Ultrasensitive PCR system for HBV DNA detection: Risk stratification for occult hepatitis B virus infection in English blood donors

Occult hepatitis B (HBV) infection (OBI), characterized by low viral loads, accounts for much of the risk of HBV transfusion-transmitted infection. With anticore antibodies (anti-HBc) screening introduced in England, the imperative to identify OBI donors has increased. We aimed to develop an ultra-sensitive PCR system and investigate risk factors for HBV DNA presence in blood donations. Seven extraction methods and three PCR assays were compared. The optimal system was sought to determine HBV DNA presence in anti-HBc-positive donations. Predictors of DNA positivity were subsequently investigated. Extraction from 5 mL of plasma increased sample representation and resulted in HBV DNA detection in low viral load samples (~0.5 IU/mL). Screening of 487 763 donations in 2022 identified two OBI donors and 2042 anti-HBc-positive donors, 412 of the latter with anti-HBs < 100 mIU/mL. Testing of 134 anti-HBc-positive donations utilizing the 5 mL extraction method identified two further HBV DNA-positive donations. Higher anti-HBc titer and anti-HBs negativity were significant predictors of DNA detectability in anti-HBc-positive donations. An ultrasensitive PCR assay identified potentially infectious donations increasing HBV DNA detection in anti-HBc-positive donors from 0.5% to 1.9%. Anti-HBc titers may further complement the risk stratification for DNA positivity in anti-HBc screening and minimize unnecessary donor deferral

Disparities in care and outcomes for primary liver cancer in England during 2008–2018: a cohort study of 8.52 million primary care population using the QResearch database

Background: Liver cancer has one of the fastest rising incidence and mortality rates among all cancers in the UK, but it receives little attention. This study aims to understand the disparities in epidemiology and clinical pathways of primary liver cancer and identify the gaps for early detection and diagnosis of liver cancer in England. Methods: This study used a dynamic English primary care cohort of 8.52 million individuals aged ≥25 years in the QResearch database during 2008–2018, followed up to June 2021. The crude and age-standardised incidence rates, and the observed survival duration were calculated by sex and three liver cancer subtypes, including hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (CCA), and other specified/unspecified primary liver cancer. Regression models were used to investigate factors associated with an incident diagnosis of liver cancer, emergency presentation, late stage at diagnosis, receiving treatments, and survival duration after diagnosis by subtype. Findings: 7331 patients were diagnosed with primary liver cancer during follow-up. The age-standardised incidence rates increased over the study period, particularly for HCC in men (increased by 60%). Age, sex, socioeconomic deprivation, ethnicity, and geographical regions were all significantly associated with liver cancer incidence in the English primary care population. People aged ≥80 years were more likely to be diagnosed through emergency presentation and in late stages, less likely to receive treatments and had poorer survival than those aged <60 years. Men had a higher risk of being diagnosed with liver cancer than women, with a hazard ratio (HR) of 3.9 (95% confidence interval 3.6–4.2) for HCC, 1.2 (1.1–1.3) for CCA, and 1.7 (1.5–2.0) for other specified/unspecified liver cancer. Compared with white British, Asians and Black Africans were more likely to be diagnosed with HCC. Patients with higher socioeconomic deprivation were more likely to be diagnosed through the emergency route. Survival rates were poor overall. Patients diagnosed with HCC had better survival rates (14.5% at 10-year survival, 13.1%–16.0%) compared to CCA (4.4%, 3.4%–5.6%) and other specified/unspecified liver cancer (12.5%, 10.1%–15.2%). For 62.7% of patients with missing/unknown stage in liver cancer, their survival outcomes were between those diagnosed in Stages III and IV. Interpretation: This study provides an overview of the current epidemiology and the disparities in clinical pathways of primary liver cancer in England between 2008 and 2018. A complex public health approach is needed to tackle the rapid increase in incidence and the poor survival of liver cancer. Further studies are urgently needed to address the gaps in early detection and diagnosis of liver cancer in England. Funding: The Early Detection of Hepatocellular Liver Cancer (DeLIVER) project is funded by Cancer Research UK (Early Detection Programme Award, grant reference: C30358/A29725)

Interferon-λ rs12979860 genotype and liver fibrosis in viral and non-viral chronic liver disease

Tissue fibrosis is a core pathologic process that contributes to mortality in ∼45% of the population and is likely to be influenced by the host genetic architecture. Here we demonstrate, using liver disease as a model, that a single-nucleotide polymorphism (rs12979860) in the intronic region of interferon-λ4 (IFNL4) is a strong predictor of fibrosis in an aetiology-independent manner. In a cohort of 4,172 patients, including 3,129 with chronic hepatitis C (CHC), 555 with chronic hepatitis B (CHB) and 488 with non-alcoholic fatty liver disease (NAFLD), those with rs12979860CC have greater hepatic inflammation and fibrosis. In CHC, those with rs12979860CC also have greater stage-constant and stage-specific fibrosis progression rates (P<0.0001 for all). The impact of rs12979860 genotypes on fibrosis is maximal in young females, especially those with HCV genotype 3. These findings establish rs12979860 genotype as a strong aetiology-independent predictor of tissue inflammation and fibrosis

