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COVID-19; Hepatitis B; Tenofovir COVID-19; Hepatitis B; Tenofovir COVID-19; Hepatitis B; Tenofovir Background and Aims HIV-positive patients on tenofovir hydroxyl fumarate (TDF)/emtricitabine have a lower risk of COVID-19 and hospitalization than those given other treatments. Our aim was to analyze the severity of COVID-19 in patients with chronic hepatitis B (CHB) on TDF or entecavir (ETV). Methods Spanish hospital databases (n = 28) including information regarding adult CHB patients on TDF or ETV for the period February 1st to November 30th 2020 were searched for COVID-19, defined as a positive SARS-CoV-2 polymerase chain reaction, and for severe COVID-19. Results Of 4736 patients, 117 had COVID-19 (2.5%), 67 on TDF and 50 on ETV. Compared to patients on TDF, those on ETV showed (p

BACKGROUND Spontaneous HDV-RNA fluctuations, assessed by nonstandardised in-house assays, have been reported during the course of chronic hepatitis delta (CHD). AIMS To evaluate changes in serum HDV-RNA concentrations in untreated CHD patients and correlate these changes with other HBV markers. METHODS A total of 323 consecutive serum samples from 56 CHD patients (detectable HDV-RNA) followed for >3 years were retested for HDV-RNA levels by a sensitive technique using the first WHO international HDV-RNA standard. Quantitative HBsAg, HBV-DNA, and HBV-RNA were also determined. RESULTS Most participants were male, middle-aged, white European, and HBeAg-negative (82%). Almost half had liver cirrhosis and 64% were receiving nucleos(t)ide analogues. At inclusion, median-HDV-RNA was 5.3 (4.2-6.5) log10 IU/mL, HBsAg 4.0 (3.5-4.3) log10 IU/mL, and HBV-DNA 1.6 (1.0-2.6) log10 IU/mL; ALT values were normal in 13 (23%). During a mean follow-up of 5.6 (3-16) years, 14 (25%) showed ≥2log10 HDV-RNA decline, including 11 (20%) who spontaneously achieved undetectable HDV-RNA. Four patients (7%) lost HBsAg, with undetectable HDV-RNA. The remaining 42 (75%) had persistently detectable HDV-RNA. During follow-up, patients with a ≥2log10 HDV-RNA decline showed a greater HBsAg drop (-0.7 ± 1.1 vs -0.09 ± 0.9 log IU/mL; P = 0.039) than those with a

[Background & Aims] Prospective drug-induced liver injury (DILI) registries are important sources of information on idiosyncratic DILI. We aimed to present a comprehensive analysis of 843 patients with DILI enrolled into the Spanish DILI Registry over a 20-year time period., [Methods] Cases were identified, diagnosed and followed prospectively. Clinical features, drug information and outcome data were collected., [Results] A total of 843 patients, with a mean age of 54 years (48% females), were enrolled up to 2018. Hepatocellular injury was associated with younger age (adjusted odds ratio [aOR] per year 0.983; 95% CI 0.974–0.991) and lower platelet count (aOR per unit 0.996; 95% CI 0.994–0.998). Anti-infectives were the most common causative drug class (40%). Liver-related mortality was more frequent in patients with hepatocellular damage aged ≥65 years (p = 0.0083) and in patients with underlying liver disease (p = 0.0221). Independent predictors of liver-related death/transplantation included nR-based hepatocellular injury, female sex, higher onset aspartate aminotransferase (AST) and bilirubin values. nR-based hepatocellular injury was not associated with 6-month overall mortality, for which comorbidity burden played a more important role. The prognostic capacity of Hy’s law varied between causative agents. Empirical therapy (corticosteroids, ursodeoxycholic acid and MARS) was prescribed to 20% of patients. Drug-induced autoimmune hepatitis patients (26 cases) were mainly females (62%) with hepatocellular damage (92%), who more frequently received immunosuppressive therapy (58%)., [Conclusions] AST elevation at onset is a strong predictor of poor outcome and should be routinely assessed in DILI evaluation. Mortality is higher in older patients with hepatocellular damage and patients with underlying hepatic conditions. The Spanish DILI Registry is a valuable tool in the identification of causative drugs, clinical signatures and prognostic risk factors in DILI and can aid physicians in DILI characterisation and management., [Lay summary] Clinical information on drug-induced liver injury (DILI) collected from enrolled patients in the Spanish DILI Registry can guide physicians in the decision-making process. We have found that older patients with hepatocellular type liver injury and patients with additional liver conditions are at a higher risk of mortality. The type of liver injury, patient sex and analytical values of aspartate aminotransferase and total bilirubin can also help predict clinical outcomes., The present study has been supported by grants of Instituto de Salud Carlos III cofounded by Fondo Europeo de Desarrollo Regional – FEDER (contract numbers: PI19/00883, PI16/01748, PI18/00901, PI18/01804, PI-0285-2016, PI-0274-2016, PI-0310-2018, PT17/0017/0020) and Agencia Española del Medicamento. CIBERehd and Plataforma ISCIII Ensayos Clinicos are funded by Instituto de Salud Carlos III. MRD holds a Joan Rodes (JR16/00015)/Acción B clinicos investigadores (B-0002-2019) and JSC a Rio Hortega (CM17/00243) research contract from ISCIII and Consejería de Salud de Andalucía.

