ORCID Profile
0000-0003-2790-8353
Current Organisation
University of Oxford
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Publisher: Springer Science and Business Media LLC
Date: 08-05-2018
DOI: 10.1038/S41598-018-25559-6
Abstract: Hepatitis C virus (HCV)-specific T cell responses are critical for immune control of infection. Viral adaptation to these responses, via mutations within regions of the virus targeted by CD8 + T cells, is associated with viral persistence. However, identifying viral adaptation to HCV-specific CD4 + T cell responses has been difficult although key to understanding anti-HCV immunity. In this context, HCV sequence and host genotype from a single source HCV genotype 1B cohort (n = 63) were analyzed to identify viral changes associated with specific human leucocyte antigen (HLA) class II alleles, as these variable host molecules determine the set of viral peptides presented to CD4 + T cells. Eight sites across the HCV genome were associated with HLA class II alleles implicated in infection outcome in this cohort (p ≤ 0.01 Fisher’s exact test). We extended this analysis to chronic HCV infection (n = 351) for the common genotypes 1A and 3A. Variation at 38 sites across the HCV genome were associated with specific HLA class II alleles with no overlap between genotypes, suggestive of genotype-specific T cell targets, which has important implications for vaccine design. Here we show evidence of HCV adaptation to HLA class II-restricted CD4 + T cell pressure across the HCV genome in chronic HCV infection without a priori knowledge of CD4 + T cell epitopes.
Publisher: Springer Science and Business Media LLC
Date: 08-05-2019
DOI: 10.1038/S41598-019-43524-9
Abstract: Advancing interventions to tackle the huge global burden of hepatitis B virus (HBV) infection depends on improved insights into virus epidemiology, transmission, within-host ersity, drug resistance and pathogenesis, all of which can be advanced through the large-scale generation of full-length virus genome data. Here we describe advances to a protocol that exploits the circular HBV genome structure, using isothermal rolling-circle lification to enrich HBV DNA, generating concatemeric licons 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 analysing reads generated from plus and minus strands. With this approach, we can achieve an improved consensus sequencing accuracy of 99.7% and resolve intra-s le sequence variants to form whole-genome haplotypes. Thus while Illumina sequencing may still be the most accurate way to capture within-s le ersity, Nanopore data can contribute to an understanding of linkage between polymorphisms within in idual virions. The combination of isothermal lification and Nanopore sequencing also offers appealing potential to develop point-of-care tests for HBV, and for other viruses.
Publisher: Cold Spring Harbor Laboratory
Date: 11-05-2021
DOI: 10.1101/2021.05.11.21256877
Abstract: Treatment of severe COVID-19 is currently limited by clinical heterogeneity and incomplete understanding of potentially druggable immune mediators of disease. To advance this, we present a comprehensive multi-omic blood atlas in patients with varying COVID-19 severity and compare with influenza, sepsis and healthy volunteers. We identify immune signatures and correlates of host response. Hallmarks of disease severity revealed cells, their inflammatory mediators and networks as potential therapeutic targets, including progenitor cells and specific myeloid and lymphocyte subsets, features of the immune repertoire, acute phase response, metabolism and coagulation. Persisting immune activation involving AP-1 38MAPK was a specific feature of COVID-19. The plasma proteome enabled sub-phenotyping into patient clusters, predictive of severity and outcome. Tensor and matrix decomposition of the overall dataset revealed feature groupings linked with disease severity and specificity. Our systems-based integrative approach and blood atlas will inform future drug development, clinical trial design and personalised medicine approaches for COVID-19.
Publisher: Centers for Disease Control and Prevention (CDC)
Date: 04-2016
Publisher: eLife Sciences Publications, Ltd
Date: 03-09-2019
DOI: 10.7554/ELIFE.42463
Abstract: 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 ersity 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.
