Outcomes of 38 patients with PFIC3: Impact of genotype and of response to ursodeoxycholic acid therapy.

Background & Aims: Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare liver disease caused by biallelic variations in ABCB4. Data reporting on the impact of genotype and of response to ursodeoxycholic acid (UDCA) therapy on long-term outcomes are scarce. Methods: We retrospectively describe a cohort of 38 patients with PFIC3 with a median age at last follow-up of 19.5 years (range 3.8-53.8). Results: Twenty patients presented with symptoms before 1 year of age. Thirty-one patients received ursodeoxycholic acid (UDCA) therapy resulting in serum liver test improvement in 20. Twenty-seven patients had cirrhosis at a median age of 8.1 years of whom 18 received a liver transplant at a median age of 8.5 years. Patients carrying at least one missense variation were more likely to present with positive (normal or decreased) canalicular MDR3 expression in the native liver and had prolonged native liver survival (NLS; median 12.4 years [range 3.8-53.8]). In contrast, in patients with severe genotypes (no missense variation), there was no detectable canalicular MDR3 expression, symptom onset and cirrhosis occurred earlier, and all underwent liver transplantation (at a median age of 6.7 years [range 2.3-10.3]). The latter group was refractory to UDCA treatment, whereas 87% of patients with at least one missense variation displayed an improvement in liver biochemistry in response to UDCA. Biliary phospholipid levels over 6.9% of total biliary lipid levels predicted response to UDCA. Response to UDCA predicted NLS. Conclusions: Patients carrying at least one missense variation, with positive canalicular expression of MDR3 and a biliary phospholipid level over 6.9% of total biliary lipid levels were more likely to respond to UDCA and to exhibit prolonged NLS. Impact and implications: In this study, data show that genotype and response to ursodeoxycholic acid therapy predicted native liver survival in patients with PFIC3 (progressive familial intrahepatic cholestasis type 3). Patients carrying at least one missense variation, with positive (decreased or normal) immuno-staining for canalicular MDR3, and a biliary phospholipid level over 6.9% of total biliary lipids were more likely to respond to ursodeoxycholic acid therapy and to exhibit prolonged native liver survival.

In Vitro Rescue of the Bile Acid Transport Function of ABCB11 Variants by CFTR Potentiators.

ABCB11 is responsible for biliary bile acid secretion at the canalicular membrane of hepatocytes. Variations in the ABCB11 gene cause a spectrum of rare liver diseases. The most severe form is progressive familial intrahepatic cholestasis type 2 (PFIC2). Current medical treatments have limited efficacy. Here, we report the in vitro study of Abcb11 missense variants identified in PFIC2 patients and their functional rescue using cystic fibrosis transmembrane conductance regulator potentiators. Three ABCB11 disease-causing variations identified in PFIC2 patients (i.e., A257V, T463I and G562D) were reproduced in a plasmid encoding an Abcb11-green fluorescent protein. After transfection, the expression and localization of the variants were studied in HepG2 cells. Taurocholate transport activity and the effect of potentiators were studied in Madin-Darby canine kidney (MDCK) clones coexpressing Abcb11 and the sodium taurocholate cotransporting polypeptide (Ntcp/Slc10A1). As predicted using three-dimensional structure analysis, the three variants were expressed at the canalicular membrane but showed a defective function. Ivacaftor, GLP1837, SBC040 and SBC219 potentiators increased the bile acid transport of A257V and T463I and to a lesser extent, of G562D Abcb11 missense variants. In addition, a synergic effect was observed when ivacaftor was combined with SBC040 or SBC219. Such potentiators could represent new pharmacological approaches for improving the condition of patients with ABCB11 deficiency due to missense variations affecting the function of the transporter.

In vitro functional rescue by ivacaftor of an ABCB11 variant involved in PFIC2 and intrahepatic cholestasis of pregnancy.

