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Following the COVID-19 pandemic, policies such as social distancing, hand washing and the use of masks were implemented which could play an important role in the reduction of infectious diseases. An observational, descriptive, cross-sectional study was conducted to observe the prevalence of respiratory infections in children under 15 years of age during the 2018-2020 period in Primary Care centres in Central Catalonia. In 2020, there was a 44.35 decrease in total consultations for respiratory infections compared to 2019. All respiratory infections exhibited a significant decrease except influenza, which increased by 41.76%. Children between the ages of 6 and 12 had the highest prevalence of influenza (87.68%), and the SARS-CoV-2-19 infection was most frequent (4.06%) among those between the ages of 12 and 15. Compared to urban centres, rural centres presented a higher prevalence of all infections except influenza and SARS-CoV-2. In conclusion, the COVID-19 pandemic caused a significant decrease in the number of consultations for respiratory infections in the paediatric population, except for influenza, which increased in cases in January and February 2020. No differences were found between sexes, although differences were found in the distribution of the different age groups.

Aim To define the seroprevalence of antibodies against SARS-CoV-2 in the municipality of Vilanova del Camí (in the sub-region of Conca d'Ódena) and to know the risk factors associated with positive seroprevalence.Method Cross-sectional descriptive study. The population of Vilanova del Camí had the opportunity to voluntarily attend two screenings for antibodies against SARS-CoV-2 (October and December 2020). Users who participated in the screening signed an informed consent form. From the 3610 attendees at the screening 2,170 patients were randomly selected. The test results and other demographic variables (sex, age, morbidity index) and users’ clinical variables (diagnoses, smoking and drugs) were analysed. Results The prevalence of antibodies against COVID19 was 9.6% (95% CI 8.4% to 10.9%) and was similar for men and women but increased with age. In complex chronic patients, the prevalence was 14.3% and in patients with advanced chronic disease, 25%. Age, AMG (Adjusted Morbidity Groups) index, COVID diagnosis and COVID contact diagnosis were risk factors for positive seroprevalence. The October analysis detected a higher seroprevalence (12.16%) than in the December analysis (8.38%). In the December screening, obesity was a risk factor for positive seroprevalence. Conclusions This study demonstrates the high seroprevalence of antibodies in the pandemic epicentre of Catalonia. The differences in the analysis of October and December show the impact of the first wave in the Conca d'Ódena sub-region.

We report on the phase behavior of a technical grade and commercially available diglycerol monoisostearate, C41V, and its use for the preparation of nanostructured liquid crystal dispersions (hexosomes). C41V in water forms a reverse hexagonal liquid crystal at room temperature and in a wide range of concentrations (0.5–95 wt%); this hexagonal liquid crystal is stable up to 70 °C. A simple and effective method has been developed to disperse hexosomes with an encapsulated active molecule (Ketoprofen) that consists of (1) producing a nano-emulsion stabilized by an amphiphilic block copolymer (Pluronic F127) and containing ethyl acetate and C41V by using ultrasounds and (2) evaporating the solvent to produce hexosomes. The size of the hexosomes and ultrasound dispersion time is markedly reduced by using ethyl acetate as an auxiliary solvent with an optimal initial ratio of C41V:ethyl acetate of 50:50. Dynamic light scattering shows that the size of the hexosomes decreases as the concentration of stabilizer F127 or encapsulated Ketoprofen is increased. The lattice parameter in the hexagonal structure is calculated from small angle scattering data to be ca. 5.3 nm and is only slightly dependent on the amount of F127 and/or encapsulated Ketoprofen. Cryo electron microscopy reveals that the samples contain hexosomes and these coexist with spherical, likely F127 micelles. Lastly, hexosomes show a pH responsive release of Ketoprofen which could be useful for target delivery in the gastrointestinal tract. © 2019 Elsevier Inc., Financial support from the Ministry of Science, Innovation and Universities (grant CTQ2017-84998-P ) and Spanish National Research Council (CSIC) (grant I-LINK 1188) is gratefully acknowledged. Synchrotron SAXS experiments were performed at BL11-NCD beamline at ALBA Synchrotron Light Facility with the collaboration of ALBA staff. DLS measurements have been performed at the Nanostructured Liquid Characterization Unit, located at the Institute of Advanced Chemistry of Catalonia (IQAC), belonging to the Spanish National Research Council (CSIC) and affiliated to the NANBIOSIS ICTS of the Biomedical Networking Research Center (CIBER-BBN). Appendix A

The classical emulsion droplet models found in the literature are based on a Volume/Area ratio, which gives the minimum droplet size of a monodisperse system at a fixed composition. These models have been revised and summarized into a general equation. Additionally, an alternative model based on a Volume/Volume ratio has been introduced which shows a non-linear behavior between droplet size and composition. At the micellar domain, the introduced droplet model shows a different behavior from the classical approach and also when combined with the flexible surface model or with the Israelachvili packing equation. To understand the differences between models, the packing of surfactant molecules at the droplet interface has been studied as a function of composition. A spherical packing parameter (Pagg) has been defined which, as a difference with Israelachvili´s concept, includes the penetration of oil and water molecules into the surfactant palisade layer. It has been described a semi-empirical method to estimate Pagg in monodisperse droplet like systems from simple size measurements. On the other hand, an equation showing the relation between Pagg and droplet composition has been deduced and applied on O/W discrete cubic liquid crystalline phases, microemulsion and nano-emulsion systems formed in selected Water / Polyethoxylated alcohol / Oil systems.

