Clinical and genetic definition of serum bilirubin levels for the diagnosis of Gilbert syndrome and hypobilirubinemia.

BACKGROUND AND AIMS: Gilbert syndrome (GS) is genotypically predetermined by UGT1A1*28 homozygosity in Europeans and is phenotypically defined by hyperbilirubinemia using total bilirubin (TB) cutoff ≥1mg/dL (17 µmol/L). The prevalence of illnesses associated with GS and hypobilirubinemia has never been studied prospectively. As TB varies with UGT1A1*28 genotyping, sex, and age, we propose stratified definitions of TB reference intervals and report the prevalence of illnesses and adjusted 15 years survival. METHODS: UK Biobank with apparently healthy liver participants (middle-aged, n=138,125) were analyzed after the exclusion of of nonhealthy individuals. The stratified TB was classified as GS when TB >90th centile; <10th centile indicated hypobilirubinemia, and between the 10th and 90th centile was normobilirubinemia. We compared the prevalence and survival rates of 54 illnesses using odds ratio (OR), logistic regression, and Cox models adjusted for confounders, and causality by Mendelian randomizations. RESULTS: In women, we identified 10% (7,741/76,809) of GS versus 3.7% (2,819/76,809) using the historical cutoff of ≥1 mg/dL (P<0.0001). When GS and hypobilirubinemia participants were compared with normobilirubinemia, after adjustment and Mendelian randomizations, only cholelithiasis prevalence was significantly higher (OR=1.50; 95% CI [1.3-1.7], P=0.001) in men with GS compared with normobilirubinemia and in causal association with bilirubin (P=0.04). No adjusted survival was significantly associated with GS or hypobilirubinemia. CONCLUSIONS: In middle-aged Europeans, the stratified TB demonstrates a careless GS underestimation in women when using the standard unisex 1 mg/dL cutoff. The prevalence of illnesses is different in GS and hypobilirubinemia as well as survivals before adjusting for confounding factors. With the exception of cholelithiasis in men, these differences were no more significant after adjustment and Mendelian randomization.

Prospective direct comparison of non-invasive liver tests in outpatients with type 2 diabetes using intention-to-diagnose analysis.

BACKGROUND: No prospective diagnostic studies have directly compared widespread non-invasive liver tests in patients with type 2 diabetes (T2D) using the intention-to-diagnose method for each of the three main histological features of metabolic dysfunction associated steatotic liver disease - namely fibrosis, metabolic dysfunction-associated steatohepatitis (MASH), and steatosis. AIMS: To compare the performance of nine tests using the intention-to-diagnose rather than the standard method, which would exclude non-evaluable participants METHODS: Biopsy was used as the reference with predetermined cut-offs, advanced fibrosis being the main endpoint. The Nash-FibroTest panel including FibroTest-T2D, SteatoTest-T2D and MashTest-T2D was optimised for type 2 diabetes. FibroTest-T2D was compared to vibration-controlled transient elastography stiffness (VCTE), two-dimensional shear-wave elastography stiffness (TD-SWE), and Fibrosis-4 blood test. NashTest-T2D was compared to aspartate aminotransferase. SteatoTest-T2D was compared to the controlled attenuation parameter and the hepatorenal gradient. RESULTS: Among 402 cases, non-evaluable tests were 6.7% for VCTE, 4.0% for hepatorenal gradient, 3.2% for controlled attenuation parameter, 1.5% for TD-SWE, 1.2% for NashTest-T2D, and 0.02% for Fibrosis-4, aspartate aminotransferase and SteatoTest-T2D. The VCTE AUROC for advanced fibrosis was over-estimated by 6% (0.83 [95% CI: 0.78-0.87]) by standard analysis compared to intention-to-diagnose (0.77 [0.72-0.81] p = 0.008). The AUROCs for advanced fibrosis did not differ significantly in intention-to-diagnose between FibroTest-T2D (0.77; 95% CI: 0.73-0.82), VCTE (0.77; 95% CI: 0.72-0.81) and TD-SWE(0.78; 0.74-0.83) but were all higher than the Fibrosis-4 score (0.70; 95% CI all differences ≥7%; p ≤ 0.03). For MASH, MashTest-T2D had a higher AUROC (0.76; 95% CI: 0.70-0.80) than aspartate aminotransferase (0.72; 95% CI: 0.66-0.77; p = 0.035). For steatosis, AUROCs did not differ significantly between SteatoTest-T2D, controlled attenuation parameter and hepatorenal gradient. CONCLUSIONS: In intention-to-diagnose analysis, FibroTest-T2D, TD-SWE and VCTE performed similarly for staging fibrosis, and out-performed Fibrosis-4 in outpatients with type 2 diabetes. The standard analysis over-estimated VCTE performance. CLINICALTRIAL: gov: NCT03634098.

Hepatocyte-derived biomarkers predict liver-related events at 2 years in Child-Pugh class A alcohol-related cirrhosis.

