Risk of end-stage liver disease, hepatocellular carcinoma, and liver-related death by fibrosis stage in the hepatitis C Alaska Cohort.

Long-term prospective studies of the outcomes associated with hepatitis C virus (HCV) infection are rare and critical for assessing the potential impact of HCV treatment. Using liver biopsy as a starting point, we analyzed the development of end-stage liver disease (ESLD), hepatocellular carcinoma (HCC), and liver-related death (LRD) according to fibrosis stage among a cohort of American Indian/Alaska Native persons in Alaska. Persons were classified as having no/mild (Ishak = 0,1), moderate (Ishak = 2), or severe (Ishak = 3,4) fibrosis or cirrhosis (Ishak = 5,6). We examined time until development of ESLD, HCC, and LRD and report survival probabilities at 3, 5, 7, and 10 years. Of 407 persons, 39% (n = 150) had no/mild fibrosis, 32% (n = 131) had moderate fibrosis, 22% (n = 88) had severe fibrosis, and 9% (n = 38) had cirrhosis. The average time of follow-up was 7.3 years. Within 5 years of biopsy, 1.7% (95% confidence interval [CI]: 0.4-6.8) of persons with no/mild fibrosis developed ESLD compared with 7.9% (95% CI, 4.0-15.2), 16.4% (95% CI, 9.6-27.2), and 49.0% (95% CI, 33.0-67.7) with moderate, severe fibrosis, and cirrhosis, respectively (P < 0.01). The 5-year outcome of HCC was 1.0% (95% CI, 0.1-7.0), 1.0% (95% CI, 0.1-6.6), 1.1% (95% CI, 0.2-7.7), and 13.4% (95% CI, 4.4-36.7) among persons with no/mild fibrosis, moderate fibrosis, severe fibrosis, and cirrhosis, respectively (P < 0.01). Five years after biopsy, 0.0% (95% CI, 0.0-14.8) of persons with no/mild fibrosis had suffered an LRD compared with 1.0% (95% CI, 0.2-7.5) of persons with moderate fibrosis, 4.7% (95% CI, 1.5-14.1) with severe fibrosis, and 16.3% (95% CI, 7.0-35.1) with cirrhosis (P < 0.01). CONCLUSION: For prevention of HCC, LRD, and ESLD in the short term, HCV therapy should target individuals who have more than mild fibrosis. (Hepatology 2017;66:37-45).

A Multicomponent Nonpharmacological Intervention to Prevent Delirium for Hospitalized People with Advanced Cancer: A Phase II Cluster Randomized Waitlist Controlled Trial (The PRESERVE Pilot Study).

Background: Delirium is a common debilitating complication of advanced cancer. Objective: To determine if a multicomponent nonpharmacological delirium prevention intervention was feasible for adult patients with advanced cancer, before a phase III (efficacy) trial. Design: Phase II (feasibility) cluster randomized controlled trial. All sites implemented delirium screening and diagnostic assessment. Strategies within sleep, vision and hearing, hydration, orientation, mobility, and family domains were delivered to enrolled patients at intervention site admission days 1-7. Control sites then implemented the intervention ("waitlist sites"). Setting: Four Australian palliative care units. Measurements: The primary outcome was adherence, with an a priori endpoint of at least 60% patients achieving full adherence. Secondary outcomes were interdisciplinary care delivery, delirium measures, and adverse events, analyzed descriptively and inferentially. Results: Sixty-five enrolled patients (25 control, 20 intervention, and 20 waitlist) had 98% delirium screens and 75% diagnostic assessments completed. Nurses (67%), physicians (16%), allied health (8.4%), family (7%), patients (1%), and volunteers (0.5%) delivered the intervention. There was full adherence for 5% patients at intervention sites, partial for 25%. Both full and partial adherence were higher at waitlist sites: 25% and 45%, respectively. One-third of control site patients (32%) became delirious within seven days of admission compared to one-fifth (20%) at both intervention and waitlist sites (p = 0.5). Mean (standard deviation) Delirium Rating Scale-Revised-1998 scores were 16.8 + 12.0 control sites versus 18.4 + 8.2 (p = 0.6) intervention and 18.7 + 7.8 (p = 0.5) waitlist sites. The intervention caused no adverse events. Conclusion: The intervention requires modification for optimal adherence in a phase III trial.

Investigation of putative multisubtype hepatitis C virus infections in vivo by heteroduplex mobility analysis of core/envelope subgenomes.