BACKGROUND: ABCB11 variations are responsible for a spectrum of rare liver diseases, including progressive familial intrahepatic cholestasis type 2 (PFIC2) and intrahepatic cholestasis of pregnancy (ICP). Current medical treatment of these conditions mostly relies on ursodeoxycholic acid with limited efficacy. We report on the in vitro study of the p.A257V missense variant of ABCB11 identified in a PFIC2 patient and in her mother who experienced ICP. RESULTS: The Ala257 residue is located outside the ATP-binding site of ABCB11. We show that the p.A257V variant of ABCB11 is correctly expressed at the canalicular membrane of HepG2 cells but that its function significantly decreased when studied in MDCK cells. This functional defect can be fully rescued by Ivacaftor. CONCLUSION: Ivacaftor could be considered as a new pharmacological tool able to respond to an unmet medical need for patients with ICP and PFIC2 due to ABCB11 variations affecting ABCB11 function, even when the residue involved is not located in an ATP-binding site of ABCB11.

RAB10 Interacts with ABCB4 and Regulates Its Intracellular Traffic.

ABCB4 (ATP-binding cassette subfamily B member 4) is an ABC transporter expressed at the canalicular membrane of hepatocytes where it ensures phosphatidylcholine secretion into bile. Genetic variations of ABCB4 are associated with several rare cholestatic diseases. The available treatments are not efficient for a significant proportion of patients with ABCB4-related diseases and liver transplantation is often required. The development of novel therapies requires a deep understanding of the molecular mechanisms regulating ABCB4 expression, intracellular traffic, and function. Using an immunoprecipitation approach combined with mass spectrometry analyses, we have identified the small GTPase RAB10 as a novel molecular partner of ABCB4. Our results indicate that the overexpression of wild type RAB10 or its dominant-active mutant significantly increases the amount of ABCB4 at the plasma membrane expression and its phosphatidylcholine floppase function. Contrariwise, RAB10 silencing induces the intracellular retention of ABCB4 and then indirectly diminishes its secretory function. Taken together, our findings suggest that RAB10 regulates the plasma membrane targeting of ABCB4 and consequently its capacity to mediate phosphatidylcholine secretion.

Molecular Regulation of Canalicular ABC Transporters.

The ATP-binding cassette (ABC) transporters expressed at the canalicular membrane of hepatocytes mediate the secretion of several compounds into the bile canaliculi and therefore play a key role in bile secretion. Among these transporters, ABCB11 secretes bile acids, ABCB4 translocates phosphatidylcholine and ABCG5/G8 is responsible for cholesterol secretion, while ABCB1 and ABCC2 transport a variety of drugs and other compounds. The dysfunction of these transporters leads to severe, rare, evolutionary biliary diseases. The development of new therapies for patients with these diseases requires a deep understanding of the biology of these transporters. In this review, we report the current knowledge regarding the regulation of canalicular ABC transporters' folding, trafficking, membrane stability and function, and we highlight the role of molecular partners in these regulating mechanisms.

Effect of CFTR correctors on the traffic and the function of intracellularly retained ABCB4 variants.

BACKGROUND & AIM: ABCB4 is expressed at the canalicular membrane of hepatocytes. This ATP-binding cassette (ABC) transporter is responsible for the secretion of phosphatidylcholine into bile canaliculi. Missense genetic variations of ABCB4 are correlated with several rare cholestatic liver diseases, the most severe being progressive familial intrahepatic cholestasis type 3 (PFIC3). In a repurposing strategy to correct intracellularly retained ABCB4 variants, we tested 16 compounds previously validated as cystic fibrosis transmembrane conductance regulator (CFTR) correctors. METHODS: The maturation, intracellular localization and activity of intracellularly retained ABCB4 variants were analyzed in cell models after treatment with CFTR correctors. In addition, in silico molecular docking calculations were performed to test the potential interaction of CFTR correctors with ABCB4. RESULTS: We observed that the correctors C10, C13, and C17, as well as the combinations of C3 + C18 and C4 + C18, allowed the rescue of maturation and canalicular localization of four distinct traffic-defective ABCB4 variants. However, such treatments did not permit a rescue of the phosphatidylcholine secretion activity of these defective variants and were also inhibitory of the activity of wild type ABCB4. In silico molecular docking analyses suggest that these CFTR correctors might directly interact with transmembrane domains and/or ATP-binding sites of the transporter. CONCLUSION: Our results illustrate the uncoupling between the traffic and the activity of ABCB4 because the same molecules can rescue the traffic of defective variants while they inhibit the secretion activity of the transporter. We expect that this study will help to design new pharmacological tools with potential clinical interest.