BACKGROUND & AIMS: In patients with compensated alcohol-related cirrhosis, reliable prognostic biomarkers are lacking. Keratin-18 and hepatocyte-derived large extracellular vesicle (lEV) concentrations reflect disease activity, but their ability to predict liver-related events is unknown. METHODS: We measured plasma keratin-18 and hepatocyte lEV concentrations in 500 patients with Child-Pugh class A alcohol-related cirrhosis. The ability of these hepatocyte-derived biomarkers, alone or combined with model for end-stage liver disease (MELD) and FibroTest scores, to predict liver-related events at 2 years was analyzed, taking into account the alcohol consumption at inclusion and during follow-up. RESULTS: Keratin-18 and hepatocyte lEV concentrations increased with alcohol consumption. In patients without active alcohol consumption at enrollment (n = 419), keratin-18 concentration predicted liver-related events at 2 years, independently of FibroTest and MELD. Patients with both keratin-18 concentrations >285 U/L and FibroTest >0.74 had a 24% cumulative incidence of liver-related events at 2 years, vs. 5% to 14% in other groups of patients. Similar results were obtained when combining keratin-18 concentrations >285 U/L with MELD >10. In patients with active alcohol consumption at enrollment (n = 81), hepatocyte lEVs predicted liver-related events at 2 years, independently of FibroTest and MELD. Patients with both hepatocyte lEV concentrations >50 U/L and FibroTest >0.74 had a 62% cumulative incidence of liver-related events at 2 years, vs. 8% to 13% in other groups of patients. Combining hepatocyte lEV concentrations >50 U/L with MELD >10 had a lower discriminative ability. Similar results were obtained when using decompensation of cirrhosis, defined according to Baveno VII criteria, as an endpoint. CONCLUSION: In patients with Child-Pugh class A alcohol-related cirrhosis, combining hepatocyte-derived biomarkers with FibroTest or MELD scores identifies patients at high risk of liver-related events, and could be used for risk stratification and patient selection in clinical trials. IMPACT AND IMPLICATIONS: In patients with compensated alcohol-related cirrhosis, reliable predictors of outcome are lacking. In patients with Child-Pugh class A alcohol-related cirrhosis, combining hepatocyte-derived biomarkers (keratin-18 and hepatocyte-large extracellular vesicles) with FibroTest or MELD scores identifies those at high risk of liver-related events at 2 years. The identified patients at high risk of liver-related events are the target-of-choice population for intensive surveillance (e.g., referral to tertiary care centers; intensive control of risk factors) and inclusion in clinical trials.

High Prevalence of NASH and Advanced Fibrosis in Type 2 Diabetes: A Prospective Study of 330 Outpatients Undergoing Liver Biopsies for Elevated ALT, Using a Low Threshold.

OBJECTIVE: Most people with type 2 diabetes (T2DM) and nonalcoholic steatohepatitis (NASH) or advanced fibrosis (AF) remain undiagnosed, resulting in missed opportunities for early intervention. This multicenter, prospective study assessed the yield of using routinely available data to identify these patients. RESEARCH DESIGN AND METHODS: A total of 713 outpatients with T2DM, screened in four diabetology clinics for nonalcoholic fatty liver disease according to American Diabetes Association criteria, were referred to hepatologists for further work-up (Fibrosis-4 and vibration-controlled transient elastography [VCTE]). A liver biopsy was proposed when ALT levels were persistently >20 IU/L in female patients or >30 IU/L in male patients, in the absence of other liver disease. RESULTS: Liver biopsies were performed in 360 patients and considered adequate for reading after central review for 330 specimens (median patient age, 59 years; male patients, 63%; median BMI and HbA1c values, 32 and 7.5%, respectively). Prevalence of NASH, AF, and cirrhosis were 58%, 38%, and 10%, respectively. Liver lesions were independently associated with the components of metabolic syndrome but not with the micro- and macrovascular complications of T2DM. Models based on routinely available data with or without VCTE had good accuracy to predict AF (respectively: area under the receiver operating characteristic curve [AUROC], 0.84 and 0.77; and correctly classified 59% and 45%) and NASH (respectively: AUROC, 0.82 and 0.81; 44% and 42%). CONCLUSIONS: Despite the use of a low ALT threshold, prevalence of NASH (58%) or AF (38%) was high. Routinely available data had a high yield in identifying patients with T2DM with AF and/or NASH requiring further liver assessment.

Clinical Interest of Serum Alpha-2 Macroglobulin, Apolipoprotein A1, and Haptoglobin in Patients with Non-Alcoholic Fatty Liver Disease, with and without Type 2 Diabetes, before or during COVID-19.

