Determination of hepatitis B phenotype using biochemical and serological markers.

The aim of this study was to assess the validity of categorization of chronic hepatitis B viral infection into stages or phases based upon measures of disease activity and viral load, assuming these phenotypes will be useful for prognostication and determining the need for antiviral therapy. We assessed the phenotype of hepatitis B of 1,390 adult participants enrolled in the Hepatitis B Research Network Cohort Study, using a computer algorithm. Only 4% were immune tolerant, while 35% had chronic hepatitis B (18% e antigen positive and 17% e antigen negative) while 23% were inactive carriers. Strikingly, 38% of participants did not fit clearly into any one of these groups and were considered indeterminant. The largest subset of indeterminants had elevated serum aminotransferases with low levels of HBV DNA (less than 10,000 iu/mL). Subsequent determination of hepatitis B phenotype on the next available laboratory tests showed that 64% remained indeterminant. These findings call into question the validity of conventional staging of hepatitis B, in large part because of the substantial proportion of patients who do not fit readily into one of the usual stages or phases. Further studies are needed of the indeterminant category of chronic hepatitis B viral infection, including assessments of whether patients in this group are perhaps in transition to another phase or if they are a distinct phenotype with a need to assess liver disease severity and need for antiviral therapy. (ClinicalTrials.gov identifier NCT01263587).

GS-9857 in patients with chronic hepatitis C virus genotype 1-4 infection: a randomized, double-blind, dose-ranging phase 1 study.

GS-9857, an inhibitor of the hepatitis C virus (HCV) nonstructural protein (NS) 3/4A, demonstrates potent activity against HCV genotypes 1-6 and improved coverage against commonly encountered NS3 resistance-associated variants (RAVs). In this study, the safety, tolerability, antiviral activity and pharmacokinetics (PK) of GS-9857 were evaluated in patients with chronic HCV genotype 1-4 infection. Patients with genotype 1-4 infection received placebo or once-daily GS-9857 at doses ranging from 50 to 300 mg for 3 days under fasting conditions. GS-9857 was well tolerated; all reported adverse events (AEs) were mild or moderate in severity. Diarrhoea and headache were the most commonly reported AEs. Grade 3 or 4 laboratory abnormalities were observed in 17% of patients receiving GS-9857; there were no Grade 3 or 4 abnormalities in alanine aminotransferase, aspartate aminotransferase or alkaline phosphatase levels. GS-9857 demonstrated potent antiviral activity in patients with chronic HCV infection, achieving mean and median maximum reductions in HCV RNA of ≥3 log10 IU/mL following administration of a 100-mg dose in patients with HCV genotype 1a, 1b, 2, 3 or 4 infection. The antiviral activity of GS-9857 was unaffected by the presence of pretreatment NS3 RAVs. In patients with genotype 1-4 infection, GS-9857 exhibited linear PK and was associated with a median half-life of 29-42 h, supporting once-daily dosing. Thus, the tolerability, efficacy and pharmacokinetic profile of GS-9857 support its further evaluation for treatment of patients with chronic HCV infection.

Prospective Multicenter Observational Study of Overt Hepatic Encephalopathy.

BACKGROUND: Overt hepatic encephalopathy (OHE) is a frequent complication of decompensated cirrhosis. AIMS: A multicenter prospective observational study was performed to assess the most commonly recorded presenting manifestations of OHE and its associated health-care burden. METHODS: Qualifying patients must have experienced ≥1 OHE episode within 30 days of enrollment (qualifying OHE) and were followed for recurrence (on-study OHE). RESULTS: Two hundred and sixty-five patients were enrolled at 30 sites and followed for up to 9 months (mean 72 days). Seventy-two patients experienced 122 on-study episodes; with 72, 23, and 13 having ≥1, ≥2, or ≥3 on-study episodes with median days to occurrence of the 1st, 2nd, and 3rd episode of 34, 19, and 11, respectively. The most frequently recorded OHE manifestations included confusion (78 %), change in mental status (57 %), disorientation (48 %), lethargy (46 %), and asterixis (45 %). West Haven grade was used inconsistently and recorded for only 28 % of episodes. Most qualifying and on-study episodes occurred on rifaximin (60 and 82 %, respectively) and were associated with hospitalization (68 and 85 %, respectively). Twenty-three patients experienced ≥2 on-study episodes within 2 months of enrollment on average (median 45 days) and accounted for 60 % of on-study episodes. CONCLUSIONS: In this prospective study, OHE's most commonly recorded presenting manifestations included confusion, altered mental status, disorientation, lethargy, and asterixis. As reflected by frequent recurrence and hospitalizations, OHE, particularly the approximately 10 % of "high-resource-utilizing" patients with frequent recurrence, continues to pose a major unmet medical need and health-care burden despite the use of rifaximin.

