Integrating viral hepatitis management into the emergency department: A further step towards viral hepatitis elimination.

Background & Aims: Many people with HCV and HBV infection are unaware of their condition, particularly at-risk and vulnerable populations who face barriers for screening and linkage to care. Emergency departments are often their only point of contact with the health system. Methods: This is a prospective study investigating HBsAg and HCV antibody testing, with reflex testing for HDV antibodies and HCV RNA, in adults attending an emergency department and requiring a blood test. Positive cases were linked to care. A cost-effectiveness analysis was performed. Results: From February 2020 to February 2022, a total of 17,560 individuals were screened. HBsAg was detected in 91 (0.5%), HCV RNA in 128 (0.7%), and HDV antibodies in two (0.01%) individuals. Nearly 40% of positive cases were unaware of their condition. Linkage to care was achieved in 42 of 56 HBsAg-positive and 45 of 69 HCV RNA-positive participants who were candidates for referral. HCV and HBV screening vs. no screening yielded 1.06 and 0.42 additional quality-adjusted life-years, respectively, with incremental cost-utility ratios of €7,629 and -€147 per quality-adjusted life-year gained, respectively, and proved even more cost-effective in patients with hepatitis C aged 40-70 years. Conclusions: On emergency department screening for hepatitis B, C, and D in Barcelona, the prevalence of HBsAg was 0.5% and HCV RNA 0.7%, approximately threefold higher than that observed in the general population. This strategy diagnosed patients with active HCV infection and no risk factors, who would not have been screened according to the current recommendations. Screening and linkage to care of viral hepatitis is cost-effective in this setting. Impact and implications: We evaluated the performance and cost-effectiveness of a viral hepatitis screening programme implemented in an emergency department, which aimed to identify and link to care people living with hepatitis B and C. Our findings reveal a threefold higher prevalence of hepatitis B and C than in the general Spanish population, possibly attributable to the role of the emergency department as the main healthcare gateway for vulnerable populations, who have a higher prevalence of viral hepatitis. Risk factors for viral hepatitis could not be identified in most people living with hepatitis B and C attending the emergency department; hence, screening beyond risk factors should be considered in hepatitis detection strategies. Emergency department screening is cost-effective for hepatitis C and is a cost-saving strategy for hepatitis B in our setting. These data should inform future updates to clinical guidelines.

Community-based screening enhances hepatitis B virus linkage to care among West African migrants in Spain.

BACKGROUND: Chronic infection with HBV is responsible for >50% of all hepatocellular cancer cases globally and disproportionately affects sub-Saharan African (sSA) countries. Migration from these countries to Europe has increased substantially in recent years, posing unique challenges to health systems. The aim of this study was to carry out a community-based intervention to increase HBV screening, vaccination, and linkage to care among sSA migrants in Catalonia, Spain. METHODS: This was a prospective cohort study. Participants ≥18 years were offered community-based HBV screening between 20/11/20 and 21/01/22. Rapid HBV testing and blood sample collection utilizing plasma separation cards were carried out and linkage to care was offered to all participants. HBV vaccination and post-test counseling were performed at a second visit in the community. The main outcome was the odds of those with current HBV infection being successfully linked to hepatology. Rates of completing the care cascade of this model were analyzed. RESULTS: In the present study, 444 people undergo screening, with 50.6% of participants showing evidence of past or current HBV infection, including an HBsAg prevalence of 9.2%. Migrants with current HBV infection exhibit 5.2 times higher odds of successful linkage to care compared to those in need of post-test counseling or vaccination. The study achieves a successful linkage to care rate of 72% for all participants, with specialist appointments arranged within 15.5 days. CONCLUSIONS: This community-based HBV screening program provides evidence of a successful model for identifying and providing care, including vaccination, to west African migrants at high risk of HBV infection who may otherwise not engage in care.