This study concerns the effect of polymer content on the properties of nano-emulsions formulated by the PIC low-energy method and on the nanoparticles prepared from nano-emulsions by solvent evaporation. The region of nano-emulsion formation, droplet size and stability as a function of polymer content were investigated. Nano-emulsions were formed in a wide range of oil-to-surfactant weight ratios (Ros) and water contents higher than 45%. For nanoparticles, droplet size, stability, encapsulation efficiency, pH, drug release and cytotoxicity effect were evaluated as a function of polymer content. Nano-emulsion droplet size (Rh = 8–75 nm) and nanoparticle size (Rh = 8–55 nm) increase with the polymer content. No desestabilization process such as creaming or sedimentation was observed by light transmittance/backscattering neither in nano-emulsions nor in nanoparticle dispersions. The morphology and size of nano-emulsions and nanoparticles were comfirmed by Cryo-TEM and TEM techniques, respectively. The pH of nanoparticle dispersions was in the range of 5–6.5 during the studied experimental time (7days), an indication of no polymer degradation. Moreover, Dexamethasone (DXM) encapsulated in the polymeric nanoparticles (0.18 wt% DXM in the oil phase of the template nano-emulsion) did not modify the nanoparticle size. No effect of polymer concentration was found in the encapsulation efficiency (close to 90%) of DXM. In vitro drug release studies indicated Fick’s diffusion as the main drug release mechanism from the nanoparticle to the receptor solution. DXM loaded and non-loaded nanoparticles showed cell viability higher than 70%. This study reports a promising system to be used as template for polymeric nanoparticle preparation suitable for biomedical applications.

Aim To determine the capacity of next-generation sequencing (NGS) for quantifying edited and unedited HDV populations, and to confirm if edition is a general phenomenon taking place along the entire HDV region analyzed, as we previously reported (Homs M et al. PLoS One 2016, 11, e0158557). Methods Four serum samples from 4 patients with chronic HDV/HBV infection were included in the study. The region selected for analysis covered 360 nucleotides (nt), positions 910–1270 of the HDV genome, which included the HDAg ORF editing site (nt 1014 within codon 196). Quantification of edited and unedited genomes was performed by molecular cloning and Sanger sequencing and by NGS. To evaluate the reliability of the NGS values obtained, we combined a clone with an edited codon and one with an unedited codon in known percentages in a series of artificial mixtures, which were then analyzed by NGS. In addition, we determined the nt changes occurring over the complete amplified region after excluding the editing codon (196) to evaluate edition along it. Results In total, 11,208 quality-filtered sequences were obtained in the 4 samples. The 95% confidence intervals for the proportions of unedited populations by molecular cloning and NGS were overlapping, and those of cloning were wider, indicating that they are comparable and that NGS is more precise than cloning. Unedited genomes predominated over edited ones in all 4 samples analyzed by NGS and in 3 of the 4 samples analyzed by molecular cloning. In total, 83,276 quality-filtered sequences were obtained from the artificial mixtures. Percentages of the two viral populations detected by NGS in these mixtures were comparable to the expected percentages. Evaluation of edition along the HDV coding region showed that transitions were more frequent than transversions, accounting for 63.09% and 36.91%, respectively. Interestingly, among the 4 possible transition-type changes, G:A and A:G accounted for 73.86% of the total. Conclusion Next-generation sequencing proved useful to quantify edited and unedited HDV genomes, and provided relevant information on the HDV quasispecies.

To investigate whether hepatitis B surface antigen (HBsAg) and hepatitis B core-related antigen (HBcrAg) levels are useful to identify inactive carriers among HBeAg-negative patients infected by different hepatitis B virus (HBV) genotypes.In all, 202 consecutive HBeAg-negative patients with chronic hepatitis B, 135 inactive carriers and 67 with HBV activity, were prospectively followed for 1 year.In HBeAg-negative patients, HBsAg levels differed across the different genotypes (p0.001). The highest levels were observed in genotypes F or H (4.2 ± 0.6 logIU/mL), followed by genotype E (3.4 ± 1.1 logIU/mL), genotype A (3.4 ± 0.8 logIU/mL), and the lowest in genotype D (2.7 ± 1.1 logIU/mL). Variations in HBsAg levels were similar in inactive carriers and patients with HBV activity. HBsAg3 logIU/mL showed good performance for identifying genotype D inactive carriers: 76% of genotype D inactive carriers met this cut-off versus ≤31% for genotypes A, E, F or H. However, in patients with genotype A, HBsAg levels ≤3.7 logIU/mL better classified inactive carriers. The combination of a single measurement of HBcrAg ≤3 logU/mL plus HBV DNA ≤2000 IU/mL yielded a positive predictive value and diagnostic accuracy85% in all HBV genotypes, except genotype H or F, with values of 62.5% and 72.7%, respectively, for the two parameters.HBsAg levels varied across genotypes in HBeAg-negative patients. HBsAg levels3 logIU/mL were only useful for identifying genotype D inactive carriers. A single HBcrAg measurement ≤3 logU/mL plus HBV DNA ≤2000 IU/mL was highly accurate for identifying inactive carriers, regardless of their HBV genotype.

