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BACKGROUND AND AIMS: We conducted haplotype analysis of complete hepatitis B virus (HBV) genomes following deep sequencing from 368 patients across multiple phases of chronic hepatitis B (CHB) infection from four major genotypes (A-D), analyzing 4,110 haplotypes to identify viral variants associated with treatment outcome and disease progression. APPROACH AND RESULTS: Between 18.2% and 41.8% of nucleotides and between 5.9% and 34.3% of amino acids were 100% conserved in all genotypes and phases examined, depending on the region analyzed. Hepatitis B e antigen (HBeAg) loss by week 192 was associated with different haplotype populations at baseline. Haplotype populations differed across the HBV genome and CHB history, this being most pronounced in the precore/core gene. Mean number of haplotypes (frequency) per patient was higher in immune-active, HBeAg-positive chronic hepatitis phase 2 (11.8) and HBeAg-negative chronic hepatitis phase 4 (16.2) compared to subjects in the "immune-tolerant," HBeAg-positive chronic infection phase 1 (4.3, P< 0.0001). Haplotype frequency was lowest in genotype B (6.2, P< 0.0001) compared to the other genotypes (A = 11.8, C = 11.8, D = 13.6). Haplotype genetic diversity increased over the course of CHB history, being lowest in phase 1, increasing in phase 2, and highest in phase 4 in all genotypes except genotype C. HBeAg loss by week 192 of tenofovir therapy was associated with different haplotype populations at baseline. CONCLUSIONS: Despite a degree of HBV haplotype diversity and heterogeneity across the phases of CHB natural history, highly conserved sequences in key genes and regulatory regions were identified in multiple HBV genotypes that should be further investigated as targets for antiviral therapies and predictors of treatment response.

Background & Aims The use of complementary and alternative medicine (CAM) in patients with chronic hepatitis B (CHB) can interact with antiviral treatment or influence health‐seeking behaviour. We aimed to study the use of individual CAM modalities in CHB and explore determinants of use, particularly migration‐related, socio‐economic and clinical factors. Methods A total of 436 CHB outpatients who attended the Toronto Centre for Liver Disease in 2015‐2016 were included in this cross‐sectional study. Using the comprehensive I‐CAM questionnaire and health records, data were collected on socio‐demographic and clinical variables and on usage of 16 CAM modalities in the last year. Results Sixty percent of patients were male, 74% were Asian and 46% were using antiviral treatment. Three‐hundred and nine (71%) patients used CAM. Vitamin/mineral preparations (45% of patients) were most commonly used. Overall CAM use and the specific use of potentially injurious CAM, such as green tea extract (9.2%) and St. John's wort (0.2%), were not associated with liver disease severity. Female sex, family history of CHB, lower serum HBV DNA, and higher socio‐economic status were independently associated with bio‐holistic CAM use, the clinically most‐relevant CAM group (P < 0.05); ethnicity, antiviral therapy use and liver disease severity were not. Conclusions CAM use among CHB patients was extensive, especially use of vitamin and mineral preparations, but without direct influence on liver disease severity. Bio‐holistic CAM use appeared to be associated with socio‐economic status rather than with ethnicity or liver disease severity. Despite the rare use of hepatotoxins, physicians should actively inquire about it.

BACKGROUND: Hepatitis B e antigen (HBeAg) seroconversion is a treatment endpoint for HBeAg-positive CHB, and a necessary precursor to HBsAg loss. Biomarkers that predict serological outcomes would be useful. AIM: To evaluate the utility of measuring HBeAg levels for predicting HBeAg seroconversion and HBsAg loss under long-term tenofovir (TDF) therapy. METHODS: A total of 266 patients were enrolled into a phase III study of TDF vs adefovir (ADV) for 48 weeks in HBeAg-positive patients, followed by open-label TDF up to 384 weeks. Serum HBeAg levels were measured for subjects with samples available at both baseline and week 24 of treatment (n = 200). Analysis compared subjects who achieved HBeAg seroconversion by week 384 vs no HBeAg seroconversion. RESULTS: HBeAg seroconversion rate was 52% by week 384. Time to HBeAg seroconversion was 80 weeks (IQR: 36-162). HBeAg decline at week 24 was associated with HBeAg seroconversion (1.63 vs 0.90 log10 PEIU/mL, P = .002). The optimal threshold for identifying HBeAg seroconversion was HBeAg decline ≥2.2 log10 PEIU/mL at week 24, with HBeAg seroconversion achieved by 76% of patients, compared to 44% if HBeAg decline <2.2 log10 (P < .0001). HBeAg decline ≥2.2 log10 PEIU/mL at week 24 was associated with HBsAg loss in genotype A or D patients (38% vs 15%, P = .03). Precore/basal core promotor variants were associated with lower baseline HBeAg levels, but not HBeAg seroconversion. CONCLUSION: Decline in HBeAg levels by week 24 was associated with HBeAg seroconversion and HBsAg loss in HBeAg-positive chronic hepatitis B patients treated with long-term TDF.