A large proportion of hepatitis B virus (HBV) infections occur within countries in sub-Saharan Africa. With recent increased migration from these countries to Catalonia Spain, the prevalence of HBV is greater in migrants than in host populations. However, migrants face additional barriers when trying to access care. We developed a community-based care pathway to provide migrants in Catalonia with access to HBV testing, post-test counseling, vaccinations, and appointments with specialists when needed. The results showed that this strategy was successful in increasing testing, linkage to care, and vaccination among at-risk migrant populations in Catalonia, Spain. It may be worthwhile implementing this strategy on a wider scale and with other at-risk populations to reduce HBV infections and improve outcomes.

In-Host HEV Quasispecies Evolution Shows the Limits of Mutagenic Antiviral Treatments.

Here, we report the in-host hepatitis E virus (HEV) quasispecies evolution in a chronically infected patient who was treated with three different regimens of ribavirin (RBV) for nearly 6 years. Sequential plasma samples were collected at different time points and subjected to RNA extraction and deep sequencing using the MiSeq Illumina platforms. Specifically, we RT-PCR amplified a single amplicon from the core region located in the open-reading frame 2 (ORF2). At the nucleotide level (genotype), our analysis showed an increase in the number of rare haplotypes and a drastic reduction in the frequency of the master (most represented) sequence during the period when the virus was found to be insensitive to RBV treatment. Contrarily, at the amino acid level (phenotype), our study revealed conservation of the amino acids, which is represented by a high prevalence of the master sequence. Our findings suggest that using mutagenic antivirals concomitant with high viral loads can lead to the selection and proliferation of a rich set of synonymous haplotypes that express the same phenotype. This can also lead to the selection and proliferation of conservative substitutions that express fitness-enhanced phenotypes. These results have important clinical implications, as they suggest that using mutagenic agents as a monotherapy treatment regimen in the absence of sufficiently effective viral inhibitors can result in diversification and proliferation of a highly diverse quasispecies resistant to further treatment. Therefore, such approaches should be avoided whenever possible.

Solved the enigma of pediatric severe acute hepatitis of unknown origin?

Hepatitis is an inflammation of the liver whose etiology is very heterogeneous. The most common cause of hepatitis is viral infections from hepatotropic viruses, including hepatitis A, B, C, D and E. However, other factors such as infections from other agents, metabolic disorders, or autoimmune reactions can also contribute to hepatitis, albeit to a lesser extent. On April 5, 2022, the United Kingdom Health Security Agency alerted the World Health Organization (WHO) on the increased incidence of severe acute hepatitis of unknown causes (not A-E) in previously healthy young children, with symptoms of liver failure that in some cases required liver transplantation. By July 2022, 1,296 cases were reported in 37 countries. Acute hepatitis of unknown causes is not an exceptional phenomenon: in fact, it represents more than 30% of cases of acute hepatitis in children, however in the present instance the large proportion of severe cases was surprising and alarming (6% of liver transplants and almost 3% mortality). Multiple hypotheses have been proposed to explain the etiology of such higher proportion of acute hepatitis, including their co-occurrence in the context of COVID-19 pandemic. This is a review of the history of a clinical threat that has put in check a world health care system highly sensitized by the current COVID-19 pandemics, and that it looks like has ended with the arguments that the severe acute pediatric hepatitis is caused by Adeno-associated virus 2 (AAV2) infection associated with a coinfection with a helper virus (human Adenovirus HAdV or human herpesvirus 6) in susceptible children carrying HLA-class II antigen HLA-DRB1*04:01.

HBsAg protein composition and clinical outcomes in chronic hepatitis D and variations across HBeAg-negative chronic HBsAg carriers.

