Carla Casarin

Long-term follow-up of non-A, non-B (type C) post-transfusion hepatitis.

Abstract One hundred and thirty-five patients who developed non-A, non-B post-transfusion hepatitis mostly after cardiac surgery, were followed for a mean (±S.D.) of 90±41 months (range: 13–180) to evaluate clinical and histological outcome. Thirty-one cases resolved within 12 months, while 104 (77%) progressed to chronicity. Twenty-one of 65 (32%) biopsied patients developed cirrhosis at the end of the follow-up, and one further progressed to hepatocellular carcinoma. One patient had a complete histological remission (1%). The remaining cases had chronic active (37%), chronic persistent (27%) or chronic lobular hepatitis (3%). About half of the cases with cirrhosis developed portal hypertension, and three of these died due to esophageal varices hemorrhage, one due to liver failure, and one due to hepatocellular carcinoma. Out of 26 patients with the initial histologic diagnosis of chronic hepatitis that were rebiopsied during follow-up, 13 (50%) progressed to cirrhosis. These patients were significantly older than patients who did not develop cirrhosis (mean age 57 and 45 years respectively; p During acute hepatitis anti-HCV was positive in all but one of the 114 patients tested. Percentages were similar for patients who recovered (95%) and those who developed chronic hepatitis (100%). However, during follow-up, 71% of the 1st generation and 21% of the 2nd generation ELISA test patients with acute resolved hepatitis became anti-HCV negative, while the same figures in chronic cases were only 8.5% ( p p =0.012). This suggests a correlation between anti-HCV antibody activity, hepatitis C virus replication, and the development of chronic liver disease.

Persistent Hepatitis C Viremia Predicts Late Relapse after Sustained Response to Interferon-α in Chronic Hepatitis C