Antigen-specific T lymphocyte proliferation decreases over time in advanced chronic hepatitis C.

To evaluate T cell immunity in advanced liver disease, antigen-specific lymphoproliferative (LP) responses were prospectively studied in the context of the Hepatitis C Antiviral Long-term Treatment against Cirrhosis trial. Peripheral blood responses to hepatitis C virus (HCV), tetanus and Candida protein antigens were measured at baseline, month 12 (M12), M24, M36 and M48 in 186 patients randomized to either low-dose peginterferon-alfa-2a (PEG-IFN) only or observation. Liver histology was evaluated at baseline, M24 and M48. Patients with cirrhosis (Ishak 5-6) were less likely to have positive LP responses to HCV at baseline than patients with fibrosis (15%vs 29%, P = 0.03) and had lower levels of HCV c100 responses at baseline, M24 and M48 (P = 0.11, P = 0.05, P = 0.02, respectively). For 97 patients with complete longitudinal data, the frequency of positive LP responses to HCV, tetanus and Candida antigens declined over time (P < 0.003), and the slope of this decline was greater in the PEG-IFN treatment group than the observation group (P < 0.02). Lower levels of tetanus LP responses were associated with fibrosis progression and clinical outcomes (P = 0.009). Poorer CD4+ T cell proliferative function was associated with more advanced liver disease in chronic hepatitis C and may be further affected by long-term PEG-IFN treatment.

Evidence for action of ribavirin through the hepatitis C virus RNA polymerase.

Hepatitis C virus (HCV) infections are treated with interferon alpha plus ribavirin, but it is unknown how ribavirin works against HCV. Ribavirin is a guanosine analogue that can be a substrate for the viral RNA polymerase. HCV is genetically variable, and this genetic variation could affect the polymerase's use of ribavirin triphosphate. Thirteen patients infected with HCV who failed interferon alpha monotherapy and were retreated with interferon alpha plus ribavirin were identified; seven were responders and six were nonresponders to combination therapy. The consensus sequences encoding the 13 polymerases plus seven sequences from treatment-naive controls were determined. The responder sequences were more genetically variable than the nonresponders and controls, the amino acid variations unique to responders had lower BLOSUM90 scores than variations in nonresponders and controls, and the amino acid variations correlated with response to therapy clustered around the RNA-binding channel of the polymerase. These data imply that that the responder enzymes were probably more functionally variable than the nonresponder enzymes. Enzymatic activity was measured for 10 recombinant polymerases; RNA synthesis activity varied by over sevenfold and polymerases from two of the responders used GTP much better than UTP, but technical limitations prevented direct measurement of ribavirin triphosphate use. Because response to combination therapy in these patients was primarily due to addition of ribavirin to the treatment regimen, these data imply that genetic variation in the polymerase may have affected the efficiency of ribavirin incorporation into the viral genome and hence may have modulated ribavirin's efficacy against HCV.

Prevention of hepatocellular carcinoma with antiviral therapy.

Chronic viral hepatitis types B and C may eventually lead to the development of hepatocellular carcinoma. Although hepatitis B is readily preventable by vaccination, there is growing evidence that antiviral therapy directed against hepatitis B may reduce the risk of liver cancer among those already infected. There is no vaccine against hepatitis C, but the evidence is now strong that antiviral therapy with sustained virological response (viral cure) reduces, but does not eliminate, the risk of hepatocellular carcinoma.

Psychometric properties of the PROMIS short form measures in a U.S. cohort of 961 patients with chronic hepatitis C prescribed direct acting antiviral therapy.