Background & Aims: HBsAg proteins are useful to identify HBV inactive carriers (ICs), but data on chronic hepatitis D (CHD) are scarce. This study aimed to describe HBsAg composition in CHD, its changes during the evolution, and the potential association with clinical outcomes. In addition, we assess the composition of HBsAg across different HBV genotypes and validate previous results on HBsAg proteins in an independent HBV cohort. Methods: Quantitative HBsAg, medium HBsAg proteins (MHBs), and large HBsAg proteins (LHBs) were measured in two cohorts. The first cohort consisted of patients with CHD. A cross-sectional study of samples from two European institutions (N = 46) was conducted. Outcomes were assessed in a retrospective-prospective study of those patients with a follow-up of >1 year (n = 36), and the longitudinal evolution of HBsAg proteins in those with samples >5 years apart (n = 12) was analysed. The second cohort consisted of patients with HBeAg-negative HBV, and a cross-sectional study was performed (N = 141). Results: Forty-one (89%) patients with CHD had detectable HDV-RNA, and the presence of HDV-RNA was associated with higher LHBs proportion (p = 0.010). Baseline MHBs (p = 0.051) and MHBs proportion (p = 0.086) tended to be higher in those developing clinical outcomes (9/36, 25%) after a median follow-up of 5.9 years. Patients in which HDV-RNA became spontaneously undetectable during follow-up (5/31, 16.1%) tended to present lower MHBs proportion (p = 0.085). In the longitudinal study, changes in LHBs proportion were observed (p = 0.041), whereas MHBs proportion remained stable (p = 0.209). Regarding HBV, ICs showed lower LHBs proportion (p = 0.027). LHBs and MHBs differed significantly according to HBV genotype, regardless of the HBV phase. Conclusions: Patients with CHD with detectable HDV-RNA presented higher LHBs proportion than those with undetectable HDV-RNA. A trend toward having higher baseline MHBs proportion was observed in patients who developed clinical outcomes or remained with detectable HDV-RNA. This study validates the different HBsAg composition in HBV ICs and reveals the HBV-genotype influence in HBsAg composition. Impact and implications: The composition of HBsAg in chronic hepatitis D differs in patients with detectable and undetectable HDV viral load and may help predict the likelihood of achieving undetectable HDV viraemia and the development of clinical events such as decompensation. The composition of the surface antigen is also useful to distinguish inactive carriers of HBV, and it varies according to HBV genotype.

Impact of very early antiretroviral therapy during acute HIV infection on long-term immunovirological outcomes.

OBJECTIVES: We aimed to determine if starting antiretroviral therapy (ART) in the first 30 days after acquiring HIV infection has an impact on immunovirological response. METHODS: Observational, ambispective study including 147 patients with confirmed acute HIV infection (January/1995-August/2022). ART was defined as very early (≤30 days after the estimated date of infection), early (31-180 days), and late (>180 days). We compared time to viral suppression (viral load [VL] <50 copies/ml) and immune recovery (IR) (CD4+/CD8+ ratio ≥1) according to the timing and type of ART using survival analysis. RESULTS: ART was started in 140 (95.2%) patients. ART was very early in 24 (17.1%), early in 77 (55.0%), and late in 39 (27.9%) cases. Integrase strand transfer inhibitor (INSTI)-based regimens were the most used in both the overall population (65%) and the very early ART group (23/24, 95.8%). Median HIV VL and CD4+/CD8+ ratio pre-ART were higher in the very early ART group (P <0.05). Patients in the very early and early ART groups and treated with INSTI-based regimens achieved IR earlier (P <0.05). Factors associated with faster IR were the CD4+/CD8+ ratio pre-ART (hazard ratio: 9.3, 95% CI: 3.1-27.8, P <0.001) and INSTI-based regimens (hazard ratio: 2.4, 95% CI: 1.3-4.2, P = 0.003). CONCLUSIONS: The strongest predictors of IR in patients who start ART during AHI are the CD4+/CD8+ ratio pre-ART and INSTI-based ART regimens.

Transfusion-transmission of hepatitis E virus through red blood cell transfusion but not through platelet concentrates: A case report from Spain.