[Abstract] A percentage of hepatitis C virus (HCV)-infected patients fail direct acting antiviral (DAA)-based treatment regimens, often because of drug resistance-associated substitutions (RAS). The aim of this study was to characterize the resistance profile of a large cohort of patients failing DAA-based treatments, and investigate the relationship between HCV subtype and failure, as an aid to optimizing management of these patients. A new, standardized HCV-RAS testing protocol based on deep sequencing was designed and applied to 220 previously subtyped samples from patients failing DAA treatment, collected in 39 Spanish hospitals. The majority had received DAA-based interferon (IFN) α-free regimens; 79% had failed sofosbuvir-containing therapy. Genomic regions encoding the nonstructural protein (NS) 3, NS5A, and NS5B (DAA target regions) were analyzed using subtype-specific primers. Viral subtype distribution was as follows: genotype (G) 1, 62.7%; G3a, 21.4%; G4d, 12.3%; G2, 1.8%; and mixed infections 1.8%. Overall, 88.6% of patients carried at least 1 RAS, and 19% carried RAS at frequencies below 20% in the mutant spectrum. There were no differences in RAS selection between treatments with and without ribavirin. Regardless of the treatment received, each HCV subtype showed specific types of RAS. Of note, no RAS were detected in the target proteins of 18.6% of patients failing treatment, and 30.4% of patients had RAS in proteins that were not targets of the inhibitors they received. HCV patients failing DAA therapy showed a high diversity of RAS. Ribavirin use did not influence the type or number of RAS at failure. The subtype-specific pattern of RAS emergence underscores the importance of accurate HCV subtyping. The frequency of “extra-target” RAS suggests the need for RAS screening in all three DAA target regions. Ministerio de Economía y Empresa; IDI-20151125 Ministerio de Ciencia, Innovación y Universidades; SAF SAF 2017-87846-R

Background COVID-19 is a potentially severe disease caused by the recently described SARS-CoV-2. Whether liver fibrosis might be a relevant player in the natural history of COVID-19 is currently unknown. We aimed to evaluate the association between FIB-4 and the risk of progression to critical illness in middle-aged patients with COVID-19. Methods In this multicenter, retrospective study with prospective follow-up of 160 patients aged 35–65 years with COVID-19, FIB-4, clinical, and biochemical variables were collected at baseline. FIB-4 ≥2.67 defined patients with risk for advanced liver fibrosis. Results Risk for advanced fibrosis was estimated in 28.1% of patients. Patients with FIB-4 ≥2.67 more frequently required mechanical ventilation (37.8% vs 18.3%; P = .009). In multivariate analysis, FIB-4 ≥2.67 (odds ratio [OR], 3.41; 95% confidence interval [CI], 1.30–8.92), cardiovascular risk factors (OR, 5.05; 95% CI, 1.90–13.39), previous respiratory diseases (OR, 4.54; 95% CI, 1.36–15.10), and C-reactive protein (OR, 1.01; 95% CI, 1.01–1.02) increased significantly the risk of ICU admission. Bootstrap confirmed FIB-4 as an independent risk factor. Conclusions In middle-aged patients with COVID-19, FIB-4 may have a prognostic role. The link between liver fibrosis and the natural history of COVID-19 should be evaluated in future studies., Patients with COVID-19 who present with FIB-4 ≥ 2.67 at diagnosis of the infection are at higher likelihood of requiring mechanical ventilation irrespective of previous history of cardiovascular risk factors, respiratory diseases, or baseline inflammatory response.

Despite new treatments for hepatitis C virus (HCV) infection, IFNα-based regimens still have clinical relevance in special populations of patients and remain the only therapeutic option for many patients. We sought to elucidate the interplay between two relevant factors (IL28B polymorphism and T cell immune responses) involved in the outcome of this therapy in HCV-infected patients. We evaluated 38 patients infected with HCV genotype 1-17 coinfected with HIV-who were undergoing a full course of pegIFNα/RBV therapy. The interdependence and roles of T cell-mediated immune responses and IL28B rs12979860 single-nucleotide polymorphism genotype as predictors of virological response to anti-HCV treatment in patients with chronic hepatitis C were evaluated using nonparametric tests. Factors associated with rapid virological response (RVR) in univariate analysis were presence of CD4 T cell response against NS3 HCV protein, low baseline HCV-RNA, and IL28B CC genotype. Factors associated with sustained virological response (SVR) in univariate analysis were IL28B CC genotype, low baseline HCV-RNA, and presence of CD4 response against NS2. In the multivariate analysis, low baseline HCV-RNA and NS3-specific CD4 response showed a clear trend toward association with RVR (P = 0.09 and P = 0.07, respectively). Regarding SVR, IL28B CC genotype was the strongest predictor (P = 0.02), with presence of NS2-specific CD4 response showing a clear trend (P = 0.09). HCV-specific T cell response influences the outcome of pegIFNα/RBV therapy regardless of IL28B genotype. HCV-specific T cell responses (adaptive immunity) seem to influence viral clearance both in the short and long term during therapy (RVR and SVR), whereas the influence of the IL28B genotype (innate immunity) may be more relevant to the long-lasting therapeutic effect (SVR).

Objetivo y metodos: estudio observacional, longitudinal, prospectivo en el que se evaluaron los cambios en la percepcion de calidad de vida en pacientes asintomaticos con hepatitis C tratados con antivirales de accion directa. Resultados: los cuestionarios SF-36 y EQ-5D-5L realizados sobre 86 pacientes tratados y 12 pacientes control demostraron mejoria de varios parametros (funcion fisica, dolor corporal, salud general, vitalidad y funcion social), sobre todo cuando se compara la percepcion antes del tratamiento y tras finalizar el seguimiento despues de la curacion. Discusion: estos datos apoyan la hipotesis de que el virus de la hepatitis C puede causar empeoramiento en la calidad de vida de los pacientes asintomaticos.

Background Antiviral treatment of patients with chronic hepatitis B (CHB) in the grey zone of treatment comands risk management in order to optimize the health outcome. In this sense, the identification of HBV mutants related with an increased risk of hepatocellular carcinoma (HCC) could be useful to identify subpopulations with potential indication of antiviral treatment. Aim To analyze the prevalence/persistence of hepatitis B virus (HBV) preS and basal core promoter (BCP)/precore/core variants associated to HCC development in CHB patients in the grey zone. Methods Work was designed as a longitudinal retrospective study, including 106 plasma samples from 31 patients with CHB in the grey zone of treatment: Hepatitis B e antigen negative, HBV-DNA levels between 12-20000 IU/mL, normal or discordant transaminase levels during follow up and mild/moderate necro-inflammatory activity in liver biopsy or Fibroscan (up to 9.5 kPa). Serum HBV-DNA was tested using the Abbott Real Time HBV Assay and the BCP/precore/core and the hepatitis B surface antigen (HBsAg) coding regions were analyzed in positive samples by PCR/bulk-sequencing to identify the HCC-related HBV mutants. Results High-risk HCC related mutants were detected in 24 (77%) patients: 19 (61%) in the BCP/precore/core, and 7 (23%) in the HBsAg coding region (2 preS1 and 5 preS2 deletions). The prevalence of preS deletions was genotype-dependent: 3/5 (60%) patients with preS2 deletions and 1/2 with preS1 deletions were infected with the HBV-E genotype. Since HBV-E was the most prevalent in sub-Saharan patients, a correlation between preS deletions and ethnicity was also found: 6/8 (75%) sub-Saharan vs 1/19 (5%) Caucasian patients had preS deletions (P = 0.00016). Remarkably, this correlation was maintained in those patients infected with HBV-A, a minor genotype in sub-Saharan patients: 2/2 patients infected with HBV-A from West Africa vs 0/6 of Caucasian origin had preS deletions. The HCC related variants were the major strains and persisted over time (up to 48 mo). Patients with preS deletions had a significant higher prevalence of F2 fibrosis stage than the negatives (57% vs 10%, P = 0.0078). Conclusion HBV genetic analysis of selected populations, like sub-Saharans infected with HBV-E/A genotypes, will allow identification of subpopulations with risk of HCC development due to accumulation of high-risk HBV variants, thus commanding their increased clinical surveillance.