In patients with non-alcoholic fatty liver disease (NAFLD) with or without type 2 diabetes mellitus (T2DM), alpha-2 macroglobulin (A2M), apolipoprotein A1 (ApoA1), and haptoglobin are associated with the risk of liver fibrosis, inflammation (NASH), and COVID-19. We assessed if these associations were worsened by T2DM after adjustment by age, sex, obesity, and COVID-19. Three datasets were used: the "Control Population", which enabled standardization of protein serum levels according to age and sex (N = 27,382); the "NAFLD-Biopsy" cohort for associations with liver features (N = 926); and the USA "NAFLD-Serum" cohort for protein kinetics before and during COVID-19 (N = 421,021). The impact of T2DM was assessed by comparing regression curves adjusted by age, sex, and obesity for the liver features in "NAFLD-Biopsy", and before and during COVID-19 pandemic peaks in "NAFLD-Serum". Patients with NAFLD without T2DM, compared with the values of controls, had increased A2M, decreased ApoA1, and increased haptoglobin serum levels. In patients with both NAFLD and T2DM, these significant mean differences were magnified, and even more during the COVID-19 pandemic in comparison with the year 2019 (all p < 0.001), with a maximum ApoA1 decrease of 0.21 g/L in women, and a maximum haptoglobin increase of 0.17 g/L in men. In conclusion, T2DM is associated with abnormal levels of A2M, ApoA1, and haptoglobin independently of NAFLD, age, sex, obesity, and COVID-19.

Validation of the Performance of A1HPV6, a Triage Blood Test for the Early Diagnosis and Prognosis of SARS-CoV-2 Infection.

BACKGROUND AND AIMS: Apolipoprotein A1 (A1) and haptoglobin (HP) serum levels are associated with the spread and severity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We have constructed and validated a multivariable risk calculator (A1HPV6) integrating A1, HP, alpha2-macroglobulin, and gamma glutamyl transferase to improve the performances of virological biomarkers. METHODS: In a prospective observational study of hospitalized patients with nonsevere SARS-CoV-2 infection, A1HPV6 was constructed in 127 patients and validated in 116. The specificity was assessed in 7482 controls representing the general population. The primary diagnostic endpoint was the area under the receiver operating characteristic curve in patients with positive SARS-CoV-2 PCR. The primary prognostic endpoint was the age-and sex-adjusted risk of A1HPV6 to predict patients with WHO-stage > 4 (W > 4) severity. We assessed the kinetics of the A1HPV6 components in a nonhuman primate model (NHP), from baseline to 7 days (D7) after SARS-CoV-2 infection. RESULTS: The area under the receiver operating characteristic curve for A1HPV6 was 0.99 (95% CI 0.97-0.99) in the validation subset, which was not significantly different from that in the construction subset, 0.99 (0.99-0.99; P = .80), like for sensitivity 92% (85-96) vs 94% (88-97; P = .29). A1HPV6 was associated with W > 4, with a significant odds ratio of 1.3 (1.1-1.5; 0.002). In NHP, A1 levels decreased (P < .01) at D2 and normalized at D4; HP levels increased at D2 and peaked at D4. In patients, A1 concentration was very low at D2 vs controls (P < .01) and increased at D14 (P < .01) but was still lower than controls; HP increased at D2 and remained elevated at D14. CONCLUSION: These results validate the diagnostic and prognostic performances of A1HPV6. Similar kinetics of apolipoprotein A1, HP, and alpha-2-macroglobulin were observed in the NHP model. ClinicalTrials.gov number, NCT01927133.

Baveno VI criteria as a prognostic factor for clinical complications in patients with compensated cirrhosis.

BACKGROUND: Combination of liver stiffness measurement and platelets count is a tool to safely rule out varices needing treatment (VNT) in patients with compensated advanced chronic liver disease (cACLD). AIMS: to evaluate 4-year liver-related complications and survival in low-risk patients according to Baveno VI criteria. METHODS: we conducted a monocentric retrospective analysis of prospectively collected data of all consecutive patients, with cirrhosis (LSM≥12.5 kPa) and without previous complication, evaluated between 2012 and 2015. Liver-related complications and survival were compared between 2 groups of patients: favourable (LSM< 20 kPa and platelet count>150.000/mm3) and unfavourable Baveno VI status patients (LSM ≥ 20 kPa or platelet count ≤150.000/mm3). RESULTS: 455 patients with cACLD were analysed. Two hundred patients had favourable Baveno VI criteria, 3.6% with VNT. The 4-year probability of being free of acute decompensation was higher in low-risk patients (94.4 ± 1.8% vs. 85.7%±2.6%, p = 0.018). Unfavourable Baveno status was independently associated with acute decompensation. The probability of being free of HCC was significantly higher in low-risk patients (94.2 ± 1.8% vs. 87.6 ± 2.4%, p = 0.048). Liver-related mortality was not different between the 2 groups (p = 0.56). CONCLUSION: The Baveno VI criteria could predict clinical outcome in cACLD.

