Biology subjects

Matthews, P. C.

Publications and source records attributed to Matthews, P. C..

13 recordsLinked to original sources

bioRxiv2023

Pan-genotypic probe-based enrichment to improve efficiency of Hepatitis B virus sequencing

Hepatitis B Virus (HBV) genome sequencing can be used to provide more complete genetic information at the population and individual level to shed light on the limitations of current interventions, and inform new strategies for elimination. HBV sequencing is challenging due to the partially dsDNA genome, high diversity, low viral loads and presence of large amounts of host genetic material in clinical samples. Here we describe the design and use of a pan-genotypic panel of 74 HBV specific capture-probes and nuclease treatment in improving sequencing efficiency. We processed 20 plasma samples (viral loads 1.98 to 4.07 log10, genotypes A-E) and three positive controls (human total brain RNA and bacteriophage lambda DNA) in triplicate to compare DNAse vs. RNAse vs. no nuclease treatment. We prepared libraries using the Takara Bio SMARTer Stranded Total RNA-Seq Kit v3, split the library in two, enriching half with the custom-designed probe panel and xGen Hybridization and Wash Kit (IDT), the other half was not enriched. Both libraries were sequenced on the NovaSeq6000 platform with 2x150nt paired-end reads. Capture resulted in a 47,970 fold increase in the number of reads mapped to the HBV genome in the "no nuclease" arm (243 HBV reads per million reads sequenced in the capture pool vs. 5x10-3 reads per million in the no-capture pool). Out of 20 samples, only 1 without capture generated HBV reads (viral load 3.89 log10 IU/ml) vs. 19 samples with capture. HBV sequence yield was increased in the capture arm and resulted in 2.30 log10 (95% confidence interval 1.99 - 2.48 log10) increase in HBV reads (per million reads sequenced) per log10 increase in viral load. The proportion of HBV reads increased a median of 12 fold with RNAse treatment. We developed a targeted pan-genotypic sequencing method using a custom panel of biotinylated oligos that increases the sequencing efficacy of HBV. This method will allow us to gain a better insight into HBV diversity.

genomics↗

bioRxiv2022

Polymorphisms Predicting Phylogeny in Hepatitis B Virus (HBV)

Hepatitis B viruses (HBV) are compact viruses with circular genomes of [~]3.2kb in length. Four genes (HBx, Core, Surface and Polymerase) generating seven products are encoded on overlapping reading frames. Ten HBV genotypes have been characterised (A-J), which may account for differences in transmission, outcomes of infection, and treatment response. However, HBV genotyping is rarely undertaken, and sequencing remains inaccessible in many settings. We used a machine learning approach based on random forest algorithms (RFA) to assess which amino acid (aa) sites in the genome are most informative for determining genotype. We downloaded 5496 genome-length HBV sequences from a public database, excluding recombinant sequences, regions with conserved indels, and genotypes I/J. Each gene was separately translated into aa, and the proteins concatenated into a single sequence (length 1614aa). Using RFA, we searched for aa sites predictive of genotype, and assessed co-variation among the sites with a Mutual Information (MI)-based method. We were able to discriminate confidently between genotypes A-H using 10 aa sites. 5/10 sites were identified in Polymerase (Pol), of which 4/5 were in the spacer domain, and a single site in reverse transcriptase. A further 4/10 sites were located in Surface protein, and a single site in HBx. There were no informative sites in Core. Properties of the aa were generally not conserved between genotypes at informative sites. Co-variation analysis identified 55 pairs of highly-linked sites. Three RFA-identified sites were represented across all pairs (two sites in spacer, and one in HBx). Residues that co-vary with these sites are concentrated in the small HBV surface gene. We also observe a cluster of sites adjacent to the Surface promoter region that co-vary with a spacer residue. Overall, we have shown that RFA analysis is a powerful tool for identifying aa sites that predict HBV lineage, with an unexpectedly high number of such sites in the spacer domain, which has conventionally been viewed as unimportant for structure or function. Our results improve ease of genotype prediction from limited regions of HBV sequence, and may have implications for understanding HBV evolution and the role of the spacer domain.

genomics↗

bioRxiv2020

Global prevalence and phylogeny of hepatitis B virus (HBV) drug and vaccine resistance mutations