Publisher: Cold Spring Harbor Laboratory
Date: 15-02-2023
DOI: 10.1101/2023.02.12.23285726
Abstract: Respiratory syncytial virus (RSV) is the leading cause of hospitalisation associated with acute respiratory infection in infants and young children, with substantial disease burden globally. The impact of additional respiratory pathogens on RSV disease severity is not completely understood. The objective of this study was to explore the associations between RSV disease severity and the presence of other respiratory pathogens. Nasopharyngeal swabs were prospectively collected from two infant cohorts: a prospective longitudinal birth cohort study and an infant cross-sectional study recruiting infants year of age with RSV infection in Spain, the UK, and the Netherlands during 2017–20 [part of the REspiratory Syncytial virus Consortium in EUrope (RESCEU) project]. The s les were sequenced using targeted metagenomic sequencing with a probe set optimised for high-resolution capture of sequences of over 100 pathogens, including all common respiratory viruses and bacteria. Viral genomes and bacterial genetic sequences were reconstructed. Associations between clinical severity and presence of other pathogens were evaluated after adjusting for potential confounders, including age, gestational age, RSV viral load, and presence of comorbidities. RSV was detected in 433 infants. Nearly one in four of the infants (24%) harboured at least one additional non-RSV respiratory virus, with human rhinovirus being the most frequently detected (15% of the infants), followed by seasonal coronaviruses (4%). In this cohort, RSV-infected infants harbouring any other virus tended to be older (median age: 4.3 vs. 3.7 months) and were more likely to require intensive care and mechanical ventilation than those who did not. Moraxella, Streptococcus , and Haemophilus species were the most frequently identified target bacteria, together found in 392 (91%) of the 433 infants ( S. pneumoniae in 51% of the infants and H. influenzae in 38%). The strongest contributors to severity of presentation were younger age and the co-detection of Haemophilus species alongside RSV. Across all age groups in both cohorts, detection of Haemophilus species was associated with higher overall severity, as captured by ReSVinet scores, and specifically with increased rates of hospitalisation and respiratory distress. In contrast, presence of Moraxella species was associated with lower ReSVinet scores and reduced need for intensive care and mechanical ventilation. Infants with and without Streptococcus species (or S. pneumoniae in particular) had similar clinical outcomes. No specific RSV strain was associated with co-detection of other pathogens. Our findings provide strong evidence for associations between RSV disease severity and the presence of additional respiratory viruses and bacteria. The associations, while not indicating causation, are of potential clinical relevance. Awareness of coexisting microorganisms could inform therapeutic and preventive measures to improve the management and outcome of RSV-infected infants.
Publisher: F1000 Research Ltd
Date: 25-01-2021
DOI: 10.12688/WELLCOMEOPENRES.16157.2
Abstract: Deep sequencing of the full-length hepatitis B virus (HBV) genome provides the opportunity to determine the extent to which viral ersity, genotype, polymorphisms, insertions and deletions may influence presentation and outcomes of disease. Increasing experience with analysis of HBV genomic data opens up the potential for using these data to inform insights into pathophysiology of infection and to underpin decision making in clinical practice. We here set out to undertake whole genome HBV sequencing from an adult who presented acutely unwell with a new diagnosis of HBV infection, and tested positive for both HBV anti-core IgM and IgG, possibly representing either acute hepatitis B infection (AHB) or chronic hepatitis B with an acute reactivation (CHB-AR). The distinction between these two scenarios may be important in predicting prognosis and underpinning treatment decisions, but can be challenging based on routine laboratory tests. Through application of deep whole-genome sequencing we typed the isolate as genotype-D1, and identified several minority variants including G1764A and G1986A substitutions in the pre-core promoter and pre-core regions, which support CHB-AR rather than AHB. In the longer term, enhanced deep sequencing data for HBV may provide improved evidence to distinguish between acute and chronic infection, to predict outcomes and to stratify treatment.
Publisher: American Society for Microbiology
Date: 10-2016
DOI: 10.1128/JCM.00330-16
Abstract: Affordable next-generation sequencing (NGS) technologies for hepatitis C virus (HCV) may potentially identify both viral genotype and resistance genetic motifs in the era of directly acting antiviral (DAA) therapies. This study compared the ability of high-throughput NGS methods to generate full-length, deep, HCV sequence data sets and evaluated their utility for diagnostics and clinical assessment. NGS methods using (i) unselected HCV RNA (metagenomics), (ii) preenrichment of HCV RNA by probe capture, and (iii) HCV pre lification by PCR implemented in four United Kingdom centers were compared. Metrics of sequence coverage and depth, quasispecies ersity, and detection of DAA resistance-associated variants (RAVs), mixed HCV genotypes, and other coinfections were compared using a panel of s les with different viral loads, genotypes, and mixed HCV genotypes/subtypes [geno(sub)types]. Each NGS method generated near-complete genome sequences from more than 90% of s les. Enrichment methods and PCR pre lification generated greater sequence depth and were more effective for s les with low viral loads. All NGS methodologies accurately identified mixed HCV genotype infections. Consensus sequences generated by different NGS methods were generally concordant, and majority RAVs were consistently detected. However, methods differed in their ability to detect minor populations of RAVs. Metagenomic methods identified human pegivirus coinfections. NGS provided a rapid, inexpensive method for generating whole HCV genomes to define infecting genotypes, RAVs, comprehensive viral strain analysis, and quasispecies ersity. Enrichment methods are particularly suited for high-throughput analysis while providing the genotype and information on potential DAA resistance.