Prospective external validation of a new non-invasive test for the diagnosis of non-alcoholic steatohepatitis in patients with type 2 diabetes.

BACKGROUND: One of the unmet needs in patients with type 2 diabetes mellitus (T2DM) is the prediction of non-alcoholic liver disease by non-invasive blood tests, for each of the three main histological features, fibrosis, non-alcoholic steatohepatitis (NASH) and steatosis. AIMS: To validate externally the performances of a recent panel, Nash-FibroTest, for the assessment of the severity of fibrosis stages, NASH grades and steatosis grades. METHODS: We prospectively analysed 272 patients with T2DM. Standard definitions of stages and grades were used, and analyses were centralised and blinded. The performances of the FibroTest, NashTest-2 and SteatoTest-2 were assessed using the Obuchowski measure (OM), the main outcome recommended as a summary measure of accuracy includeing all pairwise stages and grades comparisons, which is not provided par the extensively used binary area under the ROC curve. RESULTS: The diagnostic performance of each component of the panel was significant. OM (SE; significance) of the FibroTest, the NashTest-2 and the SteatoTest-2 was 0.862 (0.012; P < 0.001), 0.827 (0.015; P < 0.001) and 0.794 (0.020; P < 0.01), respectively. For ballooning and lobular inflammation, OM was 0.794 (0.021; P < 0.001) and 0.821 (0.017; P < 0.001), respectively. In a post hoc analysis the FibroTest outperformed VCTE by 4.1% (2.5-6.5; P < 0.001) for reliability, with a non-significant difference for OM for fibrosis staging, 0.859 (0.012) for FibroTest vs 0.870 (0.009) for VCTE. CONCLUSIONS: From a single blood sample, the panel provides non-invasive diagnosis of the stages of fibrosis, and the grades of NASH and steatosis in patients with T2DM. TRIAL REGISTRATION NUMBER: NCT03634098.

External validation of LCR1-LCR2, a multivariable HCC risk calculator, in patients with chronic HCV.

BACKGROUND & AIMS: The Liver Cancer Risk test algorithm (LCR1-LCR2) is a multianalyte blood test combining proteins involved in liver cell repair (apolipoprotein-A1 and haptoglobin), known hepatocellular carcinoma (HCC) risk factors (sex, age, and gamma-glutamyl transferase), a marker of fibrosis (alpha2-macroglobulin) and alpha-fetoprotein (AFP), a specific marker of HCC. The aim was to externally validate the LCR1-LCR2 in patients with chronic HCV (CHC) treated or not with antivirals. METHODS: Pre-included patients were from the Hepather cohort, a multicentre prospective study in adult patients with CHC in France. LCR1-LCR2 was assessed retrospectively in patients with the test components and AFP, available at baseline. The co-primary study outcome was the negative predictive value (NPV) of LCR1-LCR2 for the occurrence of HCC at 5 years and for survival without HCC according to the predetermined LCR1-LCR2 cut-offs. The cut-offs were adjusted for risk covariables and for the response to HCV treatment, and were quantified using time-dependent proportional hazards models. RESULTS: In total, 4,903 patients, 1,026 (21.9%) with baseline cirrhosis, were included in the study. Patients were followed for a median of 5.7 (IQR 4.2-11.3) years. A total of 3,788/4,903 (77.3%) patients had a sustained virological response. There were 137 cases of HCC at 5 years and 214 at the end of follow-up. HCC occurred at 5 years in 24/3,755 patients with low-risk LCR1-LCR2 compared with 113/1,148 patients with high-risk LCR1-LCR2. The NPV was 99.4% (95% CI 99.1-99.6). Similar findings (hazard ratio, 10.8; 95% CI, 8.1-14.3; p <0.001) were obtained after adjustment for exposure to antivirals, age, sex, geographical origin, HCV genotype 3, alcohol consumption, and type 2 diabetes mellitus. CONCLUSIONS: The results showed that LCR1-LCR2 can be used to successfully identify patients with HCV at very low risk of HCC at 5 years. LAY SUMMARY: Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death worldwide and the fastest growing cause of cancer death in many countries. We constructed and internally validated a new multianalyte blood test to assess this Liver Cancer Risk (LCR1-LCR2). This study confirmed the performance of LCR1-LCR2 in patients with chronic HCV in the national French cohort Hepather, and its ability to identify patients at a very low risk of HCC at 5 years. CLINICAL TRIALS REGISTRATION: The study is registered at ClinicalTrials.gov (NCT01953458).

Performance of serum apolipoprotein-A1 as a sentinel of Covid-19.

