Hepatitis B virus (HBV) whole genome sequencing (WGS) is currently limited as the DNA viral loads (VL) of many clinical samples are below the threshold required to generate full genomes using current sequencing methods. We developed two pan-genotypic viral enrichment methods, using probe-based capture and tiled amplicon PCR (HEP-TILE) for HBV WGS. We demonstrate using mock samples that both enrichment methods are pan-genotypic (genotypes A-J). Using clinical samples, we demonstrate that HEP-TILE amplification successfully amplifies full genomes at the lowest HBV VL tested (30 IU/ml), and the PCR products can be sequenced using both Nanopore and Illumina platforms. Probe-based capture with Illumina sequencing required VL > 300,000 IU/ml to generate full length HBV genomes. The capture-Illumina and HEP-TILE-Nanopore pipelines had consensus sequencing accuracy of 100% in mock samples with known DNA sequences. Together, these protocols will facilitate the generation of HBV sequence data, enabling a more accurate and representative picture of HBV molecular epidemiology, cast light on persistence and pathogenesis, and enhance understanding of the outcomes of infection and its treatment.

BACKGROUND:Outcomes of chronic Hepatitis B (CHB) infection have been increasingly associated with various metabolic syndromes, including metabolic-dysfunction associated steatotic liver disease (MASLD), with a potential for impact on liver disease progression. There is some evidence that metformin, a widely used anti-diabetic drug, may reduce hepatocellular carcinoma (HCC) incidence in people living with Hepatitis B Virus (HBV), but with little to no evidence of impact on the virus itself in vivo. However, previous in vitro studies suggest metformin may have a direct impact on HBV replication, although the mechanism remains unclear. OBJECTIVES:We aimed to investigate the impact of metformin on HBV replication in vitro. STUDY DESIGN:Hepatocyte cell lines constitutively expressing HBV (HepAD38) were treated once or thrice with escalating doses of metformin, using lamivudine and water as controls. We monitored cellular cytotoxicity as well as HBV biomarkers (HBeAg, HBsAg, HBV DNA and RNA) throughout the assay. RESULTS:We did not observe any impact of metformin on HBV replication after a single dose or three repeated treatments. CONCLUSIONS:In HepAD38 cells, HBV replication is not impacted by metformin treatment. This contrasts with prior in vitro data but is in line with clinical evidence that suggests metformin acts through an influence on liver disease progression rather than a direct antiviral impact on HBV itself.

Introduction and Objectives Chronic hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC). In African populations, HCC frequently presents at an advanced stage with poor outcomes. We applied whole genome sequencing (WGS) to compare HBV genomes in individuals with and without HCC. Materials and Methods We identified adults with HBV infection, with and without complicating HCC, in Cape Town, South Africa. We generated HBV WGS using pan-genotypic probe-based enrichment followed by Illumina sequencing. Results Compared to the non-HCC group, HCC patients were more likely to be male (p < 0.0001), older (p = 0.01), HIV-negative (p = 0.006), and have higher HBV viral loads (p < 0.0001). Among 19 HCC and 12 non-HCC patients for whom WGS was obtained, genotype A dominated (74 %), of which 96 % were subgenotype A1. PreS2 deletions (Δ38–55) were enriched in HBV sequences from HCC patients (n = 7). The sequence motif most strongly associated with HCC comprised either a deletion or polymorphism at site T53 in PreS2 – collectively coined ‘non-T53’ – together with a basal core promoter (BCP) mutation G1764A (AUROC = 0.79). Conclusions In this setting, HBV sequence polymorphisms and deletions are associated with HCC, and ‘non-T53 + G1764A’ represents a putative signature motif for HCC. Additional investigations are needed to disaggregate the impact of other demographic, clinical, and environmental influences, to ascertain the extent to which viral polymorphisms contribute to oncogenesis, and to determine whether HBV sequence is a useful biomarker for HCC risk stratification.