Irreversible Pulmonary Hypertension Associated with the use of Interferon Alpha for Chronic Hepatitis C

The interferons are a complex group of virally induced proteins produced by activated macrophages and lymphocytes, which have become the mainstay of therapy for hepatitis C infection. Sustained viral response (SVR) rates in noncirrhotic patients vary from 40–80% with interferon-based therapy. This, along with transplantation, has drastically changed the course of hepatitis C virus (HCV) infection over the last two decades. Numerous side effects associated with interferon therapy have been reported. These range from transient flu-like symptoms to serious effects such as cardiac arrhythmias, cardiomyopathy, renal and liver failure, polyneuropathy, and myelosuppression. Pulmonary side effects including pneumonitis, pulmonary fibrosis, and reversible pulmonary hypertension have been reported. Herein, we present four cases in which irreversible pulmonary hypertension was diagnosed after prolonged treatment with interferon alpha. In each case, other causes of pulmonary hypertension were systematically eliminated. Pulmonary artery hypertension, which may be irreversible, should be considered in patients being treated with interferon alpha who present with exertional dyspnea and do not have a readily identifiable inflammatory or thromboembolic cause

Real-World Outcomes of Direct-Acting Antiviral Treatment and Retreatment in United Kingdom–Based Patients Infected With Hepatitis C Virus Genotypes/Subtypes Endemic in Africa

Abstract Background Chronic hepatitis C virus (HCV) infection affects 71 million individuals, mostly residing in low- and middle-income countries (LMICs). Direct-acting antivirals (DAAs) give high rates of sustained virological response (SVR) in high-income countries where a restricted range of HCV genotypes/subtypes circulate. Methods We studied United Kingdom–resident patients born in Africa to examine DAA effectiveness in LMICs where there is far greater breadth of HCV genotypes/subtypes. Viral genome sequences were determined from 233 patients. Results Full-length viral genomic sequences for 26 known subtypes and 5 previously unidentified isolates covering 5 HCV genotypes were determined. From 149 patients who received DAA treatment/retreatment, the overall SVR was 93%. Treatment failure was associated primarily with 2 subtypes, gt1l and gt4r, using sofosbuvir/ledipasvir. These subtypes contain natural resistance-associated variants that likely contribute to poor efficacy with this drug combination. Treatment failure was also significantly associated with hepatocellular carcinoma. Conclusions DAA combinations give high SVR rates despite the high HCV diversity across the African continent except for subtypes gt1l and gt4r, which respond poorly to sofosbuvir/ledipasvir. These subtypes are widely distributed across Western, Central, and Eastern Africa. Thus, in circumstances where accurate genotyping is absent, ledipasvir and its generic compounds should not be considered as a recommended treatment option. </jats:sec

Interferon lambda 4 impacts the genetic diversity of hepatitis C virus

Hepatitis C virus (HCV) is a highly variable pathogen that frequently establishes chronic infection. This genetic variability is affected by the adaptive immune response but the contribution of other host factors is unclear. Here, we examined the role played by interferon lambda-4 (IFN-λ4) on HCV diversity; IFN-λ4 plays a crucial role in spontaneous clearance or establishment of chronicity following acute infection. We performed viral genome-wide association studies using human and viral data from 485 patients of white ancestry infected with HCV genotype 3a. We demonstrate that combinations of host genetic variants, which determine IFN-λ4 protein production and activity, influence amino acid variation across the viral polyprotein - not restricted to specific viral proteins or HLA restricted epitopes - and modulate viral load. We also observed an association with viral di-nucleotide proportions. These results support a direct role for IFN-λ4 in exerting selective pressure across the viral genome, possibly by a novel mechanism

Dominance of variant A in Human Herpesvirus 6 viraemia after renal transplantation