BACKGROUND: Since 1920, a decrease in serum cholesterol has been identified as a marker of severe pneumonia. We have assessed the performance of serum apolipoprotein-A1, the main transporter of HDL-cholesterol, to identify the early spread of coronavirus disease 2019 (Covid-19) in the general population and its diagnostic performance for the Covid-19. METHODS: We compared the daily mean serum apolipoprotein-A1 during the first 34 weeks of 2020 in a population that is routinely followed for a risk of liver fibrosis risk in the USA (212,297 serum) and in France (20,652 serum) in relation to a local increase in confirmed cases, and in comparison to the same period in 2019 (266,976 and 28,452 serum, respectively). We prospectively assessed the sensitivity of this marker in an observational study of 136 consecutive hospitalized cases and retrospectively evaluated its specificity in 7,481 controls representing the general population. RESULTS: The mean serum apolipoprotein-A1 levels in the survey populations began decreasing in January 2020, compared to the same period in 2019. This decrease was highly correlated with the daily increase in confirmed Covid-19 cases in the following 34 weeks, both in France and USA, including the June and mid-July recovery periods in France. Apolipoprotein-A1 at the 1.25 g/L cutoff had a sensitivity of 90.6% (95%CI84.2-95.1) and a specificity of 96.1% (95.7-96.6%) for the diagnosis of Covid-19. The area under the characteristics curve was 0.978 (0.957-0.988), and outperformed haptoglobin and liver function tests. The adjusted risk ratio of apolipoprotein-A1 for survival without transfer to intensive care unit was 5.61 (95%CI 1.02-31.0; P = 0.04). CONCLUSION: Apolipoprotein-A1 could be a sentinel of the pandemic in existing routine surveillance of the general population. NCT01927133, CER-2020-14.

Transportation and handling of blood samples prior to ammonia measurement in the real life of a large university hospital.

BACKGROUND: Hyperammonemia is neurotoxic and as such can be a medical emergency. Preanalytical factors greatly influence the blood ammonia concentration results. AIMS AND METHODS: Ammonia concentrations measured in the real life setting of a large hospital after pneumatic transport of blood samples and various time periods before centrifugation were compared to those based on the indications of the reagent manufacturer. In the same routine context, the effects of waiting times of centrifuged samples or after plasma storage at -20 °C and -80 °C were determined. RESULTS: Despite the pneumatic transport, the lead times for sample arrival to the lab were even longer than those recommended for their complete handling until ammonia assay. Ammonia concentration results were not affected by the pneumatic transport of blood samples and by waiting times up to a maximum of 1.75 h before their centrifugation and 1 h after centrifugation. Ammonia stability was superior when plasma was stored at -80 °C. CONCLUSION: Pneumatic transport and sample handling in the routine practice of our lab do not affect ammonia concentration results provided that waiting times are limited to 1.75 h before and 1 h after centrifugation and samples are kept cold. Otherwise, it is better to freeze plasma at -80 °C.

Hepatitis C Clearance by Direct-Acting Antivirals Impacts Glucose and Lipid Homeostasis.

BACKGROUND: Chronic hepatitis C virus (HCV) infections are causally linked with metabolic comorbidities such as insulin resistance, hepatic steatosis, and dyslipidemia. However, the clinical impact of HCV eradication achieved by direct-acting antivirals (DAAs) on glucose and lipid homeostasis is still controversial. The study aimed to prospectively investigate whether antiviral therapy of HCV with DAAs alters glucose and lipid parameters. METHODS: 50 patients with chronic HCV who were treated with DAAs were screened, and 49 were enrolled in the study. Biochemical and virological data, as well as noninvasive liver fibrosis parameters, were prospectively collected at baseline, at the end of treatment (EOT) and 12 and 24 weeks post-treatment. RESULTS: 45 of 46 patients achieved sustained virologic response (SVR). The prevalence of insulin resistance (HOMA-IR) after HCV clearance was significantly lower, compared to baseline (5.3 ± 6.1 to 2.5 ± 1.9, p < 0.001), which is primarily attributable to a significant decrease of fasting insulin levels (18.9 ± 17.3 to 11.7 ± 8.7; p = 0.002). In contrast to that, HCV eradication resulted in a significant increase in cholesterol levels (total cholesterol, low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein (HDL-C) levels) and Controlled Attenuated Score (CAP), although BMI did not significantly change over time (p = 0.95). Moreover, HOMA-IR correlated significantly with noninvasive liver fibrosis measurements at baseline und during follow-up (TE: r = 0.45; p = 0.003, pSWE: r = 0.35; p = 0.02, APRI: r = 0.44; p = 0.003, FIB-4: r = 0.41; p < 0.001). CONCLUSION: Viral eradication following DAA therapy may have beneficial effects on glucose homeostasis, whereas lipid profile seems to be worsened.

Origin, HDV genotype and persistent viremia determine outcome and treatment response in patients with chronic hepatitis delta.

