HERACLIS-HDV cohort for the factors of underdiagnosis and prevalence of hepatitis D virus infection in HBsAg-positive patients.

BACKGROUND AND AIMS: Hepatitis D virus (HDV) underdiagnosis remains common. We assessed the HDV screening and prevalence rates in HBsAg-positive patients seen at tertiary liver centres throughout Greece as well as factors affecting HDV diagnosis. METHODS: All adult HBsAg-positive patients seen within the last 5 years were included. Non-screened patients who visited or could be recalled to the clinics over a 6-month period were prospectively tested for anti-HDV. RESULTS: Of 5079 HBsAg-positive patients, 53% had anti-HDV screening (41% before and 12% after study initiation). Pre-study (8%-88%) and total screening rates (14%-100%) varied widely among centres. Screening rates were associated with older age, known risk group, elevated ALT, centre location and size and period of first visit. Anti-HDV prevalence was 5.8% without significant difference in patients screened before (6.1%) or after study initiation (4.7%, p = 0.240). Anti-HDV positivity was associated with younger age, parenteral drug use, born abroad, advanced liver disease and centre location. Overall, HDV RNA detectability rate was 71.6% being more frequent in anti-HDV-positive patients with elevated ALT, advanced liver disease and hepatitis B therapy. CONCLUSIONS: Anti-HDV screening rates and recall capabilities vary widely among Greek liver clinics being higher in HBsAg-positive patients of known risk group with active/advanced liver disease seen at smaller centres, while non-medical factors are also important. Anti-HDV prevalence varies throughout Greece being higher in patients born abroad with younger age, parenteral drug use and advanced liver disease. Viremia is more frequently but not exclusively detected in anti-HDV-positive patients with elevated ALT and advanced liver disease.

Noninvasive, Blood-Based Biomarkers as Screening Tools for Hepatic Fibrosis in People With Type 2 Diabetes.

Nonalcoholic fatty liver disease (NAFLD) is dramatically increasing in parallel with the pandemic of type 2 diabetes. Here, the authors aimed to assess the performance of the most commonly used noninvasive, blood-based biomarkers for liver fibrosis (FibroTest, NAFLD fibrosis score, BARD score, and FIB-4 Index) in subjects with type 2 diabetes. Liver stiffness measurement was estimated by two-dimensional shear wave elastography. Finally, the authors assessed the diagnostic role of ActiTest and NashTest 2 in liver fibrosis in the examined population.

Long-term clinical outcome of HBeAg-negative chronic hepatitis B patients who discontinued nucleos(t)ide analogues.

BACKGROUND & AIMS: Discontinuation of nucleos(t)ide analogues (NA) remains a debatable issue in HBeAg-negative chronic hepatitis B (CHB). This study aimed to address the outcome of HBeAg-negative CHB patients who discontinued NA therapy. METHODS: This prospective study included 57 non-cirrhotic HBeAg-negative Caucasian CHB patients who discontinued NA therapy after median virological remission of 6 years. All patients had regular blood tests. Virological relapse was defined as HBV DNA > 2000 IU/mL or >20 000 IU/mL and biochemical relapse as ALT > ULN (40 IU/mL) or >2xULN. All patients with retreatment predefined criteria restarted entecavir or tenofovir. RESULTS: Of the 57 patients, 29 remained without retreatment after median follow-up of 65 months (range: 36-87) following treatment discontinuation. At 3, 6, 12, 24, 36 and 48 months, cumulative rates of retreatment were 16%, 20%, 32%, 35%, 46% and 50%, while the proportion of patients with HBV DNA < 2000 IU/mL and ALT < ULN were 73%, 60%, 52%, 52%, 47% and 37% respectively. All patients had virological and biochemical response after retreatment. No patient developed liver failure, hepatocellular carcinoma or death. Cumulative rates of HBsAg loss were 2%, 4%, 7%, 10% and 20% at 3, 6, 12, 24 and 36 months. HBsAg levels < 100 IU/mL at the end of NA treatment could predict HBsAg loss (P = .001). CONCLUSIONS: Our study supports that NA therapy can be safely stopped in non-cirrhotic patients with HBeAg-negative CHB. Over a median follow-up of more than 5 years, half of the patients remained without retreatment with a substantial proportion of them achieving functional cure.

Liver stiffness measurements by 2-dimensional shear wave elastography compared to histological and ultrasound parameters in primary biliary cholangitis.