Background:Guidelines for Hepatitis B treatment released by the World Health Organization in 2024 include the potential for use of dual therapy, combining tenofovir with either emtricitabine or lamivudine. These fixed-dose combinations are also used for Pre-Exposure Prophylaxis (PrEP) in people at risk of Human Immunodeficiency Virus (HIV). We hypothesize that pre-existing HIV PrEP programmes can support access to HBV testing and treatment. Methods:At the Africa Health Research Institute (AHRI) in KwaZulu Natal, South Africa, we evaluated PrEP uptake and retention amongst adolescents and young adults aged 15-30 years. We reviewed HBV status, acceptance of PrEP and retention in follow-up between June 2022-Sept 2024. Results:15847 adolescents and young adults received an assessment in the community, of whom 3481/15847 (21.9 %) were eligible for sexual health prevention interventions. 3431/3481 (98.6 %) accepted HBV screening, of whom 21/3431 (0.6 %) tested positive for HBsAg. These 21 individuals had not previously been aware of their HBV status, but one was already on antiretroviral therapy for HIV infection. Amongst the others, 16/20 (80 %) were considered eligible for PrEP, and 15/16 started PrEP. When investigating retention in care, among 15 individuals due for a refill, 8/15 (53.3 %) returned at least once. Conclusion:Sexual reproductive health and PrEP programmes provide an opportunity for HBV testing and treatment. However, attrition from the care cascade at each step highlights the pressing need for interventions that address barriers to sustainable delivery of long-term care.

Background As nucleos/tide analogue (NA) therapy (e.g. entecavir and tenofovir) for chronic Hepatitis B virus (HBV) infection becomes more widely indicated and available, understanding drug resistance is essential. A systematic review to quantify resistance to these agents has not previously been undertaken. Methods We performed a systematic review and random-effects meta-analysis to estimate the risk of HBV resistance to entecavir and tenofovir. We searched nine databases up to 29-Aug-23. We included studies of HBV infection featuring >10 individuals, written in English, reporting treatment ≥48 weeks, with assessment of HBV resistance based on viral sequence data. Data were analysed according to prior exposure history to NA, and choice of NA agent. Analyses were performed in R. Findings 62 studies involving a total of 12,358 participants were included. For entecavir, in treatment-naive individuals (22 studies; 4326 individuals), resistance increased over time to 0.9 % at ≥5 years (95 %CI 0.1–2.3 %), and resistance was increased in NA-experienced individuals (18 studies; 1112 individuals), to 20.1 % (95 %CI 1.6–50.1 %) at ≥5 years. For tenofovir, pooled resistance risk was 0.0 % at all time points, whether previously NA naive (11 studies; 3778 individuals) or experienced (19 studies; 2059 individuals). There was a lack of consistent definitions, poor global representation and insufficient metadata to support subgroup analysis. Interpretation We have generated the first pooled estimates of HBV entecavir and tenofovir resistance over time. HBV resistance to entecavir in treatment-experienced groups in particular may represent a clinical and public health challenge. To date, tenofovir appears to have an excellent resistance profile, but due to data gaps, we caution that existing studies under-estimate the true real-world risk of resistance. Robust prospective data collection is crucial to reduce health inequities and reduce blind-spots in surveillance as treatment is rolled out more widely.

Introduction: The World Health Organisation (WHO) has set targets for the elimination of Hepatitis B virus (HBV), which include preventing new infections and reducing deaths. We explored beliefs, behaviours and barriers to diagnosis, prevention and treatment for people living with HBV infection (PLWHB) and those with liver disease in a rural South African population in KwaZulu-Natal, to gather information to inform research and support the development of improved clinical and public health services. Methods: Using an interdisciplinary approach (combining public engagement, social science, clinical and laboratory team members) we conducted a community dialogue with members of the Africa Health Research Institute (AHRI) Community Advisory Board (CAB). Notes from the discussions were used to write up an account from which themes were identified during a team debrief session for data analysis. Results: There was a lack of knowledge and awareness of HBV infection and transmission and prevention amongst CAB members, also reported among community members and healthcare workers. The participants recognised liver disease symptoms. Perceived causes of liver disease reported by the CAB were alcohol and non-adherence to HIV treatment. Barriers to care included stigma, poverty, and delays in referrals for HBV diagnosis and management. Conclusion: Understanding barriers to care is important to shape future services for diagnosis, treatment and prevention of HBV and liver disease which are accessible, affordable and acceptable to the local population. Education, awareness and advocacy for improved liver health care pathways are required to make them effective for local communities.

