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International review of blood donation screening for anti-HBc and occult hepatitis B virus infection.
BACKGROUND: Hepatitis B core antibody (anti-HBc) screening has been implemented in many blood establishments to help prevent transmission of hepatitis B virus (HBV), including from donors with occult HBV infection (OBI). We review HBV screening algorithms across blood establishments globally and their potential effectiveness in reducing transmission risk. MATERIALS AND METHODS: A questionnaire on HBV screening and follow-up strategies was distributed to members of the International Society of Blood Transfusion working party on transfusion-transmitted infectious diseases. Screening data from 2022 were assimilated and analyzed. RESULTS: A total of 30 unique responses were received from 25 countries. Sixteen respondents screened all donations for anti-HBc, with 14 also screening all donations for HBV DNA. Anti-HBc prevalence was 0.42% in all blood donors and 1.19% in new donors in low-endemic countries; however, only 44% of respondents performed additional anti-HBc testing to exclude false reactivity. 0.68% of anti-HBc positive, HBsAg-negative donors had detectable HBV DNA. Ten respondents did universal HBV DNA screening without anti-HBc, whereas four respondents did not screen for either. Deferral strategies for anti-HBc positive donors were highly variable. One transfusion-transmission from an anti-HBc negative donor was reported. DISCUSSION: Anti-HBc screening identifies donors with OBI but also results in the unnecessary deferral of a significant number of donors with resolved HBV infection and donors with false-reactive anti-HBc results. Whilst confirmation of anti-HBc results could be improved to reduce donor deferral, transmission risks associated with anti-HBc negative OBI donors must be considered. In high-endemic areas, highly sensitive HBV DNA testing is required to identify infectious donors.
Early mucosal responses following a randomised controlled human inhaled infection with attenuated Mycobacterium bovis BCG.
The development of an effective vaccine against Mycobacterium tuberculosis is hampered by an incomplete understanding of immunoprotective mechanisms. We utilise an aerosol human challenge model using attenuated Mycobacterium bovis BCG, in BCG-naïve UK adults. The primary endpoint of this study (NCT03912207) was to characterise the early immune responses induced by aerosol BCG infection, the secondary endpoint was to identify immune markers associated with in-vitro protection. Blinded volunteers were randomised to inhale 1 × 107 CFU aerosolised BCG or 0.9% saline (20:6); and sequentially allocated to bronchoscopy at day 2 or 7 post-inhalation (10 BCG, 3 saline each timepoint). In the bronchoalveolar lavage post-aerosol BCG infection, there was an increase in frequency of eosinophils, neutrophils, NK cells and Donor-Unrestricted T cells at day 7, and the frequency of antigen presenting cells decreased at day 7 compared with day 2. The frequency of interferon-gamma+ BCG-specific CD4+ T cells increased in the BAL and peaked in the blood at day 7 post-BCG infection compared to day 2. BAL cells at day 2 and day 7 upregulated gene pathways related to phagocytosis, MHC-II antigen loading, T cell activation and proliferation. BCG's lack of key virulence factors and its failure to induce granulomas, may mean the observed immune responses do not fully recapitulate Mycobacterium tuberculosis infection. However, human infection models can provide unique insights into early immune mechanisms, informing vaccine design for complex pathogens.
Identification of undetected SARS-CoV-2 infections by clustering of Nucleocapsid antibody trajectories.
During the COVID-19 pandemic, numerous SARS-CoV-2 infections remained undetected. We combined results from routine monthly nose and throat swabs, and self-reported positive swab tests, from a UK household survey, linked to national swab testing programme data from England and Wales, together with Nucleocapsid (N-)antibody trajectories clustered using a longitudinal variation of K-means (N = 185,646) to estimate the number of infections undetected by either approach. Using N-antibody (hypothetical) infections and swab-positivity, we estimated that 7.4% (95%CI: 7.0-7.8%) of all true infections (detected and undetected) were undetected by both approaches, 25.8% (25.5-26.1%) by swab-positivity-only and 28.6% (28.4-28.9%) by trajectory-based N-antibody-classifications-only. Congruence with swab-positivity was respectively much poorer and slightly better with N-antibody classifications based on fixed thresholds or fourfold increases. Using multivariable logistic regression N-antibody seroconversion was more likely as age increased between 30-60 years, in non-white participants, those less (recently/frequently) vaccinated, for lower cycle threshold values in the range above 30, and in symptomatic and Delta (vs. BA.1) infections. Comparing swab-positivity data sources showed that routine monthly swabs were insufficient to detect infections and incorporating national testing programme/self-reported data substantially increased detection. Overall, whilst N-antibody serosurveillance can identify infections undetected by swab-positivity, optimal use requires fourfold-increase-based or trajectory-based analysis.