BACKGROUND & AIMS: HDV infection causes severe chronic liver disease in individuals infected with HBV. However, the factors associated with poor prognosis are largely unknown. Thus, we aimed to identify prognostic factors in patients with HDV infection. METHODS: The French National Reference Centre for HDV performed a nationwide retrospective study on 1,112 HDV-infected patients, collecting epidemiological, clinical, virological and histological data from the initial referral to the last recorded follow-up. RESULTS: The median age of our cohort was 36.5 (29.9-43.2) years and 68.6% of our cohort were male. Most patients whose birthplace was known were immigrants from sub-Saharan Africa (52.5%), southern and eastern Europe (21.3%), northern Africa and the Middle East (6.2%), Asia (5.9%) and South America (0.3%). Only 150 patients (13.8%) were French native. HDV load was positive in 659 of 748 tested patients (88.1%). HDV-1 was predominant (75.9%), followed by sub-Saharan genotypes: HDV-5 (17.6%), HDV-7 (2.9%), HDV-6 (1.8%) and HDV-8 (1.6%). At referral, 312 patients (28.2%) had cirrhosis, half having experienced at least 1 episode of hepatic decompensation. Cirrhosis was significantly less frequent in African than in European patients regardless of HDV genotype. At the end of follow-up (median 3.0 [0.8-7.2] years), 48.8% of the patients had developed cirrhosis, 24.2% had ≥1 episode(s) of decompensation and 9.2% had hepatocellular carcinoma. European HDV-1 and African HDV-5 patients were more at risk of developing cirrhosis. Persistent replicative HDV infection was associated with decompensation, hepatocellular carcinoma and death. African patients displayed better response to interferon therapy than non-African patients (46.4% vs. 29.1%, p <0.001). HDV viral load at baseline was significantly lower in responders than in non-responders. CONCLUSION: Place of birth, HDV genotype and persistent viremia constitute the main determinants of liver involvement and response to treatment in chronic HDV-infected patients. LAY SUMMARY: Chronic liver infection by hepatitis delta virus (HDV) is the most severe form of chronic viral hepatitis. Despite the fact that at least 15-20 million people are chronically infected by HDV worldwide, factors determining the severity of liver involvement are largely unknown. By investigating a large cohort of 1,112 HDV-infected patients followed-up in France, but coming from different areas of the world, we were able to determine that HDV genotype, place of birth (reflecting both viral and host-related factors) and persistent viremia constitute the main determinants of liver involvement and response to treatment.

Input of serum haptoglobin fucosylation profile in the diagnosis of hepatocellular carcinoma in patients with non-cirrhotic liver disease.

BACKGROUND: Haptoglobin bifucosylated tetra-antennary glycan have been identified in patients with early stage hepatocellular carcinoma, but its specificity according to the presence or not of cirrhosis has never been assessed. The aims of this study were to determine if haptoglobin bifucosylated tetra-antennary glycan (1) could be a marker of HCC in patients without cirrhosis; (2) could increase the performance of standard alpha-fetoprotein (AFP) or recent blood tests for HCC detection, i.e., lectin-reactive alpha-fetoprotein (AFP-L3), des-gamma-carboxy prothrombin (DCP) and Liver-Cancer-Risk-test (LCR1-test). METHODS: We retrospectively selected patients, 102 with HCC (21 without cirrhosis), matched by stages with 140 controls without HCC (81 without cirrhosis). Haptoglobin fucosylation was assessed by MALDI-TOF. LCR-glycan algorithm was constructed combining components of the LCR-1 test (haptoglobin, gammaglutamyl-transpeptidase, apolipoproteinA1, alpha-2-macroglobulin) with AFP, AFP-L3, DCP and haptoglobin bifucosylated tetra-antennary glycan. RESULTS: In 102 patients without cirrhosis (21 HCC and 81 controls), the intention-to-diagnose analyses showed that haptoglobin bifucosylated tetra-antennary glycan alone had a sensitivity of 71% (15/21;95%CI 50-86), significantly better (P=0.02) than standard AFP (43%;9/21;95%CI 24-63), and a specificity of 96% (78/81;95% 90-99). The sensitivity of LCR-glycan, in patients without cirrhosis, was 86% (18/21; 95%CI 63-95) significantly better (P=0.001) than standard AFP (43%; 9/21; 95%CI 24-63), with an AUROC of 0.943 (95%CI 0.806-0.98) compared to 0.811 (95%CI 0.630-0.908) for AFP (P=0.06). CONCLUSION: Haptoglobin bifucosylated tetra-antennary glycan is associated with the presence of HCC in patients with chronic liver disease including those without cirrhosis. Its combination with existing HCC biomarkers could improve the performance of standard AFP for HCC detection.

Subclinical proximal tubulopathy in hepatitis B: The roles of nucleot(s)ide analogue treatment and the hepatitis B virus.