BACKGROUND AND AIMS: Liver stiffness measurements (LSMs) by 2-dimensional-shear-wave elastography (LSM2D-SWE) are now widely used in hepatology. However, relevant information for primary biliary cholangitis (PBC) is scant. We compare LSM2D-SWE with liver biopsy (LB) in a cohort of PBC patients in Greece. METHODS: Data of 68 LBs from 53 PBC patients were retrospectively analyzed and fibrosis stage was compared to LSM2D-SWE. Forty-six patients (86.8%) were females and at the time of LBx median (IQR) age was 62.6 (53.2-72.1). Demographic, UDCA treatment, histological and B-mode ultrasound data were tested for their influence on LSM2D-SWE estimates. RESULTS: Liver fibrosis stages F0-F4 were found in 4, 19, 19, 16 and 10 cases, respectively. Across stages F0-F4, the LSM2D-SWE was 5.6 (5.1-6.1), 7.0 (5.8-7.7), 9.1 (7.3-11.5), 10.8 (9.9-12.2) and 14.5 (11.9-25.7) kPa, respectively, with highly significant difference (p<.001). The LSM2D-SWE differed also significantly between F0 vs. F1 (p=.027), F1 vs. F2 (p=.005) and F3 vs. F4 (p=.017). The discriminatory ability of LSM2D-SWE for mild, significant, severe fibrosis and cirrhosis was highly significant in all comparisons (p<.001), with AUC2D-SWE 95.3%, 87.4%, 85.3% and 95.3% and accuracy 89.7%, 85.3%, 80.9% and 86.8%, respectively. Among 21 parameters tested, significant predictors of LSM2D-SWE by multiple linear regression were fibrosis stage, portal inflammation and parenchymal heterogeneity. The portal inflammation grade accounted for 32.2% of LSM variation with adjusted R2 0.428. CONCLUSIONS: In patients with PBC, LSM measurements by 2D-SWE can reliably discriminate between mild, significant, severe fibrosis and cirrhosis. Measurements are significantly affected by portal inflammation grade.

Assessment of biopsy-proven liver fibrosis by two-dimensional shear wave elastography: An individual patient data-based meta-analysis.

Two-dimensional shear wave elastography (2D-SWE) has proven to be efficient for the evaluation of liver fibrosis in small to moderate-sized clinical trials. We aimed at running a larger-scale meta-analysis of individual data. Centers which have worked with Aixplorer ultrasound equipment were contacted to share their data. Retrospective statistical analysis used direct and paired receiver operating characteristic and area under the receiver operating characteristic curve (AUROC) analyses, accounting for random effects. Data on both 2D-SWE and liver biopsy were available for 1,134 patients from 13 sites, as well as on successful transient elastography in 665 patients. Most patients had chronic hepatitis C (n = 379), hepatitis B (n = 400), or nonalcoholic fatty liver disease (n = 156). AUROCs of 2D-SWE in patients with hepatitis C, hepatitis B, and nonalcoholic fatty liver disease were 86.3%, 90.6%, and 85.5% for diagnosing significant fibrosis and 92.9%, 95.5%, and 91.7% for diagnosing cirrhosis, respectively. The AUROC of 2D-SWE was 0.022-0.084 (95% confidence interval) larger than the AUROC of transient elastography for diagnosing significant fibrosis (P = 0.001) and 0.003-0.034 for diagnosing cirrhosis (P = 0.022) in all patients. This difference was strongest in hepatitis B patients. CONCLUSION: 2D-SWE has good to excellent performance for the noninvasive staging of liver fibrosis in patients with hepatitis B; further prospective studies are needed for head-to-head comparison between 2D-SWE and other imaging modalities to establish disease-specific appropriate cutoff points for assessment of fibrosis stage. (Hepatology 2018;67:260-272).

Prognostic Models for Survival in Patients with Stable Cirrhosis: A Multicenter Cohort Study.