BACKGROUND: Few cases of transfusion-transmitted hepatitis E virus (HEV) have been published in Spain. Here, we describe a well-characterized lookback investigation of a transfusion-transmitted HEV case at the Community Centre for Blood and Tissues of Asturias (Spain). CASE REPORT: A female patient with chronic myeloid leukemia underwent an allogeneic bone marrow transplant in March 2019 and showed alterations in liver function shortly afterwards. This patient received blood components from 30 different donors in the 3 months before the transplant. Frozen plasma samples from these donations were investigated for the presence of HEV-RNA. One frequent donor was identified as asymptomatic HEV RNA-positive at the time of his whole blood donation. The investigation revealed that this donor's plasma unit, originally intended for the fractionation industry, had a viral RNA concentration of 1.9 × 104 copies/mL. HEV RNA was detected initially in the index patient who received the red cell concentrate from this donor 25 days after the transfusion. HEV RNA isolated from both donor and recipient were identified as subtype 3f. The recipient of platelet concentrate (PC), treated with a riboflavin-based pathogen reduction technology (PRT) was not infected, being negative for the presence of HEV IgM, IgG, and HEV RNA before and after the transfusion. CONCLUSION: This case study shows that HEV was transmitted through red cell transfusion to a recipient, while the patient who received riboflavin/UV light treated PC did not develop signs of infection. A causal relationship between PRT treatment of the PC and the non-transmission of HEV remains to be established.

Community Strategy for Hepatitis B, C, and D Screening and Linkage to Care in Mongolians Living in Spain.

Mongolia has one of the highest viral hepatitis infection (B, C, and D) rates in the world. The aims of this study were to increase awareness of this disease and promote viral hepatitis screening in the Mongolian community living in Spain. Through a native community worker, Mongolian adults were invited to a community program consisting of an educational activity, an epidemiological questionnaire, and rapid point-of-care testing for hepatitis B and C. In those testing positive, blood extraction was performed to determine serological and virological parameters. In total, 280 Mongolians were invited to the program and 222 (79%) attended the event: 139 were women (63%), mean age was 42 years, and 78 (35%) had viral hepatitis risk factors. Testing found 13 (5.8%) anti-HCV-positive individuals, 1 with detectable HCV RNA (0.5%), 8 HBsAg-positive (3.6%), and 7 with detectable HBV DNA (3.1%). One additional individual had HBV/HCV co-infection with detectable HBV DNA and HCV RNA. Two subjects had hepatitis B/D co-infection (0.9%). The knowledge questionnaire showed a 1.64/8-point (20.5%) increase in correct answers after the educational activity. In summary, a viral hepatitis community program was feasible and widely accepted. It increased awareness of this condition in the Mongolian community in Spain and led to linkage to care in 22 participants, 50% of whom were unaware of their infection.

mTOR inhibitors a potential predisposing factor for chronic hepatitis E: Results from the prospective collaborative CHES study (Chronic Hepatitis EScreening in patients with immune impairment and increased transaminases levels).

BACKGROUND: Chronic hepatitis E virus (HEV) in persons with immune impairment has a progressive course leading to a rapid progression to liver cirrhosis. However, prospective data on chronic HEV is scarce. The aim of this study was to determine the prevalence and risk factors for chronic HEV infection in subjects with immune dysfunction and elevated liver enzymes. PATIENTS AND METHODS: CHES is a multicenter prospective study that included adults with elevated transaminases values for at least 6 months and any of these conditions: transplant recipients, HIV infection, haemodialysis, liver cirrhosis, and immunosuppressant therapy. Anti-HEV IgG/IgM (Wantai ELISA) and HEV-RNA by an automated highly sensitive assay (Roche diagnostics) were performed in all subjects. In addition, all participants answered an epidemiological survey. RESULTS: Three hundred and eighty-one patients were included: 131 transplant recipients, 115 cirrhosis, 51 HIV-infected subjects, 87 on immunosuppressants, 4 hemodialysis. Overall, 210 subjects were on immunosuppressants. Anti-HEV IgG was found in 94 (25.6%) subjects with similar rates regardless of the cause for immune impairment. HEV-RNA was positive in 6 (1.6%), all of them transplant recipients, yielding a rate of chronic HEV of 5.8% among solid-organ recipients. In the transplant population, only therapy with mTOR inhibitors was independently associated with risk of chronic HEV, whereas also ALT values impacted in the general model. CONCLUSIONS: Despite previous abnormal transaminases values, chronic HEV was only observed among solid-organ recipients. In this population, the rate of chronic HEV was 5.8% and only therapy with mTOR inhibitors was independently associated with chronic hepatitis E.