BACKGROUND: The recommended monitoring tools for evaluating nucleot(s)ide analogue renal toxicity, such as estimated glomerular filtration rate (eGFR) and phosphatemia, are late markers of proximal tubulopathy. Multiple early markers are available, but no consensus exists on their use. AIM: To determine the 24 mo prevalence of subclinical proximal tubulopathy (SPT), as defined with early biomarkers, in treated vs untreated hepatitis B virus (HBV)-monoinfected patients. METHODS: A prospective, non-randomized, multicenter study of HBV-monoinfected patients with a low number of renal comorbidities was conducted. The patients were separated into three groups: Naïve, starting entecavir (ETV) treatment, or starting tenofovir disoproxil (TDF) treatment. Data on the early markers of SPT, the eGFR and phosphatemia, were collected quarterly. SPT was defined as a maximal tubular reabsorption of phosphate/eGFR below 0.8 mmoL/L and/or uric acid fractional excretion above 10%. The prevalence and cumulative incidence of SPT at month 24 (M24) were calculated. Quantitative data were analyzed using analyses of variance or Kruskal-Wallis tests, whereas chi-squared or Fisher's exact tests were used to analyze qualitative data. Multivariate analyses were used to adjust for any potential confounding factors. RESULTS: Of the 196 patients analyzed, 138 (84 naïve, 28 starting ETV, and 26 starting TDF) had no SPT at inclusion. At M24, the prevalence of SPT was not statistically different between naïve and either treated group (21.1% vs 30.7%, P < 0.42 and 50.0% vs 30.7%, P = 0.32 for ETV and TDF, respectively); no patient had an eGFR lower than 50 mL/min/1.73 m² or phosphatemia less than 0.48 mmoL/L. In the multivariate analysis, no explanatory variables were identified after adjustment. The cumulative incidence of SPT over 24 mo (25.5%, 13.3%, and 52.9% in the naïve, ETV, and TDF groups, respectively) tended to be higher in the TDF group vs the naïve group (hazard ratio: 2.283, P = 0.05). SPT-free survival at M24 was 57.6%, 68.8%, and 23.5% for the naïve, ETV, and TDF groups, respectively. The median survival time without SPT, evaluated only in the TDF group, was 5.9 mo. CONCLUSION: The prevalence and incidence of SPT was higher in TDF-treated patients compared to naïve patients. SPT in the naïve population suggests that HBV can induce renal tubular toxicity.

Clinical validation of the FLIP algorithm and the SAF score in patients with non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Histological classifications used to diagnose/stage non-alcoholic fatty liver disease (NAFLD) are based on morphology, with undetermined clinical correlates and relevance. We assessed the clinical relevance of the fatty liver inhibition of progression (FLIP) algorithm and the steatosis, activity, and fibrosis (SAF) scoring system. METHODS: One hundred and forty consecutive patients with suspected NAFLD and a separate validation cohort of 78 patients enrolled in a therapeutic trial, all with central reading of liver biopsy, were included. FLIP and SAF were used to categorize patients with non-alcoholic steatohepatitis (NASH), non-NASH NAFLD (NAFL), or non-NAFLD. The SAF activity score assessed hepatocyte ballooning and lobular inflammation; a histologically severe disease was defined as a SAF activity score of ≥3 and/or bridging fibrosis or cirrhosis. Clinical, biochemical, and metabolic data were analyzed in relation to histology. RESULTS: Patients with NASH according to the FLIP algorithm had a clinical profile distinct from those with NAFL, with a higher prevalence of metabolic risk factors (increased body mass index [BMI], central obesity, serum glucose, and glycated hemoglobin), more severe insulin resistance (fasting insulin and homeostasis model assessment for insulin resistance [HOMA-IR] values), and higher levels of aminotransferases. Similar findings were documented for patients with severe disease vs. those without. Positive linear trends existed between NASH or severe disease and increasing BMI and HOMA-IR. There was a strong association between liver fibrosis and NASH or SAF-defined scores of activity. Patients with either significant or bridging fibrosis overwhelmingly had NASH, and bridging fibrosis most often coexisted with severe activity. CONCLUSIONS: The FLIP algorithm/SAF score, although based on purely morphological grounds, are clinically relevant, as they identify patients with distinct clinical and biological profiles of disease severity. Disease activity in NAFLD is associated with fibrosis severity. LAY SUMMARY: The examination of liver tissue under the microscope (histology) serves to define the type and severity of non-alcoholic fatty liver disease morphologically, and is also used to determine improvement in therapeutic or natural history clinical trials. The FLIP algorithm/SAF classification is a new histological classification well validated on morphological but not clinical grounds. Here, we demonstrate that different disease categories defined by the FLIP/SAF classification correspond to entities of different clinical and biological severity. We also show a strong association between the activity of steatohepatitis (defined histologically) and the amount of fibrotic scar.

Performance of liver biomarkers, in patients at risk of nonalcoholic steato-hepatitis, according to presence of type-2 diabetes.