BACKGROUND: Two models are mostly used to predict survival in cirrhosis: the Child-Pugh score (CP score) and the model for end-stage liver disease score (MELD score). AIMS: The aim of this study is to evaluate the CP score and the MELD score for short- and long-term prognosis in cirrhosis, as well as CP-creatinine score, MELD-Na score, and UKELD score. METHODS: One thousand and forty-seven patients from five referral centers were included: men/women: 620/427, median age: 58 years (IQR 48-66), median follow-up: 33 months (IQR 12-74), CP (A/B/C): 493/357/147, CP score: 7 (IQR 5-9), MELD score: 12 (IQR 9-16). The performance of each score was evaluated by the Cox hazard model in terms of their: discrimination ability (C-index and Somer's D) and calibration (3, 12 months). Internal validation was done with bootstrapping (100 samples). RESULTS: Three hundred and fifty-two patients (33.6%) died. All scores were significantly associated with overall mortality, when assessed by univariate Cox analysis. CP-creatinine score performed significantly better than all other scores [bootstrap C-index 0.672, 95% CI 0.642-0.703, bootstrap Somer's D 0.344 (0.285-0.401)], apart from CP score, which showed similar performance. Inclusion in the multivariable Cox model of age together with CP-creatinine score improved the discriminative ability of the model [bootstrap C-index (95% CI) 0.700 (0.661-0.740)]. In terms of calibration, CP-creatinine score was the best for both 3- and 12-month survival in the total population. CONCLUSIONS: CP score and CP-creatinine score have better prognostic value compared to MELD score, MELD-Na score, and UKELD score for predicting short- and long-term mortality in patients with stable cirrhosis.

Changes of HBsAg and interferon-inducible protein 10 serum levels in naive HBeAg-negative chronic hepatitis B patients under 4-year entecavir therapy.

BACKGROUND & AIMS: Serum HBsAg levels might represent an important predictor of sustained off-treatment response in HBeAg-negative chronic hepatitis B (CHB). We evaluated the changes of HBsAg and interferon-inducible protein 10 (IP10) serum levels in HBeAg-negative CHB patients treated with entecavir. METHODS: 114 patients received entecavir for a median of 4.3 years. HBsAg levels were determined at baseline, 6 and 12 months and every year thereafter until year-4. IP10 levels were measured at baseline and annually until year-4 in 76 patients. RESULTS: Virological remission rates were high (year-1: 94%, after year-2: 97-98%). Compared to baseline, HBsAg levels decreased by a median of 0.03, 0.13, 0.17, 0.22, and 0.32 log10 IU/ml at 6 months and 1, 2, 3, and 4 years, respectively (p≤0.001 for all comparisons). The proportions of patients with HBsAg decline of ≥0.5 or ≥1 log10 IU/ml were 9% or 6% at year-1 and 21% or 10% at the last visit. Median IP10 levels (pg/ml) did not change from baseline to year-1 or -2 (245 vs. 229 or 251), but increased at year-3 and -4 (275 and 323, p<0.030). HBsAg drop ≥0.5 log10 was associated with baseline IP10 or IP10 >350 pg/ml (p≤0.002). HBsAg loss occurred in 4/114 (3.5%) patients or in 1/2, 3/21, and 0/91 patients with baseline HBsAg <100, 100-1000 and >1000 IU/ml, respectively (p<0.001). CONCLUSIONS: In HBeAg-negative CHB patients, 4-year entecavir therapy decreases serum HBsAg levels, but the rate of decline is rather slow. Serum IP10 levels represent a promising predictor of HBsAg decline in this setting.

Prevalence and clinical course of hepatitis delta infection in Greece: a 13-year prospective study.

BACKGROUND & AIMS: Hepatitis D virus (HDV) has decreased in Europe, but recent reports indicate a rising trend. We report the epidemiological changes, clinical progress, and effect of treatment on the natural course of HDV infection in Greece during the last 13 years. METHODS: Prospective data were extracted from the HepNet.Greece Cohort-Study. RESULTS: Since 1997, 4673 chronic HBV (CHB) cases (4527 adults, 146 children) have been followed prospectively. Two thousand one hundred thirty-seven patients were tested for anti-HDV [101 (4.7%) positive]. Anti-HDV testing in Greece decreased significantly (57.0% before 2003, 35.3% thereafter; p<0.001). Anti-HDV prevalence among HBsAg-positives was 4.2%; lower in native Greeks (2.8%) than in immigrants (7.5%) or in children (15.3%; p<0.001). Within 2.3 years of follow-up, HDV occurred in 11/2047 HBsAg-positive patients (2.2 new delta-infected adults and 8.7 children per 1000 HBsAg-positive annually). HDV-positive compared to CHB adults were younger (p=0.035) and had more active and advanced disease at baseline, as indicated by laboratory indices and the higher prevalence of cirrhosis at younger age. During a 4.2-year median observation, significantly more anti-HDV-positive than CHB adults developed a liver-related first event (20.0% vs. 8.5%, p Log-rank=0.014).Treatment was received by 46/90 (51.1%) patients, 40 of them interferon-based. In multivariable analysis, interferon significantly decreased disease progression in HDV-positive patients [HR=0.14 (95% CI: 0.02-0.86; p=0.033)]. CONCLUSIONS: In Greece, HDV serology is currently tested in only one-third of HBsAg-positive patients. HDV prevalence is lower in native Greeks compared to immigrants, who may contribute >50% of the HDV infection burden in Greece. Data show that HDV infection is a rapidly progressive disease, but interferon-based treatment may alter its course.