Emergence of Delta and Omicron variants carrying resistance-associated mutations in immunocompromised patients undergoing sotrovimab treatment with long-term viral excretion.

OBJECTIVES: To monitor the early emergence of genetic mutations related to reduced susceptibility to monoclonal anti-body (mAb)-based treatment in immunocompromised patients with long-term viral excretion using whole-genome sequencing at a tertiary university hospital in Barcelona, Spain. METHODS: Serial severe acute respiratory syndrome coronavirus 2-positive samples (mid-December 2021-mid-March 2022) from eight immunosuppressed, fully vaccinated patients (for solid-organ transplantation or haematologic malignancies) with long-term viral excretion despite undergoing mAb therapy (sotrovimab) for coronavirus disease 2019 were selected. Whole-genome sequencing was performed following the ARTIC, version 4.1, protocol on the MiSeq platform. Mutations in the coding sequence of the spike protein with a frequency of ≥5% were studied. RESULTS: A total of 37 samples from the studied cases were analysed. All the cases, except one, were confirmed to have the Omicron variant BA.1; one had Delta (AY.100). Thirty-four different mutations were detected within the receptor-binding domain of the spike protein in 62.5% of patients, eight of which were not lineage related and located in the sotrovimab target epitope (P337L, E340D, E340R, E340K, E340V, E340Q, R346T and K356T). Except for P337L, all changes showed a significant increase in frequency or fixation after the administration of sotrovimab. Some of them have been associated with either reduced susceptibility to mAb therapy, such as those at position 340, or the acquisition of a new glycosylation site (346 and 356 positions). CONCLUSIONS: This study highlights the importance of monitoring for early in vivo selection of mutations associated with reduced susceptibility to mAb therapy, especially in immunocompromised patients receiving anti-viral drugs, whose immune response is not able to control viral replication, resulting in long-term viral shedding, and those receiving selective evolution pressure. Virologic surveillance of genetically resistant viruses to available anti-viral therapies is considered a priority for both patients and the community.

The frequency of defective genomes in Omicron differs from that of the Alpha, Beta and Delta variants.

The SARS-CoV-2 Omicron variant emerged showing higher transmissibility and possibly higher resistance to current COVID-19 vaccines than other variants dominating the global pandemic. In March 2020 we performed a study in clinical samples, where we found that a portion of genomes in the SARS-CoV-2 viral population accumulated deletions immediately before the S1/S2 cleavage site (furin-like cleavage site, PRRAR/S) of the spike gene, generating a frameshift and appearance of a premature stop codon. The main aim of this study was to determine the frequency of defective deletions in prevalent variants from the first to sixth pandemic waves in our setting and discuss whether the differences observed might support epidemiological proposals. The complete SARS-CoV-2 spike gene was deeply studied by next-generation sequencing using the MiSeq platform. More than 90 million reads were obtained from respiratory swab specimens of 78 COVID-19 patients with mild infection caused by the predominant variants circulating in the Barcelona city area during the six pandemic waves: B.1.5, B.1.1, B.1.177, Alpha, Beta, Delta, and Omicron. The frequency of defective genomes found in variants dominating the first and second waves was similar to that seen in Omicron, but differed from the frequencies seen in the Alpha, Beta and Delta variants. The changing pattern of mutations seen in the various SARS-CoV-2 variants driving the pandemic waves over time can affect viral transmission and immune escape. Here we discuss the putative biological effects of defective deletions naturally occurring before the S1/S2 cleavage site during adaption of the virus to human infection.