Pharmacological Premature Termination Codon Readthrough of ABCB11 in Bile Salt Export Pump Deficiency: An In Vitro Study.

BACKGROUND AND AIMS: Progressive familial intrahepatic cholestasis type 2 (PFIC2) is a severe hepatocellular cholestasis due to biallelic mutations in ABCB11 encoding the canalicular bile salt export pump (BSEP). Nonsense mutations are responsible for the most severe phenotypes. The aim was to assess the ability of drugs to induce readthrough of six nonsense mutations (p.Y354X, p.R415X, p.R470X, p.R1057X, p.R1090X, and p.E1302X) identified in patients with PFIC2. APPROACH AND RESULTS: The ability of G418, gentamicin, and PTC124 to induce readthrough was studied using a dual gene reporter system in NIH3T3 cells. The ability of gentamicin to induce readthrough and to lead to the expression of a full-length protein was studied in human embryonic kidney 293 (HEK293), HepG2, and Can 10 cells using immunodetection assays. The function of the gentamicin-induced full-length protein was studied by measuring the [3 H]-taurocholate transcellular transport in stable Madin-Darby canine kidney clones co-expressing Na+-taurocholate co-transporting polypeptide (Ntcp). Combinations of gentamicin and chaperone drugs (ursodeoxycholic acid, 4-phenylbutyrate [4-PB]) were investigated. In NIH3T3, aminoglycosides significantly increased the readthrough level of all mutations studied, while PTC124 only slightly increased the readthrough of p.E1302X. Gentamicin induced a readthrough of p.R415X, p.R470X, p.R1057X, and p.R1090X in HEK293 cells. The resulting full-length proteins localized within the cytoplasm, except for BsepR1090X , which was also detected at the plasma membrane of human embryonic kidney HEK293 and at the canalicular membrane of Can 10 and HepG2 cells. Additional treatment with 4-PB and ursodeoxycholic acid significantly increased the canalicular proportion of full-length BsepR1090X protein in Can 10 cells. In Madin-Darby canine kidney clones, gentamicin induced a 40% increase of the BsepR1090X [3 H]-taurocholate transport, which was further increased with additional 4-PB treatment. CONCLUSION: This study constitutes a proof of concept for readthrough therapy in selected patients with PFIC2 with nonsense mutations.

Functional rescue of an ABCB11 mutant by ivacaftor: A new targeted pharmacotherapy approach in bile salt export pump deficiency.

BACKGROUND & AIM: The canalicular bile salt export pump (BSEP/ABCB11) of hepatocytes is the main adenosine triphosphate (ATP)-binding cassette (ABC) transporter responsible for bile acid secretion. Mutations in ABCB11 cause several cholestatic diseases, including progressive familial intrahepatic cholestasis type 2 (PFIC2) often lethal in absence of liver transplantation. We investigated in vitro the effect and potential rescue of a BSEP mutation by ivacaftor, a clinically approved cystic fibrosis transmembrane conductance regulator (CFTR/ABCC7) potentiator. METHODS: The p.T463I mutation, identified in a PFIC2 patient and located in a highly conserved ABC transporter motif, was studied by 3D structure modelling. The mutation was reproduced in a plasmid encoding a rat Bsep-green fluorescent protein. After transfection, mutant expression was studied in Can 10 cells. Taurocholate transport activity and ivacaftor effect were studied in Madin-Darby canine kidney (MDCK) clones co-expressing the rat sodium-taurocholate co-transporting polypeptide (Ntcp/Slc10A1). RESULTS: As the wild-type protein, BsepT463I was normally targeted to the canalicular membrane of Can 10 cells. As predicted by 3D structure modelling, taurocholate transport activity was dramatically low in MDCK clones expressing BsepT463I . Ivacaftor treatment increased by 1.7-fold taurocholate transport activity of BsepT463I (P < .0001), reaching 95% of Bsepwt activity. These data suggest that the p.T463I mutation impairs ATP-binding, resulting in Bsep dysfunction that can be rescued by ivacaftor. CONCLUSION: These results provide experimental evidence of ivacaftor therapeutic potential for selected patients with PFIC2 caused by ABCB11 missense mutations affecting BSEP function. This could represent a significant step forward for the care of patients with BSEP deficiency.