The hepatitis viruses represent a major public health problem worldwide. Procedures for characterization of the genomic composition of their populations, accurate diagnosis, identification of multiple infections, and information on inhibitor-escape mutants for treatment decisions are needed. Deep sequencing methodologies are extremely useful for these viruses since they replicate as complex and dynamic quasispecies swarms whose complexity and mutant composition are biologically relevant traits. Population complexity is a major challenge for disease prevention and control, but also an opportunity to distinguish among related but phenotypically distinct variants that might anticipate disease progression and treatment outcome. Detailed characterization of mutant spectra should permit choosing better treatment options, given the increasing number of new antiviral inhibitors available. In the present review we briefly summarize our experience on the use of deep sequencing for the management of hepatitis virus infections, particularly for hepatitis B and C viruses, and outline some possible new applications of deep sequencing for these important human pathogens.

Virus-specific CD8 T cell response seems to play a significant role in the outcome of hepatitis delta virus (HDV) infection. However, the HDV-specific T cell epitope repertoire and mechanisms of CD8 T cell failure in HDV infection have been poorly characterized. We therefore aimed to characterize HDV-specific CD8 T cell epitopes and the impacts of viral mutations on immune escape. In this study, we predicted peptide epitopes binding the most frequent human leukocyte antigen (HLA) types and assessed their HLA binding capacities. These epitopes were characterized in HDV-infected patients by intracellular gamma interferon (IFN-γ) staining. Sequence analysis of large hepatitis delta antigen (L-HDAg) and HLA typing were performed in 104 patients. The impacts of substitutions within epitopes on the CD8 T cell response were evaluated experimentally and by in silico studies. We identified two HLA-B*27-restricted CD8 T cell epitopes within L-HDAg. These novel epitopes are located in a relatively conserved region of L-HDAg. However, we detected molecular footprints within the epitopes in HLA-B*27-positive patients with chronic HDV infections. The variant peptides were not cross-recognized in HLA-B*27-positive patients with resolved HDV infections, indicating that the substitutions represent viral escape mutations. Molecular modeling of HLA-B*27 complexes with the L-HDAg epitope and its potential viral escape mutations indicated that the structural and electrostatic properties of the bound peptides differ considerably at the T cell receptor interface, which provides a possible molecular explanation for the escape mechanism. This viral escape from the HLA-B*27-restricted CD8 T cell response correlates with a chronic outcome of hepatitis D infection. T cell failure resulting from immune escape may contribute to the high chronicity rate in HDV infection. IMPORTANCE Hepatitis delta virus (HDV) causes severe chronic hepatitis, which affects 20 million people worldwide. Only a small number of patients are able to clear the virus, possibly mediated by a virus-specific T cell response. Here, we performed a systematic screen to define CD8 epitopes and investigated the role of CD8 T cells in the outcome of hepatitis delta and how they fail to eliminate HDV. Overall the number of epitopes identified was very low compared to other hepatotropic viruses. We identified, two HLA-B*27-restricted epitopes in patients with resolved infections. In HLA-B*27-positive patients with chronic HDV infections, however, we detected escape mutations within these identified epitopes that could lead to viral evasion of immune responses. These findings support evidence showing that HLA-B*27 is important for virus-specific CD8 T cell responses, similar to other viral infections. These results have implications for the clinical prognosis of HDV infection and for vaccine development.

Recent studies have shown that antiviral treatment discontinuation is safe and associated with virologic remission in HBeAg-negative patients. However, the period of viral suppression and follow-up in these studies was relatively short.To investigate whether continuous viral suppression with tenofovir disoproxil fumarate for more than 7 years is associated with HBsAg loss and sustained response after treatment discontinuation and receiving a full course of hepatitis B vaccination.Patients with HBeAg-negative chronic HBV infection and more than 7 years of persistent viral suppression with tenofovir therapy were selected for treatment discontinuation and HBV vaccination. Follow-up with monthly ALT, HBV-DNA, and HBsAg determinations lasted 72 weeks. In patients with viral relapse, the viral quasispecies in the overlapping reverse transcriptase and small surface protein regions was analysed by ultra-deep pyrosequencing.Eight of 17 HBeAg-negative patients accepted tenofovir discontinuation: 5 patients achieved sustained response (persistent HBV-DNA levels2000IU/mL and normal ALT) despite an initial virologic relapse, one lost HBsAg, and two needed re-treatment. All patients with an on-treatment HBsAg level decline5000IU/mL achieved sustained response. Patients with HBsAg level100IU/mL during an ALT flare after antiviral discontinuation achieved sustained response. Significant changes were seen in the composition of the HBV quasispecies, and half the patients showed changes in HBV genotype.Even though the majority of patients presented an initial relapse with selection of HBV variants, most achieved sustained response. Changes in HBsAg levels on and off treatment may be useful for predicting the likelihood of virologic remission.

Aims To investigate an optimal long-term prophylactic strategy for prevention of hepatitis B virus (HBV) recurrence after liver transplantation, we conducted a randomized study of 29 transplant recipients receiving a short course of hepatitis B immune globulin (HBIg) + lamivudine (LAM), followed by randomization to long-term prophylaxis with LAM with or without HBIg. Methods The efficacy and safety, and impact on survival and HBV recurrence of these 2 prophylactic regimens were compared over a mean period of 10 years. In patients with viral recurrence, the HBV quasispecies in the surface/polymerase region were studied by ultra-deep pyrosequencing (UDPS). Results The 10-year survival rate was 76% and was not affected by the type of prophylaxis. Four patients had hepatitis B surface antigen (HBsAg) recurrence within the first 48 months after orthotopic liver transplantation (OLT). HBsAg-positive and -negative patients showed similar mean survival times, with no differences between the 2 regimens. Low HBV DNA levels were transiently detected in 32% of HBsAg-negative patients. UDPS showed major changes after OLT in the HBV quasispecies of patients with viral recurrence, which may be explained by a “bottleneck” effect of OLT together with prophylactic therapy. Conclusion Long-term survival after OLT in end-stage chronic hepatitis B patients was good with both prophylactic strategies. However, low, transient HBV DNA levels were detected even in the absence of HBsAg, showing the importance of continuing HBV prophylaxis.