Hepatitis C virus (HCV)-specific CD8 + T cells suffer a progressive exhaustion during persistent infection (PI) with HCV. This process could involve the positive immune checkpoint 4-1BB/4-1BBL through the loss of its signal transducer, TRAF1. To address this issue, peripheral HCV-specific CD8 + T cells (pentamer-positive [pentamer + ]/CD8 + T cells) from patients with PI and resolved infection (RI) after treatment were studied. The duration of HCV infection and the liver fibrosis progression rate inversely correlated with the likelihood of detection of peripheral pentamer + /CD8 + cells. In PI, pentamer + /CD8 + cells had impaired antigen-specific reactivity that worsened when these cells were not detectable ex vivo . Short/midduration PI was characterized by detectable peripheral PD-1 + CD127 low TRAF1 low cells. After triggering of T cell receptors (TCR), the TRAF1 level positively correlated with the levels of CD127, Mcl-1, and CD107a expression and proliferation intensity but negatively with PD-1 expression, linking TRAF1 low to exhaustion. In vitro treatment with interleukin-7 (IL-7) upregulated TRAF1 expression, while treatment with transforming growth factor-β1 (TGF-β1) did the opposite, suggesting that the IL-7/TGF-β1 balance, besides TCR stimulation, could be involved in TRAF1 regulation. In fact, the serum TGF-β1 concentration was higher in patients with PI than in patients with RI, and it negatively correlated with TRAF1 expression. In line with IL-7 increasing the level of TRAF1 expression, IL-7 plus 4-1BBL treatment in vitro enhanced T cell reactivity in patients with short/midduration infection. However, in patients with long-lasting PI, anti-PD-L1, in addition to the combination of IL-7 and 4-1BBL, was necessary to reestablish T cell proliferation in individuals with slowly progressing liver fibrosis (slow fibrosers) but had no effect in rapid fibrosers. In conclusion, a peripheral hyporeactive TRAF1 low HCV-specific CD8 + T cell response, restorable by IL-7 plus 4-1BBL treatment, characterizes short/midduration PI. In long-lasting disease, HCV-specific CD8 + T cells are rarely detectable ex vivo , but treatment with IL-7, 4-1BBL, and anti-PD-L1 recovers their reactivity in vitro in slow fibrosers. IMPORTANCE Hepatitis C virus (HCV) infects 71 million people worldwide. Two-thirds develop a chronic disease that can lead to cirrhosis and hepatocellular carcinoma. Direct-acting antivirals clear the infection, but there are still patients who relapse. In these cases, additional immunotherapy could play a vital role. A successful anti-HCV immune response depends on virus-specific CD8 + T cells. During chronic infection, these cells are functionally impaired, which could be due to the failure of costimulation. This study describes exhausted specific T cells, characterized by low levels of expression of the signal transducer TRAF1 of the positive costimulatory pathway 4-1BB/4-1BBL. IL-7 upregulated TRAF1 expression and improved T cell reactivity in patients with short/midduration disease, while in patients with long-lasting infection, it was also necessary to block the negative PD-1/PD-L1 checkpoint. When the results are taken together, this work supports novel ways of restoring the specific CD8 + T cell response, shedding light on the importance of TRAF1 signaling. This could be a promising target for future immunotherapy.

ObjectiveRecently approved direct acting antivirals provide transformative therapies for chronic hepatitis C virus (HCV) infection. The major clinical challenge remains to identify the undiagnosed patients worldwide, many of whom live in low-income and middle-income countries, where access to nucleic acid testing remains limited. The aim of this study was to develop and validate a point-of-care (PoC) assay for the qualitative detection of HCV RNA.DesignWe developed a PoC assay for the qualitative detection of HCV RNA on the PCR Genedrive instrument. We validated the Genedrive HCV assay through a case–control study comparing results with those obtained with the Abbott RealTime HCV test.ResultsThe PoC assay identified all major HCV genotypes, with a limit of detection of 2362 IU/mL (95% CI 1966 to 2788). Using 422 patients chronically infected with HCV and 503 controls negative for anti-HCV and HCV RNA, the Genedrive HCV assay showed 98.6% sensitivity (95% CI 96.9% to 99.5%) and 100% specificity (95% CI 99.3% to 100%) to detect HCV. In addition, melting peak ratiometric analysis demonstrated proof-of-principle for semiquantification of HCV. The test was further validated in a real clinical setting in a resource-limited country.ConclusionWe report a rapid, simple, portable and accurate PoC molecular test for HCV, with sensitivity and specificity that fulfils the recent FIND/WHO Target Product Profile for HCV decentralised testing in low-income and middle-income countries. This Genedrive HCV assay may positively impact the continuum of HCV care from screening to cure by supporting real-time treatment decisions.Trial registration numberNCT02992184.