Ultrasensitive amplification refractory mutation system real-time PCR (ARMS RT-PCR) assay for detection of minority hepatitis B virus-resistant strains in the era of personalized medicine.

Resistance to antiviral treatment for chronic hepatitis B virus (HBV) has been associated with mutations in the HBV polymerase region. This study aimed at developing an ultrasensitive method for quantifying viral populations with all major HBV resistance-associated mutations, combining the amplification refractory mutation system real-time PCR (ARMS RT-PCR) with a molecular beacon using a LightCycler. The discriminatory ability of this method, the ARMS RT-PCR with molecular beacon assay, was 0.01 to 0.25% for the different HBV resistance-associated mutations, as determined by laboratory-synthesized wild-type (WT) and mutant (Mut) target sequences. The assay showed 100% sensitivity for the detection of mutant variants A181V, T184A, and N236T in samples from 41 chronically HBV-infected patients under antiviral therapy who had developed resistance-associated mutations detected by direct PCR Sanger sequencing. The ratio of mutant to wild-type viral populations (the Mut/WT ratio) was >1% in 38 (63.3%) of 60 samples from chronically HBV-infected nucleos(t)ide analogue-naive patients; combinations of mutations were also detected in half of these samples. The ARMS RT-PCR with molecular beacon assay achieved high sensitivity and discriminatory ability compared to the gold standard of direct PCR Sanger sequencing in identifying resistant viral populations in chronically HBV-infected patients receiving antiviral therapy. Apart from the dominant clones, other quasispecies were also quantified. In samples from chronically HBV-infected nucleos(t)ide analogue-naive patients, the assay proved to be a useful tool in detecting minor variant populations before the initiation of the treatment. These observations need further evaluation with prospective studies before they can be implemented in daily practice.

Virological suppression does not prevent the development of hepatocellular carcinoma in HBeAg-negative chronic hepatitis B patients with cirrhosis receiving oral antiviral(s) starting with lamivudine monotherapy: results of the nationwide HEPNET. Greece cohort study.

OBJECTIVE: To evaluate the risk and predictors of hepatocellular carcinoma (HCC) in HBeAg-negative chronic hepatitis B patients of the large HEPNET.Greece cohort study who received long-term oral antivirals starting with lamivudine monotherapy. DESIGN: Retrospective analysis of HCC incidence in HBeAg-negative chronic hepatitis B patients from a retrospective-prospective cohort who were treated with nucleos(t)ide analogue(s) starting with lamivudine monotherapy for ≥12 months. SETTING: A nationwide network of liver centres. PATIENTS: 818 patients were included: 517 with chronic hepatitis B only; 160 with compensated cirrhosis; 56 with decompensated cirrhosis; 85 with unclassified disease severity. INTERVENTIONS: All patients were treated with nucleos(t)ide analogue(s) starting with lamivudine monotherapy. MAIN OUTCOME MEASURES: Development of HCC. RESULTS: During a median follow-up of 4.7 years, HCC developed in 49 (6.0%) patients. The 5-year cumulative incidence of HCC was higher in patients with cirrhosis than in those with chronic hepatitis B only (11.5% vs 3.2%, respectively; p<0.001). HCC developed in 0.7%, 6.7% and 11.7% of patients <50, 50-60 and >60 years old, respectively (p<0.001). Virological on-therapy remission did not significantly affect the incidence of HCC in all patients or those with cirrhosis, but it showed a trend for lower HCC incidence in patients with chronic hepatitis B only (p=0.076). In multivariate analysis, age, gender and cirrhosis were independently associated with HCC risk regardless of virological remission. CONCLUSIONS: Long-term therapy with nucleos(t)ide analogue(s) starting with lamivudine monotherapy does not eliminate HCC risk in HBeAg-negative chronic hepatitis B. The risk of HCC is particularly high in patients with cirrhosis, who should remain under HCC surveillance even during effective therapy. Older age and male gender remain independent risk factors for HCC, while virological on-therapy remission does not seem to significantly reduce the overall incidence of HCC.