Quasispecies Fitness Partition to Characterize the Molecular Status of a Viral Population. Negative Effect of Early Ribavirin Discontinuation in a Chronically Infected HEV Patient.

The changes occurring in viral quasispecies populations during infection have been monitored using diversity indices, nucleotide diversity, and several other indices to summarize the quasispecies structure in a single value. In this study, we present a method to partition quasispecies haplotypes into four fractions according to their fitness: the master haplotype, rare haplotypes at two levels (those present at <0.1%, and those at 0.1−1%), and a fourth fraction that we term emerging haplotypes, present at frequencies >1%, but less than that of the master haplotype. We propose that by determining the changes occurring in the volume of the four quasispecies fitness fractions together with those of the Hill number profile we will be able to visualize and analyze the molecular changes in the composition of a quasispecies with time. To develop this concept, we used three data sets: a technical clone of the complete SARS-CoV-2 spike gene, a subset of data previously used in a study of rare haplotypes, and data from a clinical follow-up study of a patient chronically infected with HEV and treated with ribavirin. The viral response to ribavirin mutagenic treatment was selection of a rich set of synonymous haplotypes. The mutation spectrum was very complex at the nucleotide level, but at the protein (phenotypic/functional) level the pattern differed, showing a highly prevalent master phenotype. We discuss the putative implications of this observation in relation to mutagenic antiviral treatment.

Implementation of anti-HDV reflex testing among HBsAg-positive individuals increases testing for hepatitis D.

Background & Aims: Although EASL guidelines recommend anti-HDV testing in all HBsAg-positive individuals, HDV infection remains an underdiagnosed condition. We describe the impact of an HDV screening program by reflex anti-HDV testing in all HBsAg-positive samples and compare the results before and after its implementation. Methods: In total, 2,236 HBsAg-positive determinations were included from January 2018 to December 2021. Only the first sample from each participant was evaluated: 1,492 samples before reflex anti-HDV testing (2018-2020) and 744 samples after (2021). Demographic and clinical characteristics of anti-HDV-positive patients were collected. Results: Before reflex testing, anti-HDV had been tested in 7.6% (114/1492) of HBsAg-positive individuals: 23% (91/390) attended in an academic hospital and only 2% (23/1,102) in primary care centres. After reflex testing was established, 93% (691/744) of HBsAg-positive cases were evaluated for anti-HDV: 91% (533/586) in the academic hospital and 100% (158/158) in primary care. The anti-HDV-positive prevalence was similar before and after reflex testing: 9.6% (11/114) and 8.1% (56/691), respectively. However, the absolute number of anti-HDV-positive patients increased. Most anti-HDV-positive patients were young, HBeAg-negative, Caucasian males. HDV-RNA was detectable in 35 (65%) of 54 tested, HBV-DNA was undetectable in 64%, and alanine aminotransferase levels were normal in 48%. Conclusions: Anti-HDV reflex testing quintupled the absolute number of diagnoses of chronic hepatitis D infection. Before the reflex test, a large percentage of HBsAg-positive individuals had not undergone any anti-HDV determination. Implementation of reflex testing increases the diagnosis of patients with chronic hepatitis D. Lay summary: Chronic hepatitis delta (CHD) is a viral disease caused by HDV, which requires the presence of HBV to propagate. HDV infection can cause rapid progression to cirrhosis, among other severe complications. The prevalence of CHD worldwide is controversial, and the infection often goes unrecognised, mainly because of unawareness among physicians. Use of reflex testing in other viral hepatitis has proven to increase detection and linking-to-care of infected patients. Implementation of anti-HDV testing in all HBsAg-positive patients has led to a 5-fold increase in the number of HDV diagnoses in an academic hospital and primary care centres.

Prevalence of Hepatitis C Virus Infection, Genotypes and Subtypes in Migrants from Pakistan in Barcelona, Spain.