BACKGROUND: To better understand symptoms experienced by patients infected with chronic hepatitis C virus (HCV), valid and reliable patient-reported outcome (PRO) measures are needed. AIM: To assess the reliability and validity of 10 patient-reported outcomes measurement information system (PROMIS) measures and the Headache Impact Test-6 (HIT-6) in a large national sample of patients with HCV. METHODS: Pre-treatment data from 961 patients with HCV starting direct acting antiviral therapy at 11 U.S. liver centers were analyzed. Internal reliability was evaluated using Cronbach's alpha coefficient; frequency distributions were examined for floor and ceiling effects; structural validity was investigated via item-response-theory models; convergent validity was evaluated using correlations with theoretically-similar items from the HCV-PRO and memorial symptom assessment scale (MSAS); and known-groups validity was investigated by observing PRO differences by liver disease status and number of comorbidities. RESULTS: The HIT-6 and the majority of the PROMIS measures yielded excellent reliability (alphas ≥ 0.87). Ceiling effects were infrequent ( < 4%), while 30%-59% of patients reported no symptoms (floor effects). The data supported structural validity of the HIT-6 and most PROMIS measures. The PROMIS measures showed moderate to strong correlations with theoretically-similar items from the HCV-PRO and MSAS (0.39-0.77). Trends were observed between worse PRO scores and advanced cirrhosis and greater number of comorbidities, lending support for known-groups validity. CONCLUSIONS: The psychometric properties of the HIT-6 and PROMIS measures performed satisfactorily in this large cohort of patients with HCV starting direct acting antiviral therapy. Opportunities exist for further refinement of these PROs. Evaluation of performance over time and in under-represented subgroups is needed.

Detection of replicative intermediates of hepatitis C viral RNA in liver and serum of patients with chronic hepatitis C.

The hepatitis C virus is a positive stranded hepatotropic RNA virus. We describe a method of detecting positive and negative strands of hepatitis C viral RNA using the polymerase chain reaction. We tested serum and liver tissue from nine patients with chronic hepatitis C. The positive RNA strand of HCV was detected in the sera and livers of all nine, the negative strand was detected in the livers of eight (89%), and in the sera of five (55%). Titers of both strands of HCV RNA were determined by serial endpoint dilutions. The amount of the negative strand in the serum and liver was usually 10-100 times less than the positive strand. Predigestion of serum with ribonucleases did not alter the detection of the negative strand. This suggests that the negative strand found in the serum may be protected from digestion by being associated with virions.

All-oral direct-acting antiviral therapy in HCV-advanced liver disease is effective in real-world practice: observations through HCV-TARGET database.

BACKGROUND: Chronic hepatitis C virus therapy in patients with advanced liver disease remains a clinical challenge. HCV-TARGET collects data in patients treated at tertiary academic and community centres. AIM: To assess efficacy of all-oral HCV therapy in advanced liver disease. METHODS: Between December 2013 and October 2014, 240 patients with a MELD score of ≥10 initiated HCV treatment with an all-oral regimen. Data from the 220 patients who completed 12-week follow-up were analysed. RESULTS: Genotype 1 (GT1) patients had higher sustained virological response (SVR) when treated with sofosbuvir plus simeprevir ± ribavirin than with sofosbuvir plus ribavirin (66-74% vs. 54%); GT1b vs GT1a (84% vs. 64%). SVR for GT2 was 72% with sofosbuvir plus ribavirin, while GT3 patients had a substantially lower response (35%). A decrease in MELD score was not clearly related to SVR over the short course of follow-up although some had improvements in MELD score, serum bilirubin and albumin. A predictor of virological response was albumin level while negative predictors were elevated bilirubin level and GT1a. Most patients with GT1 were treated with approximately 12-week duration of sofosbuvir and simeprevir ± ribavirin therapy while GT2 and GT3 patients were treated with approximately 12 and 24 weeks of sofosbuvir plus ribavirin respectively. CONCLUSIONS: All-oral therapies are effective among patients with advanced liver disease with high levels of success in GT2 and GT1b, and may serve to reduce the severity of liver disease after SVR. Treatment for GT3 patients remains an unmet need. Clinical trial number: NCT01474811.

Virological outcomes and treatment algorithms utilisation in observational study of patients with chronic hepatitis C treated with boceprevir or telaprevir.

