Mutations in the nonstructural 5A region of hepatitis C virus and response of chronic hepatitis C to interferon alfa

BACKGROUND & AIMS: Mutations in hepatitis C virus (HCV) nonstructural protein 5A (NS5A) may correlate with response to interferon in Japanese patients with chronic hepatitis C. The aim of this study was to examine whether these findings could be expanded to European patients infected with genotypes associated to low (1b) or high (3a) response rates. METHODS: Pretreatment serum samples of 66 patients with chronic HCV infection, 48 infected with genotype 1b and 18 with 3a, were analyzed. RESULTS: Among patients infected with genotype 3a, 1 of 7 long-term responders and none of 11 nonresponders showed NS5A amino acid mutations. Among patients infected with genotype 1b, all 7 long-term responders, but also 27 of 41 nonresponders, showed NS5A mutations. There was no correlation between number of mutations and response to therapy. In 10 patients, sequences obtained before and after treatment were compared and failed to show any change. Serum HCV RNA levels did not differ between patients with and without mutations in NS5A sequence. CONCLUSIONS: No significant correlation was found in patients infected with genotypes 1b or 3a between NS5A sequence and response to interferon alfa. NS5A mutations do not correlate with viral load. Changes in this region were not found during interferon alfa treatment.     

Effectiveness of relative low-dose interferon treatment for patients with chronic hepatitis C

To demonstrate effectiveness by relative low-dose of interferon treatment for patients with chronic hepatitis C, we investigated the histological activity index (HAI) score of liver tissue, hepatitis C virus (HCV) genotype by polymerase chain reaction (PCR) with type-specific primers, HCV RNA levels by competitive PCR, serum aminotransferase, sex, age, history of blood transfusion and history of hepatitis in patients with chronic hepatitis C prior to treatment. Twenty-two patients were treated with human lymphoblastoid interferon (3 MU daily, 2 weeks, 3 MU thrice a week, 6 weeks, 1.5 MU thrice a week, 16 weeks) for 24 weeks, of whom 10 (45.5%) were responders within a 6 month follow-up. HAI score before treatment was significantly lower in responders than in a combined group of relapse patients and nonresponders (7.5 +/- 3.4 vs. 12.0 +/- 3.1, p < 0.01). The prevalence of responders in patients with genotypes III and IV was significantly higher than in those with genotype II (85.7% vs. 21.4%, p < 0.05). HCV RNA level (logarithmic transformed copy per 50 microliter of serum) was significantly lower in responders than in a combined group of relapse patients and nonresponders (4.8 +/- 1.2 vs. 5.7 +/- 0.8, p < 0.05). In case of other pretreatment factors, there were no significant differences between responders and a combined group relapse patients and nonresponders. Thus severe histological changes in the liver, HCV genotype II and high HCV RNA levels are markers of unfavorable effects of interferon treatment for patients with chronic hepatitis C.     

Large dose natural interferon alpha therapy for patients with chronic hepatitis C

To evaluate the efficacy of large dose interferon treatment for patients with chronic hepatitis C virus (HCV) infection, we studied 99 Japanese patients treated with either 6 million units (MU) or 9 MU natural interferon alpha. Serum samples were tested for HCV RNA by polymerase chain reaction (PCR). HCV RNA genotypes were determined by PCR with type-specific preimers, and the HCV RNA level was measured by competitive PCR. HCV RNA was detected in all patients, prior to the initiation of treatment. We examined interleukin-1 receptor antagonist (IL-1 Ra) by enzyme-linked immunosorbent assay. Forty-four patients were treated with 9 MU natural interferon alpha for 24 weeks (group A), and fifty-five patients were treated with 6 MU natural interferon alpha for 24 weeks (group B). There were no significant differences in HCV RNA levels, HCV RNA genotype or histological activity index (HAI) score between the two groups. Of the 94 patients who completed this treatment, nine (23.1%) in group A and 14 (25.5%) in group B sustained elimination of HCV RNA throughout a 6-month follow-up. There were no differences in the rate of complete response when comparing HCV RNA genotype, levels and HAI score and no significant differences in elevation of IL-1 Ra levels between the two groups. Five of group A patients refused further treatment because of severe side effects such as retinal hemorrhage, while no patient in group B had severe side effects. Thus, large dose natural interferon alpha treatment confers no additional benefit to the patient, compared with the current use of a lower dose.     