Chronic hepatitis B virus (HBV) infection results from inadequate HBV-specific immunity. BDCA3(+) dendritic cells (DCs) are professional antigen presenting cells considered to be important for antiviral responses because of specific characteristics, including high interferon-lambda production. BDCA3+ DCs may thus also have a role in the immune response against HBV, and immunotherapeutic strategies aiming to activate DCs, including BDCA3(+) DCs, in patient livers may represent an interesting treatment option for chronic HBV. However, neither the effect of chronic hepatitis B (CHB) infection on the frequency and function of BDCA3(+) DCs in liver and blood, nor the effect of the viral surface protein (HBsAg) that is abundantly present in blood of infected individuals are known. Here, we provide an overview of BDCA3(+) DC frequency and functional capacity in CHB patients. We find that intrahepatic BDCA3(+) DC numbers are increased in CHB patients. BDCA3(+) DCs from patient blood are not more mature at steady state, but display an impaired capacity to mature and to produce interferon-lambda upon polyI: C stimulation. Furthermore, in vitro experiments exposing blood and intrahepatic BDCA3(+) DCs to the viral envelope protein HBsAg demonstrate that HBsAg does not directly induce phenotypical maturation of BDCA3(+) DCs, but may reduce IFN-lambda production via an indirect unknown mechanism. These results suggest that BDCA3(+) DCs are available in the blood and on site in HBV infected livers, but measures may need to be taken to revive their function for DC-targeted therapy.

Prevalence of non-alcoholic fatty liver disease is increased in patients with psoriasis. However, it is not known how liver fibrosis correlates with psoriasis. This study investigated the association between psoriasis and liver fibrosis compared with participants without psoriasis within the population-based Rotterdam Study. All participants were screened for liver fibrosis using transient elastography. Liver stiffness > 9.5 kPa suggested advanced liver fibrosis. Psoriasis was identified using a validated algorithm. A total of 1,535 participants were included (mean age +/- standard deviation 70.5 +/- 7.9 years; 50.8% female; median body mass index 26.4 kg/m(2) (interquartile range 24.2-28.9)) of whom 74 (4.7%) had psoriasis. Prevalence of advanced liver fibrosis was 8.1% in psoriasis patients compared with 3.6% in the reference group (p = 0.05). The risk of advanced liver fibrosis in psoriasis patients remained comparable after adjustment for demographics, lifestyle characteristics and laboratory findings (odds ratio 2.57 (95% confidence interval 1.00-6.63). This study suggests that elderly people with psoriasis are twice as likely to have advanced liver fibrosis irrespective of common risk factors.