Hybrid B- and T-Cell Immunity Associates With Protection Against Breakthrough Infection After Severe Acute Respiratory Syndrome Coronavirus 2 Vaccination in Avon Longitudinal Study of Parents and Children (ALSPAC) Participants.
BACKGROUND: Immunological memory to vaccination and viral infection involves the coordinated action of B and T cells; thus, integrated analysis of these 2 components is critical for understanding their respective contributions to protection against breakthrough infections (BIs) after vaccination. METHODS: We investigated cellular and humoral immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and/or vaccination in 300 adult participants from the Avon Longitudinal Study of Parents and Children (ALSPAC). Participants were grouped by those with (cases) and without (controls) a history of SARS-CoV-2 infection. To provide a quantitative correlate for protection against BI in the 8-month period after the study, Youden index thresholds were calculated for all immune measures analyzed. RESULTS: The magnitude of antibody and T-cell responses following the second vaccine dose was associated with protection against BI in participants with a history of SARS-CoV-2 infection (cases), but not in infection-naive controls. Over 8 months of follow-up, 2 threshold combinations provided the best performance for protection against BI in cases: (i) anti-spike immunoglobulin G (IgG) (≥666.4 binding antibody units [BAU]/mL) combined with anti-nucleocapsid pan-immunoglobulin (pan-Ig) (≥0.1332 BAU/mL) and (ii) spike 1-specific T cells (≥195.6 spot-forming units/106 peripheral blood mononuclear cells) combined with anti-N pan-Ig (≥0.1332 BAU/mL). Both combinations offered 100% specificity for detecting cases without BI, with sensitivities of 83.3% and 72.2%, respectively. CONCLUSIONS: Collectively, these results suggest that hybrid B- and T-cell immunity offers superior protection from BI after coronavirus disease 2019 (COVID-19) vaccination, and this finding has implications for designing next-generation COVID-19 vaccines that are capable of eliciting immunity to a broader repertoire of SARS-CoV-2 proteins.
Immune mechanisms mediating the heterologous effects of BCG vaccination: a systematic review
IntroductionBCG vaccination can have heterologous or non-specific effects (NSE) that confer resistance against pathogens other than its target Mycobacterium tuberculosis, but the underlying mechanisms are not fully understood.MethodsWe conducted a systematic review synthesising existing literature on immune mechanisms mediating the heterologous/NSE of BCG. Searches were conducted using MEDLINE and Scopus.Results1032 original records were identified, of which 67 were deemed eligible. Several potentially relevant immune pathways were identified, although there may be variation by pathogen. Recent studies have focused on trained immunity whereby innate cells, or the hematopoietic stem and progenitor cells from which they are derived, undergo epigenetic and metabolic reprogramming allowing them to respond more effectively to antigen exposures unrelated to the original stimulus. However, other processes such as granulopoiesis and cross-reactive adaptive immunity may also play a role. Heterologous immunity and NSEs may be influenced by several endogenous and exogenous variables.DiscussionWe discuss the quality of available data, the importance of understanding mechanisms of heterologous protection, and its implications for vaccination strategies.Systematic review registrationhttps://www.crd.york.ac.uk/PROSPERO/view/CRD42023400375, identifier CRD42023400375.
Development and application of viral whole genome sequencing for Severe Acute Respiratory Syndrome Coronavirus 2 and Hepatitis B virus
During my DPhil, I set out to develop and apply sequencing techniques to Hepatitis B Virus (HBV), a pathogen which causes over a million deaths a year and for which molecular epidemiology is poorly resolved. However, this work was eclipsed by the COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) which rapidly became the world’s biggest viral killer. I rapidly applied the tools and approaches planned for endemic HBV to tackle pandemic SARS-CoV, implementing whole genome sequencing (WGS) techniques as a translational tool. I established SARS-CoV-2 WGS in my local hospital and explored how sequencing can help track the spread of SARS-CoV-2 in healthcare settings. By combining WGS with epidemiological data, I showed that most hospital-acquired infections result from superspreading events, providing insights to inform infection prevention control (IPC) practice. Returning to work on HBV, I optimised WGS tools, developing methods for HBV WGS, including the easy to use, open-access HEP-TILE protocol. To explore the clinical need for sequencing, I conducted a systematic review and meta-analysis to determine the epidemiology of drug resistance, demonstrating the high genetic barrier for tenofovir resistance compared to entecavir. Using HEP-TILE, I generated HBV genomes from adults with CHB in Uganda, Kenya, South Africa and the Democratic Republic of Congo. I described new HBV strains including recombinants and established the prevalence of resistance associated mutations (RAMs). I also discovered a potential link between a specific HBV polymorphism (S13T) and liver disease (fibrosis/cirrhosis). To further develop insights into HBV treatment, I explored the application of HBV treatment guidelines in Uganda, highlighting the limitations of current non-invasive tests. My thesis demonstrates how viral WGS can be applied to different viruses to enhance public health management. Globally, sequencing expertise and infrastructure gained during the COVID-19 pandemic can be applied to HBV, to support progress towards 2030 elimination targets for viral hepatitis.
