P. Colombatto

Transient elastography: a new surrogate marker of liver fibrosis influenced by major changes of transaminases.

Summary.  Liver stiffness was measured by transient elastography (FibroScan®) in 228 consecutive patients with chronic viral hepatitis, with (115) or without cirrhosis (113), to study its correlations with serum transaminases [alanine aminotransferase (ALT)], fibrosis stage and surrogate noninvasive markers of fibrosis (APRI, FORNS, FibroTest and hyaluronic acid). The dynamic profiles of serum transaminases and liver stiffness were compared by multiple testing in 31 patients during a 6‐month follow‐up. We identified 8.3 and 14 kPa as the fibrosis ≥F2 and cirrhosis cut‐offs, respectively: their sensitivities were 85.2%/78.3%; specificities 90.7%/98.2%; positive predictive values 93.9%/97.8%; negative predictive values 78.8%/81.6%; diagnostic accuracies 87.3%/88.2%. FibroScan® performed better than the other surrogate markers of fibrosis (P < 0.001). Other than fibrosis, other factors independently associated with liver stiffness were ALT for all patients and chronic hepatitis patients (P < 0.001), and 12‐month persistently normal ALT (biochemical remission, P < 0.001) in cirrhotics. In patients with biochemical remission either spontaneous or after antiviral therapy (48 of 228, 21%), liver stiffness was lower than in patients with identical fibrosis stage, but elevated ALT (P < 0.001). The liver stiffness dynamic profiles paralleled those of ALT, increasing 1.3‐ to 3‐fold during ALT flares in 10 patients with hepatitis exacerbations. Liver stiffness remained unchanged in 21 with stable biochemical activity (P = 0.001). In conclusion, transient elastography is a new liver parameter that behaves as a reliable surrogate marker of fibrosis in chronic viral hepatitis patients, provided that its relationship with major changes of biochemical activity is taken into account.

Outcome of anti-HBe positive chronic hepatitis B in alpha-interferon treated and untreated patients: a long term cohort study.

BACKGROUND/AIMS We studied the influence of biochemical and virologic patterns and interferon on the outcome of anti-HBe positive chronic hepatitis B in 164 (103 treated) consecutive patients, followed-up prospectively for a mean of 6 years (21 months-12 years). METHODS Histology, biochemical and virologic profiles were characterized by monthly monitoring during the first 12 months of follow-up. Thereafter patients underwent blood and clinical controls every 4 and 6 months, respectively. Cirrhosis at follow-up histology or end stage complications of cirrhosis served as end points for the analysis of factors influencing disease progression in patients with baseline chronic hepatitis or cirrhosis, respectively. RESULTS Disease progression was associated with older age (P<0.001), absence of previous HBeAg history (P=0.017) and higher serum HBV-DNA levels (P=0.009) (more frequently observed in unremitting disease profile, P=0.012) at multivariate analysis. Fluctuations of IgM anti-HBc levels (associated with disease exacerbations, P=0.045) correlated with end stage complications in cirrhotics (P=0.011). Disease improved in 14.6 and 1.6% of treated and untreated patients, respectively (P=0.015): interferon slowed disease progression (P<0.001). CONCLUSIONS The outcome of anti-HBe positive chronic hepatitis B is worsened by older age and persistent viral replication or hepatitis exacerbations in chronic hepatitis or in cirrhotic patients, respectively. Interferon reduces by 2.5-folds disease progression.

Hepatitis B surface antigen serum levels help to distinguish active from inactive hepatitis B virus genotype D carriers.

BACKGROUND & AIMS The accurate identification of inactive (serum HBV-DNA persistently <or=2000 IU/mL) hepatitis B virus (HBV) carriers (IC) is difficult because of wide and frequent HBV-DNA fluctuations. We studied whether hepatitis B surface antigen (HBsAg) serum levels (HBsAgsl) quantification may contribute to diagnosis of HBV phases in untreated hepatitis B e antigen-negative genotype D asymptomatic carriers. METHODS HBsAgsl were measured at baseline and end of follow-up and correlated with virologic and biochemical profiles of 209 consecutive carriers followed-up prospectively (median, 29; range, 12-110 months). HBV phases were defined after 1-year monthly monitoring of HBV-DNA and transaminases. RESULTS HBsAgsl were significantly lower in 56 inactive carriers (IC) than 153 active carriers (AC): median, 62.12 (range, 0.1-4068) vs median, 3029 (range, 0.5-82,480) IU/mL; P<.001. Among AC, HBsAgsl were lower in 31 AC whose viremia remained persistently <20,000 IU/mL (AC1) than in 122 AC with fluctuations>or=20,000 IU/mL (AC2): 883 (0.5-7838) vs 4233 (164-82,480) IU/mL, P=.002. HBV infection was less productive in IC and AC1 than AC2 (log10 HBV-DNA/HBsAgsl ratios 0.25 and 0.49 vs 2.06, respectively, P<.001) and in chronic hepatitis than cirrhosis (1.97 vs 2.34, respectively; P=.023). The combined single point quantification of HBsAg (<1000 IU/mL) and HBV-DNA (<or=2000 IU/mL) identified IC with 94.3% diagnostic accuracy, 91.1% sensitivity, 95.4% specificity, 87.9% positive predictive value, 96.7% negative predictive value. During follow-up, HBsAgsl were stable in AC but declined in IC (yearly median decline, -0.0120 vs -0.0768 log10 IU/mL, respectively, P<.001), 10 of whom cleared HBsAg. CONCLUSIONS HBsAgsl vary during chronic hepatitis B e antigen-negative genotype D infection and are significantly lower in IC. Single-point combined HBsAg and HBV-DNA quantification provides the most accurate identification of IC, comparable with that of long-term tight monitoring.