Background: Hepatitis C virus (HCV) is a major cause of chronic liver infection with 71 million people infected worldwide. Pakistan has the second highest prevalence of HCV infection and more than half (52%) of Pakistani living in Spain reside in Barcelona. The aim of this study was to analyse the seroprevalence and viraemic rate and determine the genotypes and subtypes of HCV among Pakistanis living in the southern metropolitan area of Barcelona. Methods: We included all Pakistani patients seeking primary healthcare in the southern metropolitan area of Barcelona from August 2011 to July 2014. Serum samples were screened for HCV antibodies. HCV viral load was determined by reverse transcription polymerase chain reaction and genotypes and subtypes were performed using Versant HCV Genotype and/or deep-sequencing. Screening for hepatitis B virus (HBV) was also carried out. Results: Among 5877 Pakistani patients, 565 (9.61%) were screened for anti-HCV antibodies, with 68 (12.04%) being positive. The viral load was determined in 65, with 31 presenting active infection and the viraemic rate was 47.69% (95% confidence interval 36.02-59.62). HCV genotyping and subtyping were performed in 24 individuals. Most infections corresponded to HCV genotype 3 (91.67%), and high resolution HCV subtyping was performed in 18 samples, 16 of which presented subtype 3a. One subject presented HBV coinfection with undetectable HBV DNA. During the study period, we identified a possible case of HCV vertical transmission followed by spontaneous viraemia clearance in a chronically infected mother with a C/T IL28B genetic polymorphism. Conclusion: These results suggest that general HCV screening protocols in patients from high prevalence countries, such as Pakistan, would be helpful to identify and treat active HCV infections. This could avoid further transmission and contribute to building targeted health policies for micro-elimination of HCV infection in specific communities.

Hepatitis B Virus Variants with Multiple Insertions and/or Deletions in the X Open Reading Frame 3' End: Common Members of Viral Quasispecies in Chronic Hepatitis B Patients.

Deletions in the 3' end region of the hepatitis B virus (HBV) X open reading frame (HBX) may affect the core promoter (Cp) and have been frequently associated with hepatocellular carcinoma (HCC). The aim of this study was to investigate the presence of variants with deletions and/or insertions (Indels) in this region in the quasispecies of 50 chronic hepatitis B (CHB) patients without HCC. We identified 103 different Indels in 47 (94%) patients, in a median of 3.4% of their reads (IQR, 1.3-8.4%), and 25% (IQR, 13.1-40.7%) of unique sequences identified in each quasispecies (haplotypes). Of those Indels, 101 (98.1%) caused 44 different altered stop codons, the most commonly observed were at positions 128, 129, 135, and 362 (putative position). Moreover, 39 (37.9%) Indels altered the TATA-like box (TA) sequences of Cp; the most commonly observed caused TA2 + TA3 fusion, creating a new putative canonical TATA box. Four (8%) patients developed negative clinical outcomes after a median follow-up of 9.4 (8.7-12) years. In conclusion, we observed variants with Indels in the HBX 3' end in the vast majority of our CHB patients, some of them encoding alternative versions of HBx with potential functional roles, and/or alterations in the regulation of transcription.

Standardized Hepatitis B Virus RNA Quantification in Untreated and Treated Chronic Patients: a Promising Marker of Infection Follow-Up.