Chronic hepatitis B infection (CHB) affects 300 million people worldwide and is being targeted by the United Nations 2030 Sustainable Development Goals (SDGs) and the World Health Organisation (WHO), working towards elimination of hepatitis B virus (HBV) as a public health threat. In this piece, we explore the evidence and potential impact of peer support to enhance and promote interventions for people living with CHB. Peer support workers (PSWs) are those with lived experience of an infection, condition or situation who work to provide support for others, aiming to improve education, prevention, treatment and other clinical interventions and to reduce the physical, psychological and social impacts of disease. Peer support has been shown to be a valuable tool for improving health outcomes for people living with human immunodeficiency virus (HIV) and hepatitis C virus (HCV), but to date has not been widely available for communities affected by HBV. HBV disproportionately affects vulnerable and marginalised populations, who could benefit from PSWs to help them navigate complicated systems and provide advocacy, tackle stigma, improve education and representation, and optimise access to treatment and continuity of care. The scale up of peer support must provide structured and supportive career pathways for PSWs, account for social and cultural needs of different communities, adapt to differing healthcare systems and provide flexibility in approaches to care. Investment in peer support for people living with CHB could increase diagnosis, improve retention in care, and support design and roll out of interventions that can contribute to global elimination goals.

Aim Chronic hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC) particularly in African populations, in whom malignancy frequently presents at an advanced stage with poor outcomes. We derived HBV whole genome sequences (WGS) from individuals with HCC and compared them to sequences from individuals without HCC. Methods: We identified adults with HBV infection, with and without complicating HCC, in Cape Town, South Africa and utilized pan-genotypic probe-based enrichment followed by Illumina sequencing to derive HBV WGS. Results Compared to the non-HCC group, HCC patients were more likely to be male (p < 0.0001), older (p = 0.01), HIV-negative (p = 0.006), and to have higher HBV viral loads (p < 0.0001). Among 19 HCC and 12 non-HCC patients, genotype A dominated (74%), of which 96% were subtype A1. PreS2 deletions (Δ38–55) were enriched in HBV sequences from HCC patients (n = 7). The sequence motif most strongly associated with HCC comprised either deletion or polymorphism at site T53 in PreS2 – collectively coined ‘non-T53’ – together with a basal core promoter (BCP) mutation G1764A (AUROC 0.79). Conclusions In this setting, HBV sequence polymorphisms and deletions are associated with HCC, and ‘non-T53 + G1764A’ represents a putative signature motif for HCC. Additional investigations are needed to disaggregate the impact of age, sex and HIV status, to ascertain the extent to which viral polymorphisms contribute to oncogenesis, and to determine whether HBV sequence is a useful biomarker for risk stratification. ### Competing Interest Statement PCM has participated in projects supported by GSK, outside the direct scope of the work presented here. MIA has participated in projects supported by Prenetics, J&J and Pfizer outside the direct scope of this work. ### Funding Statement PCM received funding support from the Wellcome Trust (grant ref 110110/Z/15/Z), UCL NIHR Biomedical Research Centre and the Francis Crick Institute. TGM received support from the Poliomyelitis Research Foundation, the Harry Crossley Foundation and Columbia University South Africa Training Program for Research on AIDS-related Malignancies through the National Cancer Institute, NIH (Grant # 1D43CA153715). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethical approval was granted from the health research ethics committee at the University of Stellenbosch (S13/04/072 and N11/09/284). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Consensus sequences are available on line at data produced in the present study are available upon reasonable request to the authors.

The WHO African region bears a disproportionate burden of morbidity and mortality related to chronic hepatitis B virus (HBV) infection and accounts for an estimated 70% of new HBV infections worldwide. We investigated the extent to which HBV clinical trials represented populations in this region by searching the WHO International Clinical Trials Registry Platform and ClinicalTrials.gov for interventional clinical trials published in English between database inception and May 29, 2023, using the search term “Hepatitis B”. We identified 1804 unique clinical trials, of which 18 (1·0%) recorded involvement of the WHO African region. There is no evidence that the number of HBV clinical trials in this region has improved over time. The diversity of new interventions and industry sponsorship in the WHO African region were low, with trials of HBV comparing poorly with those of other endemic infectious diseases (eg, malaria, HIV, and SARS-CoV-2). HBV research and clinical trial investigations have neglected the WHO African region, leading to profound health inequities. HBV clinical trials are urgently needed to evaluate the efficacy of newly discovered therapeutics and to ensure that interventions can be equitably distributed and deployed as they become available.