Association of hepatitis C virus envelope proteins with exosomes

As the human tetraspanin CD81 binds hepatitis C virus (HCV) envelope glycoprotein E2, we addressed the role CD81 may play in cellular trafficking of HCV envelope proteins. Studies on HCV life cycle are complicated by the lack of a robust cell culture system; we therefore transfected mammalian cells with HCV E1‐E2 cDNA, with or without human CD81 (huCD81) cDNA. In the absence of huCD81, HCV envelope proteins are almost completely retained in the endoplasmic reticulum. Instead, when huCD81 is present, a fraction of HCV envelope proteins passes through the Golgi apparatus, matures acquiring complex sugars and is found extracellularly associated with exosomes. These are 60–100‐nm membrane vesicles enriched in tetraspanins, released into the extracellular milieu by many cell types and having fusogenic activity. We also report that human plasma contains exosomes and that in HCV patients, viral RNA is associated with these circulating vesicles. We propose that the HCV‐CD81 complex leaves cells in the form of exosomes, circulates in this form and exploits the fusogenic capabilities of these vesicles to infect cells even in the presence of neutralizing antibodies.

Hepatitis G virus RNA in the serum of patients with elevated gamma glutamyl transpeptidase and alkaline phosphatase: a specific liver disease

SUMMARY. We tested the sera of 67 consecutive patients for hepatitis G virus (HGV) RNA by reverse transcriptase‐polymerase chain reaction (RT‐PCR). These patients (42 males and 2 5 females, median age 35 years, range 13–64 years) had liver disease of unknown aetiology and were without markers of hepatitis (A‐E) viruses or signs of genetically determined, autoimmune, alcoholic or drug‐induced liver disease. The controls in this study were 110 patients (50 females and 60 males, median age 45 years, range 9–65 years) with chronic hepatitis B virus (HBV) infection (19 patients) or hepatitis C virus (HCV) infection (91 patients). Ten of 67 (14.9%) patients with cryptogenic disease were positive for HGV RNA by at least three separate tests; HGV RNA was also detected in one of 19 (5.3%) hepatitis B surface antigen (HBsAg) carriers and in nine of 91 (16.6%) patients with antibody to HCV. These data suggest that HGV occurs as frequently in HCV infected patients as in those with cryptogenic disease. Elevated serum gamma glutamyl transpeptidase (γ‐GT) (higher than twice the normal value) and alkaline phosphatase levels were found in eight of 10 (80%) HGV RNA positive patients and in six of 57 (10.5%) HGV RNA negative patients (P < 0.0001). Five (50%) HGV RNA positive patients had non‐specific inflammatory bile duct lesions. A statistically significant difference was observed between HGV RNA positive and negative patients with chronic HBV or HCV infections (P < 0.029). Therefore, the spectrum of liver disease associated with HGV is wide, but a characteristic lesion of the bile duct leading to elevation of cholestatic enzymes might be specific for this virus.

Long term response to therapy of chronic anti-HBe-positive hepatitis B is poor independent of type and schedule of interferon.

Objective:The response rate to alpha interferon (IFN) of chronic anti-HBe–positive hepatitis B is variable. We studied whether type, dose, and schedule of IFN, and type and frequency of posttreatment monitoring, influence the response rate.Methods:Seventy-two consecutive anti-HBe–positive chronic hepatitis B patients (59 male and 13 female, median age 41 yr) stratified by sex and histology were randomly allocated to three treatment arms. Twenty-seven patients (A) received 10 million units alpha-N1 IFN i.m. t.w. for 24 wk (total dose: 720 million units); 21 (B) received 9 million units alpha-2a IFN i.m. t.w. for 4 wk, followed by 18 million units for 12 wk and 9 million units for 8 wk (972 million units); 24 (C) received 2 alpha-2a IFN courses (9 million units i.m. t.w. for 16 and 12 wk separated by a 6-month interval [756 million units]). Primary response was defined by normal ALT and serum HBV-DNA levels below 10 pg/ml at the end of therapy and sustained response by normal ALT (tested monthly), undetectable HBV-DNA and IgM anti-HBc (<7 I.U. Paul Ehrlich Institute) (tested every 3 months) during the posttreatment follow-up.Results:At the end of treatment, 12, 8, and 13 patients from groups A, B, and C, respectively, were responders. At the 18-month follow-up, two patients in group A and only one in groups B and C maintained the response. Overall, after 34 months (median posttreatment follow-up), three patients were long term responders, whereas three showed a sustained remission after relapse.Conclusions:The rate of long term response to interferon of anti-HBe–positive chronic hepatitis B is poor, independent of IFN type, dose, or schedule; the more stringent the monitoring, the higher the relapse rate.