Whole genome sequencing of hepatitis B virus using tiled amplicon (HEPTILE) and probe based enrichment on Illumina and Nanopore platforms.
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.
A putative hepatitis B virus sequence motif associated with hepatocellular carcinoma in South African adults.
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.
Temporal correlations between RBD-ACE2 blocking and binding antibodies to SARS-CoV-2 variants in CoronaVac-vaccinated individuals and their persistence in COVID-19 patients.
Antibodies play a crucial role in protection against SARS-CoV-2. Understanding the correlation between binding and functional antibodies is essential to determine whether binding antibody levels can reliably predict neutralizing activity. We assessed antibody responses in 111 individuals vaccinated with the inactivated vaccine CoronaVac and 111 COVID-19 patients in Thailand. Plasma levels of ACE2-blocking antibodies targeting the receptor-binding domain (RBD) of SARS-Co-V2 variants were measured before vaccination and at 14 and 28 days after the second dose using a multiplex surrogate virus neutralization test. Anti-spike and anti-nucleocapsid antibodies were quantified by electrochemiluminescence immunoassay, and anti-RBD IgG by ELISA. After vaccination, blocking, anti-spike, and IgG antibody levels increased but declined rapidly within a month, whereas antibody levels in COVID-19 patients increased and persisted. Blocking and anti-spike antibody correlated at day 14 post-vaccination but not at day 28. In COVID-19 patients, correlations were moderate at day 14, and stronger at day 28. Correlations were weaker for Omicron subvariants than for the ancestral strain and non-Omicron variants. The weak correlation between blocking and anti-RBD IgG suggests binding antibodies might not predict neutralizing activity. These findings highlight the temporal nature of CoronaVac-induced immunity and the need for booster doses and variant-adapted vaccine.
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
The immune-epithelial-stromal interactions underpinning intestinal damage in celiac disease (CD) are incompletely understood. To address this, we performed single-cell transcriptomics (RNA sequencing; 86,442 immune, parenchymal and epithelial cells; 35 participants) and spatial transcriptomics (20 participants) on CD intestinal biopsy samples. Here we show that in CD, epithelial populations shifted toward a progenitor state, with interferon-driven transcriptional responses, and perturbation of secretory and enteroendocrine populations. Mucosal T cells showed numeric and functional changes in regulatory and follicular helper-like CD4+ T cells, intraepithelial lymphocytes, CD8+ and γδ T cell subsets, with skewed T cell antigen receptor repertoires. Mucosal changes remained detectable despite treatment, representing a persistent immune-epithelial 'scar'. Spatial transcriptomics defined transcriptional niches beyond those captured in conventional histological scores, including CD-specific lymphoid aggregates containing T cell-B cell interactions. Receptor-ligand spatial analyses integrated with disease susceptibility gene expression defined networks of altered chemokine and morphogen signaling, and provide potential therapeutic targets for CD prevention and treatment.
Hepatitis B virus resistance to nucleos(t)ide analogue therapy: WHO consultation on questions, challenges, and a roadmap for the field.
In this Review, we summarise outputs from a multidisciplinary consultation convened by WHO between July 11 and 13, 2023, to discuss hepatitis B virus (HBV) drug resistance (HBVDR). Treatment of chronic HBV infection with highly effective nucleos(t)ide analogue agents, tenofovir and entecavir, is a crucial intervention that supports the global goal of elimination of HBV infection as a public health threat. The risk of HBVDR as a threat to treatment outcomes is currently considered low from a public health perspective; however, drug resistance can influence individual outcomes, particularly among those who are treatment-experienced. We highlight the need to develop appropriate prevention, monitoring, and surveillance approaches for HBVDR, to support investment in the global scale-up of HBV diagnosis and treatment. Recommendations for the HBVDR field will ultimately be incorporated into a WHO integrated Global Action Plan for drug-resistant HIV, viral hepatitis, and priority sexually transmitted infections.