Chronic infection with the hepatitis C virus (HCV) is a major cause of cirrhosis and hepatocellular carcinoma worldwide. In many countries, interferon- therapy is the only licensed method for treating this condition. It has been licensed on the basis of randomized, controlled trials that have shown its effectiveness in reducing disease activity and virus replication [1, 2]. However, few treated patients achieve a complete response that is maintained after therapy, and many others have a transient response or no response at all [3, 4]. Serum alanine aminotransferase activity is the conventional marker used to monitor response to therapy, and sustained biochemical response is defined as normalization that lasts for 6 to 12 months after cessation of interferon- therapy. More recently, serum HCV RNA testing has been recognized as an important component of assessing response to treatment; patients who respond at the biochemical level and maintain normal alanine aminotransferase levels after therapy may remain viremic, indicating that interferon- has suppressed liver disease activity but has not eradicated the virus [5]. There is increasing agreement that patients with biochemical response should be tested for serum HCV RNA to more accurately define the effect and efficacy of treatment. However, little is known of the clinical significance and long-term outcome of persistent viremia with normal alanine aminotransferase activity after interferon- therapy. It is still unclear whether this profile reflects delayed virus clearance or persistence of a noncytopathic type or load of HCV or whether it should instead be regarded as a predictor of reactivation of liver damage and progression of disease. To clarify these issues, we investigated the long-term outcome of a large series of consecutive patients with chronic hepatitis C who had persistently maintained normal alanine aminotransferase activity for at least 1 year after cessation of interferon- therapy. Methods Patients Between January 1990 and November 1993, 440 consecutive patients with chronic hepatitis C were treated with interferon- therapy in three consecutive randomized trials [6, 7] using different schedules of treatment. Inclusion and exclusion criteria were identical for the three studies, and the clinical characteristics of patient subgroups were similar [8]. Enrolled patients were between 18 and 65 years of age and had a persistent (lasting more than 6 months) elevation of alanine aminotransferase levels of more than twice the upper limit of normal positive result on testing for antibody to HCV, and histologic evidence of chronic hepatitis with or without Child grade A cirrhosis. Patients who were also infected with hepatitis B virus or human immunodeficiency virus who had autoimmunity or comorbid conditions were not included. The total interferon- dose ranged from 234 MU to 740 MU, administered for 6 or 12 months. Alanine aminotransferase levels were tested monthly during therapy. Complete biochemical response was defined as normalization of liver enzyme levels during therapy, maintained until withdrawal of therapy. After cessation of interferon- therapy, patients who had responded were monitored for serum alanine aminotransferase at 3-month intervals; sustained biochemical response was defined as enzyme levels that were normal for as long as 12 months after therapy. Of 440 treated patients, 259 (59%) showed complete biochemical response during treatment, but 141 (54%) of the 259 had relapse within 12 months after therapy was discontinued. Of the 118 patients with alanine aminotransferase levels that were normal for as long as 12 months after therapy, 107 could be tested for serum HCV RNA and followed prospectively for an additional 6 to 36 months (mean follow-up after therapy, 35 18 months). In the remaining 11 patients who had responded to interferon-, serum samples were not available for HCV RNA testing 1 year after therapy. Testing Serum HCV RNA was tested using nested polymerase chain reaction [9]; the sensitivity limit of the assay was between 103 and 104 copies/mL. The virus genotype was identified using spot hybridization with genotype-specific oligonucleotide probes [9] and was classified according to the system of Simmonds and colleagues [10]. Liver biopsies were done during the 6 months before therapy. Sixty-three of the 107 patients with persistently normal alanine aminotransferase levels consented to have liver biopsy, done 8 to 12 months after cessation of interferon therapy. The histologic evaluation was made by a pathologist who was blinded to clinical data and to the sequence in which biopsy specimens had been obtained. Continuous variables were compared by using the t-test, and proportions were compared by using the chi-square test. The probability of maintaining a sustained response to therapy in different groups was analyzed by using the Kaplan-Meier method and was compared by using the log-rank test. Results One year after therapy, serum HCV RNA was detectable in 27 (25%) of the 107 patients with sustained biochemical response; 80 patients were negative for HCV RNA. Retrospective analysis of stored serum samples showed that 13 of 27 (48%) patients positive for HCV RNA were viremic at completion of therapy. In the remaining 14 patients (52%), HCV RNA had reappeared either during the first 6 months (6 patients) or the second 6 months (8 patients) after therapy. Patients who were positive for HCV RNA differed substantially from those who were negative for HCV RNA; the former were older and were treated with a smaller total dose of interferon-. Furthermore, a statistically significant difference was seen in the distribution of virus genotypes; HCV RNA-positive patients had a higher prevalence of HCV genotype 2 and a lower prevalence of HCV genotype 3 than did HCV RNA-negative patients. After treatment, a liver biopsy could be done while alanine aminotransferase levels were normal in 14 HCV RNA-positive patients and 49 nonviremic patients. Chronic hepatitis with piecemeal necrosis was seen in 8 of 14 (57%) HCV RNA-positive patients; the remaining patients had chronic hepatitis with portal inflammation but no periportal necrosis (4 patients) or minimal changes (2 patients). In contrast, active histologic findings were seen in only 6 of 49 (12%) HCV RNA-negative patients (P = 0.01); the remaining patients had chronic hepatitis with portal inflammation but no piecemeal necrosis (25 patients) or either normal liver histologic findings or minimal changes (18 patients). When the 107 patients were followed prospectively beyond the first year after therapy had been discontinued, the outcomes in viremic and nonviremic patients clearly differed. All HCV RNA-negative patients maintained normal alanine aminotransferase levels and remained negative for HCV RNA, whereas a biochemical relapse was seen in 8 of the 27 (30%) viremic patients. The estimated probability of a return of elevated alanine aminotransferase levels 4 years after cessation of treatment was 53% in viremic patients and 0% in the negative patients (P < 0.001) (Figure 1). No characteristics could be used to distinguish patients with early (within 12 months) or late (beyond 12 months) relapse after therapy. However, when all patients who had relapse were compared with all patients who had sustained biochemical and virologic long-term responses, the former were older (P < 0.01), had a longer duration of disease (P < 0.01), and had a different distribution of HCV genotype (P < 0.05) with increased prevalence of genotype 1 and decreased prevalences of genotypes 2 and 3. Figure 1. Probability of maintaining normal serum alanine aminotransferase levels at long-term follow-up (Kaplan-Meier curves). P Discussion Our study shows that serum HCV RNA testing is clinically useful when done 1 year after interferon- therapy in patients with chronic hepatitis C who have responded to therapy with persistently normal aminotransferase levels. If the test result is negative 1 year after therapy, the response is permanent; in patients who are still viremic 1 year after therapy, liver disease is often still histologically active and the risk for a late reactivation of hepatitis is substantial (53% of patients in our study had probability of reactivation by 4 years after therapy). Our results are consistent with previous reports on the discrepancy between biochemical and virologic responses to interferon- in chronic hepatitis C and on the possibility of late relapse after a prolonged biochemical response [11, 12]. Only one HCV RNA test was done 1 year after therapy, and we cannot exclude the possibility of some false-negative results caused by intermittent viremia or HCV-RNA levels below the sensitivity of our assay. Furthermore, viremia may not accurately reflect the presence or absence of the virus in the liver. Despite these limitations and the fact that not all of our patients who responded to interferon could have a liver biopsy done after treatment, our results clearly indicate that serum HCV RNA should be tested in all patients who have developed a sustained biochemical response to interferon- to establish whether the response is complete and permanent. Patients who are negative for HCV RNA may be considered cured. Viremic patients with normal alanine amino-transferase levels should be followed carefully well beyond the first year after cessation of therapy, because many will eventually relapse. Studies must be done to determine whether treatment with larger interferon- doses could reduce the prevalence of residual viremia after therapy and whether patients who remain positive for HCV RNA after interferon- therapy could benefit from early retreatment. Dr. Belussi: Divisione Malattie Infettive, O. Le Grazie, 5501 San Marco, 30124 Venezia, Italy. Dr. Martinelli: Divisione Medica, O.C. Cittadella (PD), via Circonvallazione, 35013 Cittadella (PD), Italy.