Co-infection with hepatitis B (HBV) and D virus (HDV) is associated with the most severe course of liver disease. Interferon represents the only treatment currently approved. However, knowledge about the impact of interferons on HDV in human hepatocytes is scant. Aim was to assess the effect of pegylated interferon alpha (peg-IFNα) and lambda (peg-IFNλ), compared to the HBV-polymerase inhibitor entecavir (ETV) on all HDV infection markers using human liver chimeric mice and novel HDV strand-specific qRT-PCR and RNA in situ hybridization assays, which enable intrahepatic detection of HDV RNA species. Peg-IFNα and peg-IFNλ reduced HDV viremia (1.4 log and 1.2 log, respectively) and serum HBsAg levels (0.9-log and 0.4-log, respectively). Intrahepatic quantification of genomic and antigenomic HDV RNAs revealed a median ratio of 22:1 in untreated mice, resembling levels determined in HBV/HDV infected patients. Both IFNs greatly reduced intrahepatic levels of genomic and antigenomic HDV RNA, increasing the amounts of HDAg- and antigenomic RNA-negative hepatocytes. ETV-mediated suppression of HBV replication (2.1-log) did not significantly affect HBsAg levels, HDV productivity and/or release. In humanized mice lacking adaptive immunity, IFNs but not ETV suppressed HDV. Viremia decrease reflected the intrahepatic reduction of all HDV markers, including the antigenomic template, suggesting that intracellular HDV clearance is achievable.

Current measures of health literacy have been criticized on a number of grounds, including use of a limited range of content, development on small and atypical patient groups, and poor psychometric characteristics. In this paper, we report the development and preliminary validation of a new computer-administered and -scored health literacy measure addressing these limitations. Items in the measure reflect a wide range of content related to health promotion and maintenance as well as care for diseases. The development process has focused on creating a measure that will be useful in both Spanish and English, while not requiring substantial time for clinician training and individual administration and scoring. The items incorporate several formats, including questions based on brief videos, which allow for the assessment of listening comprehension and the skills related to obtaining information on the Internet. In this paper, we report the interim analyses detailing the initial development and pilot testing of the items (phase 1 of the project) in groups of Spanish and English speakers. We then describe phase 2, which included a second round of testing of the items, in new groups of Spanish and English speakers, and evaluation of the new measure’s reliability and validity in relation to other measures. Data are presented that show that four scales (general health literacy, numeracy, conceptual knowledge, and listening comprehension), developed through a process of item and factor analyses, have significant relations to existing measures of health literacy., Video abstract

Objectives This study aimed to characterize the chronically infected general hepatitis C virus (HCV) population in Barcelona using a highly sensitive subtyping method that can identify the 67 recognized HCV subtypes and diagnose mixed infection by various genotypes/subtypes in a single individual. The resulting information has implications for selecting optimal direct-acting antiviral (DAA) treatment for each patient and establishing public healthcare policies in our setting. Methods Consecutive HCV patients (treatment-naive or interferon-based failures) attending Vall d'Hebron Hospital outpatient clinics from February 2015 to May 2016 ( N =1473) were included in the study. Patient samples were characterized using HCV subtyping by next-generation ultra-deep pyrosequencing. Results The following genotypes (G) were found: G1 (1126/1473 (76.4%)), G4 (145/1473 (9.8%)), G3 (135/1473 (9.2%)), G2 (51/1473 (3.5%)), and G5 (1/1473 (0.1%)). Twenty-two subtypes were seen: 1b (790/1473 (53.6%)), 1a (332/1473 (22.5%)), 3a (133/1473 (9.0%)), 4d (105/1473 (7.1%)), 4a (29/1473 (2.0%)), and 2c (25/1473 (1.7%)), with 16 low-prevalence subtypes accounting for the remaining 3.0% (44/1473). There was a worrisome 1.0% (15/1473) of mixed infections. G2 (51/1473 (3.5%)) showed a high level of heterogeneity. Analyses by age groups showed a predominance of G1b over G1a (428/506 (84.6%) vs. 24/506 (4.7%)) in patients born before 1950 ( N =506/1473), and similar percentages of these subtypes in those born between 1951 and 1975 ( N =834/1473) (315/834, 37.8% vs. 266/834, 31.9%) and after 1976 ( N =133/1473) (47/133, 35.3% vs. 42/133, 31.6%). Conclusions Subtype distribution showed a higher level of heterogeneity than was expected, particularly for G2. Prevalence of mixed infections was around 1%. HCV subtype distribution related to patient age group suggested that patients born from 1936 to 1975 in our setting should undergo screening for the infection. Next-generation sequencing enabled better classification of candidates for DAA-based treatment.