Hepatitis C virus (HCV)-specific CD8+ T cells suffer a progressive exhaustion during persistent infection (PI) with HCV. This process could involve the positive immune checkpoint 4-1BB/4-1BBL through the loss of its signal transducer, TRAF1. To address this issue, peripheral HCV-specific CD8+ T cells (pentamer-positive [pentamer+]/CD8+ T cells) from patients with PI and resolved infection (RI) after treatment were studied. The duration of HCV infection and the liver fibrosis progression rate inversely correlated with the likelihood of detection of peripheral pentamer+/CD8+ cells. In PI, pentamer+/CD8+ cells had impaired antigen-specific reactivity that worsened when these cells were not detectable ex vivo. Short/midduration PI was characterized by detectable peripheral PD-1+ CD127low TRAF1low cells. After triggering of T cell receptors (TCR), the TRAF1 level positively correlated with the levels of CD127, Mcl-1, and CD107a expression and proliferation intensity but negatively with PD-1 expression, linking TRAF1low to exhaustion. In vitro treatment with interleukin-7 (IL-7) upregulated TRAF1 expression, while treatment with transforming growth factor-β1 (TGF-β1) did the opposite, suggesting that the IL-7/TGF-β1 balance, besides TCR stimulation, could be involved in TRAF1 regulation. In fact, the serum TGF-β1 concentration was higher in patients with PI than in patients with RI, and it negatively correlated with TRAF1 expression. In line with IL-7 increasing the level of TRAF1 expression, IL-7 plus 4-1BBL treatment in vitro enhanced T cell reactivity in patients with short/midduration infection. However, in patients with long-lasting PI, anti-PD-L1, in addition to the combination of IL-7 and 4-1BBL, was necessary to reestablish T cell proliferation in individuals with slowly progressing liver fibrosis (slow fibrosers) but had no effect in rapid fibrosers. In conclusion, a peripheral hyporeactive TRAF1low HCV-specific CD8+ T cell response, restorable by IL-7 plus 4-1BBL treatment, characterizes short/midduration PI. In long-lasting disease, HCV-specific CD8+ T cells are rarely detectable ex vivo, but treatment with IL-7, 4-1BBL, and anti-PD-L1 recovers their reactivity in vitro in slow fibrosers.

El antigeno de superficie del virus de la hepatitis B (HBsAg), descubierto en 1965 por Blumberg, es el marcador fundamental para el diagnostico de la infeccion por el virus de la hepatitis B (VHB). La sintesis del HBsAg es muy compleja, se realiza en el hepatocito por mas de una via y su produccion excede lo que es necesario para el ensamblaje de los viriones1. El HBsAg puede encontrarse en el plasma formando parte de estructuras filamentosas o esfericas sin capacidad infecciosa que pueden formar inmunocomplejos circulantes con antiHBs. La cuantificacion del HBsAg incluye las 3 formas (viriones, esferas y filamentos)2, y el interes en su determinacion surgio hace unos anos, cuando algunos estudios lo asociaron a los niveles intrahepaticos de cccDNA. Aunque los primeros intentos de cuantificacion se realizaron en los anos ochenta, no ha sido hasta la ultima decada cuando se ha dispuesto de tecnicas reproducibles y estandarizadas para la cuantificacion del HBsAg. En la actualidad hay 2 pruebas comerciales, denominadas Architect QT assay® (Abbott Diagnostic) y Elecsys HBsAg II Quant assay® (Roche

A total of 1,220 subjects from Equatorial Guinea living in Spain (median age = 41 years; 453 male and 767 female) was examined for antibodies to human immunodeficiency virus (HIV) and Hepatitis B (HBV), C (HCV), and D (HDV) viruses. Extracted RNA and DNA from the positive samples were used to quantify viral load. The prevalence of HIV antibodies, HCV RNA, and HBV surface antigen (HBsAg) was 10.8% (N = 132), 11.6% (N = 141), and 7.9% (N = 96), respectively. The most prevalent HIV variant was CRF02_AG (38.5%; N = 40). HCV genotype 4 (60%; N = 36) and HBV genotype A3 (32%; N = 8) were the hepatitis variants most frequently found. Superinfection with HDV was seen in 20.9% (N = 24) of HBsAg carriers. A control group of 276 immigrants from other sub-Saharan countries showed similar rates of HIV and HBsAg, although no HCV cases were found. Immigrants constitute a major source of HIV and hepatitis viruses in Spain; therefore, it is important that control measures are intensified.

Summary. The efficacy of current hepatitis C therapy in HIV/HCV-coinfected patients is largely dependent on HCV genotype. The annual prevalence of HCV genotypes/subtypes and their influence on HCV clearance with antiviral treatment were examined in a dynamic cohort of HIV/HCV-coinfected patients followed up in Madrid since 2000. Patients entered the cohort at first visit and left the cohort when HCV clearance was achieved with HCV therapy or when follow-up was interrupted for any reason, including death. A total of 672 HIV/HCV-coinfected patients constituted the cohort. The mean follow-up time was 5.5 years, corresponding to 4108 patient-years. Mean age at entry was 37 years, and 73% were men and 86% were intravenous drug users. Overall distribution of HCV genotypes was as follows: 57.1% HCV-1 (1a: 29.2%, 1b: 20.4%, unknown: 7.6%), 1.3% HCV-2, 25.4% HCV-3 and 15.9% HCV-4. A total of 274 (40.8%) patients were treated with peginterferon–ribavirin, of whom 116 (42.3%) achieved HCV clearance following 1–3 courses of therapy. The proportion of HCV-1/4 rose from 71.7% in 2000 to 76.8% in 2008, whereas the proportion of HCV-2/3 fell from 28.1% in 2000 to 23.2% in 2008. The yearly prevalence increased for HCV-1 (R2: 0.92, b: 0.59, P