Plasma vitronectin concentrations in patients with chronic hepatitis C treated with interferon alpha

Benzidine and 4-aminobiphenyl (4-ABP) are promutagenic bicyclic aromatic amines that are activated into frameshift and base pair substitution mutagens by plant systems. Using the plant cell/microbe coincubation assay, plant-activated benzidine from 0 to 50 microM induced a concentration-response in Salmonella typhimurium. At concentrations above 5 microM, plant-activated benzidine induced frameshift and base pair substitution mutations in the N- or O-acetyltransferase over-expressing strains, DJ460, YG1024, and YG1029. With plant-activated 4-ABP, concentrations above 250 microM induced a significant mutagenic response in strains YG1024 and YG1029. A tobacco cell-free mixture, TX1MX, activated benzidine and 4-ABP into mutagenic metabolites in S. typhimurium strains YG1024, YG1029, and DJ460. The mutagenic sensitivities of plant-activated benzidine and 4-ABP were the same with two different types of plant activation systems, TX1 suspension cells and TX1MX cell-free medium. The plant activation of these aromatic amines is mediated by tobacco cell peroxidase. Plant-activated benzidine and 4-ABP are converted into intermediates that serve as substrates for bacterial or humanacetylCoA: N-hydroxyarylamine N-acetyltransferase to generate the ultimate mutagenic products.     

Responsiveness to interferon alpha treatment in patients with chronic hepatitis C coinfected with hepatitis G virus

BACKGROUND/AIMS: Patients with chronic hepatitis C are often coinfected with the new identified Flaviviridae-like agent, termed hepatitis G virus (HGV). The aim of the study was to investigate the responsiveness of hepatitis G virus to interferon alpha and to evaluate whether a hepatitis G virus coinfection negatively influences the outcome of treatment in chronic hepatitis C. METHODS: One hundred and fifteen patients with histologically proven chronic hepatitis C were treated with interferon alpha and investigated for the presence of hepatitis G virus coinfection by nested polymerase chain reaction with primers from the helicase region of hepatitis G virus. All patients received at least 3 MU (range 3-6) interferon alpha thrice weekly for at least 6 months (mean 8, range 6-12). Polymerase chain reaction products of seven pre- and post-treatment hepatitis G virus positive patients were directly sequenced for identification of sequence variability during the follow-up. RESULTS: Eighteen (16%) patients were coinfected with hepatitis G virus. Although nine (50%) of these patients became HGV RNA negative during interferon alpha therapy, only three patients (17%) remained HGV RNA negative at the end of follow-up (mean 24 months). The rate of sustained response of chronic hepatitis C was not significantly different between patients with hepatitis C virus infection and HCV/HGV coinfection (19% vs 28%). Severity of liver disease as determined by alanine aminotransferase levels, histology and hepatitis C virus viremia was not significantly different in patients with hepatitis C virus or HCV/HGV coinfection. Sequence analysis of the helicase region revealed that our isolates all belonged to the hepatitis G virus and not to the GBV-C like genotype. No amino acid exchanges during the observation period of up to 48 months were observed, indicating that this region is highly conserved. CONCLUSIONS: The responsiveness of hepatitis G virus to interferon alpha in chronic HCV/HGV coinfected patients is similar to that observed in chronic hepatitis C. Hepatitis G virus coinfection seems not to interfere with the efficacy of interferon alpha treatment in patients with chronic hepatitis C.     

Negative strand of hepatitis C virus RNA in the liver of patients with chronic hepatitis C after interferon treatment

In patients receiving interferon therapy for chronic hepatitis C, serum hepatitis C virus (HCV) RNA often reverts from an undetectable to a detectable form after completion of treatment. Detection of the negative strand of HCV-RNA in liver tissue is regarded as an index of viral proliferation. Therefore, we investigated changes in the hepatic negative-strand HCV-RNA following interferon therapy to determine whether this parameter could predict the long-term response to treatment. The subjects of this study were 27 patients with chronic active hepatitis C. Serum positive-strand and hepatic tissue negative-strand HCV-RNA were detected using polymerase chain reaction. At the completion of interferon treatment, serum HCV-RNA was not detected in 21 patients. One year following treatment it remained undetectable in 14 of these patients but it had reverted to a detectable form in seven. The 14 patients in whom hepatic negative-strand RNA was not detected between 2 weeks and 12 months after treatment, had not relapsed after another year. In the 13 remaining patients, negative-strand RNA was found in liver tissue and serum RNA either reverted to a detectable form or remained detectable throughout. From these findings, we conclude that the detection of negative-strand HCV-RNA in liver tissue 2 weeks after the completion of interferon therapy is useful for predicting the long-term effect of therapy.     