Is high AgNOR quantity in hepatocytes associated with increased risk of hepatocellular carcinoma in chronic liver disease

AIMS--To evaluate whether high numbers of silver staining nucleolar organiser regions (AgNORs) in hepatocytes are associated with increased risk of hepatocellular carcinoma in chronic liver disease. METHODS--The quantitative distribution of AgNORs was studied in the liver biopsy specimens of 33 patients with chronic liver disease, 11 of whom developed hepatocellular carcinoma. The interval between liver biopsy and diagnosis of hepatocellular carcinoma was 26 months (range one to 61 months); the mean follow up of patients without hepatocellular carcinoma was 45 months (range 24-59 months). Quantitative evaluation of AgNORs was carried out on silver stained routine sections by morphometric analysis, using a computer assisted image analysis system. RESULTS--High interphase AgNOR values (> 3 microns2) were found in hepatocytes of nine out of the 11 (82%) patients in whom neoplastic transformation occurred. Of the remaining 22 patients, only seven (31%) had AgNOR values higher than > 3 microns2 (chi 2 4.83; p = 0.036). CONCLUSIONS--These results indicate that high numbers of interphase AgNORs are associated with increased risk of hepatocellular carcinoma in patients with chronic liver disease.

Viral load 1 week after liver transplantation, donor age and rejections correlate with the outcome of recurrent hepatitis C

Background: Early identification of patients at a higher risk of rapidly progressive recurrent hepatitis post liver transplantation (LT) could help to tailor antiviral therapy.

Treatment of HBeAg-negative chronic hepatitis B with interferon or pegylated interferon

Anti-HBe-positive chronic hepatitis B was first described and characterized in patients of the Mediterranean area, where about 20% of hepatitis B surface antigen (HBsAg) carriers with antibody to hepatitis B ‘e’ antigen (HBeAg) show detectable serum levels of hepatitis B virus (HBV) DNA by hybridization assays and intrahepatic necro-inflammation [1,2]. They are infected with HBV variants with mutations in the pre-core region which hamper HBeAg production. The G to A mutation at nucleotide 1896 of the pre-core region is the most commonly described [3,4]. Liver disease runs an indolent course for 3–4 decades reaching the stage of histological cirrhosis at a median age of 45 years [5]; thereafter about 25% of the patients progress to end stage complications within 10 years [5]. In recent years several reports suggest a worldwide incidence of HBeAg-negative chronic hepatitis B [6,7]. However, further virological and clinical studies are needed to warrant a complete characterization of the disease and to understand whether it shares common features with the anti-HBe-positive chronic hepatitis B observed in Mediterranean patients, who are infected with HBV genotype D, bearing the stop codon at nucleotide 1896 [8]. Because of the progressive course of HBeAgnegative/anti-HBe-positive chronic hepatitis B, alphainterferon (IFN) treatment was attempted as soon as the drug became available for treatment of chronic HBV infection [9–12]. Most studies about interferon treatment of anti-HBe-positive chronic hepatitis B were performed in southern Europe [9–14] and more recently a few reports had been published from Asia [15–17]. 2. Aims of antiviral treatment and monitoring criteria

Lamivudine-resistant HBV strain rtM204V/I in acute hepatitis B

AIMS To detect HBV rtM204V/I lamivudine-resistant strains in serum of patients with acute hepatitis B and to assess their biological and clinical significance. METHODS Eighty HBV DNA-positive patients with symptomatic acute hepatitis B observed from 1999 to 2010 were enrolled. A plasma sample obtained at the first observation was tested for HBV mutants in the polymerase region by direct sequencing; the antiviral drug-resistant rtM204V/I mutations, the most frequent HBV mutants in Italy, were also sought by the more sensitive allele-specific polymerase chain reaction (PCR). RESULTS No HBV mutation associated with resistance to nucleos(t)ide analogues was identified by direct sequencing, whereas allele-specific PCR identified HBV strains carrying the substitution rtM204V/I in 11 (13.7%) patients. Compared with those with the HBV wild strain, patients with rtM204V/I more frequently showed severe acute hepatitis B (36.4% vs 8.7%; p < 0.05) and lower values of serum HBV DNA (1.77 × 10(6) ± 4.76 × 10(6) vs. 1.68 × 10(8) ± 5.46 × 10(8)). In addition, a multivariate analysis identified the presence of a pre-existing HCV chronic infection as independently associated with severe acute hepatitis B (p < 0.05). CONCLUSIONS HBV rtM204V/I lamivudine-resistant strains were detected in serum of 11 (13.7%) patients with acute hepatitis B by allele-specific polymerase chain reaction. The frequent association of rtM204V/I with a more severe acute hepatitis B and with a lower viral load may suggest that greater and/or more prolonged immune pressure might have induced their selection.