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

Chronic HDV infection can cause a severe form of viral hepatitis for which there is no specific treatment. Characterization of the hepatitis B or C viral quasispecies has provided insight into treatment failure and disease recurrence following liver transplantation, has proven useful to understand hepatitis B e antigen seroconversion, and has helped to predict whether hepatitis C infection will resolve or become chronic. It is likely that characterization of the hepatitis delta virus (HDV) quasispecies will ultimately have similar value for the management of this infection. This study sought to determine the RNA evolution rates in serum of chronic hepatitis delta (CHD) treatment-naïve patients, using next-generation sequencing methods. The region selected for study encompassed nucleotide positions 910 to 1270 of the genome and included the amber/W codon. Amber/W is a substrate of the editing process by the ADAR1 host enzyme and is essential for encoding the 2 delta antigens (HDAg). The amber codon encodes the small (unedited) HDAg form and the W codon the large (edited) HDAg form. The evolution rate was analyzed taking into account the time elapsed between samples, the percentage of unedited and edited genomes, and the complexity of the viral population. The longitudinal studies included 29 sequential samples from CHD patients followed up for a mean of 11.5 years. In total, 121,116 sequences were analyzed. The HDV evolution rate ranged from 9.5x10-3 to 1.2x10-3 substitutions/site/year and showed a negative correlation with the time elapsed between samples (p

The specific drugs available for chronic hepatitis B infection include standard and pegylated IFN-α, and nucleoside/nucleotide analogs that directly inhibit the reverse transcriptase. The main goal of current hepatitis B virus therapy is to achieve sustained suppression of viral replication to prevent the development of chronic liver disease, but favorable long-term tolerability and resistance profiles are also desirable. This article reviews the chemistry and the mechanisms of action of tenofovir disoproxil fumarate, but it also focuses on the related clinical trails designed to date, in which clinical efficacy has been analyzed attending to HBe antigen status. In addition, studies including patients that have been previously treated with lamivudine or adefovir are discussed.

SUMMARY. Epidemiological, viral and host factors are associated with the outcome of hepatitis C virus (HCV) infection, and strong host immune responses against HCV favour viral clearance. Recently, genome-wide association studies have shown a strong correlation between single-nucleotide polymorphisms (SNPs) near the interleukin-28B (IL28B) gene and spontaneous or treatment-induced HCV clearance. We have investigated whether protective IL28B genetic variants are associated with HCV-specific T-cell responses among Spanish blood donors. The rs12979860 IL28B haplotype was determined in 69 anti-HCV-positive blood donors (21 HCV RNA negative and 48 HCV RNA positive) and 30 seronegative donors. In all cases, HCV-specific CD4 + T-cell responses to HCV recombinant proteins (core, NS3 and NS3 helicase) were assessed by ex vivo interferon-c ELISpot assay. The rs12979860-CC genotype was highly overrepresented in donors with spontaneous HCV clearance when compared to those with chronic infection (76.2% vs 29.2%, P < 0.001; odds ratio, 7.77; 95% confidence interval, 2.4–25.3, P < 0.001). HCV-specific CD4 + T-cell responses were detected in 16 (76.2%) spontaneous resolvers especially towards nonstructural proteins, but with no correlation with IL28B genotype. Chronic individuals had a significantly lower overall T-cell response again irrespective of IL28B genotype. When spontaneous resolvers and chronic individuals were stratified according to their IL28B genotype, significantly stronger T-cell responses were only observed among those with non-CC haplotypes. Although the protective rs12979860 IL28B CC genotype is associated with spontaneous HCV clearance, stronger CD4 + T-cell responses towards NS3 were only evident among those with non-CC haplotypes.

Resumen El tratamiento de la hepatitis cronica C (HCC) se basa en la combinacion de peginterferon alfa 2a o 2b y ribavirina que consiguen una respuesta virologica persistente (RVP) en mas del 50% de los casos. Esta RVP es superior en los pacientes infectados por los genotipos 2 y 3, pero los casos infectados por el genotipo 1, la RVP es inferior al 50%, lo que hace necesario el desarrollo de nuevos tratamientos. A partir de los conocimientos del ciclo replicativo del virus de la hepatitis C (VHC) y de la caracterizacion de los enzimas virales se han definido dianas terapeuticas que inhiben los enzimas del VHC. En esta revision se analizan los dos primeros inhibidores de la proteasa NS3/NS4 aprobados para el tratamiento de la HCC, boceprevir y telaprevir. Ambos consiguen incrementar la tasa de RVP, tanto en pacientes naives como en pacientes previamente tratados con la ventaja de que en ocasiones es posible acortar el tratamiento. Sin embargo, estos farmacos se asocian a nuevos efectos adversos como eritema cutaneo e incremento de la anemia.

Long-term changes in the frequency and outcome of hepatitis delta virus (HDV) infection have seldom been analysed. This retrospective, longitudinal study includes 398 consecutive hepatitis B surface antigen (HBsAg)-positive patients with anti-HDV antibodies who attended our institution between 1983 and 2008. At enrolment, 182 patients had acute and 216 chronic hepatitis. Patients were grouped into two periods. Those who attended between 1983 and 1995 and those between 1996 and 2008. The former group was significantly younger, mainly intravenous drugs users, and had a greater incidence of acute HDV and HIV and HCV coinfection. Patients with acute HBV/HDV coinfection cleared both infections in 90% of cases, while all patients with HDV superinfection evolved to chronic disease. One hundred and fifty-eight patients with chronic HDV were followed for a median period of 158months. Seventy-two per cent of the patients remained stable, 18% had hepatic decompensation, 3% developed hepatocellular carcinoma, and 8% cleared HBsAg. Liver-related death was observed in 13% of patients and mainly occurred in patients from the first period (P=0.012). These results indicate an outbreak of HDV at the end of the 1980s and the beginning of the 1990s, with a large number of acute HDV cases affecting predominately young, male intravenous drug users. Currently, patients with chronic HDV disease are older, and factors associated with worse prognosis include the presence of cirrhosis and age at the time of diagnosis.