Treatment options to support the elimination of hepatitis C: an open-label, factorial, randomised controlled non-inferiority trial.
BACKGROUND: WHO recommends treating hepatitis C infection with one of three antiviral combinations for 8-12 weeks. No randomised trials have compared these regimens, and high cure rates might be achievable with shorter durations of therapy. We aimed to compare sofosbuvir-daclatasvir with sofosbuvir-velpatasvir, and to evaluate potential novel treatment strategies. METHODS: We conducted a multi-arm, open-label, randomised controlled non-inferiority trial in two public hospitals in Viet Nam. Adults (aged ≥18 years) with chronic hepatitis C infection and mild-to-moderate liver fibrosis were eligible. Recruitment was stratified by centre and viral genotype (1-5 vs 6) with 1:1 random allocation to an oral fixed-dose combination of sofosbuvir 400 mg plus daclatasvir 60 mg (sofosbuvir-daclatasvir) or sofosbuvir 400 mg plus velpatasvir 100 mg (sofosbuvir-velpatasvir). Participants were simultaneously factorially randomly assigned to one of four treatment strategies: 12 weeks' standard of care (SOC); 4 weeks' therapy with four weekly PEGylated interferon alfa-2a subcutaneous injections; induction and maintenance therapy with 2 weeks' standard therapy followed by 10 weeks' therapy 5 days a week; and response-guided therapy (RGT) for 4, 8, or 12 weeks determined by viral load on day 7. The primary outcome was sustained virological response (SVR) 12 weeks after treatment completion, analysed in all evaluable participants regardless of actual treatment received. We chose a 5% non-inferiority margin for the drug comparison, and a 10% non-inferiority margin for the treatment strategy comparisons. Safety was assessed in all randomised participants. This trial is registered with ISRCTN, 61522291, and is completed. FINDINGS: Between June 19, 2020, and May 10, 2023, 624 participants were randomised (470 [75%] were male and 154 [25%] were female). 296 (47%) had genotype 6 and 328 (53%) had genotypes 1-5. The primary outcome was assessable in 609 (98%) participants. SVR occurred in 294 (97%) of 302 participants in the sofosbuvir-daclatasvir group and 292 (95%) of 307 participants in the sofosbuvir-velpatasvir group (risk difference 2·2%, 90% credible interval [CrI] -0·2 to 4·8, within the 5% non-inferiority margin; 93% probability that sofosbuvir-daclatasvir is superior to sofosbuvir-velpatasvir). SVR occurred in 148 (99%) of 150 in the SOC group, 143 (94%) of 152 in the 4-week antiviral plus interferon group (-4·5%, 90% CrI -8·3 to -1·3), 151 (99%) of 152 in the induction-maintenance group (0·6%, -1·1 to 2·7), and 144 (93%) of 155 in the RGT group (-5·7%, -9·6 to -2·3); all risk differences were within the 10% non-inferiority margin. Serious adverse events were rare (11 [4%] of 313 participants in the sofosbuvir-velpatasvir group vs six [2%] of 311 in the sofosbuvir-daclatasvir group; risk difference -1·6% [95% CrI -4·2 to 0·8]) with no evidence of differences between regimens or strategies, but adverse reactions were very common in the 4-week antiviral plus interferon group compared with the other treatment strategies (risk difference vs SOC group, 66·8% [59·2 to 74·0]; p<0·0001). INTERPRETATION: Sofosbuvir-daclatasvir was non-inferior to sofosbuvir-velpatasvir. High efficacy was seen with novel strategies, which might help to inform approaches to treatment for harder-to-reach populations. FUNDING: Wellcome Trust.
Influence of context on engagement with COVID-19 testing: a scoping review of barriers and facilitators to testing for healthcare workers, care homes and schools in the UK.