The equilibrative nucleoside transporter 1 (ENT1) is the main protein involved in ribavirin cellular uptake. Polymorphisms at the ENT1 gene may influence ribavirin activity as part of hepatitis C virus (HCV) therapy. A retrospective study was conducted in 109 human immunodeficiency virus (HIV)-infected patients who were infected with HCV genotypes 1 or 4 who had received pegylated interferon (pegIFN)-ribavirin. Single nucleotide polymorphisms (SNPs) at the ENT1 gene were examined using TaqMan 5'-nuclease assays. In the study population, allelic frequencies at rs760370 were as follows: A3 (43 [39%] of 109 patients), AG (50 [46%] of 109 patients), and GG (16 [15%] of 109 patients). Achievement of rapid virological response was more frequent in GG carriers than in AA/AG carriers (50% vs 17%, respectively; P = .007). In multivariate analysis, the GG genotype (odds ratio [OR], 15.9; 95% confidence interval [CI], 2.8-92.2; P.002), a baseline serum HCV-RNA level600,000 IU/mL (OR, 45.7; 95% CI, 8.7-240.5; P.001) and a serum ribavirin trough concentration2.5 μg/mL (OR, 4.8; 95% CI, 1.3-17.1; P.016) were associated with rapid virological response. When 2 or more of these factors were present, positive and negative predictive values of rapid virological response were 65% and 91%, respectively. In summary, a SNP rs760370A→G at the ENT1 gene influences the chance of rapid virological response to pegIFN-ribavirin therapy in HIV-infected patients with chronic HCV infection due to HCV genotypes 1 or 4, most likely modulating intracellular ribavirin exposure within hepatocytes.

Background Longitudinal assessment of liver fibrosis with transient elastometry (TE) in patients with chronic viral hepatitis is becoming routine clinical practice in many clinics, as this procedure is non-invasive, easy to perform and relatively inexpensive, allowing early detection of cirrhosis. Herein, we examine the incidence of cirrhosis, using TE assessment, in HIV-infected individuals with chronic hepatitis B or C receiving highly active anti-retroviral therapy (HAART). Methods A longitudinal study was performed on a cohort of HIV-infected patients with chronic hepatitis B or C who were followed since 2004 at Hospital Carlos III (Madrid, Spain) with periodic TE assessments. The primary outcome was the development of cirrhosis, defined as liver stiffness >12.5 KPa. Results A total of 508 HIV-infected patients were examined, of whom 54 developed liver cirrhosis during a mean ±sd follow-up of 2.6 ±1.0 years (overall incidence was 41.13 cases per 1,000 person-years). The risk of developing cirrhosis was significantly higher in 297 HCV-RNA-positive patients (either untreated or non- responders to hepatitis C therapy) compared with 55 patients who had cleared HCV with therapy (odds ratio 3.73, 95% confidence interval 1.06–13.17; P=0.04). By contrast, the risk of developing cirrhosis was low and similar in 24 HIV–HBV-coinfected patients under long-term suppressive HBV therapy (mainly tenofovir disoproxil fumarate), 132 HIV-infected patients without chronic liver disease and those who had cleared HCV with therapy. Conclusions Development of liver cirrhosis in HIV- infected individuals in the HAART era is mainly associated with active HCV coinfection. The risk of developing cirrhosis is negligible in patients who cleared HCV with therapy, as well as in HIV–HBV-coinfected patients on long-term suppressive tenofovir disoproxil fumarate therapy.

Background Direct acting antivirals (DAA) targeting the HCV serine protease and RNA polymerase have recently entered clinical development. Information about primary resistance to these compounds in HIV–HCV-coinfected patients is scarce. Methods All individuals newly diagnosed with HIV-1 at several clinics in Madrid between 2000 and 2010 were tested for serum HCV antibody and HCV RNA. The NS3 protease and NS5B polymerase genes were sequenced in all HCV viraemic patients with genotype 1 (G1). Results From 1,684 individuals newly diagnosed with HIV-1 during the 10-year study period, 141 (8.4%) were positive for serum HCV RNA. Overall, 58% were infected with G1, being 1a in 64.2% of them. Altogether, 62% of G1a and 30% of G1b harboured HCV drug-resistant changes, with the most common being prQ80K ( n=9), prV55A ( n=2), polC316Y/N ( n=3) and polV499A ( n=24). Although no primary resistance mutations were identified for HCV protease inhibitors or nucleoside analogues, mutations C316Y/N and V499A conferring resistance to some non-nucleoside analogues were found in 6% and 51% of G1 patients, respectively. Conclusions Natural DAA resistance-associated mutations are frequently seen in HIV–HCV-coinfected individuals. Changes polV499A and prQ80K seem to be natural polymorphisms and their effect on treatment outcomes warrants further examination. However, drug resistance testing in HCV drug-naive individuals coinfected with HIV currently does not seem to be warranted before using HCV protease inhibitors and nucleoside analogues. More information is needed for HCV non-nucleoside analogues.

Information on the rate and timing of hepatitis C virus (HCV) relapse after treatment with pegylated interferon plus ribavirin is scarce. Among 604 patients treated for chronic hepatitis C, the 386 who were human immunodeficiency virus (HIV) positive attained an end-of-treatment response less frequently and experienced relapse more often than did the 218 who were HIV negative. However, episodes of HCV relapse occurred before week 12 in most cases, regardless of HIV status.

Chronic hepatitis C virus (HCV) infection remains a global health threat with approximately 200 million carriers worldwide. Current treatment consists of the use of peginterferon (pegIFN)/ribavirin (RBV) for 24 or 48 weeks depending on HCV genotype. Serious side effects and the fact that less than half of patients infected with HCV genotypes 1 and 4 (which are the most common) accomplish sustained virological response with this medication warrant the need for novel anti-HCV therapies.Description of specifically targeted antiviral therapies for hepatitis C (STAT-C) designed to inhibit the serine protease and the RNA-dependent HCV-RNA polymerase.Review of available data reported in peer-reviewed journals and medical conferences.Early preclinical studies using these compounds produced encouraging results, but the initial enthusiasm has been hampered by toxicity issues and rapid selection of resistance. Therefore, combination therapy with a backbone of pegIFN/RBV, or perhaps in the future using several of these small molecules, preferably having distinct modes of action and resistance profiles, will be required.