IntroductionVaccination and anti-viral therapy with nucleos(t)ide analogues (NAs) are key approaches to reducing the morbidity, mortality and transmission of hepatitis B virus (HBV) infection. However, the efficacy of these interventions may be reduced by the emergence of drug resistance-associated mutations (RAMs) and/or vaccine escape mutations (VEMs). We have assimilated data on the global prevalence and distribution of HBV RAMs/VEMs from publicly available data and explored the evolution of these mutations. MethodsWe analysed sequences downloaded from the Hepatitis B Virus Database, and calculated prevalence of 41 RAMs and 38 VEMs catalogued from published studies. We generated maximum likelihood phylogenetic trees and used treeBreaker to investigate the distribution of selected mutations across tree branches. We performed phylogenetic molecular clock analyses using BEAST to estimate the age of mutations. ResultsRAM M204I/V had the highest prevalence, occurring in 3.8% (109/2838) of all HBV sequences in our dataset, and a significantly higher rate in genotype C sequence at 5.4% (60/1102, p=0.0007). VEMs had an overall prevalence of 1.3% (37/2837) and had the highest prevalence in genotype C and in Asia at 2.2% (24/1102; p=0.002) and 1.6% (34/2109; p=0.009) respectively. Phylogenetic analysis suggested that most RAM/VEMs arose independently, however RAMs including A194T, M204V and L180M formed clusters in genotype B. We show evidence that polymorphisms associated with drug and vaccine resistance may have been present in the mid 20th century suggesting that they can arise independently of treatment/ vaccine exposure. DiscussionHBV RAMs/VEMs have been found globally and across genotypes, with the highest prevalence observed in genotype C variants. Screening for the genotype and for resistant mutations may help to improve stratified patient treatment. As NAs and HBV vaccines are increasingly being deployed for HBV prevention and treatment, monitoring for resistance and advocating for better treatment regimens for HBV remains essential.

evolutionary biology↗

bioRxiv2019

Analysis of genomic-length HBV sequences to determine genotype and subgenotype reference sequences

Hepatitis B virus (HBV) is a diverse, partially double-stranded DNA virus, with 9 genotypes (A-I), and a putative 10th genotype (J), thus far characterised. Given the broadening interest in HBV sequencing, there is an increasing requirement for a consistent, unified approach to HBV genotype and subgenotype classification. We set out to generate an updated resource of reference sequences using the diversity of all genomic-length HBV sequences available in public databases. We collated and aligned genomic-length HBV sequences from public databases and used maximum-likelihood phylogenetic analysis to identify genotype clusters. Within each genotype, we examined the phylogenetic support for currently defined subgenotypes, as well as identifying well-supported clades and deriving reference sequences for them. An alignment of these reference sequences and maximum-likelihood phylogenetic trees of the sequences are provided to simplify classification. Based on the phylogenies generated, we present a comprehensive set of HBV reference sequences at the genotype and subgenotype level.

microbiology↗

bioRxiv2019

Metagenomic Nanopore sequencing of influenza virus direct from clinical respiratory samples

Influenza is a major global public health threat as a result of its highly pathogenic variants, large zoonotic reservoir, and pandemic potential. Metagenomic viral sequencing offers the potential of a diagnostic test for influenza which also provides insights on transmission, evolution and drug resistance, and simultaneously detects other viruses. We therefore set out to apply Oxford Nanopore Technology to metagenomic sequencing of respiratory samples. We generated influenza reads down to a limit of detection of 102-103 genome copies/ml in pooled samples, observing a strong relationship between the viral titre and the proportion of influenza reads (p = 4.7x10-5). Applying our methods to clinical throat swabs, we generated influenza reads for 27/27 samples with high-to-mid viral titres (Cycle threshold (Ct) values <30) and 6/13 samples with low viral titres (Ct values 30-40). No false positive reads were generated from 10 influenza-negative samples. Thus Nanopore sequencing operated with 83% sensitivity (95% CI 67-93%) and 100% specificity (95% CI 69-100%) compared to the current diagnostic standard. Coverage of full length virus was dependent on sample composition, being negatively influenced by increased host and bacterial reads. However, at high influenza titres, we were able to reconstruct >99% complete sequence for all eight gene segments. We also detected Human Coronavirus and generated a near complete Human Metapneumovirus genome from clinical samples. While further optimisation is required to improve sensitivity, this approach shows promise for the Nanopore platform to be used in the diagnosis and genetic analysis of influenza and other respiratory viruses.

microbiology↗

bioRxiv2019

HBV seroepidemiology data for Africa provides insights into transmission and prevention