The frequency that multiple different subtypes of hepatitis C virus (HCV) simultaneously infect a given individual is controversial. To address this question, heteroduplex mobility analysis (HMA) of portions of the HCV core and envelope 1 region was optimized for sensitive and specific detection of mixtures of HCV genomes of different genotype or subtype. Using the standard HCV genotyping approach of 5'-untranslated region (UTR) analysis, 28 of 374 (7.5%) chronic hepatitis C research subjects were classified as having either multiple-subtype HCV infections (n = 21) or switching HCV subtypes over time (n = 7), the latter pattern implying viral superinfection. Upon retesting of specimens by HMA, 25 of 28 multiple-subtype results could not be reproduced. All three patients with positive results were injection drug users with potential multiple HCV exposures. To address the hypothesis of tissue sequestration of multiple-subtype HCV infections, liver (n = 22), peripheral blood mononuclear cell (n = 13), perihepatic lymph node (n = 16), and serum (n = 19) specimens from 23 subjects with end-stage hepatitis C were collected and analyzed by the HMA technique. Whereas 5'-UTR results implicated mixed-subtype HCV infections in 2 subjects, HMA testing revealed no evidence of a second HCV subtype in any tissue compartment (0 of 70 compartments [0%]) or within any given subject (0 of 23 subjects [0%]). In summary, a large proportion of mixed-genotype and switching-genotype patterns generated by 5'-UTR analysis were not reproducible using the HMA approach, emphasizing the need for additional study.

Polyomavirus-Negative Merkel Cell Carcinoma: A More Aggressive Subtype Based on Analysis of 282 Cases Using Multimodal Tumor Virus Detection.

Previous studies have reached conflicting conclusions regarding the proportion of Merkel cell carcinomas (MCCs) that contain the Merkel cell polyomavirus (MCPyV) and the clinical significance of tumor viral status. To address these controversies, we detected MCPyV large T antigen using immunohistochemistry with two distinct antibodies and MCPyV DNA using quantitative PCR. Tumors were called MCPyV-positive if two or more of these three assays indicated presence of this virus. A total of 53 of 282 (19%) MCC tumors in this cohort were virus-negative using this multimodal system. Immunohistochemistry with the CM2B4 antibody had the best overall performance (sensitivity = 0.882, specificity = 0.943) compared with the multimodal classification. Multivariate analysis including age, sex, and immunosuppression showed that, relative to MCC patients with virus-positive tumors, virus-negative MCC patients had significantly increased risk of disease progression (hazard ratio = 1.77, 95% confidence interval = 1.20-2.62) and death from MCC (hazard ratio = 1.85, 95% confidence interval = 1.19-2.89). We confirm that approximately 20% of MCCs are not driven by MCPyV and that such virus-negative MCCs, which can be quite reliably identified by immunohistochemistry using the CM2B4 antibody alone, represent a more aggressive subtype that warrants closer clinical follow-up.

Effects of the hepatitis C virus core protein on innate cellular defense pathways.

The hepatitis C virus (HCV) core protein is thought to contribute to HCV pathogenesis through its interaction with various signal transduction pathways. In this study, we explored the interaction of the core protein with innate defense pathways (interferon [IFN] regulatory factor [IRF], Jak-Stat, and inducible nitric oxide synthase [iNOS]) in HeLa and Huh7 human cell lines. Expression of a patient-derived genotype 1b core protein activated human IRF-1 and guanylate-binding protein-2 (GBP-2) promoters, induced IRF-1 mRNA, but failed to induce IRF-3 phosphorylation. HCV core protein caused dose-dependent induction of the IFN-beta promoter and IFN-beta mRNA but not the IFN-alpha1 and IFN-alpha4 promoters. In the presence of IFN-alpha, core expression was associated with increased IFN-stimulated gene factor 3 (ISGF3) binding to the IFN-stimulated response element (ISRE) and tyrosine phosphorylation of Stat1. Core expression resulted in dose-dependent activation of the ISRE and gamma activated sequence (GAS) promoters, in both the absence and the presence of either IFN-alpha or IFN-gamma. Core stimulated the human iNOS promoter and induced iNOS protein. The data indicate that HCV core can modulate IRF, Jak-Stat, and iNOS pathways and suggest mechanisms by which core could affect HCV persistence and pathogenesis.

Genetic and functional heterogeneity of the hepatitis C virus p7 ion channel during natural chronic infection.

The present study describes natural genetic heterogeneity of hepatitis C virus (HCV) p7 protein, the ion channel that plays a critical role in assembly and release of HCV, within 299 variants isolated from serum specimens of 27 chronically infected patients, 12 of whom with human immunodeficiency virus (HIV) co-infection. Liver fibrosis stage was inversely correlated with p7 synonymous substitutions (dS) (p=0.033), and indices of p7 genetic diversity were significantly higher in HIV-negative subjects compared to HIV-positive subjects (dS, p=0.005; non-synonymous substitutions (dN), p=0.002; dN/dS ratio, p=0.024; amino acid distances, p=0.007). Six p7 genes with naturally occurring unique amino acid variations were selected for in vitro study. The variants demonstrated diversified functional heterogeneity in vitro, with one variant from a subject with severe liver disease displaying hyperactive ion channel function, as well as other variants presenting altered pH-activated channel gating activities.

Genetic diversity of near genome-wide hepatitis C virus sequences during chronic infection: evidence for protein structural conservation over time.