BACKGROUND: HCV-TARGET is a longitudinal observational study of chronic hepatitis C virus (HCV) patients treated with direct-acting anti-viral agents (DAAs) in a US consortium of 90 academic and community medical centres. AIM: To assess utilisation of response-guided therapy (RGT) and sustained virological response (SVR) of a large cohort of patients. METHODS: Patients received peginterferon (PEG-IFN), ribavirin and either telaprevir or boceprevir. Demographical, clinical and virological data were collected during treatment and follow-up. RGT and treatment futility stopping rules was assessed at key time points. RESULTS: Of 2084 patients, 38% had cirrhosis and 56% had received prior treatment for HCV. SVR rates were 31% (95% CI: 24-40) and 50% (95% CI: 44-56) in boceprevir patients with and without cirrhosis, respectively. SVR rates were 46% (95% CI: 42-50) and 60% (95% CI: 57-64) in telaprevir patients with and without cirrhosis, respectively. Early clearance of virus, IL28B genotype, platelet counts and diabetes were identified as predictors of SVR among boceprevir patients, while early clearance of virus, IL28B, cirrhosis, HCV subtype, age, haemoglobin, bilirubin and albumin levels were identified as predictors of SVR for telaprevir patients. CONCLUSIONS: In academic and community centres, triple therapy including boceprevir or telaprevir led to SVR rates somewhat lower than those noted in large phase 3 clinical trials. Response rates were consistently higher among patients without cirrhosis compared to those with cirrhosis regardless of DAA used and prior treatment response. Trial registration clinicaltrials.gov NCT01474811.

The effect of cholesterol-lowering agents on hepatic and plasma cholesterol in Niemann-Pick disease type C.

Niemann-Pick disease type C (NP-C) is a neurovisceral lipidosis characterized by defective intracellular trafficking of cholesterol and lysosomal accumulation of unesterified cholesterol, believed to be an offending metabolite. We studied the effect of cholesterol-lowering agents on hepatic and plasma cholesterol levels in NP-C by randomly assigning 25 patients with NP-C to one of five treatment regimens containing different combinations of cholestyramine, lovastatin, nicotinic acid, or dimethyl sulfoxide (DMSO). Unesterified cholesterol content was measured in liver biopsies before and after 4 months' treatment. All drug regimens except DMSO alone reduced hepatic and plasma cholesterol levels. Toxicity was limited and did not prevent any patient from completing the study. The combination of cholestyramine, lovastatin, and nicotinic acid lowered cholesterol levels in liver and blood with minimal side effects. A controlled clinical study will be necessary to determine if this regimen influences the rate of neurologic progression.

Hepatitis C--virology and future antiviral targets.

The hepatitis C virus is a single-stranded RNA virus with a genome approximately 9,000 nucleotides in length. The genome consists of a single, large open reading frame (ORF) and 5' and 3' untranslated regions. The highly conserved 5' untranslated region is 341 nucleotides in length with a complex secondary structure and may function as an internal ribosomal entry site (IRES). The 3' untranslated region is approximately 500 nucleotides in length and contains a hypervariable region, followed by a poly(U) sequence and a highly conserved 98-nucleotide element with a stable secondary structure. The ORF codes form a single polyprotein that is processed into as many as 10 polypeptides, including a capsid protein (core), two envelope proteins (E1 and E2), and nonstructural proteins (NS2, NS3, NS4, and NS5). Potentially suitable antiviral targets include the IRES, protease, helicase, and RNA polymerase. In vitro studies show that antisense oligonucleotides can inhibit the production of structural HCV proteins and may be therapeutically useful if the problems of stability and delivery can be solved. The binding of HCV envelope proteins to CD81, a potential receptor for viral entry into hepatocytes, has recently been described and also raises the possibility of agents to block the binding to CD81 or the entry of the virus into cells.

Chronic hepatitis C viral infection in patients with normal serum alanine aminotransferases.