International goals for elimination of hepatitis B virus (HBV) infection set ambitious targets for 2030. In many African populations, HBV prevalence remains high ([≥]8%) despite the roll-out of infant HBV immunisation from the mid-1990s onwards. Enhanced efforts are now urgently required to improve an understanding of population epidemiology, in order to determine which interventions are most likely to be effective in advancing populations towards elimination goals. In populations with a high prevalence of infection, catch-up HBV vaccination of adults has sometimes been deployed as a preventive strategy. An alternative approach of test and treat could be applied as a tool to interrupt transmission. We used a systematic approach to investigate the relationship between prevalence of HBV infection (HBsAg) and exposure (anti-HBc) in Africa, and then applied a mathematical model to investigate the impact of catch-up vaccination and a test and treat strategy in Uganda, representing a high prevalence setting. We demonstrate a strong relationship between the prevalence of HBsAg and anti-HBc (p<0.0001), but with region-specific differences that may reflect different patterns of transmission. In high prevalence settings, catch-up vaccination may have a transient effect but this intervention does not contribute to a sustained decline in prevalence. In contrast, diagnosing and treating infection has a marked impact on reducing prevalence, equivalent to that of infant immunisation. Conclusion: We have developed a high-resolution picture of HBV epidemiology across Africa. Developing insights into regional differences provides an evidence base for the most effective interventions. In combination with robust neonatal immunisation programmes, testing and treating infection is likely to be of most impact in making advances towards elimination targets.

epidemiology↗

bioRxiv2018

Nanopore metagenomic sequencing of full length human metapneumovirus (HMPV) within a unique sub-lineage

Human metapneumovirus (HMPV) has been recognized as an important pathogen which can cause a spectrum of respiratory tract disease. Here, we report Nanopore metagenomic sequencing of the first full length HMPV genome directly from a throat swab from a UK patient with complex lung disease and immunocompromise. We found a predominance (26.4%) of HMPV reads in the metagenomic sequencing data and consequently assembled the full genome at a high depth of coverage (mean 4,786). Through phylogenetic analyses, we identified this HMPV strain to originate from a unique genetic group in A2b, showing the presence of this group in the UK. Our study demonstrated the effectiveness of Nanopore metagenomic sequencing for diagnosing infectious diseases and recovering complete sequences for genomic characterization, highlighting the applicability of Nanopore sequencing in clinical settings.\n\nImportanceNanopore metagenomic sequencing has the potential to evolve as a point-of-care test for a range of infectious diseases. Here, we report the first full length human metapneumovirus (HMPV) genome in the UK sequenced by Nanopore from a non-invasive sample from an immunocompromised patient. We demonstrate the presence of HMPV from a unique genetic group not previously reported from the UK. Our study demonstrates the effectiveness of Nanopore sequencing for diagnosing an infection that was not detected by routine first-line tests in the clinical microbiology laboratory. We report sufficient genomic data to provide insight into the epidemiology of infection and with the potential to inform treatment decisions.

microbiology↗

bioRxiv2018

Data from an electronic health informatics pipeline to describe clearance dynamics of Hepatitis B surface antigen (HBsAg) and e-Antigen (HBeAg) in chronic HBV infection

HBsAg and HBeAg have gained traction as biomarkers of control and clearance during monitoring of chronic hepatitis B virus infection (CHB). An improved understanding of the correlates of clearance of these proteins could help inform improvements in patient-stratified care and advance insights into the underlying mechanisms of disease control, thus underpinning new cure strategies. We collected electronic clinical data via an electronic pipeline supported by the National Institute for Health Research Health Informatics Collaborative (NIHR-HIC), adopting an unbiased approach to generating a robust longitudinal dataset for adults testing HBsAg-positive from a large UK teaching hospital over a six year period (2011-2016 inclusive). From 553 individuals with CHB, longitudinal data were available for 319, representing >107,000 weeks of clinical follow-up. Among these 319 individuals, 13 (4%) cleared HBsAg completely. HBsAg clearance rate was similar in individuals on NA therapy (n=4, median clearance time 150 weeks) vs those not on NA therapy (n=9, median clearance time 157 weeks). Those who cleared HBsAg were significantly older, and less likely to be on NA therapy compared to non-clearers (p=0.003 and p=0.001, respectively). Chinese ethnicity was associated with HBeAg positivity (p=0.025). HBeAg clearance occurred both on NA therapy (n=24, median time 49 weeks) and off NA therapy (n=19, median time 52 weeks). Improved insights into the dynamics of these biomarkers can underpin better prognostication and patient-stratified care. Our systematised approach to data collection paves the way for scaling up efforts to harness clinical data to address research questions and underpin improvements in clinical care provision.\n\nIMPORTANCEAdvances in the diagnosis, monitoring and treatment of hepatitis B virus (HBV) infection are urgently required if we are to meet international targets for elimination by the year 2030. Here we demonstrate how routine clinical data can be harnessed through an unbiased electronic pipeline, showcasing the significant potential for amassing large clinical datasets that can help to inform advances in patient care, and provide clues that inform new cure strategies. Our cohort from a large UK hospital includes adults from diverse ethnic groups that have previously been under-represented in the literature. Tracking two protein biomarkers that are used to monitor chronic HBV infection, we provide new insights into the timelines of HBV clearance, both on and off treatment. These results contribute to improvements in individualised clinical care and may provide important clues into the immune events that underpin disease control.