Given that little is known about the prevalence of, and factors associated with, liver fibrosis in the general population, we aimed to investigate this in a large, well-characterized cohort by means of transient elastography (TE). This study was part of the Rotterdam Study, a population-based study among individuals 45 years. All participants underwent abdominal ultrasound and TE. Liver stiffness measurement (LSM) 8.0 kilopascals (kPa) was used as a cutoff suggesting clinically relevant fibrosis. Of 3,041 participants (age, 66.0 +/- 7.6 years) with reliable LSM, 169 (5.6%) participants had LSM 8.0 kPa. Age (odds ratio [OR]: 2.40; 95% confidence interval [CI]: 1.72-3.36; P<0.001), alanine aminotransferase (ALT; OR, 1.24; 95% CI: 1.12-1.38; P<0.001), smoking (OR, 1.77; 95% CI: 1.16-2.70; P=0.008), spleen size (OR, 1.23; 95% CI: 1.09-1.40; P=0.001), hepatitis B surface antigen, or anti-hepatitis C virus positivity (OR, 5.38; 95% CI: 1.60-18.0; P=0.006), and combined presence of diabetes mellitus (DM) and steatosis (OR, 5.20; 95% CI: 3.01-8.98; P<0.001 for combined presence) were associated with LSM 8.0 kPa in multivariable analyses. The adjusted predicted probability of LSM 8.0 kPa increased per age decade, with probabilities ranging from 1.4% (0.9-3.6) in participants ages 50-60 years to 9.9% (6.8-14.5) in participants >80 years. Participants with both DM and steatosis had the highest probabilities of LSM 8.0 kPa (overall probability: 17.2% [12.5-23.4]; this probability did not increase with age [P=0.8]). Conclusion: In this large population-based study of older adults, LSM 8.0 kPa, suggestive of clinically relevant fibrosis, was present in 5.6% and was strongly associated with steatosis and DM. In the context of an aging population and an increased prevalence of DM and obesity, this study illustrates that liver fibrosis may become a more prominent public health issue in the near future. (Hepatology 2016;63:138-147)

Background/Objective Genetic polymorphisms in the inosine triphosphatase (ITPA) gene have been associated with the protection from early ribavirin(RBV)-induced hemolytic anemia among patients with chronic hepatitis C virus (HCV) infection. The aim of the present study was to investigate the association between the functional ITPA variants and hematological side effects during antiviral therapy with pegylated interferon (PegIFN) and RBV. Patients and Methods This cohort study included all consecutive Caucasian patients treated for chronic HCV infection with PegIFN and RBV between 2000 and 2009 for whom a serum sample was available for genetic testing. The predicted inosine triphosphate pyrophosphatase (ITPase) activity was based on the genotypes of the SNPs rs1127354 and rs7270101. Decline in hemoglobin (Hb) during antiviral therapy, as well as dose reductions, blood transfusions and use of erythropoietin were assessed. Results In total, 213 patients were included. The predicted ITPase activity was normal among 152 (71%) patients; 61 (29%) patients had ITPase deficiency. By multivariable linear regression, RBV dose in mg per kilogram (Beta 0.09, 95% CI 0.04-0.13, p<0.001) and normal ITPase activity (Beta 0.89, 95% CI 0.64-1.14, p<0.001) were associated with more Hb decline at week 4 of treatment. Patients with normal ITPase activity underwent more dose adjustments of RBV than patients with ITPase deficiency (19(13%) vs 1(2%), p = 0.014) and received erythropoietin more frequently (12 (8%) vs 0 (0%), p = 0.024). Conclusion Genetic variants in the ITPA gene protected against RBV treatment-induced anemia among Caucasian patients with chronic HCV infection. Patients with normal ITPase activity underwent more dose reductions of RBV and received erythropoietin more frequently.

Background and Purpose This study evaluated the cost-effectiveness of different noninvasive imaging strategies in patients with possible basilar artery occlusion. Methods A Markov decision analytic model was used to evaluate long-term outcomes resulting from strategies using computed tomographic angiography (CTA), magnetic resonance imaging, nonenhanced CT, or duplex ultrasound with intravenous (IV) thrombolysis being administered after positive findings. The analysis was performed from the societal perspective based on US recommendations. Input parameters were derived from the literature. Costs were obtained from United States costing sources and published literature. Outcomes were lifetime costs, quality-adjusted life-years (QALYs), incremental cost-effectiveness ratios, and net monetary benefits, with a willingness-to-pay threshold of $80 000 per QALY. The strategy with the highest net monetary benefit was considered the most cost-effective. Extensive deterministic and probabilistic sensitivity analyses were performed to explore the effect of varying parameter values. Results In the reference case analysis, CTA dominated all other imaging strategies. CTA yielded 0.02 QALYs more than magnetic resonance imaging and 0.04 QALYs more than duplex ultrasound followed by CTA. At a willingness-to-pay threshold of $80 000 per QALY, CTA yielded the highest net monetary benefits. The probability that CTA is cost-effective was 96% at a willingness-to-pay threshold of $80 000/QALY. Sensitivity analyses showed that duplex ultrasound was cost-effective only for a prior probability of 0.02 and that these results were only minimally influenced by duplex ultrasound sensitivity and specificity. Nonenhanced CT and magnetic resonance imaging never became the most cost-effective strategy. Conclusions Our results suggest that CTA in patients with possible basilar artery occlusion is cost-effective.