A substantial proportion of patients with chronic hepatitis C virus (HCV) infection (HCV RNA positive) have persistently normal serum alanine aminotransferases (ALT). These patients currently pose a therapeutic dilemma, as it is not clear how best to deal with them. Response rates to interferon are low, and in some cases interferon therapy has been associated with an increase in serum aminotransferases. Therefore, the NIH Consensus Conference recommended against treating them with interferon. Although most patients with persistently normal serum ALT levels have mild disease on liver biopsy, the consensus panel did think it appropriate to do liver biopsies in these subjects. Few data are available on therapy with the combination of interferon and ribavirin for such patients. At Saint Louis University, 24 patients with normal ALT were treated with interferon/ribavirin. Preliminary analysis of the results shows that 33% of the patients had a sustained response 24 weeks after therapy. Thus, uncertainties remain about patients with chronic HCV infection and persistently normal serum ALT with the currently available therapies. However, if a more effective and better tolerated therapy were to become available, such patients might best be treated to eliminate the chronic viral infection.

Multifocal verruciform xanthoma of the upper aerodigestive tract in a child with a systemic lipid storage disease.

We report the clinical, light-microscopic, and ultrastructural features of a case of multifocal verruciform xanthoma in the upper aerodigestive tract of a child with a systemic lipid disorder. Lipid storage cells were found in liver, bone marrow, and as a component of verruciform xanthomas. To our knowledge this represents the first case of verruciform xanthoma reported in (a) a child, (b) as a multifocal lesion in the upper aerodigestive tract, (c) associated with a systemic lipid disorder, and (d) with ultrastructural evidence of lipid accumulation within endothelial cells. Although this patient presented with lesions involving the tongue and larnyx, subsequently lesions were found in the bone marrow and liver. Two months later more lesions were discovered on the epiglottis, posteior tongue, right glottis, and in grossly normal peritonsillar mucosa. Six months later a new oral lesion developed. Based upon these observations, we speculate that the pathogenesis of verruciform xanthoma involves accumulation of excess lipid in subepithelial sites which is scavenged by macrophages. Lipid-laden macrophages release epithelial growth factors that lead to epithelial hyperplasia. Depending on the degree of epithelial hyperplasia, the gross appearance of verruciform xanthomas may be flat, sessile, papillary, or verrucous.

Hepatitis C virus infection in the mothers and infants cohort study.

OBJECTIVES: To estimate the hepatitis C virus (HCV) vertical transmission rate, the effect of potential risk factors, and the pattern of HCV antibody response and viremia in HCV-infected infants. STUDY DESIGN: The Mothers and Infants Cohort Study enrolled both human immunodeficiency virus (HIV)-seropositive and HIV-seronegative pregnant women at five obstetric clinics in New York City in a prospective cohort study between January 1986 and January 1991. HCV-infected mothers and their 122 offspring were followed-up for a minimum of 12 months for evidence of HCV infection as determined by persistent HCV antibodies or detection of HCV RNA by reverse transcription polymerase chain reaction. Comparisons among groups for categorical variables were performed using the Fisher's exact test. RESULTS: Seven (6%; 95% confidence interval, 2%-11%) of the 122 infants were HCV-infected. There was a tendency for increased risk of transmission with maternal viral and obstetrical factors, such as coinfection with HIV (7% vs 4%), high HIV viral load (13% vs 6%), HCV viremia (8% vs 3%), vaginal delivery (6% vs 0%), and female gender of offspring (8% vs 3%), although none of the associations reached statistical significance. After loss of maternal antibody, HCV antibody seroconversion occurred at a mean age of 26 months in 3 HIV-coinfected infants compared with 7 months of age in 4 HCV-infected HIV-uninfected infants. Serial samples showed that HCV RNA persisted in 6 infants for at least 18 to 54 months. CONCLUSIONS: Our study is in accordance with other studies that have shown low overall HCV vertical transmission risk and a trend toward higher risk with maternal risk factors such as HIV-coinfection or HCV viremia. A delay in infant HCV antibody response may be associated with HIV coinfection although larger studies are needed to confirm these findings.

Seroprevalence of viral hepatitis in an older nursing home population.