OBJECTIVE: The UK government's response to the COVID-19 pandemic included a 'test, trace and isolate' strategy. Testing services for healthcare workers, care homes and schools accounted for the greatest spend and volume of tests. We reviewed relevant literature to identify common and unique barriers and facilitators to engaging with each of these testing services. DESIGN: Scoping review. SEARCH STRATEGY: PubMed, Scopus and the WHO COVID-19 Research Database were searched for evidence published between 1 January 2020 and 7 November 2022. This was supplemented by evidence identified via free-text searches on Google Scholar and provided by the UK Health Security Agency (UKHSA). DATA EXTRACTION AND SYNTHESIS: Data were extracted by a team of reviewers and synthesised thematically under the broad headings of perceptions, experiences, barriers and facilitators to engaging with the COVID-19 testing programme. RESULTS: This study included 40 sources, including 17 from projects that informed UKHSA's decisions during the pandemic. Eight themes emerged and were used to categorise barriers and facilitators to engaging with the testing services for healthcare workers, care homes and schools: (1) perceived value, (2) trust in the tests and public bodies, (3) importance of infrastructure, (4) impact of media and social networks, (5) physical burden of the test, (6) perceived capability to undertake testing, (7) importance of relevant information and 8) consequences of testing. CONCLUSIONS: Universal barriers and facilitators to engagement with the testing programme related to the core elements of each testing service, such as uncomfortable specimen collection and the influence of media and peers; these could be mitigated or leveraged to increase engagement across settings. However, the individuals involved, perceptions of value and available resources differed across services, leading to unique experiences between settings. Thus, consideration of context is crucial when designing and implementing a testing programme in response to a pandemic.
Use of polyethylene glycol precipitation and ultracentrifugation to enhance the sensitivity of hepatitis B virus DNA detection.
BACKGROUND: Sensitive molecular detection of hepatitis B virus (HBV) DNA is crucial for diagnosing and managing occult hepatitis. To improve the sensitivity of HBV DNA detection, we compared the effectiveness of polyethylene glycol (PEG) precipitation and ultracentrifugation to concentrate DNA prior to extraction. METHODS: Twenty-three HBV DNA-positive samples with low viral loads were compared between the extraction of standard (0.2 mL) and larger volumes (5 mL) of plasma, through PEG precipitation of 10 mL and 20 mL of plasma, and ultracentrifugation from 35 mL of plasma. The effectiveness of the methods for HBV DNA detection was assayed by quantitative PCR. For genetic characterisation, Sanger sequencing of amplicons and targeted Illumina sequencing were used. Costs, sample capacities, and turnaround times were compared. RESULTS: DNA was detected in a greater number of samples using PEG and ultracentrifugation (detecting up to all 23 samples) compared to more standard extraction methods (detecting at least 18 samples). Efficiencies of recovery of HBV DNA from samples were comparable in all concentration methods. HBV and other DNA viruses, such as human herpesviruses and anelloviruses, were detected in samples and at higher read counts with PEG concentration than without. The availability, cost, relative simplicity, and throughput of PEG precipitation conferred further advantages to ultracentrifugation. CONCLUSIONS: PEG precipitation from large volumes of plasma is a practical and economical alternative to ultracentrifugation and could be a similarly effective concentration method for low viral load samples in blood donation and clinical virology laboratories.
Mass testing for discovery and control of COVID-19 outbreaks in adult social care: an observational study and cost-effectiveness analysis of 14 805 care homes in England.
INTRODUCTION: We retrospectively evaluated the impact of COVID-19 testing among residents and staff in social care homes in England. METHODS: We obtained 80 million reported PCR and lateral flow device (LFD) test results, from 14 805 care homes (residents and staff) in England, conducted between October 2020 and March 2022. These testing data were then linked to care home characteristics, test costs and 24 500 COVID-19-related deaths of residents. We decomposed the mechanism of outbreak mitigation into outbreak discovery and outbreak control and used Poisson regressions to investigate how reported testing intensity was associated with the size of outbreak discovered and to uncover its association with outbreak control. We used negative binomial regressions to determine the factors influencing COVID-19-related deaths subsequent to outbreaks. We performed a cost-effectiveness analysis of the impact of testing on preventing COVID-19-related deaths of residents. RESULTS: Reported testing intensity generally reflected changes in testing policy over time, although there was considerable heterogeneity among care homes. Client type was the strongest determinant of whether COVID-19-related deaths in residents occurred subsequent to testing positive. Higher staff-to-resident ratios were associated with larger outbreak sizes but rapid outbreak control and a decreased risk of COVID-19-related deaths. Assuming our regression estimates represent causal effects, care home testing in England was cost-effective at preventing COVID-19-related deaths among residents during the pandemic and approximately 3.5 times more cost-effective prior to the vaccine rollout. CONCLUSIONS: PCR and LFD testing was likely an impactful intervention for detecting and controlling COVID-19 outbreaks in care homes in England and cost-effective for preventing COVID-19-related deaths among residents. In future pandemics, testing must be prioritised for care homes, especially if severe illness and death particularly affect older people or individuals with characteristics similar to care home residents, and an efficacious vaccine is unavailable.