MicroRNA-based diagnostic tools for advanced fibrosis and cirrhosis in patients with chronic hepatitis B and C.

Staging fibrosis is crucial for the prognosis and to determine the rapid need of treatment in patients with chronic hepatitis B (CHB) and C (CHC). The expression of 13 fibrosis-related microRNAs (miRNAs) (miR-20a, miR-21, miR-27a, miR-27b, miR-29a, miR-29c, miR-92a, miR-122, miR-146a, miR-155, miR-221, miR-222, and miR-224) was analyzed in 194 serums and 177 liver biopsies of patients with either CHB or CHC to develop models to diagnose advanced fibrosis and cirrhosis (Metavir F3-F4). In CHB patients, the model (serum miR-122, serum miR-222, platelet count and alkaline phosphatase) was more accurate than APRI and FIB-4 to discriminate in between mild and moderate fibrosis (F1-F2) and F3-F4 (AUC of CHB model: 0.85 vs APRI: 0.70 and FIB-4: 0.81). In CHC patients, the model (hepatic miR-122, hepatic miR-224, platelet count, albumin and alanine aminotransferase) was more accurate than both APRI and FIB-4 to discriminate in between patients with F3-F4 and F1-F2 (AUC of the CHC model = 0.93 vs APRI: 0.86 and FIB-4: 0.79). Most of the miRNAs tested were differentially expressed in patients with CHB and CHC. In particular, serum miR-122 was 28-fold higher in patients with CHB than in those with CHC. Both CHB and CHC models may help for the diagnosis of advanced fibrosis and cirrhosis (F3-F4).

Accumulation of Intrahepatic TNF-α-Producing NKp44+ NK Cells Correlates With Liver Fibrosis and Viral Load in Chronic HCV Infection.

In the setting of chronic hepatitis C virus (HCV) infection, changes in natural killer (NK) cells have been shown to reflect activation in response to virus stimulation. The contribution of individual natural cytotoxicity receptors to HCV infection remains to be clarified. NKp44 is the sole specific natural cytotoxicity receptor expressed only on activated NK cells.In this study, peripheral blood and liver NK-cell subsets were purified from 31 patients with chronic C hepatitis or nonalcoholic steatohepatitis, and then characterized by flow cytometry. Their polyfunctional activity was determined by expression of the CD107a degranulation marker, together with intracellular cytokine production.Unlike the patients with nonalcoholic steatohepatitis, patients with chronic HCV infection had a higher frequency of NKp44 NK cells in the liver than in their peripheral blood (P < 0.0001). Intrahepatic NKp44 NK cells from HCV individuals produced higher levels of tumor necrosis factor-α than did NKp44 NK cells (P = 0.0011). Importantly, the frequency of intrahepatic NKp44 NK cells was correlated with both HCV-RNA levels (P = 0.0234) and stage of fibrosis (P = 0.0003).Our findings suggest that the accumulation of intrahepatic tumor necrosis factor-α-producing NKp44 resident NK cells play a role in the liver damage associated with chronic HCV infection.

IFI35, mir-99a and HCV genotype to predict sustained virological response to pegylated-interferon plus ribavirin in chronic hepatitis C.

