Elisabetta Bernardinello

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.

Evidence for an association between the aetiology of cirrhosis and pattern of hepatocellular carcinoma development.

BACKGROUND Patients with liver cirrhosis are at significant risk of hepatocellular carcinoma (HCC) that may develop as well defined nodular lesions or as more aggressive infiltrating tumours. AIM To compare prospectively risk factors associated with nodular or infiltrating HCC in cirrhotic patients. PATIENTS AND METHODS We studied 370 patients with cirrhosis, followed prospectively by periodic ultrasound (US) of the liver, for a mean period of 74.6 (SD 32.4) months to define the incidence and patterns of HCC development. Patients who developed HCC were compared according to tumour pattern using univariate and multivariate analysis. RESULTS Sixty one (16.5%) patients developed HCC: HCC was classified as nodular in 49 (80.3%) and infiltrating in 12 (19.7%) according to US and computerised tomography (CT) imaging. The five and 10 year cumulative probabilities were 8.1% (95% confidence interval (CI) 5.2%-11%) and 25.2% (15.0–35.4%) for nodular HCC and 2.1% (0.5–3.7%) and 6.9% (2.1–11.7%) for infiltrating HCC. Patients with infiltrating HCC were younger than those with nodular HCC (59.5 v66.2 years, 95% CI 55.2–63.8 and 64.1–68.3 years; p=0.014). Using multivariate analysis, development of nodular HCC was associated with older age (p=0.0002; relative risk (RR) 3.1; 95% CI 1.6–5.2), longer duration (p=0.09; RR 2.6; 95% CI 1.8–3.4), and more advanced stage (p=0.002; RR 2.5; 95% CI 1.3–4.5) of cirrhosis but not with the aetiology of liver disease. In contrast, development of infiltrating HCC appeared to be unrelated to age or disease duration or stage, while it was associated with hepatitis B virus infection (p=0.07; RR 3.96; 95% CI 1.1–5.2) and with hepatitis B/hepatitis C virus coinfection (p=0.0007; RR 16.9; 95% CI 3.8–36.7). CONCLUSIONS In liver cirrhosis, we identified two patterns of HCC developing with distinct risk factors. Nodular HCC was related to the cirrhotic process per se independent of aetiological factors and may depend on the proliferative activity within regenerative nodules, while the infiltrating form of HCC was linked to hepatitis B virus infection and may reflect more direct virus induced carcinogenesis.

Progressive increase of SCCA-IgM immune complexes in cirrhotic patients is associated with development of hepatocellular carcinoma.

About 3–4% of cirrhotic patients develop primary liver cancer every year. Specific serologic markers have not yet been identified for screening of high risk patients. The serpin squamous cell carcinoma antigen (SCCA) is overexpressed in liver cancer and circulating SCCA‐IgM complexes have been described in patients with hepatocellular carcinoma (HCC). The aim of the present study was to assess the behavior of SCCA‐IgM in relation to HCC development in patients with cirrhosis. A retrospective, longitudinal study was conducted in a cohort of prospectively followed cirrhotic patients. Two groups with similar clinical profile at presentation were studied : group A included 16 patients who developed HCC during a median follow up of 4 years; group B included 17 patients who did not develop HCC during the same time interval. Circulating SCCA‐IgM immune complexes were determined using a recently standardized ELISA assay. At presentation similar levels of SCCA‐IgM complexes [mean ± SD: 267.40 ± 382.25 U/ml vs. 249.10 ± 446.90 U/ml, p = 0.9006] and of alpha‐fetoprotein [AFP; 24.11 ± 59.04 IU/ml vs. 10.91 ± 23.34 IU/ml, p = 0.3995] were detected in group A and in group B. The increase over time (ϕ) of SCCA‐IgM, assessed within at least one year before clinical diagnosis of HCC, was remarkably higher in group A than in group B (mean ± SD = 280.05 ± 606.71 (U/ml)/year vs. −37.92 ± 95.94 (U/ml)/year, p = 0.0408), while AFP increase was not significantly different (11.89 ± 23.27 (IU/ml)/year vs. 3.67 ± 11.46 (IU/ml)/year, p = 0.2179). Receiver operating characteristic (ROC) curves were plotted for the rate of change in the levels of both markers and the diagnostic accuracy measured as AUROC was higher for SCCA‐IgM ϕ (0.821) than for AFP ϕ (0.654). In conclusion, the progressive increase of SCCA‐IgM over time was associated with liver tumor development, suggesting that monitoring the behavior of SCCA‐IgM might become useful to identify cirrhotic patients at higher risk of HCC development.

Effects of PEG-interferon alpha plus ribavirin on tryptophan metabolism in patients with chronic hepatitis C.