HepatitisCvirus (HCV)is classified into seven major genotypesand67 subtypes. Recent studies haveshownthat inHCVgenotype 1-infected patients, response rates to regimens containingdirect-acting antivirals(DAAs)are subtype dependent. Currently available genotypingmethods have limited subtyping accuracy.Wehave evaluated theperformanceof adeep-sequencing-basedHCVsubtyping assay, developed for the 454/GS-Junior platform, incomparisonwith thoseof twocommercial assays (VersantHCVgenotype 2.0andAbbott Real-timeHCVGenotype II)andusingdirectNS5Bsequencing as a gold standard (direct sequencing), in 114 clinical specimenspreviously tested by first-generation hybridization assay (82 genotype 1 and32 with uninterpretable results). Phylogenetic analysis of deep-sequencing reads matched subtype 1 callingbypopulation Sanger sequencing (69%1b,31%1a) in81 specimensandidentified amixed-subtype infection (1b/3a/1a) inone sample. Similarly,amongthe 32previously indeterminate specimens, identical genotypeandsubtype results were obtained by directanddeep sequencing inall but four samples with dual infection. In contrast, both VersantHCVGenotype 2.0andAbbott Real-timeHCVGenotype II failed subtype 1 calling in13 (16%)samples eachandwere unable to identify theHCVgenotype and/or subtype inmore thanhalfof the nongenotype 1 samples.Weconcluded that deep sequencing ismore efficient forHCVsubtyping than currently available methods andallows qualitative identificationofmixed infectionsandmay bemorehelpfulwith respect toinforming treatment strategies withnewDAA-containing regimens across allHCVsubtypes

To study the hepatitis B virus (HBV) and hepatitis D virus (HDV) replication interferences in patients with chronic hepatitis delta infected with different HBV genotypes.We conducted a transversal study including 68 chronic hepatitis delta (CHD) (37 HIV-positive) patients and a control group of 49 chronic hepatitis B (CHB) (22 HIV-positive) patients. In addition, a dynamic follow-up was performed in 16 CHD patients. In all the samples, the surface antigen of hepatitis B (HBsAg) serum titers were analyzed with the Monolisa HBsAg Ultra system (Bio-Rad), using as quantification standard a serial dilution curve of an international HBsAg standard. Serum HBV-DNA titers were analyzed using the Roche Cobas TaqMan (Roche, Barcelona, Spain), and the serum HDV-RNA using an in-house real-time qRT-PCR method, with TaqMan probes. HBV genotype was determined with the line immunoassay LiPA HBV genotyping system (Innogenetics, Ghent, Belgium). In those patients negative for LiPA assay, a nested PCR method of complete HBsAg coding region, followed by sequence analysis was applied.No differences in the HBV-DNA levels were found in CHB patients infected with different HBV genotypes. However, in CHD patients the HBV-DNA levels were lower in those infected with HBV-A than in those with HBV-D, both in HIV negative [median (IQR): 1.25 (1.00-1.35) vs 2.95 (2.07-3.93) log10 (copies/mL), P = 0.013] and HIV positive patients [2.63 (1.24-2.69) vs 7.25 (4.61-7.55) log10 (copies/mL), P0.001]. This was confirmed in the dynamic study of the HBV/HDV patients. These differences induce an under-estimation of HBV-A incidence in patients with CHD analyzed with LiPA assay. Finally, the HBsAg titers reflected no significant differences in CHD patients infected with HBV-A or D.Viral replication interference between HBV and HDV is HBV-genotype dependent, and more evident in patients infected with HBV-genotype A, than with HBV-D or E.

[Background & Aims]: Chronic hepatitis C is a leading cause of chronic liver disease, cirrhosis and hepatocellular carcinoma. DNA methylation and histone covalent modifications constitute crucial mechanisms of genomic instability in human disease, including liver fibrosis and hepatocellular carcinoma. The present work studies the consequences of HCV-induced histone modifications in early stages of infection. [Methods]: Human primary hepatocytes and HuH7.5 cells were transiently transfected with the core protein of hepatitis C virus (HCV) genotypes 1a, 1b, and 2a. Infectious genotype 2a HCV in culture was also used. [Results]: We show that HCV and core protein inhibit the phosphorylation of Serine 10 in histone 3. The inhibition is due to the direct interaction between HCV core and Aurora B kinase (AURKB) that results in a decrease of AURKB activity. HCV and core significantly downregulate NF-κB and COX-2 transcription, two proteins with anti-apoptotic and proliferative effects implicated in the control of the inflammatory response. AURKB depletion reduced HCV and core repression of NF-κB and COX-2 gene transcription and AURKB overexpression reversed the viral effect. AURKB abrogation increased HCV specific infectivity which was decreased when AURKB was overexpressed. [Conclusions]: The core-mediated decrease of AURKB activity may play a role in the inflammatory pathway during the initial steps of viral infection, while ensuring HCV infectivity., This work was supported by grants BFU2009-11071 and BFU2011-23604 from Ministerio de Ciencia e Innovación, FIS: PI12/02146 and CIBERehd (Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas) from Instituto de Salud Carlos III, Epistem Ltd and Fundación Ramón Areces. Dr Julie Sheldon was supported by a Juan de la Cierva contract from CSIC and Irene Francisco by a fellowship from Spanish Cancer Association (AECC) and Postgraduate studies from the Universidad Autónoma de Madrid.

All Rights Reserved. HepatitisCvirus(HCV)is classified into seven major genotypesand67 subtypes. Recent studies haveshownthat inHCVgenotype 1-infected patients, response rates to regimens containingdirect-acting antivirals(DAAs)are subtype dependent. Currently available genotypingmethods have limited subtyping accuracy.Wehave evaluated theperformanceof adeep-sequencing-basedHCVsubtyping assay, developed for the 454/GS-Junior platform, in comparisonwith thoseof two commercial assays (VersantHCVgenotype 2.0andAbbott Real-timeHCVGenotype II)andusingdirectNS5Bsequencing as a gold standard (direct sequencing), in 114 clinical specimenspreviously tested by first-generation hybridization assay (82 genotype 1and32 with uninterpretable results). Phylogenetic analysis of deep-sequencing reads matched subtype 1 callingbypopulation Sanger sequencing(69%1b,31%1a) in 81 specimensandidentified amixed-subtype infection (1b/3a/1a) in one sample. Similarly,amongthe 32previously indeterminate specimens, identical genotypeandsubtype results were obtained by directanddeep sequencing in all but four samples with dual infection. In contrast, both VersantHCVGenotype 2.0andAbbott Real-timeHCVGenotype II failed subtype 1 calling in 13 (16%) samples eachandwere unable to identify theHCVgenotype and/or subtype inmore than half of the nongenotype 1 samples.Weconcluded that deep sequencing ismore efficient forHCVsubtyping than currently available methodsandallows qualitative identificationofmixed infectionsandmay bemorehelpfulwith respect to informing treatment strategies withnewDAA-containing regimens across allHCVsubtypes., This study has been supported by CDTI (Centro para el Desarrollo Tecnológico Industrial), Spanish Ministry of Economics and Competitiveness (MINECO), IDI-20110115; MINECO projects SAF 2009-10403; and also by the Spanish Ministry of Health, Instituto de Salud Carlos III (FIS) projects PI10/01505, PI12/01893, and PI13/00456. CIBERehd is funded by the Instituto de Salud Carlos III, Madrid, Spain. Work at CBMSO was supported by grant MINECO-BFU2011-23604, FIPSE, and Fundación Ramón Areces.