Although, the treatment of chronic hepatitis C (CHC) greatly improved with the use of direct antiviral agents, pegylated-interferon (PEG-IFN) plus ribavirin remains an option for many patients, worldwide. The intra-hepatic level of expression of interferon stimulated genes (ISGs) and the rs12979860 CC genotype located within IFNL3 have been associated with sustained virological response (SVR), in patients with CHC. The aim of the study was to identify micro-RNAs associated with SVR and to build an accurate signature to predict SVR. Pre-treatment liver biopsies from 111 patients, treated with PEG-IFN plus ribavirin, were studied. Fifty-seven patients had SVR, 36 non-response (NR) and 18 relapse (RR). The expression of 851 human miRNAs and 30 selected mRNAs, including ISGs, was assessed by RT-qPCR. In the first group of patients (screen), 20 miRNAs out of the 851 studied were deregulated between NRs and SVRs. From the 4 miRNAs validated (mir-23a, mir-181a*, mir-217 and mir-99a), in the second group of patients (validation), 3 (mir-23a, mir-181a* and mir-99a) were down-regulated in NRs as compared to SVRs. The ISGs, studied, were accumulated in SVRs and IFNL3 rs12979860 CT/TT carriers compared respectively to NRs and CC carriers. Combining, clinical data together with the expression of selected genes and micro-RNAs, we identified a signature (IFI35, mir-99a and HCV genotype) to predict SVR (AUC:0.876) with a positive predictive value of 86.54% with high sensibility (80%) and specificity (80.4%). This signature may help to characterize patients with low chance to respond to PEG-IFN/ribavirin and to elucidate mechanisms of NR.

Precore/Core promoter variants to predict significant fibrosis in both HBeAg positive and negative chronic hepatitis B.

BACKGROUND & AIMS: Assessing fibrosis is essential in patients with chronic hepatitis B (CHB). The objective was to investigate the relationship between fibrosis, host and viral factors to identify non-invasive markers of significant fibrosis in a large cohort of unselected, well-characterized, treatment-naïve CHB patients. METHODS: Three hundred and seventy-seven HBsAg-positive patients (97 HBeAg-positive and 280 HBeAg-negative, genotypes A to E) who had liver biopsy were consecutively included. Host and viral factors (ALT, HBsAg and HBV-DNA levels, HBV genotype and precore (PC)/basal core promoter (BCP) variants) were determined on the day of the biopsy. Fibrosis stage was assessed using METAVIR score. RESULTS: Thirty-nine percent of the patients had significant fibrosis (METAVIR F ≥ 2). On univariate analysis, the stages of fibrosis F ≥ 2 were associated with older age (P < 0.0001), male gender (P = 0.01), higher ALT and HBV-DNA levels (P < 0.0001 and P = 0.0003, respectively), the presence of BCP (P < 0.0001) and BCP/PC variants (P < 0.0001). On multivariate analysis, age (P < 0.0001), the presence of HBV variants (P < 0.0001), HBV-DNA level (P = 0.0006) and ALT level (P = 0.02) were independently associated with significant fibrosis. The diagnostic accuracy of the combination (age, ALT, HBV-DNA, HBV variants) in predicting fibrosis F ≥ 2 was evidenced by a c-index of 0.76 (CI 95% 0.71-0.81). CONCLUSIONS: We identified strong independent risk factors (age, ALT, HBV-DNA, HBV variants) predicting significant fibrosis (F ≥ 2) independently of HBeAg status in patients with CHB. Patients with BCP variants have a higher risk of severe liver disease. The detection of these mutants may help to predict significant fibrosis (F ≥ 2).

Using pharmacokinetic and viral kinetic modeling to estimate the antiviral effectiveness of telaprevir, boceprevir, and pegylated interferon during triple therapy in treatment-experienced hepatitis C virus-infected cirrhotic patients.