Treatment with interferon(s) of community-acquired chronic hepatitis and cirrhosis type C

Two hundred and thirty-four patients with chronic non-A, non-B hepatitis, 86% positive for anti-HCV by ELISA, were treated with recombinant interferon-alpha 2a or with natural (human-leukocytes-derived) interferon-alpha using different dosage and periods of administration. Interim analysis of follow-up data indicate that 65-70% of patients treated initially with 6 MU, thrice weekly, of recombinant interferon-alpha 2a achieved a complete biochemical response (normalization of alanine aminotransferase: ALT) during therapy compared to 56-58% of those treated with 3 MU, thrice weekly, of recombinant or natural interferon-alpha. A 12-month schedule of interferon administration appeared superior to a 6-month schedule in reducing the probability of reactivation of liver disease after therapy withdrawal, although further data are needed to confirm such a conclusion. The probability of response to interferon in terms of maintaining normal ALT after withdrawal did not appear to be influenced by sex, while it was significantly higher in patients aged below 45 years and in those without cirrhosis.

Interferon antibodies in patients with chronic hepatitic C virus infection treated with recombinant interferon alpha-2α

Patients treated with alpha-2a interferon for chronic hepatitis C may produce anti-interferon antibodies whose effect, if any, on the individual response to therapy has not been fully clarified. The prevalence and kinetics of anti-interferon, including those of neutralizing type, have been studied in 60 patients with chronic hepatitis C enrolled in a randomized controlled trial of recombinant alpha-2a interferon. Thirty patients received interferon while 30 were untreated controls. Two different methods, an enzyme immunoassay and an antiviral neutralization bioassay, were used and serial serum samples from each patient were analyzed. Enzyme immunoassay-positive anti-interferon appeared in 60.7% of treated patients within 6 months of therapy; antiviral neutralization bioassay-positive anti-interferon appeared in 52.9% of these enzyme immunoassay-positive patients, and was associated with high enzyme immunoassay reactivity and long-term persistence. Anti-interferon was detected in 75% of patients showing no response to interferon. Antibodies were also detected in three out of six patients who showed alanine aminotransferase normalization persisting up to the end of treatment and in 8 out of 14 patients who showed an initial marked reduction or even normalization of alanine aminotransferase, followed by reactivation of liver damage during treatment. Interestingly, patients who became anti-interferon positive before complete alanine aminotransferase normalization later showed reactivation of liver damage independently of interferon dose reduction, while patients who became positive for anti-interferon after complete alanine aminotransferase normalization either did not reactivate or did so only after interferon dose reduction.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between serum HCV markers and response to interferon therapy in chronic hepatitis C: Evaluation of HCV genotypes during and after long-term follow-up