Prediction of response during interferon alfa 2b therapy in chronic hepatitis C patients using viral and biochemical characteristics: a comparison

Patients with chronic hepatitis C (n = 103) were treated for 24 weeks with interferon alfa 2b and followed up for 24 weeks after cessation of therapy (week 48). When hepatitis C virus (HCV) RNA at week 48 was used to assess interferon response, 15 (14.6%) were virological complete responders, and all have remained HCV RNA negative for a mean of 3 years. At week 48, 3 of 15 virological complete responders had elevated alanine transaminase (ALT) values. When serum ALT level was used at week 48 to determine response to interferon, 20 (19.4%) were biochemical complete responders. However, 8 of the 20 patients with normal ALT levels were HCV RNA positive at week 48, and 7 of these individuals have had a recurrence of elevated ALT levels within 3 years after cessation of treatment. These findings indicate that measurement of HCV RNA was more accurate than ALT in determining true responses to interferon therapy. Identification of nonresponders early during the course of interferon treatment showed that an elevated ALT level at week 12 was 92% predictive (odds ratio 3.7) but misidentified 33% (5 of 15) of the patients who were virological complete responders at week 48. In contrast, a positive HCV RNA at week 12 of treatment was 98% predictive (odds ratio 35.5) and misidentified only 6.7% (1 of 15) of the virological complete responders. Thus, positive HCV RNA at week 12 of therapy was more accurate in identifying eventual virological nonresponders than measurement of ALT at this time. Termination of interferon therapy in patients who were HCV RNA positive at week 12 would result in a 27% reduction in the direct medical costs and keep patients from undergoing unnecessary treatment. Therefore, testing for HCV RNA at week 12 to identify nonresponders and then discontinuing their treatment is practical, cost-efficient and beneficial both to patients and to third-party payers.     

The incidence of hepatocellular carcinoma in patients with chronic hepatitis C after interferon treatment

To determine whether serum hepatitis C virus (HCV) RNA disappearance after interferon (IFN) treatment prevents development of hepatocellular carcinoma (HCC), we evaluated retrospectively the incidence of HCC in patients with chronic hepatitis C. A total of 213 patients were monitored for more than 6 months after completion of IFN treatment. Sixty-three of the 213 patients (29.6%) achieved a complete response (CR) to treatment and 150 (70.4%) had no response (NR). HCC developed in 12 (5.6%), all of whom were NR. Logistic analysis showed age, alpha -fetoprotein, and staging of histological finding before IFN treatment were independent factors to development of HCC. The fact that there was no HCC development from CR provides a basis for IFN treatment in chronic HCV infection.     

Cellular immunity to hepatitis C virus core protein and the response to interferon in patients with chronic hepatitis C

To investigate the involvement of T-cell response against hepatitis C virus (HCV) antigens in viral clearance after interferon therapy, we measured interleukin-2 (IL-2) production by peripheral mononuclear cells in response to HCV core in patients with chronic hepatitis C. In a cohort of 43 patients, we investigated the frequency of circulating core-specific T-helper (Th) cell precursors by the limiting-dilution assay, and in a second cohort of 60 patients, we analyzed the response to specific core epitopes using 52 synthetic 15-mer overlapping peptides. We observed that the frequency of core-specific Th cell precursors was significantly higher in patients with sustained biochemical and virological response (SR) after interferon (IFN) therapy (median, 1/55,736) than in untreated patients (1/274,023) or that in patients who remained viremic after completion of the treatment-nonresponders (NR) plus transient responders (TR) (1/1,909,972). Patients who failed to respond to IFN (NR) and those who relapsed after IFN discontinuation (TR) had a similarly low number of precursors. The number of core peptides recognized by SR, TR, NR, UT, and healthy controls was 8.2 +/- 1.5, 6.5 +/- 1.2, 2.0 +/- 0.5, 2.7 +/- 0.9, and 0.3 +/- 0.2, respectively. In SR, the intensity of the proliferative response to core peptides as estimated by the summation of stimulation indexes (sigmaSI) was significantly higher than in NR and than in UT, but not different from that of TR. Our results indicate that both expansion of HCV-specific Th cell precursors and Th cell recognition of multiple core epitopes seem to be important in the elimination of HCV after IFN therapy.     