Triple therapy combining a protease inhibitor (PI) (telaprevir or boceprevir), pegylated interferon (PEG-IFN), and ribavirin (RBV) has dramatically increased the chance of eradicating hepatitis C virus (HCV). However, the efficacy of this treatment remains suboptimal in cirrhotic treatment-experienced patients. Here, we aimed to better understand the origin of this impaired response by estimating the antiviral effectiveness of each drug. Fifteen HCV genotype 1-infected patients with compensated cirrhosis, who were nonresponders to prior PEG-IFN/RBV therapy, were enrolled in a nonrandomized study. HCV RNA and concentrations of PIs, PEG-IFN, and RBV were frequently assessed in the first 12 weeks of treatment and were analyzed using a pharmacokinetic/viral kinetic model. The two PIs achieved similar levels of molar concentrations (P=0.5), but there was a significant difference in the 50% effective concentrations (EC50) (P=0.008), leading to greater effectiveness for telaprevir than for boceprevir in blocking viral production (99.8% versus 99.0%, respectively, P=0.002). In all patients, the antiviral effectiveness of PEG-IFN was modest (43.4%), and there was no significant contribution of RBV exposure to the total antiviral effectiveness. The second phase of viral decline, which is attributed to the loss rate of infected cells, was slow (0.19 day(-1)) and was higher in patients who subsequently eradicated HCV (P=0.03). The two PIs achieved high levels of antiviral effectiveness. However, the suboptimal antiviral effectiveness of PEG-IFN/RBV and the low loss of infected cells suggest that a longer treatment duration might be needed in cirrhotic treatment-experienced patients and that a future IFN-free regimen may be particularly beneficial in these patients.

Reduction of microRNA 122 expression in IFNL3 CT/TT carriers and during progression of fibrosis in patients with chronic hepatitis C.

UNLABELLED: The microRNA miR-122 is highly expressed in the liver and stimulates hepatitis C virus (HCV) replication in vitro. IFNL3 (lambda-3 interferon gene) polymorphisms and the expression of miR-122 have been associated with sustained virological response (SVR) to treatment with pegylated interferon plus ribavirin in patients with chronic hepatitis C (CHC). We investigated, in vivo, the relationship between miR-122 expression, IFNL3 polymorphism, fibrosis, and response to PEG-IFN plus ribavirin. Pretreatment liver biopsy specimens and serum samples from 133 patients with CHC were included. Sixty-six patients achieved SVR, and 64 failed to respond to the treatment (43 nonresponders [NR] and 21 relapsers [RR]). All stages of fibrosis were represented, with 39, 50, 23, and 19 patients, respectively, having Metavir scores of F1, F2, F3, and F4. miR-122 expression was assessed by real-time quantitative PCR (RT-qPCR) and IFNL3 rs12979860 by direct sequencing. Hepatic miR-122 expression was higher in patients with the IFNL3 CC genotype than in those with the IFNL3 CT or TT genotype, in all patients (P = 0.025), and in NRs plus RRs (P = 0.013). Increased hepatic miR-122 was more strongly associated with complete early virological response (cEVR) (P = 0.003) than with SVR (P = 0.016). In multivariate analysis, increased hepatic miR-122 was only associated with the IFNL3 CC genotype. miR-122 was decreased in patients with advanced fibrosis (Metavir scores of F3 and F4) compared to its levels in patients with mild and moderate fibrosis (F1 and F2) (P = 0.01). Serum and hepatic expression of miR-122 were not associated. The association between miR-122 and IFNL3 was stronger than the association between miR-122 and response to treatment. miR-122 may play a role in the early viral decline that is dependent on IFNL3 and the innate immune response. IMPORTANCE: miR-122 plays a crucial role during HCV infection. Moreover, it was reported that miR-122 binding within the HCV genome stimulates its replication. Moreover, miR-122 is highly expressed within hepatocytes, where it regulates many cellular pathways. A reduction of miR-122 expression has been suggested to be associated with responsiveness to IFN-based therapy in patients with chronic hepatitis C. Several independent genome-wide association studies reported a strong association between IFNL3 polymorphism and responsiveness to IFN-based therapy. We report here a strong association between the expression of miR-122 and IFNL3 polymorphism that is independent of the response to the treatment. Our data suggest that modification of miR-122 expression may play an important role in the molecular mechanism associated with IFNL3 polymorphism. Moreover, we report a reduction of miR-122 at more advanced stages of fibrosis in patients with chronic hepatitis C.

HBsAg quantification: useful for monitoring natural history and treatment outcome.