The currently recommended therapy for chronic hepatitis C (HCV) is a combination of pegylated interferon-alpha (PEG-IFN alpha) and ribavirin. Psychiatric disorders, including depression, are frequent in HCV patients under therapy. We investigated the effect of the antiviral treatment on tryptophan (Trp) metabolism along both serotonin pathway (via 5-hydroxytryptophan, 5-HTP) and kynurenine (Kyn) pathway and on the onset of depressive symptoms in patients with HCV. The key enzyme of the Kyn pathway is indoleamine 2,3-dioxygenase (IDO), an intracellular haem protein enzyme expressed in several tissues. It was also investigated the influence of the therapy with PEG-IFN-alpha-2a or PEG-IFN-alpha-2b plus oral ribavirin and possible differences between genders. Free and total Trp, 5-hydroxytryptophan (5-HTP) and Kyn serum concentrations and the presence of depressive symptoms [Beck Depression Inventory (BDI) scores] were evaluated in 45 patients with HCV infection treated with PEG-IFN alpha-2a or -2b at four different times: baseline (before treatment), 1 and 6 months during therapy, and 3 months after the end of therapy. The concentration of serum total TRP (free+protein bound) as well as that of 5-HTP significantly decreased after 1 and 6 months of therapy, and then returned to baseline values 3 months after the end of therapy, while the levels of free TRP did not vary significantly during and after the therapy. On the contrary, the time course of Kyn markedly arose during treatment, paralleled by a significant increase of [Kyn/Trp]×10(3) ratio, an index used to measure IDO activity. No significant difference was detected between males and females neither between PEG-IFN-alpha-2a or -2b treatment. The BDI scores significantly increased during therapy, and returned to baseline values 3 months after the end of therapy. Our results support the hypothesis that the increased IDO-mediated tryptophan metabolism along the Kyn pathway, leading to plasma Trp depletion and a decline of serotonin pathway, concurs to the development of depressive symptoms observed in HCV patients undergoing IFN-alpha therapy.

Monitoring SCCA-IgM complexes in serum predicts liver disease progression in patients with chronic hepatitis.

Summary.  About 30% of the patients with chronic hepatitis develop a progressive liver disease and one of the most intriguing issues is the detection of noninvasive markers for fibrosis stage and disease progression. High levels of squamous cell carcinoma antigen (SCCA)‐immunoglobulin M (IgM) are detectable in hepatocellular carcinoma and their increase in cirrhotic patients can predict tumour development. As SCCA‐IgM can also be detectable at low percentages in patients with chronic hepatitis, the aim of this study was to assess SCCA‐IgM complexes in relation to disease outcome in this group of patients. An ELISA assay was used to determine the presence of SCCA‐IgM in 188 patients with chronic hepatitis and in 100 controls. An additional serum sample was available after a median period of 6 years in 57 untreated patients: these patients were subdivided in group A, including eight patients with a fibrosis score increase ≥2 in a second liver biopsy and group B, including 49 patients without fibrosis progression during a similar follow up. SCCA‐IgM complexes were detectable in 63 of 188 (33%) patients but in none of the controls. A significant increase of SCCA‐IgM levels over time was observed in patients with fibrosis progression (mean ± SD: 117 ± 200 U/mL/year), but not in those without histologic deterioration (mean ± SD: –8.8 ± 31 U/mL/year, P < 0.0001). In conclusion, monitoring SCCA‐IgM levels over time appears a useful approach to identify patients with chronic hepatitis at higher risk for cirrhosis development.

The pattern of response to interferon alpha (α-IFN) predicts sustained response to a 6-month α-IFN and ribavirin retreatment for chronic hepatitis C

Abstract Background/Aims: In chronic hepatitis C, interferon-alpha (α-IFN) and ribavirin combination therapy improves sustained response compared to α-IFN monotherapy, both in naive patients and in previous α-IFN relapsers, but the efficacy of such therapy remains limited in non-responder cases. The aim of this study was to assess whether the pattern of response to α-IFN alone may predict sustained response to combination therapy during retreatment. Methods: Fifty previous α-IFN relapsers and 50 previous α-IFN non-responders were retreated with a high α-IFN dose (6 MU/thrice weekly for 2 months; induction phase) and then randomised to continue with α-IFN alone (3 MU/thrice weekly) or to receive combination therapy (3 MU/thrice weekly of α-IFN and 1000–1200 mg/daily of ribavirin) for an additional 6 months according to the biochemical response to α-IFN shown after the induction phase. All patients were also evaluated for virological and histological response. Results: Eleven of 25 (44%) relapsers treated with combination therapy and 4/25 (16%) treated with α-IFN alone achieved a sustained response. The corresponding figures among non-responders were 1/25 (4%) and 0/25, respectively. Among 26 patients with a complete ALT and HCV-RNA response after 2 months of α-IFN, sustained response was seen in 11/14 (79%) treated with combination therapy and in 4/12 (33%) treated with α-IFN alone ( p =0.05). On the other hand, of 74 cases still HCV-RNA positive after 2 months of α-IFN alone, biochemical and virological end of therapy response was better with combination therapy (11/36; 30.5%) compared to α-IFN alone (4/38; 10.5%), but only one patient developed a sustained response (1/36; 3%). Conclusions: The retreatment with a 6-month combination therapy was associated with a high rate of sustained response only in patients showing a complete biochemical and virological response to α-IFN alone. Longer retreatment with combination therapy may be needed to achieve a sustained response in patients without a prompt virological response to α-IFN.