microbiology↗

bioRxiv2018

Modelling cost-effectiveness of tenofovir for prevention of mother to child transmission of hepatitis B virus infection in South Africa

In light of sustainable development goals for 2030, an important priority for Africa is to have affordable, accessible and sustainable hepatitis B virus (HBV) prevention of mother to child transmission (PMTCT) programmes, delivering screening and treatment for antenatal women and implementing timely administration of HBV vaccine for their babies. We developed a decision-analytic model simulating 10,000 singleton pregnancies to assess the cost-effectiveness of three possible strategies for deployment of tenofovir in pregnancy, in combination with routine infant vaccination: S1: no screening nor antiviral therapy; S2: screening and antiviral prophylaxis for all women who test HBsAg-positive; S3: screening for HBsAg, followed by HBeAg testing and antiviral prophylaxis for women who are HBsAg-positive and HBeAg-positive. Our outcome was cost per infant HBV infection avoided and the analysis followed a healthcare perspective. S1 predicts 45 infants would be HBV-infected at six months of age, compared to 21 and 28 infants in S2 and S3, respectively. Relative to S1, S2 had an incremental cost of $3,940 per infection avoided. S3 led to more infections and higher costs. Given the long-term health burden for individuals and economic burden for society associated with chronic HBV infection, screening pregnant women and providing tenofovir for all who test HBsAg+ may be a cost-effective strategy for South Africa.

epidemiology↗

bioRxiv2018

Illumina and Nanopore methods for whole genome sequencing of hepatitis B virus (HBV)

Advancing interventions to tackle the huge global burden of hepatitis B virus (HBV) infection depends on improved insights into virus epidemiology, transmission, within-host diversity, drug resistance and pathogenesis, all of which can be facilitated by the large-scale generation of full-length virus genome data. Here we describe advances to a protocol to exploit the circular HBV genome structure, using isothermal rolling-circle amplification to enrich for HBV DNA and to generate concatemeric amplicons containing multiple successive copies of the same genome. We show that this product is suitable for Nanopore sequencing as single reads, as well as for generating short-read Illumina sequences. Nanopore reads can be used to implement a straightforward method for error correction that reduces the per-read error rate, by comparing multiple genome copies combined into a single concatemer and by comparing reads generated from plus and minus strands. Thus we can achieve improved consensus sequencing accuracy of 99.7% and resolve intra-sample sequence variants to form whole-genome haplotypes. The combination of isothermal amplification and Nanopore sequencing offers the longer-term potential to develop point-of-care tests for HBV, which could also be adapted for other viruses.

genomics↗

bioRxiv2018

A systematic review of Hepatitis B virus (HBV) drug and vaccine escape mutations in Africa: a call for urgent action