Aliment Pharmacol Ther 2010; 32: 97–104 Summary Background Some patients continue to have detectable HBV-DNA levels with liver disease progression after hepatitis B e antigen (HBeAg) loss. It is important to identify these patients, candidates for long-term treatment. Aims To evaluate hepatitis B virus (HBV) genotype and the main mutations in the basic core promoter (BCP, A1762T/G1764A) and precore (G1896A) sequences as markers of persistent HBV-DNA after HBeAg loss. Methods We analysed 60 serum samples from 20 Caucasian, HBeAg-positive, chronic hepatitis B patients, who lost HBeAg and were followed-up longitudinally. HBV genotype and precore and BCP mutations were determined before, at the time of, and after HBeAg loss. Results After HBeAg loss, eight (40%) patients continued to have undetectable HBV-DNA and 12 (60%) had persistent HBV-DNA (median level 4.7 log10 copies/mL). The presence of BCP mutations prior to therapy was the only variable associated with persistently detectable viraemia (P = 0.017). Four patients with genotype A and no mutations in the BCP region experienced hepatitis B surface antigen (HBsAg) loss after a mean period of 35 months from baseline. Conclusions Main BCP mutations in HBeAg-positive patients are useful markers to identify patients who will not have sustained virological suppression after HBeAg loss and therapy discontinuation and could benefit from long-term treatment.

Background/Aims The frequency of mixed hepatitis B virus (HBV) genotypes in chronic HBV (CHB) and genotype changes during natural disease evolution and as a result of antiviral therapy were investigated. Methods Serum samples from 103 CHB patients were included in a cross-sectional study. Longitudinal study of HBV genotypes was performed in 22 patients, 17 of them under antiviral therapy (lamivudine and/or adefovir). HBV genotyping was done by the INNO-LiPA HBV assay. Results Genotypes observed in the cross-sectional study: A 32% of cases, D 42%, C 2%, F 2%, and mixed genotypes 22% (mainly A/D, followed by A/G). Genotype G was found in 7% of patients, always combined with other genotypes. In the longitudinal study, genotype changes were observed only in treated patients (9 cases). Genotype A strains were positively selected in 6 of them, mainly as mixed A/D. In 6 patients, selection coincided with a decrease in HBV-DNA levels. Conclusions A high frequency of mixed HBV genotypes was observed in our setting. Selection of genotype A strains during treatment is likely an indication that sensitivity to therapy differs between genotypes A and D. The absence of changes in untreated patients suggests that HBV genotype is stable without external factors.

The aqueous self-assembly behavior of the dyes Quinaldine red acetate and Pyronin Y in a wide range of concentrations is reported here for the first time. (1)H NMR spectroscopy, polarized-light optical microscopy, and small and wide X-ray scattering were used to get insight into molecular interactions, phase boundaries and aggregate structure. Quinaldine red acetate and Pyronin Y self-organize into unimolecular stacks driven by attractive aromatic interactions. At high concentrations, spatial correlation among the molecular stacks gives rise to nematic liquid crystals in both systems. Quinaldine red acetate additionally produces a rare chromonic O phase built of columnar aggregates with anisotropic cross-section ordered in a rectangular lattice. The O phase changes into a columnar lamellar structure as a result of a temperature-induced phase transition. Results open the possibility of finding chromonic liquid crystals in other commercially available dyes with a similar molecular structure. This would eventually expand the availability of these unique soft materials and thus introduce new applications for marketed dyes.

This study assesses the presence and outcome of genotype mixtures in the polymerase/surface and X/preCore regions of the HBV genome in patients with chronic hepatitis B virus (HBV) infection. Thirty samples from ten chronic hepatitis B patients were included. The polymerase/surface and X/preCore regions were analyzed by deep sequencing (UDPS) in the first available sample at diagnosis, a pre-treatment sample, and a sample while under treatment. HBV genotype was determined by phylogenesis. Quasispecies complexity was evaluated by mutation frequency and nucleotide diversity. The polymerase/surface and X/preCore regions were validated for genotyping from 113 GenBank reference sequences. UDPS yielded a median of 10,960 sequences per sample (IQR 16,645) in the polymerase/surface region and 11,595 sequences per sample (IQR 14,682) in X/preCore. Genotype mixtures were more common in X/preCore (90%) than in polymerase/surface (30%) (p

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.

Hepatitis D virus (HDV) is a defective RNA virus that requires the surface antigens of hepatitis B virus (HBV) (HBsAg) for viral assembly and replication. Several commercial and in-house techniques have been described for HDV RNA quantification, but the methodologies differ widely, making a comparison of the results between studies difficult. In this study, a full-length genomic RNA standard was developed and used for HDV quantification by two different real-time PCR approaches (fluorescence resonance energy transfer [FRET] and TaqMan probes). Three experiments were performed. First, the stability of the standard was determined by analyzing the effect of thawing and freezing. Second, because of the strong internal base pairing of the HDV genome, which leads to a rod-like structure, the effect of intense thermal shock (95°C for 10 min and immediate cooling to −80°C) was tested to confirm the importance of this treatment in the reverse transcription step. Lastly, to investigate the differences between the DNA and RNA standards, the two types were quantified in parallel with the following results: the full-length genomic RNA standard was stable and reliably mimicked the behavior of HDV-RNA-positive samples, thermal shock enhanced the sensitivity of HDV RNA quantification, and the DNA standard underquantified the HDV RNA standard. These findings indicate the importance of using complete full-length genomic RNA and a strong thermal-shock step for optimal HDV RNA quantification.