Infection with hepatitis C virus (HCV) is one of the leading causes of chronic hepatitis, liver cirrhosis and end-stage liver disease worldwide. The genetics of HCV infection in humans and the disease course of chronic hepatitis C are both remarkably variable. Although the response to interferon treatment is largely dependent on HCV genotypes, whether or not a relationship exists between HCV genome variability and clinical course of hepatitis C disease still remains unknown. To more thoroughly understand HCV genome evolution over time in association with disease course, near genome-wide HCV genomes present in 9 chronically infected participants over 83 total study years were sequenced. Overall, within HCV genomes, the number of synonymous substitutions per synonymous site (d(S)) significantly exceeded the number of non-synonymous substitutions per site (d(N)). Although both d(S) and d(N) significantly increased with duration of chronic infection, there was a highly significant decrease in d(N)/d(S) ratio in HCV genomes over time. These results indicate that purifying selection acted to conserve viral protein structure despite persistence of high level of nucleotide mutagenesis inherent to HCV replication. Based on liver biopsy fibrosis scores, HCV genomes from participants with advanced fibrosis had significantly greater d(S) values and lower d(N)/d(S) ratios compared to participants with mild liver disease. Over time, viral genomes from participants with mild disease had significantly greater annual changes in d(N), along with higher d(N)/d(S) ratios, compared to participants with advanced fibrosis. Yearly amino acid variations in the HCV p7, NS2, NS3 and NS5B genes were all significantly lower in participants with severe versus mild disease, suggesting possible pathogenic importance of protein structural conservation for these viral gene products.

Genetic diversity of hepatitis C virus predicts recurrent disease after liver transplantation.

Approximately 20% of patients receiving liver transplants for end-stage hepatitis C rapidly develop severe allograph fibrosis within the first 24 months after transplant. Hepatitis C virus (HCV) variants were studied in 56 genotype-1-infected subjects with end-stage hepatitis C disease at the time before and 12 months after liver transplant, and post-transplant outcome was followed with serial liver biopsies. In 15 cases, pre-transplant HCV genetic diversity was studied in detail in liver (n=15), serum (n=15), peripheral blood mononuclear cells (n=13), and perihepatic lymph nodes (n=10). Our results revealed that pre-transplant HCV genetic diversity predicted the histological outcome of recurrent hepatitis C disease after transplant. Mild disease recurrence after transplant was significantly associated with higher genetic diversity and greater diversity changes between the pre- and post-transplant time points (p=0.004). Meanwhile, pre-transplant genetic differences between serum and liver were related to a higher likelihood of development of mild recurrent disease after transplant (p=0.039).

Hepatitis C virus envelope glycoprotein co-evolutionary dynamics during chronic hepatitis C.

Hepatitis C virus (HCV) envelope glycoprotein co-evolution was studied in 14 genotype 1-infected and treatment-naive subjects, including 7 with mild and 7 with severe liver disease. Cassettes encoding the envelope 1 gene (E1) and hypervariable region (HVR1) of the envelope 2 gene were isolated at 38 different time points over 81 follow-up years. There were no significant differences in age, gender, alcohol use, or viral load between the mild and severe disease groups. Virus from subjects with severe disease had significantly slower evolution in HVR1, and significant divergent evolution of E1 quasispecies, characterized by a preponderance of synonymous mutations, compared to virus from subjects with mild disease. Phylogenetic comparisons indicated higher similarity between amino acid sequences of the E1 and HVR1 regions with mild disease versus severe disease (r=0.44 versus r=0.17, respectively; P=0.01). In summary, HCV envelope quasispecies co-evolution differs during mild versus severe disease.

Hepatitis C virus dynamics during natural infection are associated with long-term histological outcome of chronic hepatitis C disease.

BACKGROUND: The long-term dynamics of hepatitis C virus (HCV) infection and their association with hepatitis C disease are unknown. METHODS: Fifty-two treatment-naive subjects with chronic HCV genotype 1 infection were selected from the Alaska Natives and American Indians cohort. Viral RNA levels were measured in 223 specimens (mean, 4.3 specimens/subject) over 457 patient-years. Viral quasispecies diversity was analyzed in 187 specimens (mean, 3.6 specimens/subject) over 365 patient-years. RESULTS: Thirty-three subjects had minimal hepatic fibrosis, and 19 developed bridging fibrosis or cirrhosis. There was no significant difference in host variables, including alcohol consumption, between disease groups. Subjects with mild disease had higher serum RNA levels after 2 decades of infection (P=.013), greater fluctuations in RNA levels over time (P=.04), higher intraspecimen quasispecies diversity (P=.001), and higher rates of quasispecies diversification (P=.004) than did subjects with severe disease. On multivariate analysis, the odds of having severe disease were 15.3 (95% confidence interval, 2.3-99.6) times higher among persons with low quasispecies diversification rates compared with the odds among persons with high diversification rates. CONCLUSIONS: Histological progression of hepatitis C is tightly associated with homogenization of HCV quasispecies, perhaps reflecting immune failure and/or selective outgrowth of aggressive viral variants.