PNPLA3 rs 738409 and Other Nongenetic Factors Associated with Hepatic Steatosis Estimated by Magnetic Resonance Imaging Proton Density Fat Fraction in Adult Greek Subjects with Type 2 Diabetes Mellitus.

Objectives: Nonalcoholic fatty liver disease is dramatically increasing in parallel with the pandemic of type 2 diabetes mellitus. We investigated factors associated with hepatic steatosis (HS) in adult Greek individuals with established type 2 diabetes mellitus. Materials and Methods: We investigated 120 consecutive people with type 2 diabetes attending the Diabetic Outpatient Clinic at an Academic Hospital in Athens, Greece. All of them had demographic, clinical, and biochemical data recorded. HS was estimated by magnetic resonance imaging determined by proton density fat fraction software and defined as the percentage of total liver fat divided by the liver volume. HS of >5% was considered abnormal. The PNPLA3 (I148M) variant was evaluated as a genetic factor by standard molecular techniques. FibroMax™ was also calculated. Results: Of the 120 participants, median age was 61.7, 46% were females, diabetes duration was 10 years, and HbA1c (glycated hemoglobin) was 6.7%. The median value of HS was 7.8. The PNPLA3 rs738409 CC/CG/GG genotype frequencies were 54.2%, 35%, and 10.8%, respectively. In multivariate analysis, PNPLA3 rs738409 (ß = 0.425, P = 0.001), waist circumference (ß = 2.448, P = 0.001), and female sex (ß = 0.419, P = 0.002) had a direct association with HS, while duration of diabetes (ß = -0.179, P = 0.011) had an inverse association with HS. Conclusions: HS in type 2 diabetes is the sum of interplay of various factors exerting a direct or an inverse association, the most prominent among them being abdominal obesity and PNPLA3 molecular variability.

HERACLIS-TAF: a multi-centre prospective cohort study on 2-year safety and efficacy of tenofovir alafenamide in patients with chronic hepatitis B with renal and/or bone disorders or risks.

BACKGROUND: Tenofovir alafenamide (TAF) has exhibited a favourable safety profile on estimated glomerular filtration (eGFR) and bone mineral density (BMD), but has not been extensively studied in patients with renal impairment and/or BMD disorders. AIMS: To assess predictors of eGFR changes and other safety and efficacy outcomes during 24-month TAF therapy in patients with chronic hepatitis B with renal and/or BMD disorders/risks. METHODS: Adult patients who started TAF at 13 clinics throughout Greece were prospectively included. Main exclusion criteria were hepatitis D, active malignancy and bisphosphonates recent use. MDRD formula was used for eGFR estimation. RESULTS: TAF was initiated in 176 patients (91% switched from another agent). At 12 and 24 months, HBV DNA was undetectable in 97% and 100%, and ALT was normal in 96% and 95% of patients. Median ALT decreased from baseline to month 12/24 (p < 0.001). Mean eGFR decreased from previous treatment initiation to baseline (p < 0.001), increased at 12 months and remained stable at 24 months (p ≤ 0.001). An increase in eGFR of >3 ml/min at 12 month was observed in 50% of patients and was associated mainly with baseline eGFR 30-60 ml/min. In patients with baseline phosphate <2.5 mg/dl, mean serum phosphate increased at month-12/24 (p < 0.001). Median BMD did not change significantly from baseline to 12 months but improved at 24 months (p = 0.001). CONCLUSIONS: In mostly switched patients with renal and/or BMD disorders/risks, eGFR improved after 12-24 months of TAF treatment, especially in patients with baseline eGFR 30-60 ml/min. TAF may also improve low serum phosphate, BMD and ALT, whereas it maintains or induces virological suppression.

Hepatitis B surface antigen: relation to hepatitis B replication parameters in HBeAg-negative chronic hepatitis B.