BACKGROUND Acute and chronic hepatitis E have been associated with high mortality and development of cirrhosis, particularly in solid-organ recipients and patients infected by human immunodeficiency virus. However, data regarding the epidemiology of hepatitis E in special populations is still limited. AIMS Investigate seroprevalence and possible factors associated with HEV infection in a large cohort of immunosuppressed patients. METHODS Cross-sectional study testing IgG anti-HEV in serum samples from 1373 consecutive individuals: 332 liver-transplant, 296 kidney-transplant, 6 dual organ recipients, 301 non-transplanted patients with chronic liver disease, 238 HIV-infected patients and 200 healthy controls. RESULTS IgG anti-HEV was detected in 3.5% controls, 3.7% kidney recipients, 7.4% liver transplant without cirrhosis and 32.1% patients who developed post-transplant cirrhosis (p

SUMMARY. Hepatitis delta is considered the most severe form of viral hepatitis, but variables associated with disease progression are poorly defined. This study aimed to identify risk factors associated with worse clinical outcome in patients with hepatitis delta and to develop a clinical score to determine their risk of experiencing liverrelated morbidity or mortality. We followed 75 HBsAg– anti-HDV-positive patients with hepatitis delta for up to 16 years (median 5 years). The baseline-event-anticipation score (BEA score) was developed based on variables associated with the development of liver-related clinical complications. Age, region of origin, presence of cirrhosis, albumin, INR, hyperbilirubinemia and thrombocytopenia were all associated with the development of an event in the training cohort. The BEA score included age, sex, region of origin, bilirubin, platelets and INR. Points were allocated according to hazard ratios, and three risk groups were defined: BEA-A mild risk, BEA-B moderate risk and BEA-C high risk. Hazard ratios of BEA-B and BEA-C patients for liver-related clinical endpoints were 9.01 and 25.27 vs BEA-A with an area under curve of the receiving operating characteristic curve of 0.88. The accuracy of the BEA score was confirmed in two independent validation cohorts followed in Barcelona (n = 77) and D€ (n = 62). Delta hepatitis is associated with a very severe long-term outcome. The BEA score is easy to apply and predicts with a very high accuracy the development of liver-related complications in patients with hepatitis delta.

Hepatitis B virus (HBV) is a DNA virus with complex replication, and high replication and mutation rates, leading to a heterogeneous viral population. The population is comprised of genomes that are closely related, but not identical; hence, HBV is considered a viral quasispecies. Quasispecies variability may be somewhat limited by the high degree of overlapping between the HBV coding regions, which is especially important in the P and S gene overlapping regions, but is less significant in the X and preCore/Core genes. Despite this restriction, several clinically and pathologically relevant variants have been characterized along the viral genome. Next-generation sequencing (NGS) approaches enable high-throughput analysis of thousands of clonally amplified regions and are powerful tools for characterizing genetic diversity in viral strains. In the present review, we update the information regarding HBV variability and present a summary of the various NGS approaches available for research in this virus. In addition, we provide an analysis of the clinical implications of HBV variants and their study by NGS.

In this study, the reliability and reproducibility of viral quasispecies quantification by three ultra-deep pyrosequencing (UDPS) methods (FLX+, FLX, and Junior) were investigated and results compared with the conventional cloning technique. Hepatitis B virus (HBV) infection was selected as the model. The preCore/Core region, the least overlapped HBV region, was analyzed in samples from a chronic hepatitis B patient by cloning and by UDPS. After computation filtering of the UDPS results, samples A1 and A2 (FLX+) and sample B (FLX) yielded the same 20 polymorphic positions. Junior yielded 18 polymorphic positions that coincided with the FLX results. In contrast, 50 polymorphic positions were detected by cloning. Quasispecies complexity plotted on graphs showed superimposed patterns and the quantitative parameters were similar between FLX+, FLX, Junior, and the cloning sequences. Twenty-two haplotypes were detected by Junior, and 37, 40, and 39 were detected by FLX A1, A2, and B, respectively. These differences may be attributable to methodological differences between FLX and Junior. By cloning, 47 haplotypes were detected. Eight clones with insertions and deletions that induced de novo stop codons were not observed by UDPS because the UDPS filter discarded them. Our results indicate that UDPS is an optimal alternative to molecular cloning for quantitative study of the viral quasispecies. Nonetheless, specific mutations, such as insertions and deletions, were only detected by cloning. A filter should be designed to analyze cloning sequences, and UDPS filters should be improved to include the specific mutations.