Diagnosing and treating chronic hepatitis B virus (HBV) infection are key interventions to support progress towards elimination of viral hepatitis by 2030. Although nucleos/tide analogue (NA) therapy is typically highly effective, challenges remain for viral load (VL) suppression, including medication access, incomplete adherence and drug resistance. We present a case of a long-term HBV and HIV coinfected adult prescribed with sequential NA therapy regimens, with episodes of breakthrough viraemia. Multiple factors contribute to virological breakthrough, including exposure to old NA agents, initial high HBV VL, therapy interruptions, intercurrent illnesses and potential contribution from resistance mutations. The case underscores the importance of individualised treatment approaches and adherence support in achieving HBV suppression. Furthermore, it emphasises the need for improved clinical pathways addressing education, support and access to care, particularly for marginalised populations. Comprehensive data collection inclusive of under-represented individuals is crucial for maintaining retention in the care cascade and informing effective interventions.

Introduction: Hepatitis B virus (HBV) infection is a pressing global public health threat, responsible for increasing mortality with a disproportionate impact on populations in the WHO African region. There is a need for evaluation of new biomarkers that may be able to provide insights into risk stratification and disease pathogenesis. We therefore set out to evaluate Hepatitis B core related antigen (HBcrAg), in cohorts in the United Kingdom (UK) and South Africa (SA). Methods: We undertook a cross-sectional retrospective observational study, using serum samples obtained from adults living with chronic HBV infection enrolled at Oxford University Hospitals (OUH) NHS Foundation Trust in the UK (n=142), and from a clinical cohort in Cape Town and Bloemfontein, SA (n=211). We recorded routine clinical and laboratory parameters gathered at each site, and undertook quantification of HBcrAg and host immune biomarkers, IL-21, IP-10 and PD-1. We report on HBcrAg distribution, relationship with other clinical and immunological biomarkers, and performance as a risk stratification tool in each setting. Results: Sex and age were comparable between SA and UK cohorts (p>0.05). There were significant differences between cohorts in ethnicity (p <0.001), HIV coinfection (2.3% in UK vs 56% in SA, p<0.001), HBeAg-positivity (12% in the UK vs 29% in SA, p<0.001), and proportion with HBV DNA >200,000 IU/ml (6% in the UK vs. 22% in SA, p<0.001). Host immune markers were all significantly higher in the SA vs UK cohorts (p<0.001 for all); (i) HBcrAg was positively associated with HBV DNA in both cohorts (p<0.0001), and in the UK this relationship was mediated by HBeAg-positivity. (ii) HBcrAg was also positively associated with overall liver inflammation assessed by ALT (p<0.001 in UK, p<0.01 in SA), however there was no specific significant correlation with more precise host immune markers, IP-10, IL-21 or PD-1 (p>0.05 in all cases). (iii) In univariable and multivariable analysis, there was no association between HBcrAg and liver fibrosis using elastography, APRI or FIB-4 scores in both cohorts. Discussion: In spite of similar HBcrAg levels in both the UK and SA cohorts, prediction of HBV VL seems to differ depending on settings, suggesting a more tailored and personalised approach to HBcrAg testing is required. In our cohorts, HBcrAg did not correlate with any liver disease outcomes. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This research was funded in whole, or in part, by the Wellcome Trust [220549/Z/20/Z and 110110/Z/15/Z). LOD is funded by a Wellcome Trust Grant (ref. 225485/Z/22/Z) and Oxford University John Fell Fund (award number 0012112). SFL is funded by a Wellcome doctoral fellowship (ref 220549/Z/20/Z ). PCM receives funding from Wellcome (ref 110110/Z/15/Z), the Francis Crick Institute (ref CC2223) and University College London NIHR Biomedical Research Centre. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Oxford Research Ethics Committee A (reference 09/H0604/20 and N17/01/013) of Oxford University (UK) gave ethical approval for this work. University of Oxford Tropical Research Ethics Committee (ref. OXTREC 01 18) of Oxford University (UK); Stellenbosch University Health Research Ethics Committee (HREC ref. N17/01/013) of the Stellenbosch University (South Africa), and the University of the Free State Research Ethics Committee (HSREC ref. 203/2016) of the University of the Free State (South Africa) gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript or available upon reasonable request to the authors