and recombinant core protein detected throughout the follow-up was significantly more vigorous in genotype 2c- than in genotype 1b-infected patients. This difference was the result of a greater enhancement of the T-cell response caused by IFN treatment in genotype 2c- compared with genotype 1b-infected patients. The different IFN modulatory effect on T cells from genotype 1b- and genotype 2c-infected patients illustrates an aspect of the virus-host interaction, which may contribute toward the explanation of why different genotypes differ in responsiveness to IFN treatment.

Outbreak of hepatitis C virus infection in patients with hematologic disorders treated with intravenous immunoglobulins: different prognosis according to the immune status.

The influence of immunodeficiency on the course of hepatitis C virus (HCV) infection is still debated, although a worsening effect has been suggested. We compared the characteristics of hepatitis C in two groups of hematologic patients with different levels of immunocompetence who acquired the same virus strain after treatment with contaminated intravenous immunoglobulins (IVIG). Indications for IVIG therapy were idiopathic thrombocytopenic purpura (ITP) in six patients and hypogammaglobulinemia in 7 patients with various hematologic disorders, who were defined immunodeficient (ID). Infection rate was 100%. Five ID patients never developed HCV antibodies despite serum HCV-RNA positivity. The same HCV genotype was shown in 10 patients tested. Moreover, E1-E2 gene partial nucleotide sequencing, performed in four patients, showed identical or closely related amino acid sequences, thus strongly supporting the hypothesis of a common source of infection. Clinical acute infection did not differ significantly between the two groups, but subsequent liver failure developed in five of the seven ID patients and in none of the ITP patients (P = .04). Liver biopsy, performed in three cases, documented HCV as the only cause of liver damage. Six ID patients died, with liver disease being the primary cause of death in four cases and a contributory cause in two cases. Their median survival after IVIG was 12 months, significantly worse than that of ITP patients (P = .0028). We conclude that immunodeficiency markedly worsens the course of IVIG-acquired HCV infection in hematologic patients.

Virological characterization and liver histology in HCV positive subjects with normal and elevated ALT levels.

We analyzed HCV genotype and RNA titer in 36 chronically infected subjects, 20 with persistently normal or near-normal alanine aminotransferase (ALT) activity and 16 with raised ALT activity. All subjects underwent liver biopsy and evaluation of the histological activity index (HAI) by both Knodell's and Ishak's scoring systems. Genotype 2 was detected in most subjects with normal ALT activity, whereas genotype 1 was more frequent among subjects with raised ALT activity. HCV-RNA titer was higher in subjects with increased ALT. Histological evidence of chronic hepatitis was documented in all cases, but higher scores for grading and for staging were associated with increased ALT activity. HCV genotype had no statistical relationship with RNA titer or with liver histology. In logistic regression analysis, viral genotype, RNA titer or with liver histological scores for grading and staging were correlated independently with the ALT profile. The evidence of chronic hepatitis in all subjects with persistently normal ALT activity suggests that healthy HCV carriage is a rare event.

Comparison of competitive and non-competitive reverse transcription-polymerase chain reaction (RT-PCR) for the quantification of hepatitis C virus (HCV) RNA.

A competitive reverse transcription (RT)-nested polymerase chain reaction (PCR) assay for HCV RNA was compared with the Roche Amplicor HCV Monitor assay, based on non-competitive, single step RT-PCR. A total of 83 serum samples were tested in parallel by both assays. All samples could be quantified by competitive RT-PCR (cPCR), whereas seven were negative by the non-competitive assay (ncPCR). The HCV RNA titre of the discordant samples assessed by cPCR was significantly lower than that of the remaining 76 (P < 0.001). Absolute HCV RNA titres were higher by cPCR than by ncPCR (P < 0.001), even if the results of the two methods were statistically correlated (P < 0.001). HCV RNA titre tested by cPCR was not significantly different between samples infected with genotype 1 or 2. However, values obtained by ncPCR were higher in samples with genotype 1 (P < 0.001). Furthermore, all seven discordant samples were infected with genotype 2. When both methods were used to measure serial dilutions of standard HCV RNA, we observed a bias to lower measurements with the ncPCR kit. This study shows a good correlation between the results of two PCR-based methods for the quantification HCV RNA. However, the degree of sensitivity and the absolute HCV RNA titre measured may vary according to the assay used.

Hepatitis C virus genotypes in northern Italy: clinical and virological features.

We analyzed the characteristics of subjects from the same area who were infected with hepatitis C virus genotypes 1 through 4 and subtypes 1a and 1b. Our data are consistent with a rapid evolution in the epidemiology of HCV genotypes and argue against different pathogenic potentials for genotypes 1b and 2.

Molecular analysis of mixed infection with hepatitis C virus and human immunodeficiency virus in a patient infected simultaneously.

A case of simultaneous infection with HIV and HCV characterized by a rapidly progressive clinical course was studied retrospectively over 3.5 years. Molecular analysis indicated interference between HIV and HCV and between HCV subtypes 1a and 1b. An ineffective immune response was suggested by the persistence and sequence conservation of the HCV HVR1 variants isolated during the follow-up.

Emergence of hepatitis B virus S gene mutant in a liver transplant recipient.

Immunological and genomic analysis of the "a" determinant was carried out in seven patients with concurrent HBsAg and anti-HBs, four of whom were immunized against hepatitis B virus at liver transplant, two with histologically characterized chronic hepatitis B virus infection, and one HBsAg healthy carrier. The immune reactivity of the HBsAg "a" determinant was evaluated by binding to specific monoclonal antibodies, and the corresponding genomic sequence was studied by differential hybridization in microtiter plates and nucleotide sequence analysis. A double mutation generating an amino acid change (glycine to lysine) at residue 145, able to impair recognition by monoclonal antibodies, was observed in the post-transplant serum from one patient. No significant alteration of the "a" determinant sequence or reactivity was detected in the other patients. Amino acid residue 145 appears therefore to be critical for the recognition by anti-HBs antibodies. A previously undescribed glycine to lysine substitution at this level interferes with the immune reactivity of the "a" determinant.

Quantification of hepatitis C virus RNA by competitive amplification of RNA from denatured serum and hybridization on microtiter plates.

The direct detection of hepatitis C virus (HCV) RNA by PCR is widely used to determine the presence of circulating virions. The most relevant limit of this approach is the lack of quantitative information about the viral titer. We report a technique of competitive amplification allowing the estimation of HCV RNA copy number in biological samples. We constructed a standard competitive RNA template containing only two point mutations compared with its wild-type counterpart. The competitor was added in titrated amounts to the target RNA, and the mixture was then reverse transcribed and amplified in the same reaction tube. The relative amounts of target and competitor were determined by differential hybridization on microtiter plates with nonradioactive probes. The evaluation of HCV RNA titer required a single coamplification with the competitor and could be read from a standard curve. Furthermore, this method proved suitable for amplification of HCV RNA directly from serum, thus avoiding the intrinsic variability of the RNA extraction step.