The measurement and interpretation of HBV DNA and RNA levels in HBV infected patients treated with antiviral therapy supports the objective of HBV disease management. Here, we quantified circulating HBV RNA through a standardized and sensitive assay in follow-up samples from both naive and treated patients as a marker of infection evolution. HBV DNA (HBV DNA for use in Cobas 6800/8800 Automated Roche Molecular Systems), RNA (Roche HBV RNA Investigational Assay for use in the Cobas 6800/8800; Roche), HBeAg and HBsAg (Elycsys HBsAg chemiluminescence immunoassay by Cobas 8000; Roche), and core-related antigen (Lumipulse G chemiluminescence assay; Fujirebio) levels were measured in cohorts of untreated or nucleos(t)ide treated, HBV-infected subjects in an outpatient hospital setting. HBV DNA levels in untreated people were 3.6 log10 higher than corresponding RNA levels and were stable over 5 years of observation. While only five of 52 treated patients had DNA levels below the lower limit of quantification (10 IU/mL) at the end of follow-up, 13 had HBV RNA levels persistently above this limit, including eight with undetectable DNA. In samples with undetectable core-related antigen we observed a median HBsAg titer 2.7-fold higher than in samples with undetectable RNA (adjusted P = 0.012). Detectable HBV RNA with undetectable HBV DNA was a negative predictor of HBsAg decrease to a level ≤100 IU/mL (P = 0.03). In naive patients the difference between HBV DNA and RNA was higher than previously reported. HBV RNA rapidly decreased during treatment. However, in some cases, it was detectable even after years of effective therapy, being a negative predictor of HBsAg decrease. The investigational RNA assay for use on the Cobas 6800/8800 instruments is a sensitive and standardized method that could be applied in general management of HBV infection. IMPORTANCE This study focused on the quantification of circulating HBV RNA by using a standardized and sensitive assay. Thanks to this system we observed a higher difference between circulating HBV DNA and RNA than previously reported. In treated patients, HBV RNA decreased together with DNA, although some patients presented detectable levels even after years of successful antiviral treatment, suggesting a persistent viral transcription. Of note, the detection of viral RNA when HBV DNA is undetectable was a negative predictor of HBsAg decrease to a level ≤100 IU/mL. This assay could be extremely helpful in HBV patients management to study viral transcription and to identify those treated patients that may achieve sustained viral suppression.

Inspecting the Ribozyme Region of Hepatitis Delta Virus Genotype 1: Conservation and Variability.

The hepatitis delta virus (HDV) genome has an autocatalytic region called the ribozyme, which is essential for viral replication. The aim of this study was to use next-generation sequencing (NGS) to analyze the ribozyme quasispecies (QS) in order to study its evolution and identify highly conserved regions potentially suitable for a gene-silencing strategy. HDV RNA was extracted from 2 longitudinal samples of chronic HDV patients and the ribozyme (nucleotide, nt 688-771) was analyzed using NGS. QS conservation, variability and genetic distance were analyzed. Mutations were identified by aligning sequences with their specific genotype consensus. The main relevant mutations were tested in vitro. The ribozyme was conserved overall, with a hyper-conserved region between nt 715-745. No difference in QS was observed over time. The most variable region was between nt 739-769. Thirteen mutations were observed, with three showing a higher frequency: T23C, T69C and C64 deletion. This last strongly reduced HDV replication by more than 1 log in vitro. HDV Ribozyme QS was generally highly conserved and was maintained during follow-up. The most conserved portion may be a valuable target for a gene-silencing strategy. The presence of the C64 deletion may strongly impair viral replication, as it is a potential mechanism of viral persistence.

Assessing Liver Fibrosis Using the FIB4 Index in the Community Setting.

Liver disease is frequently asymptomatic, challenging early identification in the primary care setting. The fibrosis 4 (FIB4) index is a liver fibrosis biomarker that is a potential alternative to liver biopsy for diagnosing and managing liver disease. This study aimed to calculate the FIB4 index for screening individuals at high risk of liver disease at the community level. This was a retrospective real-world study analyzing blood and serum test results from a central laboratory. The primary outcome was the number of individuals within each risk category for hepatic fibrosis: high risk (FIB4 ≥ 3.25) and low risk (FIB4 < 1.3). The analysis included samples from 31,753 patients, of which 18,102 were aged 40 to 75 years. In these patients, the FIB4 index had been explicitly requested in 1852 (10.2%) cases and estimated ad hoc in the rest. Of the 263 (1.5%) cases with FIB4 ≥ 3.25, the FIB4 index was requested in 46 (17.5%), and 52 (19.8%) showed evidence of liver fibrosis in their medical records, while the rest did not report any data regarding liver fibrosis. FIB4 is a simple score that can play a role as a "red flag" for early identification of patients at high risk of advanced liver fibrosis and their referral to specialized care.