In the treatment of chronic hepatitis B (CHB), the ultimate goal is preventing hepatitis B virus (HBV)-associated liver disease, including hepatocellular carcinoma (HCC). Recently published studies show that in CHB patients treated with the currently recommended first-line nucleos(t)ide analogs (NAs) entecavir or tenofovir, annual HCC incidences range from 0.01% to 1.4% in non-cirrhotic patients, and from 0.9% to 5.4% in those with cirrhosis. In Asian studies including matched untreated controls, current NA therapy consistently resulted in a significantly lower HCC incidence in patients with cirrhosis, amounting to an overall HCC risk reduction of similar to 30%; in non-cirrhotic patients, HCC risk reduction was overall similar to 80%, but this was only observed in some studies. For patients of Caucasian origin, no appropriate comparative studies are available to date to evaluate the impact of NA treatment on HCC. Achievement of a virologic response under current NA therapy was associated with a lower HCC risk in Asian, but not Caucasian studies. Studies comparing entecavir or tenofovir with older NAs generally found no difference in HCC risk reduction between agents, except for one study which used no rescue therapy in patients developing lamivudine resistance. Overall, these data indicate that with the current, potent NAs, HCC risk can be reduced but not eliminated, probably due to risk factors that are not amenable to change by antiviral therapy, or events that may have taken place before treatment initiation. Validated pre- and on-therapy HCC risk calculators that inform the best practice for HCC surveillance and facilitate patient counseling would be of great practical value. (C) 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Background: Infection with hepatitis E virus (HEV) in immunocompromised patients can lead to severe liver disease. Treatment options for HEV include peginterferon or ribavirin, routinely also used for the treatment of hepatitis C virus (HCV) infection. We determined the prevalence and clinical consequences of HEV in United States (US) based patients who underwent liver transplantation (LT) for chronic HCV. Methods: Seroprevalence of HEV in 145 US LT recipients with a history of chronic HCV was determined pre-LT, 1, 3 and 5 years post-LT. All last available samples and all samples in IgM positive patients and post-LT IgG seroconverters were tested for HEV RNA. Results: Overall anti-HEV seroprevalence was 42 %. Five patients were HEV IgM positive pre-LT, one patient had IgM seroconversion post-LT and eight patients had IgG seroconversion post-LT. None of the tested samples were positive for HEV RNA. Eight out of nine of the post-LT seroconverters had been treated for HCV recurrence before or at the moment of seroconversion. Conclusions: LT recipients in the US are at risk of acquiring HEV. Post-LT HCV treatment with interferons and/or ribavirin may have protected patients against chronic HEV. With the arrival of new direct antiviral agents for the treatment of HCV and the elimination of peginterferon and ribavirin from HCV treatment regimens, the prevalence of chronic HEV in this population may rise again.

Individuals who are chronically infected with the hepatitis B virus (HBV) are highly heterogenous with respect to serum levels of HBV DNA, HBV particles and viral proteins. Since circulating leukocytes, such as monocytes, are constantly exposed to these viral components, it is likely that the functionality of these cells is affected. However, at present, little information is available on the consequences of the interaction between monocytes and viral components. Therefore, we examined the in vitro effects of HBV surface antigen (HBsAg) on monocytes and evaluated whether these effects were reflected in vivo. We observed that in vitro HBsAg exposure of monocytes induced robust production of IL-6 and TNF. However, between chronic HBV patients with distinct levels of serum HBsAg, HBV early antigen (HBeAg), and HBV DNA, TLR-induced monocyte cytokine production did not differ. Importantly, HBsAg-induced cytokine production by monocytes was similar between patients and healthy controls showing that earlier in vivo exposure to HBsAg does not affect the in vitro response. Additionally, we show that IL-10 is able to inhibit cytokine production by HBsAg-induced monocytes. In conclusion, we demonstrate that monocytes can recognize and respond to HBsAg, resulting in vigorous pro-inflammatory cytokine production in vitro. However, phenotype and function of the monocyte compartment in chronic HBV patients are not influenced by differences in levels of serum viral components, suggesting that regulatory mechanisms are active to avoid excessive in vivo monocyte activation.