Pilot study on the efficacy of intravenous natural β-interferon therapy in Italian patients with chronic hepatitis C and relation to the HCV genotype

Abstract Eight Italian patients with chronic hepatitis C were treated intravenously with natural β-interferon (β-IFN) to assess primary biochemical and virological response and also to investigate the relation to the genotype of infecting HCV. Each patient received 6 MU of β-IFN daily for 6 days a week for a period of 2 months. Five patients (62.5%) showed complete normalization of alanineaminotransferase (ALT) and 6 cases (including all 5 with normal ALT) became HCV-RNA negative. ALT normalization occurred within 7–40 days of therapy. Seven patients were infected by HCV-1b and 4 (58%) of them became HCV-RNA negative with normal ALT at the end of therapy while one patient, infected by HCV-2a, became HCV-RNA negative but maintained elevated ALT values. Six months after cessation of β-IFN all 8 patients were HCV-RNA positive in serum but 3 of them had normal ALT, including one case in whom normalization had occurred after therapy. These results indicate that HCV activity, including that of HCV-1b can be efficiently suppressed by intravenous β-IFN therapy in Caucasian patients with chronic hepatitis C, as previously described in similar cases in Japan. The rate of sustained biochemical and virologic response was, however, lower in our patients compared to what has been described previously, suggesting that further studies are needed to define the optimum regimen to achieve eradication of HCV infection.

Comparison of thrice weekly vs daily human leucocyte interferon-alpha therapy for chronic hepatitis C. TVVH Study Group.

Standard treatment for chronic hepatitis C currently consists of 3–6 million units (MU) of interferon‐α (IFN‐α) given thrice weekly (t.i.w.) for 12 months, obtaining rates of sustained response (SR) that usually do not exceed 15–25%. Some recent reports have suggested that daily administration of IFN‐α may be more efficacious. More than 7 years ago, when standard therapy for hepatitis C was usually given for 6 months, we conducted a randomized clinical trial comparing daily vs t.i.w. treatment. In this study, 149 patients with chronic hepatitis C were randomized to received 3 MU of IFN‐α either t.i.w. for 6 months or daily for 3 months followed by t.i.w. for 3 months. All patients were treated with human leucocyte IFN‐α and were followed‐up for up to 72 months after inclusion. Overall, patients treated daily or t.i.w. had similar rates of virological response after 3 months of induction [24/49 (50%) vs 40/100 (40%)], at the end of therapy [15/49 (31%) vs 36/100 (36%)] and at the end of follow‐up [6/49 (12%) vs 9/100 (9%)]. However, when patients infected with HCV types other than HCV‐1 were studied, there was a trend favouring the daily schedule that was associated with a higher [5/20 (25%) vs 5/48 (10%)] rate of long‐term SR. All patients with a virological response – hepatitis C virus (HCV) RNA negative in serum as determined using the polymerase chain reaction – at 6 months after therapy remained in biochemical and virological remission at long‐term follow‐up, while seven of eight subjects who had normal alanine aminotransferase (ALT) levels but were serum positive for HCV RNA at 6 months, relapsed later, indicating that serum HCV RNA is better than ALT at predicting long‐term cure after IFN‐α therapy in chronic hepatitis C.

Squamous cell carcinoma antigen-1 (SERPINB3) polymorphism in chronic liver disease

BACKGROUND The serpin squamous cell carcinoma antigen (SCCA, SERPINB3) has been found over-expressed in primary liver cancer and at lower extent in cirrhosis and chronic hepatitis. A novel SCCA-1 variant (SCCA-PD), presenting a single mutation in the reactive centre (Gly351Ala), has been recently identified (rs3180227). AIM To explore SCCA-1 polymorphism in patients with HCV infection as single etiologic factor and different extent of liver disease. METHODS One hundred and fourty-eight patients with chronic HCV infection (45 chronic hepatitis, 53 cirrhosis, 50 HCC) and 50 controls were evaluated. SCCA-1 polymorphism was studied by restriction fragment length polymorphism and confirmed randomly by direct sequencing. Circulating SCCA-IgM complex was determined by ELISA. RESULTS SCCA-PD was detected with higher frequency in cirrhotic patients (45.3%, odds ratio=2.62; 95%CI 1.13-6.10, p=0.038) than in patients with chronic hepatitis or in controls (24.4% and 24%, respectively). Intermediate figures were found in hepatocarcinoma (36.0%). SCCA-IgM in serum was lower in patients carrying SCCA-PD than in wild type patients and the difference was statistically significant in cirrhotic patients (mean+/-S.D.=117.45+/-54.45 U/ml vs. 268.52+/-341.27 U/ml, p=0.026). CONCLUSIONS The newly identified SCCA-PD variant was more frequently found in liver cirrhosis, suggesting that patients carrying this polymorphism are more prone to develop progressive liver fibrosis.