<p>Abstract</p> <p>Background</p> <p>Human herpesvirus 6 (HHV-6), mostly variant B reactivation in renal transplant patients has been published by other authors, but the pathogenetic role of HHV-6 variant A has not been clarified. Our aims were to examine the prevalence of HHV-6, to determine the variants, and to investigate the interaction between HHV-6 viraemia, human cytomegalovirus (HCMV) infection and clinical symptoms.</p> <p>Methods</p> <p>Variant-specific HHV-6 nested PCR and quantitative real-time PCR were used to examine blood samples from renal transplant patients and healthy blood donors for the presence and load of HHV-6 DNA and to determine the variants. Active HHV-6 infection was proved by RT-PCR, and active HCMV infection was diagnosed by pp65 antigenaemia test.</p> <p>Results</p> <p>HHV-6 viraemia was significantly more frequent in renal transplant patients compared to healthy blood donors (9/200 vs. 0/200; p = 0.004), while prevalence of HHV-6 latency was not significantly different (13/200 vs. 19/200; p > 0.05). Dominance of variant A was revealed in viraemias (8/9), and the frequency of HHV-6A was significantly higher in active infections compared with latency in renal transplant patients (8/9 vs. 2/13; p = 0.0015). Latency was established predominantly by HHV-6B both in renal transplant patients and in healthy blood donors (11/13 and 18/19). There was no statistical significant difference in occurrence of HCMV and HHV-6 viraemia in renal transplant patients (7/200 vs. 9/200). Statistical analysis did not reveal interaction between HHV-6 viraemia and clinical symptoms in our study.</p> <p>Conclusions</p> <p>Contrary to previous publications HHV-6A viraemia was found to be predominant in renal transplant patients. Frequency of variant A was significantly higher in cases of active infection then in latency.</p

An analysis of national target groups for monovalent 2009 pandemic influenza vaccine and trivalent seasonal influenza vaccines in 2009-10 and 2010-11

<p>Abstract</p> <p>Background</p> <p>Vaccination is generally considered to be the best primary prevention measure against influenza virus infection. Many countries encourage specific target groups of people to undertake vaccination, often with financial subsidies or a priority list. To understand differential patterns of national target groups for influenza vaccination before, during and after the 2009 influenza pandemic, we reviewed and analyzed the country-specific policies in the corresponding time periods.</p> <p>Methods</p> <p>Information on prioritized groups targeted to receive seasonal and pandemic influenza vaccines was derived from a multi-step internet search of official health department websites, press releases, media sources and academic journal articles. We assessed the frequency and consistency of targeting 20 different groups within populations which are associated with age, underlying medical conditions, role or occupations among different countries and vaccines. Information on subsidies provided to specific target groups was also extracted.</p> <p>Results</p> <p>We analyzed target groups for 33 (seasonal 2009 and 2009-10 vaccines), 72 (monovalent pandemic 2009-10 vaccine) and 34 (seasonal 2010 and 2010-11 vaccines) countries. In 2009-10, the elderly, those with chronic illness and health care workers were common targets for the seasonal vaccine. Comparatively, the elderly, care home residents and workers, animal contacts and close contacts were less frequently targeted to receive the pandemic vaccine. Pregnant women, obese persons, essential community workers and health care workers, however, were more commonly targeted. After the pandemic, pregnant women, obese persons, health care and care home workers, and close contacts were more commonly targeted to receive the seasonal vaccine compared to 2009-10, showing continued influence from the pandemic. Many of the countries provided free vaccines, partial subsidies, reimbursements or national health insurance coverage to specific target groups and over one-third of the countries offered universal subsidy regarding the pandemic vaccine. There was also some inconsistency between countries in target groups.</p> <p>Conclusions</p> <p>Differences in target groups between countries may reflect variable objectives as well as uncertainties regarding the transmission dynamics, severity and age-specific immunity against influenza viruses before and after vaccination. Clarification on these points is essential to elucidate optimal and object-oriented vaccination strategies.</p

Selection of Anti-Sulfadimidine Specific ScFvs from a Hybridoma Cell by Eukaryotic Ribosome Display

BACKGROUND:Ribosome display technology has provided an alternative platform technology for the development of novel low-cost antibody based on evaluating antibiotics derived residues in food matrixes. METHODOLOGY/PRINCIPAL FINDINGS:In our current studies, the single chain variable fragments (scFvs) were selected from hybridoma cell lines against sulfadimidine (SM(2)) by using a ribosome library technology. A DNA library of scFv antibody fragments was constructed for ribosome display, and then mRNA-ribosome-antibody (MRA) complexes were produced by a rabbit reticulocyte lysate system. The synthetic sulfadimidine-ovalbumin (SM(2)-OVA) was used as an antigen to pan MRA complexes and putative scFv-encoding genes were recovered by RT-PCR in situ following each panning. After four rounds of ribosome display, the expression vector pCANTAB5E containing the selected specific scFv DNA was constructed and transformed into Escherichia coli HB2151. Three positive clones (SAS14, SAS68 and SAS71) were screened from 100 clones and had higher antibody activity and specificity to SM(2) by indirect ELISA. The three specific soluble scFvs were identified to be the same molecular weight (approximately 30 kDa) by Western-blotting analysis using anti-E tag antibodies, but they had different amino acids sequence by sequence analysis. CONCLUSIONS/SIGNIFICANCE:The selection of anti-SM(2) specific scFv by in vitro ribosome display technology will have an important significance for the development of novel immunodetection strategies for residual veterinary drugs