BACKGROUND & AIMS: Translation of HBsAg depends on transcription of the appropriate mRNAs from cccDNA, but its relation to other hepatitis B virus (HBV) replication parameters is not known, inasmuch as integrated sequences of HBV-DNA may also contribute to its serum levels, especially in HBeAg-negative chronic hepatitis B (CHB) patients. METHODS: We investigated HBsAg serum levels, its hepatocellular expression, and their relationship to HBV replicative- and host-response parameters before treatment in 54 HBeAg-negative CHB patients and in 15 of them after 40.1±33.3months of virological response on oral antiviral (NUC) therapy also. Liver cccDNA and HBV-DNA quantitation, HBsAg- and HBcAg-immunostaining were performed in the same needle biopsy material, while serum HBsAg and HBV-DNA levels were measured in samples drawn on the day of liver biopsy. RESULTS: In untreated patients, serum HBsAg correlated positively with HBsAg-positive hepatocytes/mm(2) (p=0.003) and weakly with serum HBV-DNA, but not with cccDNA, liver HBV-DNA, HBcAg-positive hepatocytes/mm(2), or ALT. cccDNA correlated significantly with liver HBV-DNA (p<0.00001), ALT (p=0.001), and serum HBV-DNA levels (p=0.012) but not with liver HBsAg or HBcAg. Antiviral therapy decreased serum HBsAg levels by 79.6% (p=0.012) and liver HBV-DNA by 84.4% (p=0.026) in paired comparisons and, as expected, significantly decreased serum HBV-DNA and ALT levels, but not cccDNA. CONCLUSIONS: In untreated HBeAg-negative CHB, serum HBsAg levels reflect liver HBsAg, but not cccDNA or liver HBV-DNA, suggesting that they are not solely dependent on the replicative cycle of HBV. Effective NUC therapy for 3.34 years significantly lowers serum HBsAg and liver HBV-DNA, but not cccDNA.

Effect of antiviral therapy on the immunohistochemical expression of bcl-xL and bax protein in patients with HBeAg-negative chronic hepatitis B.

The effect of antiviral treatment on apoptosis in chronic hepatitis B (CHB) has not been clarified. We evaluated the hepatic immunohistochemical expression of the pro-apoptotic bax and the antiapoptotic bcl-xL protein in HBeAg-negative CHB patients before and after treatment. In our study we included 72 paired biopsies from 36 HBeAg-negative CHB patients: 29 treated (interferon-alfa: 17, adefovir: 12) and 7 untreated. Changes in expression of apoptotic proteins (D-bax, D-bcl-xL), necroinflammation and fibrosis (D-grade/D-stage) (Ishak classification) were evaluated. We found that Bax-positive compared to bax-negative biopsies had worse necroinflammation (8.2 vs. 6.7, P = 0.05) and fibrosis score (3.9 vs. 3, P = 0.036). bcl-xL-positive compared to bcl-xL-negative biopsies had lower intralobular inflammation (1.6 vs. 2.2, P = 0.03). Decreased compared to stable/increased D-bax was associated with greater improvement in necroinflammation only in treated patients (D-grade: -4.6 vs. -1.6, P = 0.05) and greater fibrosis improvement in interferon treated patients (D-stage: -0.4 vs. 0.55, P = 0.05). Increased compared to stable/decreased total apoptotic trend [D-apoptosis: (D-bax)-(D-bcl-xL)], was associated with worsening fibrosis, particularly in adefovir treated patients (D-stage: 2.3 vs. 0, P = 0.004). In the 11 patients without significant changes from 1st to 2nd biopsy, increased apoptosis was more frequent in treated than untreated cases (P = 0.046). In multivariate analysis, bax change was independently associated with change of grade (P = 0.038) and antiviral therapy (P = 0.015). In conclusions, in HBeAg-negative CHB, histological improvement after treatment is associated with decreased hepatocyte apoptosis. In patients without substantial histological changes, treatment seems to increase the apoptosis of hepatocytes, thus having a possible protective effect on hepatocarcinogenesis.

Long-term adefovir plus lamivudine therapy does not decrease creatinine clearance in HBeAg-negative chronic hepatitis B patients.

BACKGROUND/AIMS: As there are concerns about potential nephrotoxicity of nucleotide analogues, we evaluated renal function parameters during long-term adefovir and lamivudine combination therapy. METHODS: Forty-six HBeAg-negative patients with lamivudine-resistance treated with adefovir and lamivudine for up to 90 months were included. Renal function was assessed by estimated creatinine clearance (eC(CR) ) and compared with a matched control group of untreated inactive hepatitis B virus carriers. RESULTS: Serum HBV DNA became undetectable in 39 (85%) patients after a mean of 37 ± 21 months. Three (6.5%) patients developed virological breakthrough. Adefovir resistance was detected in two patients. At the end of follow up, there was a significant decrease in mean eC(CR) (95 ± 31-83 ± 30 ml/min, P = 0.003) in the treated patients with 16% presenting aeC(CR) decrease >30%. Similar changes in eC(CR) were observed in the control group (108 ± 28-96 ± 26 ml/min, P = 0.003). In multiple regression analysis, age and baseline eC(CR) were independent predictors of eC(CR) reduction. CONCLUSIONS: Adefovir and lamivudine combination therapy is not an independent factor for significant renal dysfunction in HBeAg-negative patients with lamivudine-resistance. Baseline age and creatinine clearance are the only independent predictors of worsening renal function.