Background: Selection of amino acid substitutions associated with resistance to nucleos(t)ide-analog (NA) therapy in the hepatitis B virus (HBV) reverse transcriptase (RT) and their combination in a single viral genome complicates treatment of chronic HBV infection and may affect the overlapping surface coding region. In this study, the variability of an overlapping polymerase-surface region, critical for NA resistance, is investigated before treatment and under antiviral therapy, with assessment of NA-resistant amino acid changes simultaneously occurring in the same genome (linkage analysis) and their influence on the surface coding region. Methodology/Principal Findings: Serum samples obtained from chronic HBV-infected patients at pre-treatment and during sequential NA treatment with lamivudine, adefovir, and entecavir were analyzed by ultra-deep pyrosequencing (UDPS) using the GS-FLX platform (454 Life Sciences-Roche). The pre-treatment HBV quasispecies was not enriched with NA-resistant substitutions. The frequencies of this type of substitutions at pre-treatment did not predict the frequencies observed during lamivudine treatment. On linkage analysis of the RT region studied, NA-resistant HBV variants (except for rtA181T) were present in combinations of amino acid substitutions that increased in complexity after viral breakthrough to entecavir, at which time the combined variant rtL180M-S202G-M204V-V207I predominated. In the overlapping surface region, NA-resistant substitutions caused selection of stop codons in a significant percentage of sequences both at pre-treatment and during sequential treatment; the rtA181T substitution, related to sW172stop, predominated during treatment with lamivudine and adefovir. A highly conserved RT residue (rtL155), even more conserved than the essential residues in the RT catalytic motif YMDD, was identified in all samples. Conclusions: UDPS methodology enabled quantification of HBV quasispecies variants, even those harboring complex combinations of amino acid changes. The high percentage of potentially defective genomes, especially in the surface region, suggests effective trans-complementation of these variants. © 2012 Rodriguez-Frías et al., Centro para el Desarrollo Tecnológico Industrial; Ministry of Economy and Competitiveness

The current treatment of Chronic Hepatitis C (CHC) is based on the combination of peginterferon alpha 2a or 2b and ribavirin. This treatment achieves Sustained Virological Response (SVR) rates in more than 50% of the patients. The SVR rates are higher in genotype 2 and 3 patients than in genotype 1, which account for more than 50%. For this reason, the development of new drugs is needed. The knowledge of Hepatitis C Virus (HCV) replication cycle and the characterisation of the viral enzymes allow targets to be defined with the ability to inhibit HCV enzymes. The present overview analyses the first two NS3/NS4 protease inhibitors approved for the treatment of CHC, boceprevir and telaprevir. Both increase rates of SVR in naïve and in previously treated patients with the advantage that in some cases therapy can be shortened. However, these drugs have new side effects such as a rash and an increase in the rate of anaemia.

Background HCV is a major cause of morbidity and mortality in HIV-coinfected patients. Several observational studies have suggested that HCV response to pegylated interferon and ribavirin is lower in HIV-coinfected patients treated with abacavir. It has been postulated that abacavir could compete with ribavirin to be phosphorylated, leading to a reduction in the active form of the drug (triphosphorylated ribavirin). Here, we studied the effect of abacavir, tenofovir or lamivudine addition on the suppressive activity of ribavirin in an HCV RNA replicon system. Methods We used the human hepatoma HuH-7 cell clone 9B containing the HCV genotype 1b replicon I389/ NS3-3’. Cells were treated for 24 h with ribavirin (0, 10 and 50 μM) plus abacavir, tenofovir or lamivudine at doses of 0, 10 and 50 μM and HCV RNA production was quantified by real-time PCR in triplicate assays. Results were expressed as mean ±sd of the HCV RNA produced per cell (log10 IU/cell). Means were compared using the Student's t-test. Results Ribavirin treatment produced a dose-dependent suppression of HCV RNA production by the replicon system. Combination of ribavirin and interferon resulted in an additive antiviral activity. The addition of abacavir did not modify the suppressive activity of ribavirin on the replicon HCV RNA expression. Similar results were obtained when ribavirin was used in combination with tenofovir or lamivudine. Conclusions In a subgenomic HCV RNA replicon system, the antiviral effect of ribavirin was not modified by the addition of abacavir, tenofovir or lamivudine.

Background Amino acid (AA) changes in specific hepatitis B core antigen (HBcAg) regions were assessed in patients infected with chronic hepatitis B (CHB) after a 12-month untreated period and after receiving antiviral therapy (interferon, lamivudine or adefovir dipivoxil), and in inactive hepatitis B surface antigen-positive carriers. Methods Samples corresponding to different time points in 76 CHB cases (64 on-treatment) and 4 inactive carriers were included. The main precore mutation, T-helper immunodominant epitope at AA 50–69 (Th50–69), minor T-helper epitope (Th28–47), B-cell immunodominant epitope (B74–84) and a conserved region of HBcAg at AA 1–11 (AA1–11) were directly sequenced. For comparisons, the average number of AA changes in each region was standardized to 12 months (Av12). Results AA changes clustered mainly in immunodominant regions (69%). The highest percentage of cases (% n) with changes and highest Av12 changes were detected after interferon treatment (% n=73%, Av12=3.1 in Th50–69 and % n=86%, Av12=2.7 in B74–84). At baseline, immunodominant regions had higher Av12 changes in hepatitis B e antigen-negative patients and those with main precore mutations. Changes in the Th28–47 region were more frequent after nucleoside/nucleotide analogue treatment (40%) than before treatment (9%). Codons 74 and 77 were the most polymorphic, and the double change E64D-N67T was significantly observed. Codon 84 substitutions were mainly associated with interferon treatment ( P=0.05). Conclusions Natural and treatment-induced substitutions in HBV core protein, occurring especially with interferon treatment, were characterized. Some immune-stimulating activity related to the minor Th28–47 epitope might be associated with nucleoside/nucleotide analogues; this activity was also seen in inactive carriers.