Background/Aims: There are no data about the influence of handling conditions of liver biopsies on the integrity of viral RNAs. We studied the influence of the time delay between obtaining and freezing the liver biopsy on the stability of intrahepatic positive and negative hepatitis C virus RNA (HCV-RNA) strands. Methods: Liver samples from 30 anti-HCV patients were included. For each case, one portion of the liver biopsy (first sample) was immediately frozen (20–28 s), while the other section (second sample) was kept at room temperature (1–30 min) before freezing. Each experimental time point was performed in triplicate using liver samples from three different patients. Semi-quantitative analysis of the positive and negative HCV-RNA strands and of the α1-antitrypsin mRNA was performed by a Tth-based reverse-transcription polymerase chain reaction. Results: A significant time-related decrease in both positive ( r =−0.8412, p =0.001) and negative ( r = −0.8539, p =0.001) HCV-RNA strand titres was found in the second liver fractions. There were no appreciable changes in RNA titres in those samples frozen after less than 3 min. The RNA titres decreased in all but two samples incubated for 4–30 min. Thus, 3/15 (20%) and 7/11 (64%) of these samples lost positive and negative HCV-RNA strands, respectively. Alpha-1-antitrypsin mRNA titres decreased significantly ( r =−0.8935, p =0.01) in those samples kept at room temperature for more than 4 min. Conclusion: Freezing of liver samples immediately after extraction is crucial to avoid false negative HCV-RNA detection results, especially for the anti-genomic RNA strand.

Background/Aims: In this study, the inhibition of hepatitis delta virus replication mediated by trans-ribozyme and antisense probes, alone or in combination with recombinant interferon alpha-2a, has been assayed. Methods: A 60-nucleotide-long designed trans-ribozyme, which contains the catalytic core of the hammerhead ribozyme, and a 163-nucleotide-long antisense probe were directed against the same region of the viral genome in in vitro and cell culture systems. Results: The ribozyme activity, assayed in a chemically isolated system, resulted in the trans-cleavage of 10–20% of the 40-nucleotide-long RNA substrate. A 5-nucleotide deletion in one of the flanking arms, obtained by random mutagenesis, resulted in enhancement of the trans-cleavage activity in as many as 40–60% of the substrate molecules. The efficiency of the optimized trans-ribozyme and antisense probes against the complete viral genome was assayed in a cell culture system. The inhibitory efficacy (25%) of the trans-ribozyme is lower than that of the antisense probe (35%) or interferon at 1000 U/ml (47%). An enhancement of the interferon efficacy was achieved when it was administered in cells having a previous basal expression of ribozyme (70%) or antisense probes (83%). Conclusions: These results suggest that the combination of ribozyme or antisense probes with interferon could be a promising approach to the treatment of RNA virus infections.

Long-term effects after blood or bone marrow transplantation (BMT) are emerging as an important issue, as more patients are included in BMT programmes and as this procedure becomes more successful. Long-term liver dysfunction, mainly due to chronic graft-versus-host disease or hepatitis C virus infection, is a well-known complication. Nevertheless, the diagnosis of liver disease in this patient group is sometimes difficult and, despite adequate studies, it may remain undetected. A novel hepatitis-associated virus, hepatitis G virus (HGV), has recently been identified. The virus belongs to the Flaviviridae family and is known to be parenterally transmitted, although there is no clear evidence to implicate this agent in causing acute or chronic hepatitis. We report a patient who developed mild, but persistent, abnormalities in transaminases for 2 years after an autologous BMT. HGV RNA was detected in both serum and liver. HGV RNA persisted in serum for at least 8 months. No other known hepatitis virus was found. This report provides the first direct evidence of a patient with long-term liver abnormalities after a BMT in whom the only known hepatitis virus isolated was the HGV.

Background Soluble CD36 (sCD36) clusters with insulin resistance, but no evidence exists on its relationship with hepatic fat content. We determined sCD36 to assess its link to steatosis in nonalcoholic fatty liver disease (NAFLD) and chronic hepatitis C (CHC) patients. Materials and methods Two hundred and twenty-seven NAFLD, eighty-seven CHC, and eighty-five patients with histologically normal liver (NL) were studied. Steatosis was graded by Kleiner's histological scoring system. Serum sCD36 and hepatic CD36 expression was assessed by immunoassay and immunohistochemistry, respectively. Results In NAFLD, serum sCD36 levels were significantly higher in simple steatosis than in NL (361·4 ± 286·4 vs. 173·9 ± 137·4 pg/mL, respectively; P

We examined the efficacy of decreasing high doses (beginning at 18 MU/day) of interferon-α2a vs. that of daily low doses (3 MU) in the treatment of chronic hepatitis delta virus infection. Patients treated with 18 MU had a somewhat higher frequency of normalization of serum ALT levels than patients treated with low doses (31% and 12%, respectively, on an intention-to-treat basis). A decrease in the percentage of hepatitis D virus RNA positivity was observed in both groups at the end of treatment. Thus, whereas in baseline samples 10 (62%) of the patients in each group were positive for hepatitis D virus RNA in serum on slot-blot hybridization, these numbers decreased to 5 (31%) and 4 (25%) patients in groups 1 and 2, respectively, at the end of therapy. However, hepatitis D virus RNA, detected by means of nested polymerase chain reaction, remained in all but two (one in each group) patients who completed the treatment. Finally, during posttreatment follow-up, hepatitis D virus RNA levels returned to baseline values, and only one patient remained negative for this marker. The beneficial effect of inter-feron-a was only transient. Only two patients (one from each treatment group) had persistently normal serum ALT levels after 18 mo of follow-up. Finally, the presence of serum hepatitis D virus RNA at the end of therapy, detected with nested polymerase chain reaction, might be a good marker for the prediction of viral replication relapse. (HEPATOLOGY 1994;19:1331–1336.)