Serum apoptotic caspase activity in chronic hepatitis C and nonalcoholic Fatty liver disease.

BACKGROUND: Apoptotic caspases are substantially activated in liver and serum caspase activity has been suggested as a marker of early liver injury. AIM: To investigate whether serum levels of caspase-generated fragments of cytokeratin-18 are associated with the severity of histologic lesions in chronic hepatitis C virus (HCV) infection and nonalcoholic fatty liver disease (NAFLD). METHODS: We included 134 patients with chronic HCV infection and 58 patients with NAFLD, who consecutively underwent liver biopsy, and 40 healthy controls. Caspase-generated cytokeratin-18 fragment levels were blindly measured in stored serum samples. RESULTS: Median cytokeratin-18 fragment levels were lower in HCV-positive patients with minimal/mild than patients with moderate/severe histologic lesions (174 U/L vs. 223 U/L, P<0.001) offering moderate accuracy for differentiation between the 2 groups (c-statistic: 0.74). Cytokeratin-18 fragments levels were lower in healthy subjects (148 U/L) than patients with simple fatty liver (174 U/L, P=0.013) than patients with nonalcoholic steatohepatitis (355 U/L, P<0.001) offering excellent diagnostic accuracy for differentiation between the 2 latter groups (c-statistic: 0.87). CONCLUSIONS: Serum apoptotic caspase activity is associated with the severity of liver histologic lesions in both chronic HCV infection and NAFLD, but it has excellent diagnostic accuracy in NAFLD and moderate accuracy in chronic HCV patients.

Is there a meaningful serum hepatitis B virus DNA cutoff level for therapeutic decisions in hepatitis B e antigen-negative chronic hepatitis B virus infection?

UNLABELLED: The diagnosis of hepatitis B e antigen (HBeAg)-negative chronic hepatitis B indicating therapeutic intervention currently requires serum hepatitis B virus (HBV) DNA >or=2,000 IU/mL. We evaluated the severity of liver histology and the presence of histological indication for treatment in patients with HBeAg-negative chronic HBV infection focusing on those with low viremia and/or normal alanine aminotransferase (ALT). In total, 399 patients with increased ALT and detectable serum HBV DNA (chronic hepatitis B patients) and 35 cases with persistently normal ALT and HBV DNA >2,000 IU/mL (inactive carriers) were included. Histological indication for treatment (grading score >or=7 and/or stage >or=2 in Ishak's classification) was found in 91% (185/203), 82% (75/91), 75% (47/63), and 62% (26/42) of chronic hepatitis B patients with HBV DNA >or=200,000, 20,000-199,999, 2,000-19,999, and <2,000 IU/mL, respectively (P < 0.001). Histological indication for treatment was more frequent in chronic hepatitis B patients with persistently elevated ALT (86% or 275/321), but it was also found in 74% (58/78) of those with transiently normal ALT (P = 0.025). All inactive carriers had HBV DNA <20,000 IU/mL. Histological indication for treatment was present in 17% (6/35) of inactive carriers always due to moderate (stage 2) fibrosis without active necroinflammation. CONCLUSION: HBeAg-negative chronic HBV patients with persistently or transiently increased ALT and HBV DNA >or=20,000 IU/mL almost always require therapeutic intervention, but histological indications for treatment are also present in the majority of such cases with HBV DNA <20,000 and even <2,000 IU/mL. In contrast, minimal histological lesions are observed in the majority of HBeAg-negative patients with persistently normal ALT and HBV DNA >2,000 IU/mL, who may not require immediate liver biopsy and treatment but only close follow-up.

Circulating and liver tissue levels of retinol-binding protein-4 in non-alcoholic fatty liver disease.