The template of hepatitis B virus transcription, the covalently closed circular DNA (cccDNA), plays a key role in the life cycle of the virus and permits the persistence of infection. It has been suggested that hepatitis B surface antigen (HBsAg) quantification reflects the concentration of cccDNA in the liver. In hepatitis B e antigen (HBeAg) positive chronic hepatitis B, HBsAg levels are higherduring the immune tolerance phase than during the immune clearance phase. During the natural history of chronic hepatitis B, serum HBsAg declines progressively from the immune-tolerant to the low replicative phase. In HBeAg negative patients, the combination of low hepatitis B virus (HBV) DNA (<2000 IU/ml) and low HBsAg levels (<1000 IU/ml) can predict inactive carrier status, low risk of hepatocellular carcinoma, and the probability of HBsAg loss. HBsAg in combination with HBV DNA predicts the outcome of Peg-Interferon therapy: An absence of decline at week 12 is a good predictor of non-response and to stop therapy. Any decline at week 24, suggests that therapy should be continued to 48 weeks. Although the decrease in HBsAg decline slow with nucleos(t)ide analogue therapy, a rapid decline can predict future HBsAg seroclearance. A level <100 IU/ml during six consecutive months could be a marker of a sustained response after treatment cessation.

Prediction of disease reactivation in asymptomatic hepatitis B e antigen-negative chronic hepatitis B patients using baseline serum measurements of HBsAg and HBV-DNA.

UNLABELLED: Differentiating 'inactive carriers' (ICs) of hepatitis B virus (HBV) from hepatitis B e antigen-negative (HBeAg[-]) patients in remission is challenging. We investigated whether serum-based monitoring of hepatitis B surface antigen (HBsAg) and HBV-DNA in asymptomatic HBeAg(-) patients could distinguish these groups. DESIGN: 129 HBeAg(-) chronic hepatitis B (CHB) patients (HBV genotypes A-E) with normal alanine aminotransferase (ALT) levels at baseline were classified after 1 year of follow-up as either IC (HBV-DNA ≤2000 IU/mL) or 'active carrier' (AC, HBV-DNA >2000 IU/mL) if they exhibited normal ALT throughout, or classified as 'reactivation patient' (RP) if they exhibited marked, transient increases in ALT and HBV-DNA. RESULTS: There were 64%, 18%, and 19% patients in the IC, AC, and RP groups, respectively. Combined HBsAg and HBV-DNA cutoffs (>1000 IU/mL and >200 IU/mL, respectively) differentiated RPs with 92% sensitivity and negative predictive value (NPV) of 96%. HBsAg sero-clearance was associated with baseline HBsAg <1000 IU/mL, annual decrease of ≥0.3 log IU/mL (NPV 95%: PPV 89%) and IFNL3 genotype CC. CONCLUSION: Applying combined HBsAg and HBV-DNA cutoffs to baseline measurements accurately differentiated RPs. These results suggest that HBsAg should be included in the monitoring of asymptomatic HBeAg(-) CHB patients.

Hepatitis B surface antigen serum level is associated with fibrosis severity in treatment-naïve, e antigen-positive patients.

BACKGROUND & AIMS: Little is currently known about the association between serum HBsAg or HBV DNA levels and the severity of liver disease in chronic hepatitis B (CHB) patients. Therefore, we investigated these relationships in a large cohort of unselected, well-characterized, treatment-naïve CHB patients. METHODS: CHB patients were assessed at the Hôpital Beaujon in Paris, France, between 2000 and 2008. Serum samples and liver biopsies were obtained on the same day. HBsAg, HBV DNA, and HBV genotype were investigated using commercial diagnostic assays and liver histology was scored using the METAVIR system. RESULTS: 406 patients were included in this cross-sectional study. Serum HBsAg and HBV DNA levels in hepatitis B e antigen-positive (HBeAg[+]) patients showed strong correlation (r=0.44, p<0.0001), as did serum HBsAg levels and fibrosis severity (r=0.43, p<0.0001). HBeAg(+) patients with moderate to severe fibrosis exhibited significantly lower serum HBsAg and HBV DNA levels compared with patients with no or mild fibrosis. Modeling analysis suggested a serum HBsAg cut-off of 3.85 logIU/ml would provide a theoretical sensitivity of 100% (95% CI: 0-100), theoretical specificity of 86% (95% CI: 50-100), and a negative predictive value of 100% (95% CI: 67-100) in HBeAg(+) patients infected with HBV genotype B or C. CONCLUSIONS: We found an association between low serum HBsAg levels and moderate to severe fibrosis in HBeAg(+) CHB patients. Furthermore, we described a serum HBsAg cut-off for the prediction of fibrosis severity in CHB patients infected with HBV genotype B or C.