The use of genetic amplification of the hepatitis delta virus (HDV) genome reveals the existence of different HDV replicative behaviours during the natural history of chronic HDV infection. While some of the patients (8/19, 42%) presented high and long-term maintained levels of HDV replication, as detected by slot-blot hybridization, others showed fluctuations from positive to negative, and in 5/7 (71%) polymerase chain reaction (PCR) demonstrated the presence of the HDV genome. Finally, 4 patients were persistently slot-blot-negative and in 3 of them HDV-RNA was detected by PCR in all samples tested. The correlation observed between the low levels of HDV replication and the ALT values, as well as the reactivation observed in one of the patients, suggests that PCR is useful in the virological surveillance of HDV infection, and indicates its usefulness in evaluating the effectiveness of antiviral therapy for chronic hepatitis D.

Background: Universal vaccination and antiviral therapy have reduced chronic hepatitis B virus (HBV) in natives in the Western world. However, immigration from high HBV endemic areas continues to maintain a relatively stable prevalence of chronic hepatitis B in most developed countries. Methods: All foreigners attending a referral infectious diseases department in Madrid, Spain, from January 2007 to December 2008, were evaluated for serum HBV surface antigen (HBsAg). Positive cases underwent further virological characterization. Results: A total of 1718 foreigners were examined, of whom 1322 (77%) were sub-Saharan Africans. Serum HBsAg was positive in 121 (7%), HIV in 135 (7.9%) and hepatitis C virus antibodies in 212 (12.3%). HBV subgenotype A3, which so far had only been reported in people originating from Cameroon, was found in nearly half (14/29) of the tested specimens with detectable serum HBV-DNA. Interestingly, the lamivudine resistance mutation rtM204V was found in two Africans (6.9%), one infected with HBV-A3 and the other with HBV-E. Lack of prior exposure to antiviral therapy in these two patients was confirmed retrospectively. Conclusions: Circulation of uncommon HBV variants, including strains with primary drug resistance, may follow large immigrant flows from HBV endemic regions to Western countries. Close surveillance of this population is warranted, as early diagnosis and early antiviral therapy may reduce transmission and prevent clinical complications.

Occult hepatitis B virus (HBV) infection (OBI) is defined as the presence of HBV DNA in the liver (with or without detectable HBV DNA in serum) for individuals testing HBV surface antigen negative. Until recently, the clinical effect of OBI was unclear on the progression of liver disease; on the development of hepatocellular carcinoma; and on the risk for reactivation or transmission of HBV infection. Several studies suggest a high prevalence of OBI among patients with cryptogenic chronic liver disease, but its role in the progression to cirrhosis remains unclear. Although OBI has been well documented in human immunodeficiency virus (HIV)-positive patients, especially among those coinfected with hepatitis C virus, further studies are needed to determine its current clinical impact in HIV setting.

The influence of ribavirin trough concentrations (RBVCtrough) on the risk of hepatitis C virus (HCV) relapse was retrospectively analyzed in 99 HIV-HCV-coinfected patients who achieved end-of-treatment response with pegylated alpha interferon plus weight-based RBV. The independent predictors (odds ratio [OR] [95% confidence interval (CI)]) of HCV relapse were RBV plasmaCtroughof Ctroughmay permit early adjustment of RBV dosage to avoid HCV relapse.

From 1519 newly diagnosed HIV individuals seen in Madrid between the years 2000 and 2008, 65 (4.3%) were HBsAg+. Two HIV/HBV-coinfected patients showed the lamivudine resistance mutation M204V in HBV while no drug resistance mutations were recognized in HIV. None of them admitted prior exposure to antiretroviral drugs. Thus, HIV/HBV-coinfected patients might benefit from baseline drug resistance testing for both HIV and HBV to optimize the selection of anti-HBV active antiviral therapy.

Sir, Hepatitis B virus (HBV) infection is a major health problem worldwide. The World Health Organization estimates that more than 400 million people are affected. Chronic hepatitis B frequently leads to end-stage liver disease and is the leading cause of liver cancer worldwide. Current HBV therapies are inadequate to eradicate chronic infection. However, anti-HBV drugs may ameliorate liver disease progression in the short term (with normalization of transaminases, reduction in serum HBV-DNA levels and/or HBeAg seroreversion) and in the long term (with prevention of liver cirrhosis and/or hepatocellular carcinoma). After many years of lamivudine being the only oral drug to treat chronic hepatitis B, marketing of adefovir was very welcome. Moreover, this nucleotide analogue was shown to be also effective in lamivudine-experienced patients, with a relatively low incidence of resistance, at least within the first 2 years of therapy but increasing to � 30% at 5 years. 1 Suboptimal response to adefovir, and subsequent selection of resistance mutations, has been associated with pharmacological issues, mainly derived from the low dose (10 mg daily) approved in an attempt to minimize renal toxicity. Adefovir resistance has been classically linked to a single mutation (rtN236T) within the D domain of the HBV polymerase gene. 2 A number of other mutations, such as V84M and S85A (A domain), A181T/V (B

In this paper, we studied the usefulness of polymer ase chain reaction (PCR) in HDV-RNA detection. Using serial dilutions of serum samples of known concentrations of HDV-RNA, PCR was 10 000-times more sensitive than slot-blot hybridization. PCR was used for the detection of HDV-RNA in 33 serum samples negative to HDV-RNA by conventional slot-blot hybridization. HDV-RNA was detected in 1833 (54%) of the samples included in this study using PCR. When positivity to a viral genome was related to other viral replication markers, it was found that among the 18 patients positive to the viral genome, 13 (72%) had hepatitis delta antigen in the liver, and five (28%) were negative. In conclusion, HDV-RNA detection by gene amplification is 10 000-times more sensitive than slot-blot hybridization, and allows the detection of viral replication in patients without ether viral replication markers.