Retreatment of chronic hepatitis C (CHC) with sequential interferon-ribavirin combination (IFN-RIBA) therapy

Most patients with CHC have transient response (Relapsers-R) or no response (NR) when treated with IFN. To assess efficacy of IFN-RIBA in these patients, 50 R and 50 NR after IFN therapy were randomized to be retreated with IFN alone or with IFN-RIBA. IFN was given to all cases at 6MU tiw and at 2 mo patients were randomized to continue with IFN alone or to add RIBA 1800-1200 mglday) for 6 mo. ALT and HCV-RNA (PCR) End Theraov Resoonse and Sustained (12 mo) Resoonse are shown in the table. Previous NR (ALT) ETR(RNA) (ALT) SR (RNA) IFN monotherapy (24) 5(21 %I 3(12%) 0 0 IFN-RIBA (241 12(50%) 4(16%) 1(4%1 0 Previnus R IFN monotherapy (26) 1 16(61X) 10(38%) 1 6(23%) 4(15%) IFN.RIBA (28) 1 22(85%1 18(69%) 1 14(54%) 12(46%) IFN-RIBA significantly improved rate of SR in R (P=O.O3) but not in NR, in which only ALT ETR was improved. IFN-RIBA vs IFN improved SR by 27% with HCV-1 and by 4% with HCV-213. SR rates were 27% and 40% respectively with IFN alone and IFN.RIBA in R after 3MU x 6 mo IFN while the corresponding figures for R after higher/longer IFN therapy were 7% and 57%. Addition of RIBA to IFN improved SR only in patients with normal ALT at randomization. In conclusion, IFN-RIBA combination therapy was superior to IFN monotherapy in retreating previous IFN relapsers, particularly those with HCV-1 and those who had already being treated with high dose IFN during the I” cycle. Our schedule of IFN.RIBA was not effective in previous IFN non responders. A NOVEL HEPATITIS E (HEY) ISOLATED FROM A PATIENT WITH ACUTE nA-C HEPATITIS IN ITALY L. Roman& E. Tanzi. A. Zanetti. G. Sl *Viral Discovery Group, Abbott Laboratories, USA Objective: To assess the aetiological role of HEV in acute nA-C hepatitis and to characterize a viral isolate from a patient with no history of travel to areas where HEV is endemic. P&t&s and methods: We studied 216 patients with nA-C hepatitis (negativity for IgM anti-HAV, HBsAg, IgM anti-HBc, anti-HCV, HCV-RNA and exclusion of autoimmunity, alcohol or hepatotoxic drugs). All sera were tested by EIA for both IgG (Abbott Labs) and IgM (in house assay) anti-HEV. Sera and stools (when available) collected during the acute phase were examined by nested RT-PCR using primers derived from the ORFl region. Oligonucleotide primers based on the 5’-end of the ORFl of HEV-US1 were used to identified an isolate from a patient with no known risk factors. Results: 22/216 (10.2%) patients were found HEV-RNA and IgM positive during acute phase (ALT mean peak 2768 IUil, range 396-12290). The acute disease had a benign course with ALT normalization in 3-5 weeks in 20 (91%) patients. One patient with a history of chronic hepatitis C died from fulminant hepatitis and 1 patient (co-infected with HAV) had a clinically severe course (ALT 12290 IU/l, AST 17870 IUil) with ALT normalization within 8 weeks. 17 (77.3%) acute hepatitis E infections occurred in patients who travelled in endemic areas and 5 (22.7%) in patients with no history of travel. Pairwaise alignments of the nucleotide sequence from a HEV F’CR positive patient with no history of travel indicate that the isolate is significantly divergent from the two original isolates of HEV Burma and Mexico (79.9 and 80.7% respectively) and also significantly divergent from the new US isolates (85.8-88.6%). Phylogenetic analysis indicates that the Italian isolate represents a fourth branch distinct from those represented by the Burmese, Mexican and US isolates. Conclusions: HEV is responsible of about 10% of acute nA-C hepatitis in Italy. The identification of a new HEV variant may be important in understanding the epidemiology of HEV infection in our country.