Hepatitis C virus is the most frequent cause of chronic non-A, non-B hepatitis, and the antibodies to structural and nonstructural proteins encoded by viral genome have been suggested to be markers of ongoing HCV infection. We studied the behavior of these antibodies during interferon therapy in 18 patients with chronic hepatitis C and also during a follow-up period of at least four years. A significant decrease of anti-HCV titer was found only in patients who had shown positive response to therapy and all of them were anti-HCV negative at the end of follow-up. Analysis by recombinant immunoblotting assay showed that only anti-c100 were affected by interferon therapy, whereas anti-c22 and anti-c33 were not modified. Using polymerase chain reaction to detect small amounts of HCV genome in serum, we could confirm that the behavior of HCV-RNA during and after interferon therapy is similar to that of anti-HCV and the loss of anti-c100 seems to be closely related to HCV-RNA disappearance from serum. Our patients with chronic hepatitis C were found to be of type 1b and 2, according to the recent score of Simmonds, and the clearance of serum HCV-RNA during treatment and its sustained negative status are closely related to genotype 2 and to long-term positive response to interferon.

Characteristics of hepatitis C virus before and after interferon treatment in patients with ongoing viraemia but sustained biochemical response.

In hepatitis C virus (HCV) infection, persistent viraemia can occur after successful biochemical response to interferon treatment. To assess whether this unusual profile might be due to trivial amounts of remaining virus or to the emergence of less pathogenic HCV strains, pre‐ and posttreatment sera from 27 patients who remained with HCV‐RNA, despite sustained transaminase normalisation after interferon therapy, were investigated. All but one had infection by genotype 2 (P < 0.0001), and levels of HCV‐RNA were not decreased after therapy. Sequence comparison of the 5′ untranslated region revealed mixed viral populations and “not compensatory” nucleotide transitions localised at the stem level of the secondary structure of this region in samples taken before and after treatment. Neither quantitative nor qualitative viral changes, at least for the 5′ untranslated region, are responsible for interferon‐induced biochemical remission in these patients typically infected by genotype 2. J. Med. Virol. 54:7–11, 1998.

Hepatitis C Viremia and Serologic Profile in Post-Transfusion Non-A, Non-B Hepatitis

Serial serum samples from 19 patients with post-transfusion hepatitis C, followed for an average of 8 years, were tested for HCV antibody by individual synthetic peptide-based ELISA (core, NS3/4, El, E2/NS1 and NS5) and four-antigen RIB A, and for HCV-RNA by PCR amplification with nested primers from 5′ noncoding region and type-specific primers from core and NS5 regions. Ten patients progressed from acute to chronic hepatitis and 9 recovered. An expanded antibody pattern seems to correlate with a higher frequency of HCV-RNA-positive patients. The disappearance with time of HCV antibody was observed only in patients who cleared HCV. The persistence of HCV-RNA in 5 of 9 recovered patients suggests the existence of a ‘healthy’ carrier state of HCV. Most patients with chronic hepatitis had type I or II HCV infection, whereas most asymptomatic HCV carriers were infected with type IV HCV.

Long-term Follow-up of Non-A, Non-B (Type C) Post-transfusion Hepatitis

One hundred and thirty-five patients who developed non-A, non-B post-transfusion hepatitis mostly after cardiac surgery, were followed for a mean (+/- S.D.) of 90 +/- 41 months (range: 13-180) to evaluate clinical and histological outcome. Thirty-one cases resolved within 12 months, while 104 (77%) progressed to chronicity. Twenty-one of 65 (32%) biopsied patients developed cirrhosis at the end of the follow-up, and one further progressed to hepatocellular carcinoma. One patient had a complete histological remission (1%). The remaining cases had chronic active (37%), chronic persistent (27%) or chronic lobular hepatitis (3%). About half of the cases with cirrhosis developed portal hypertension, and three of these died due to esophageal varices hemorrhage, one due to liver failure, and one due to hepatocellular carcinoma. Out of 26 patients with the initial histologic diagnosis of chronic hepatitis that were rebiopsied during follow-up, 13 (50%) progressed to cirrhosis. These patients were significantly older than patients who did not develop cirrhosis (mean age 57 and 45 years respectively; p < 0.01). During acute hepatitis anti-HCV was positive in all but one of the 114 patients tested. Percentages were similar for patients who recovered (95%) and those who developed chronic hepatitis (100%). However, during follow-up, 71% of the 1st generation and 21% of the 2nd generation ELISA test patients with acute resolved hepatitis became anti-HCV negative, while the same figures in chronic cases were only 8.5% (p < 0.0001) and 1.4% (p = 0.012). This suggests a correlation between anti-HCV antibody activity, hepatitis C virus replication, and the development of chronic liver disease.