OBJECTIVES: To assess the prevalence of current or previous infection with viral hepatitis agents in an older nursing home population. DESIGN: A prospective cohort study. SETTING: Three nursing homes in the greater St. Louis area affiliated with Saint Louis University. SUBJECTS: Older residents admitted to these facilities. MEASUREMENTS: Residents were interviewed and examined for evidence of hepatitis or liver disease. Serum samples were tested for hepatitis B surface antigen (HBsAg), antibody to hepatitis B core and surface antigens (anti-HBc and anti-HBs), antibody to hepatitis A virus (anti-HAV), antibody to hepatitis C virus (anti-HCV), and hepatitis G virus RNA (HGV RNA). RESULTS: Of 329 residents queried, 199 gave consent and were able to participate. The seroprevalence of hepatitis was: HBsAg 0%, anti-HBc 24.1%, anti-HBs 19.5%, anti-HAV 79.9%, anti-HCV 4.5%, and HGV-RNA 10.6%. Frequency of HAV infection increased significantly with age whereas HBV infection correlated with ethnic status and former occupation as a manual worker. A history of blood transfusion was associated with a higher rate of anti-HCV. End stage renal disease, present in 17 patients, was associated with anti-HBc, anti-HCV, and HGV RNA positivity but not with anti-HBs or anti-HAV positivity CONCLUSIONS: The seroprevalence of anti-HCV was surprisingly high in this population residing in skilled nursing facilities, and we recommend that all new patients admitted to this type of institution be screened for anti-HCV. The prevalence of HGV RNA was higher than in the general US blood donor population, but the significance of this finding remains uncertain.

Hepatitis C viral RNA in serum of patients with chronic non-A, non-B hepatitis: detection by the polymerase chain reaction using multiple primer sets.

The recently introduced antibody test for hepatitis C virus (HCV) infection has proven to have certain limitations. Since HCV itself is usually present in clinical specimens at very low titers, a useful assay for the virus must have very high sensitivity. We have developed a simple, highly sensitive assay for HCV RNA based on the polymerase chain reaction (PCR). In this test, RNA extracted from HCV infected serum or plasma is used as the template for double PCR with nested primers. Sensitivity studies demonstrate that this assay is able to detect HCV at or beyond the sensitivity level of chimpanzee infectivity. We tested, with several sets of nested primers, 40 patients with chronic non-A, non-B hepatitis (36 seropositive and 4 seronegative) and found that 35/40 were PCR positive including all 4 seronegative patients. Normal human plasma and plasma from hepatitis B infected patients did not react in this test. This assay has proven to be valuable for determining the presence of HCV in various samples; furthermore, it offers the possibility of diagnosis of HCV infection in seronegative patients.

Hepatitis B.

The hepatitis B virus is a hepatotropic virus that can produce a variety of clinical syndromes in patients ranging in age from infants to elderly adults. Worldwide, it is among the leading causes of fulminant hepatic failure, cirrhosis, and hepatocellular carcinoma. Recent advances have led to effective antiviral treatments using interferon and nucleoside analogues. Highly effective vaccinations also are used widely and ultimately may lead to eradication of this life-threatening virus.

A comparison between the phenol-chloroform method of RNA extraction and the QIAamp viral RNA kit in the extraction of hepatitis C and GB virus-C/hepatitis G viral RNA from serum.

Viral RNAs are detected commonly in serum by hybridization and polymerase chain reaction (PCR) methods for both clinical and research purposes. Two methods of extracting viral RNA were evaluated prior to amplification by PCR. One was a conventional phenol-chloroform method and the other used a standardized, manufactured kit. The efficiency of extraction was tested by semi-quantitative amplification of hepatitis C viral and GB virus-C/hepatitis G viral RNAs. The standarized commercial method, although more time efficient, resulted in an approximately ten fold less sensitivity. Thus, in situations where maximum sensitivity is needed, the more labor intenstive phenol choloroform method is recommended.

Detection and quantitation of hepatitis C virus RNA in serum using the polymerase chain reaction and a colorimetric enzymatic detection system.

A sensitive, non-isotopic method for detecting and quantifying hepatitis C virus (HCV) RNA in serum using the reverse transcriptase-polymerase chain reaction (RT-PCR) and a hybridization specific, colorimetric biotin-avidin peroxidase detection system has been developed. The sensitivity of the PCR-colorimetric system was determined using RNA synthesized from cloned HCV cDNA. The assay could detect as few as 10 molecules of HCV RNA, comparable to the sensitivity achieved with double PCR using nested primers. Thus, this colorimetric assay can detect low levels of HCV RNA in serum and appears to be quantitative, suggesting that this technique may be applied to rapid screening of large numbers of samples and to monitor the effect of antiviral therapy.