Chronic hepatitis C and interferon alfa therapy: predictors of long term response

OBJECTIVE: To identify independent patient, disease and viral characteristics that predict a sustained biochemical or viral response to interferon alfa therapy in patients with chronic hepatitis C. Design: Comparison of interferon responders and non-responders by univariate and multivariate analysis. SETTING: The hepatitis clinic of the Alfred Hospital, Melbourne (a tertiary referral hospital), between July 1989 and June 1994. SUBJECTS: All patients with chronic hepatitis C who were treated with interferon alfa (IFN-alpha; 3 million IU, three times a week or more) for at least 12 weeks. OUTCOME MEASURES: Patient demographic and epidemiologic characteristics, pretreatment serum alanine aminotransferase (ALT) and 2-gamma-glutamyl transpeptidase (GGT) levels, histological grading of hepatic steatosis, necroinflammatory activity and fibrosis, serum hepatitis C virus (HCV) RNA titres and genotype and post-treatment serum ALT levels and presence of HCV RNA. Results: Of 58 patients, 13 (22%) had a sustained (six months or longer) biochemical response to IFN-alpha therapy, including 12 (21%) with a sustained viral response. Univariate analysis showed that young patients with a normal serum GGT level, grade 0-1 steatosis and fibrosis, low viral titre and infection with genotypes 3a and 2a were more likely to have a sustained response. Infection with genotypes other than 1a and 1b was the only independent variable associated with both a sustained biochemical and viral response. After adjusting for genotype, a hepatic fibrosis grade of 0-1 was also independently associated with viral response. This logistic regression model accurately predicted the virological response in 80% of cases. Conclusion: In Australian patients with chronic hepatitis C, a sustained viral response to IFN-alpha therapy is most likely in those infected with a genotype other than 1a or 1b and with minimal hepatic fibrosis.     

Suicidal impulses in patients with chronic viral hepatitis C during or after therapy with interferon alpha

OBJECTIVE: The objective of this study was to evaluate key outcomes of a universal hearing screen/rescreen program for all births with transient evoked otoacoustic emissions in all 8 maternity hospitals in the state of Rhode Island over a 4-year period. STUDY DESIGN: This was a retrospective analysis of the hearing screen/rescreen refer data collected prospectively for 53,121 survivors born in Rhode Island between January 1, 1993, and December 31, 1996. Primary outcomes included the first-stage refer rates, rescreen compliance, diagnostic referral rates, identification rates, and the age of amplification. RESULTS: During this 4-year time period 11 infants were identified with permanent hearing loss, resulting in an impairment rate of 2 per 1000. The mean age of hearing loss confirmation decreased from 8.7 months to 3.5 months, and the age at amplification declined from 13.3 months to 5.7 months. CONCLUSION: We conclude that time and experience are important factors in the development and refinement of a universal hearing screen program. Hearing screen outcome data collected over a 4-year period in Rhode Island reveal a steady improvement in the percent of infants completing the 2-stage screen process, the stage 1 and stage 2 refer rates, compliance with rescreen and diagnostic testing, and significant improvement in the age of identification and age of amplification.     

Evaluation of interferon treatment in cirrhotic patients with hepatitis C

The aim of this study was to examine the effects of interferon on cirrhotic patients with hepatitis C and the incidence of adverse reactions. The subjects were 35 cirrhotic patients, and 29 chronic active hepatitis patients without cirrhosis (CAH) served as controls. The cirrhotic patients received 3 or 6 million units of human lymphoblastoid interferon daily for one or two weeks and then three times a week for 22 or 23 weeks, while the CAH patients received 6 million units daily for 2 weeks and then three times a week for 14 or 16 weeks. Discontinuation of interferon treatment or dose reduction was required in the 7 cirrhotic patients. The most frequent reason was thrombocytopenia. Dose reduction alone was necessary in two CAH patients. Five cirrhotic patients (14.3%) and nine CAH patients (31.0%) were classified as complete responders to interferon treatment. In all five complete responders with cirrhosis, the hepatitis C virus RNA level before treatment was less than 5 log copies/50 microliters. The results of this study confirm the beneficial effect of interferon in selected patients with cirrhosis on basis of pre-treatment virus levels and platelet count.     

The effect of HLA alleles on response to interferon therapy in patients with chronic hepatitis C

The objective of this study was to determine the degree of leukocyte activation, as measured by cytokine release, in circulating blood during experimental extracorporeal circulation. Complete in vitro extracorporeal membrane oxygenation (ECMO) circuits were used, and 9 experiments were performed. Whole blood stored at 37 degrees C was used as the control. Blood samples were withdrawn before the start of perfusion and at 24 h of perfusion. Statistically significant releases of interleukin (IL)-1beta, IL-8, and IL-1 receptor antagonist were observed in the perfusion circuits compared to both the control blood and baseline values. Also, increases in plasma tumor necrosis factor (TNF)alpha and IL-6 were seen after 24 h of perfusion although these changes did not reach statistical significance. These results indicate that extracorporeal circulation induced leukocyte activation and cytokine release. These reactions might, as an additional trauma, deteriorate the situation in an already severely ill patient. A search for methods to counteract this untoward activation seems warranted.