OBJECTIVE: There is a controversy about the performance of blood tests for the diagnostic of metabolic liver disease in patients with type-2-diabetes in comparison with patients without type-2-diabetes. These indirect comparisons assumed that the gold-standard is binary, whereas fibrosis stages, steatosis and nonalcoholic-steato-hepatitis (NASH) grades use an ordinal scale. The primary aim was to compare the diagnostic performances of FibroTest in type-2-diabetes vs. controls matched on gender, age, fibrosis stages and obesity, and taking into account the spectrum effect by Obuchowski measure. METHODS: Data were retrospectively compared among patients prospectively included, with simultaneous biopsy and blindly assessed FibroTest, SteatoTest-2 and NashTest-2. The secondary aim was to construct an index (SpectrumF3F4-Index) to predict an adjusted-area under the receiver operating curve (AUROC) for F3F4 diagnosis from the prevalences of fibrosis stages, permitting to reduce the spectrum effect when performances of FibroTest, transient elastography and magnetic resonance elastography are indirectly compared. RESULTS: In 505 patients at risk of NASH, the Obuchowski measures [95% confidence interval (CI)] of FibroTest, SteatoTest-2 and NashTest-2 were all equivalent in 136 type-2-diabetes cases vs. 369 matched controls: 0.871 (0.837-0.905), vs. 0.880 (0.879-0.881), 0.835 (0.797-0.873) vs. 0.806 (0.780-0.832) and 0.829 (0.793-0.865) vs. 0.855 (0.829-0.869), respectively. Standard-AUROCs (95% CI) were 0.932 (0.898-0.965), 0.872 (0.837-0.907) and 0.834 (0.699-0.969) and reduced after adjustment by SpectrumF3F4-Index to 0.794 (0.749-0.838), 0.767 (0.750-0.783) and 0.773 (0.725-0.822) for transient, magnetic resonance elastography and FibroTest, respectively. CONCLUSIONS: When compared by Obuchowski measures, the performances of tests were not different in patients with T2-diabetes vs. patients without T2-diabetes. When individual data are not available, adjusted-AUROCs reduced the spectrum effect.

Evaluation of Point Shear Wave Elastography Using Acoustic Radiation Force Impulse Imaging for Longitudinal Fibrosis Assessment in Patients with HBeAg-Negative HBV Infection.

BACKGROUND: Accurate assessment of hepatic fibrosis in patients with chronic HBeAg-negative Hepatitis B is of crucial importance not only to predict the long-term clinical course, but also to evaluate antiviral therapy indication. The aim of this study was to prospectively assess the utility of point shear wave elastography (pSWE) for longitudinal non-invasive fibrosis assessment in a large cohort of untreated patients with chronic HBeAg-negative hepatitis B virus (HBV) infection. METHODS: 407 consecutive patients with HBeAg-negative HBV infection who underwent pSWE, transient elastography (TE) as well as laboratory fibrosis markers, including fibrosis index based on four factors (FIB-4), aspartate to platelet ratio index (APRI) and FibroTest, on the same day were prospectively followed up for six years. Patients were classified into one of the three groups: inactive carriers (IC; HBV-DNA <2000 IU/mL and ALT <40 U/L); grey zone group 1 (GZ-1; HBV DNA <2000 IU/mL and ALT >40 U/L); grey zone group 2 (GZ-2; HBV-DNA >2000 IU/mL and ALT <40 U/L). RESULTS: pSWE results were significantly correlated with TE (r = 0.29, p < 0.001) and APRI (r = 0.17; p = 0.005). Median pSWE values did not differ between IC, GZ-1 and GZ-2 patients (p = 0.82, p = 0.17, p = 0.34). During six years of follow-up, median pSWE and TE values did not differ significantly over time (TE: p = 0.27; pSWE: p = 0.05). CONCLUSION: Our data indicate that pSWE could be useful for non-invasive fibrosis assessment and follow-up in patients with HBeAg-negative chronic HBV infection.

Identification of an Endoglin Variant Associated With HCV-Related Liver Fibrosis Progression by Next-Generation Sequencing.

Despite the astonishing progress in treating chronic hepatitis C virus (HCV) infection with direct-acting antiviral agents, liver fibrosis remains a major health concern in HCV infected patients, in particular due to the treatment cost and insufficient HCV screening in many countries. Only a fraction of patients with chronic HCV infection develop liver fibrosis. While there is evidence that host genetic factors are involved in the development of liver fibrosis, the common variants identified so far, in particular by genome-wide association studies, were found to have limited effects. Here, we conducted an exome association study in 88 highly selected HCV-infected patients with and without fibrosis. A strategy focusing on TGF-ß pathway genes revealed an enrichment in rare variants of the endoglin gene (ENG) in fibrosis patients. Replication studies in additional cohorts (617 patients) identified one specific ENG variant, Thr5Met, with an overall odds ratio for fibrosis development in carriers of 3.04 (1.39-6.69). Our results suggest that endoglin, a key player in TGF-ß signaling, is involved in HCV-related liver fibrogenesis.