AIM: Retinol-binding protein-4 (RBP4) has been proposed as a new adipokine that regulates insulin action in muscles and the liver, and contributes to the pathogenesis of insulin resistance. As non-alcoholic fatty liver disease (NAFLD) is related to insulin resistance, we aimed to evaluate RBP4 levels in the serum and liver of patients with NAFLD. METHODS: Serum RBP4 was measured in 30 NAFLD patients and 30 matched healthy controls. RBP4 expression in the liver of NAFLD patients was shown by immunohistochemistry. RESULTS: Serum RPB4 was significantly lower in NAFLD patients compared with controls (25.15 vs 34.66 microg/mL, P < 0.001) and there was no correlation with metabolic parameters or insulin resistance. RBP4 liver tissue immunostaining was more extensive and intense in NAFLD liver compared with normal liver and the RBP4 immunohistochemical score was positively correlated with the grade of steatosis, grade of non-alcoholic steatohepatitis activity and stage of fibrosis. CONCLUSIONS: In NAFLD patients, serum RBP4 was significantly lower as compared with controls and did not correlate with insulin resistance. In contrast, RBP4 liver tissue expression was enhanced and correlated with NAFLD histology.

Collagen proportionate area is an independent predictor of long-term outcome in patients with non-alcoholic fatty liver disease.

BACKGROUND: Collagen proportionate area (CPA) measurement is a technique that quantifies fibrous tissue in liver biopsies by measuring the amount of collagen deposition as a proportion of the total biopsy area. CPA predicts clinical outcomes in patients with HCV and can sub-classify cirrhosis. AIM: To test the ability of CPA to quantify fibrosis and predict clinical outcomes in patients with NAFLD. METHODS: We assessed consecutive patients with biopsy-proven NAFLD from three European centres. Clinical and laboratory data were collected at baseline and at the time of the last clinical follow-up or death. CPA was performed at two different objective magnifications, whole biopsy macro and ×4 objective magnification, named standard (SM) and high (HM) magnification respectively. The correlation between CPA and liver stiffness was assessed in a sub-group of patients. RESULTS: Of 437 patients, 32 (7.3%) decompensated and/or died from liver-related causes during a median follow-up of 103 months. CPA correlated with liver stiffness and liver fibrosis stage across the whole spectrum of fibrosis. HM CPA was significantly higher than SM CPA in stages F0-F3 but similar in cirrhosis, reflecting a higher ability to capture pericellular/perisinusoidal fibrosis at early stages. Age at baseline (HR: 1.04, 95% CI: 1.01-1.08), HM CPA (HR: 1.04 per 1% increase, 95% CI: 1.01-1.08) and presence of advanced fibrosis (HR: 15.4, 95% CI: 5.02-47.84) were independent predictors of liver-related clinical outcomes at standard and competing risk multivariate Cox-regression analysis. CONCLUSIONS: CPA accurately measures fibrosis and is an independent predictor of clinical outcomes in NAFLD; hence it merits further evaluation as a surrogate endpoint in clinical trials.

Serum adipokine levels in chronic liver diseases: association of resistin levels with fibrosis severity.

OBJECTIVE: Leptin and adiponectin have been implicated in the pathogenesis and progression of non-alcoholic steatohepatitis (NASH) and chronic hepatitis C (CHC), but little is known about the role of resistin in chronic liver diseases. The objective of this study was to investigate serum levels of the above three adipokines in relation to the etiology of liver disease and to determine their associations with histological severity. MATERIAL AND METHODS: We prospectively evaluated 146 patients (HBeAg-negative chronic hepatitis B (CHB): 52, CHC: 70, NASH: 24) who consecutively underwent liver biopsy. Detailed epidemiological, anthropometric and laboratory data were recorded. Histological lesions were evaluated blindly according to the Ishak and the Brunt classifications for CHB/CHC and NASH, respectively. RESULTS: Serum adipokine levels were similar between CHB and CHC patients, while CHB/CHC patients had significantly lower leptin levels compared with NASH patients (8.3+/-7.3 versus 17.6+/-16.6 ng/ml, p=0.012) and higher adiponectin (10.2+/-5.1 versus 7.5+/-4 microg/ml, p=0.018) and resistin levels (7.1+/-2.5 versus 5.7+/-2.8 ng/ml, p=0.016). In CHB/CHC, there was no significant association between steatosis or necroinflammation and levels of adipokines, while the presence of moderate/severe fibrosis (stages 4-6) was associated with higher leptin and adiponectin levels in male but not in female patients and with lower resistin levels irrespective of gender or other factors (adjusted odds ratio=0.788, p=0.035). CONCLUSIONS: Serum adipokine levels depend on the etiology of liver disease differing between chronic viral hepatitis and NASH, but not between CHB and CHC. In CHB/CHC, resistin levels are independently associated with fibrosis severity, whereas in the association of leptin and adiponectin levels with fibrosis, it seems to be a gender effect.