International sustainable development goals for the elimination of viral hepatitis as a public health problem by 2030 highlight the pressing need to optimize strategies for prevention, diagnosis and treatment. Selected or transmitted resistance associated mutations (RAMs) and vaccine escape mutations (VEMs) in hepatitis B virus (HBV) may reduce the success of existing treatment and prevention strategies. These issues are particularly pertinent for many settings in Africa where there is high HBV prevalence and co-endemic HIV infection, but lack of robust epidemiological data and limited education, diagnostics and clinical care. The prevalence, distribution and impact of RAMs and VEMs in these populations are neglected in the current literature. We therefore set out to assimilate data for sub-Saharan Africa through a systematic literature review and analysis of published sequence data, and present these in an on-line database (https://livedataoxford.shinyapps.io/1510659619-3Xkoe2NKkKJ7Drg/). The majority of the data were from HIV/HBV coinfected cohorts. The commonest RAM was rtM204I/V, either alone or in combination with compensatory mutations, and identified in both reportedly treatment-naive and treatment-experienced adults. We also identified the suite of mutations rtM204V/I + rtL180M + rtV173L, that has been associated with vaccine escape, in over 1/3 of cohorts. Although tenofovir has a high genetic barrier to resistance, it is of concern that emerging data suggest polymorphisms that may be associated with resistance, although the precise clinical impact of these is unknown. Overall, there is an urgent need for improved diagnostic screening, enhanced laboratory assessment of HBV before and during therapy, and sustained roll out of tenofovir in preference to lamivudine alone. Further data are needed in order to inform population and individual approaches to HBV diagnosis, monitoring and therapy in these highly vulnerable settings.\n\nAuthors summaryThe Global Hepatitis Health Sector Strategy is aiming for the elimination of viral hepatitis as a public health threat by 2030. However, mutations associated with drug resistance and vaccine escape may reduce the success of existing treatment and prevention strategies. In the current literature, the prevalence, distribution and impact of hepatitis B virus (HBV) mutations in many settings in Africa are neglected, despite the high prevalence of HBV and co-endemic HIV infection. This systematic review describes the frequency, prevalence and co-occurrence of mutations associated with HBV drug resistance and vaccine escape mutations in Africa. The findings suggest a high prevalence of these mutations in some populations in sub-Saharan Africa. Scarce resources have contributed to the lack of HBV diagnostic screening, inconsistent supply of drugs, and poor access to clinical monitoring, all of which contribute to drug and vaccine resistance. Sustainable long-term investment is required to expand consistent drug and vaccine supply, to provide screening to diagnose infection and to detect drug resistance, and to provide appropriate targeted clinical monitoring for treated patients.

epidemiology↗

bioRxiv2017

Hepatitis B Virus Infection as a Neglected Tropical Disease

BACKGROUND BACKGROUND CURRENT STRATEGIES FOR HBV... APPLICATION OF NTD CRITERIA... RECOMMENDATIONS BASED ON NTD... CONCLUSIONS SUPPORTING INFORMATION LEGEND REFERENCES The Global Hepatitis Health Sector Strategy is aiming for elimination of viral hepatitis as a public health threat by 2030 [1], while enhanced elimination efforts for hepatitis are also promoted under the broader remit of global Sustainable Development Goals (SDGs) [2]. This is an enormous challenge for hepatitis B virus (HBV) given the estimated global burden of 260 million chronic carriers, of whom the majority are unaware of their infection [3] (Figure 1).\n\nWe here present HBV within the framework for ...

microbiology↗

bioRxiv2017

Utilising a Cohort Study of Hepatitis B Virus (HBV) Vaccine-Mediated Immunity in South African Children to Model Infection Dynamics: Can We Meet Global Targets for Elimination by 2030?

BackgroundSustainable Development Goals set a challenge for the elimination of hepatitis B virus (HBV) infection as a public health concern by the year 2030. Deployment of a robust prophylactic vaccine and enhanced interventions for prevention of mother to child transmission (PMTCT) are cornerstones of elimination strategy. However, in light of the estimated global burden of 290 million cases, enhanced efforts are required to underpin optimisation of public health strategy. Robust analysis of population epidemiology is particularly crucial for populations in Africa made vulnerable by HIV co-infection, poverty, stigma and poor access to prevention, diagnosis and treatment.\n\nMethodsWe here set out to evaluate the current and future role of HBV vaccination and PMTCT as tools for elimination. We first investigated the current impact of paediatric vaccination in a cohort of children with and without HIV infection in Kimberley, South Africa. Second, we used these data to inform a new model to simulate the ongoing impact of preventive interventions. By applying these two approaches in parallel, we are able to determine both the current impact of interventions, and the future projected outcome of ongoing preventive strategies over time.\n\nResultsExisting efforts have been successful in reducing paediatric prevalence of HBV infection in this setting to <1%, demonstrating the success of the existing vaccine campaign. Our model predicts that, if consistently deployed, combination efforts of vaccination and PMTCT can significantly reduce population prevalence (HBsAg) by 2030, such that a major public health impact is possible even without achieving elimination. However, the prevalence of HBV e-antigen (HBeAg)-positive carriers will decline more slowly, representing a persistent population reservoir. We show that HIV co-infection significantly reduces titres of vaccine-mediated antibody, but has a relatively minor role in influencing the projected time to elimination. Our model can also be applied to other settings in order to predict time to elimination based on specific interventions.\n\nConclusionsThrough extensive deployment of preventive strategies for HBV, significant positive public health impact is possible, although time to HBV elimination as a public health concern is likely to be substantially longer than that proposed by current goals.

microbiology↗