Background. Alanine aminotransferase (ALT) flares occur frequently during peginterferon (PEG-IFN) therapy. We related occurrence of flares to presence of precore (PC) and/or basal core promoter (BCP) mutants and studied kinetics of hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) levels during flares. Methods. Fifty of 214 (23%) patients treated with PEG-IFN +/- lamivudine for 52 weeks experienced flares. Flares were host-induced (ALT elevation followed by HBV DNA decline, n = 19), virus-induced (HBV DNA increase with subsequent ALT elevation, n = 17) or indeterminate (n = 14). Presence of wild-type (WT) or non-WT (detectable PC/BCP mutants) was studied by lineprobe assay. Results. Fifty-eight percent of host-induced flares occurred in WT HBV patients, whereas 94% of virus-induced flares occurred in patients with PC and/or BCP mutants (P = .003). HBsAg loss was only achieved in patients with a host-induced flare, and WT patients with a host-induced flare cleared HBsAg in 64% of cases. Serum HBsAg levels declined after a host-induced flare, whereas virus-induced flares were accompanied by stable or increasing levels of HBsAg. Patients with a host-induced flare achi Conclusions. Host-induced flares are associated with WT virus and may result in decline and clearance of HBV DNA, HBeAg, and HBsAg. Monitoring of HBsAg levels during and after flares may help predict a favorable treatment outcome.

In humans, clearance of hepatitis C virus (HCV) infection is associated with genetic variation near the IL-28B gene and the induction of interferon-stimulated genes, like IP-10. Also in chimpanzees spontaneous clearance of HCV is observed. To study whether similar correlations exist in these animals, a direct comparison of IP-10 and IL-28B polymorphism between chimpanzees and patients was performed. All chimpanzees studied were monomorphic for the human IL-28B SNPs which are associated with spontaneous and treatment induced HCV clearance in humans. As a result, these particular SNPs cannot be used for clinical association studies in chimpanzees. Although these human SNPs were absent in chimpanzees, gene variation in this region was present however, no correlation was observed between different SNP-genotypes and HCV outcome. Strikingly, IP-10 levels in chimpanzees correlated with HCV-RNA load and gamma GT, while such correlations were not observed in humans. The correlation between IP-10, cGT and virus load in chimpanzees was not found in patients and may be due to the lack of lifestyle-related confounding factors in chimpanzees. Direct comparison of IP-10 and IL-28B polymorphism between chimpanzees and patients in relation to HCV infection, illustrates that the IFN-pathways are important during HCV infection in both species. The Genbank EMBL accession numbers assigned to chimpanzees specific sequences near the IL-28B gene are HE599784 and HE599785.

Specific inhibitory mechanisms suppress the T-cell response against the hepatitis C virus (HCV) in chronically infected patients. However, the relative importance of suppression by IL-10, TGF-beta and regulatory T-cells and the impact of pegylated interferon-alpha and ribavirin (PegIFN-alpha/ribavirin) therapy on these inhibitory mechanisms are still unclear. We revealed that coregulation of the HCV-specific T-cell responses in blood of 43 chronic HCV patients showed a highly heterogeneous pattern before, during and after PegIFN-alpha/ribavirin. Prior to treatment, IL-10 mediated suppression of HCV-specific IFN-gamma production in therapy-naive chronic HCV patients was associated with higher HCV-RNA loads, which suggests that protective antiviral immunity is controlled by IL-10. In addition, as a consequence of PegIFN-alpha/ribavirin therapy, negative regulation of especially HCV-specific IFN-gamma production by TGF-beta and IL-10 changed dramatically. Our findings emphasize the importance of negative regulation for the dysfunctional HCV-specific immunity, which should be considered in the design of future immunomodulatory therapies. Citation: Claassen MAA, de Knegt RJ, Turgut D, Groothuismink ZMA, Janssen HLA, et al. (2012) Negative Regulation of Hepatitis C Virus Specific Immunity Is Highly Heterogeneous and Modulated by Pegylated Interferon-Alpha/Ribavirin Therapy. PLoS ONE 7(11): e49389. doi:10.1371/journal.pone.0049389