Quantification of hepatitis B e antigen between Elecsys HBeAg and Architect HBeAg assays among patients infected with hepatitis B virus.

BACKGROUND: Among patients infected with hepatitis B virus (HBV), quantification of hepatitis B e antigen (HBeAg) is accruing substantial clinical relevance as a marker for HBeAg-loss during treatment. No direct comparison has been made between assays that quantify HBeAg. OBJECTIVES: To compare the performance of HBeAg quantification (qHBeAg) between Architect and Elecsys HBeAg assays among 183 patients with chronic HBV infection (94 treatment-naïve HBV-monoinfected and 89 antiretroviral-experienced HIV-HBV co-infected). STUDY DESIGN: qHBeAg was determined in Paul Erlich Institute Units (PEIU)/mL using previously designed protocols. Values were compared with correlation and linear regression. Bland-Altman analysis was used to compare mean differences (d¯) between Elecsys and Architect assays and limits of agreement (LOA) (±2 standard deviations [SD]). RESULTS: Between-assay correlation was significant overall (r = 0.970), yet stronger for qHBeAg < 1000 (n = 131) versus > 1000PEIU/mL (n = 52) as determined by the Elecsys assay (r = 0.969 vs. 0.880, respectively). On average, the Elecsys assay reported qHBeAg at 13.3PEIU/mL lower than the Architect assay (LOA: -415.9, 389.3), while LOA between assays were much wider at higher levels (< 1000: -198.2, 147.9; ≥ 1000PEIU/mL: -688.4, 721.5). Further analysis indicated that d¯ did not change substantially with respect to HBV genotype, precore mutation, and CD4+ cell count, regardless of HBeAg-level. Nevertheless, seven of eight patients with highly divergent between-assay results had HBV-DNA> 2000IU/mL. CONCLUSIONS: Elecsys and Architect assays report similar qHBeAg units with high correlation. Since qHBeAg was performed using an in-house approach, a commercially-available assay could reduce between-assay discrepancies, especially at higher HBeAg-levels.

The role of HBsAg quantification for monitoring natural history and treatment outcome.

Since its discovery by Blumberg in 1965, the hepatitis B virus antigen (HBsAg) is used as the fingerprint of hepatitis B infection. The HBsAg level is a reflection of the transcriptional activity of cccDNA. It is an important marker that not only indicates active hepatitis B infection but can also predict clinical and treatment outcomes. Assays for HBsAg quantification are fully automated and have high output. HBsAg titres are higher in HBe antigen (HBeAg)(+) than in HBeAg(-) patients and are negatively correlated with liver fibrosis in HBeAg(+) patients. In HBeAg(-) chronic hepatitis B, an HBsAg level <1000 IU/ml and an HBV DNA titre <2000 IU/ml accurately identify inactive carriers. During PEG-IFN treatment, HBsAg quantification is used to identify patients who will not benefit from therapy as early as week 12 on therapy, so that treatment may be stopped or switched- 'week 12 stopping rule'. With nucleos(t)ide analogues (NA), the role of HBsAg quantification must be clarified. Several studies show that baseline and on-treatment HBsAg levels might identify patients that can be treated with no subsequent risk of reactivation. In clinical practice, HBsAg quantification is a simple and reproducible tool that can be used in association with HBV DNA to classify patients during the natural history of HBV and to monitor therapy.