Publisher: Oxford University Press (OUP)
Date: 29-11-2012
Publisher: F1000 Research Ltd
Date: 14-10-2020
DOI: 10.12688/WELLCOMEOPENRES.16157.1
Abstract: Deep sequencing of the full-length hepatitis B virus (HBV) genome provides the opportunity to determine the extent to which viral ersity, genotype, polymorphisms, insertions and deletions may influence presentation and outcomes of disease. Increasing experience with analysis of HBV genomic data opens up the potential for using these data to inform insights into pathophysiology of infection and to underpin decision making in clinical practice. We here set out to undertake whole genome HBV sequencing from an adult who presented acutely unwell with a new diagnosis of HBV infection, and tested positive for both HBV anti-core IgM and IgG, possibly representing either acute hepatitis B infection (AHB) or chronic hepatitis B with an acute reactivation (CHB-AR). The distinction between these two scenarios may be important in predicting prognosis and underpinning treatment decisions, but can be challenging based on routine laboratory tests. Through application of deep whole-genome sequencing we typed the isolate as genotype-D1, and identified several minority variants including G1764A and G1986A substitutions in the pre-core promoter and pre-core regions, which support CHB-AR rather than AHB. In the longer term, enhanced deep sequencing data for HBV may provide improved evidence to distinguish between acute and chronic infection, to predict outcomes and to stratify treatment.
Publisher: Cold Spring Harbor Laboratory
Date: 06-07-2023
DOI: 10.1101/2023.07.05.23292232
Abstract: Detection of HIV drug resistance is vital to successful anti-retroviral therapy (ART). HIV drug resistance (HIVDR) testing to determine drug resistance mutations (DRMs) is routinely performed in Australia to guide ART choice in either newly diagnosed people living with HIV or in cases of treatment failure. In 2022, our Australian clinical microbiology laboratory sought to validate a Next-generation sequencing (NGS)-based HIVDR assay to replace the previous Sanger sequencing (SS)-based ViroSeq assay. NGS solutions for HIVDR offer higher throughput, lower costs and higher sensitivity for variant detection. We sought to validate the previously described low-cost probe-based NGS method (veSEQ-HIV) for HIV-1 recovery and HIVDR testing in a diagnostic setting. The implemented veSEQ-HIV assay displayed 100% and 98% accuracy in major and minor mutation detection respectively and 100% accuracy of subtyping (provided mapped reads were obtained). Pairwise comparison exhibited low inter-and intra-run variability across the whole genome (Jaccard similarity coefficient [J] =0.993 J=0.972) and limited to the Pol gene only (J=0.999 J=0.999) respectively. The veSEQ-HIV assay met all our pre-set criteria based on the WHO “Recommended methods for validating an in-house genotyping assay for surveillance of HIV drug resistance” and has successfully replaced the ViroSeq assay in our laboratory.Scaling-down the veSEQ-HIV assay to a limited batch size and sequencing on the Illumina iSeq100, allowed easy implementation of the assay into the workflow of a small sequencing laboratory with minimal staff and equipment and the ability to meet clinically relevant test turn-around times.
Publisher: Springer Science and Business Media LLC
Date: 10-04-2017
DOI: 10.1038/NG.3835
Publisher: Cold Spring Harbor Laboratory
Date: 28-07-2019
DOI: 10.1101/716902
Abstract: The routine identification of pathogens during infection remains challenging because it relies on multiple modalities such as culture and nucleic acid lification and tests that tend to be specific for very few of an enormous number of possible infectious agents. Metagenomics promises single-test identification, but shotgun sequencing remains unwieldy and expensive or in many cases insufficiently sensitive to detect the amount of pathogen material in a clinical s le. Here we present the validation and application of Castanet , a method for metagenomic sequencing with enrichment that exploits clinical knowledge to construct a broad panel of relevant organisms for detection at low cost with sensitivity comparable to PCR. Castanet targets both DNA and RNA, works with small s le volumes, and can be implemented in a high-throughput diagnostic setting. We used Castanet to analyse plasma s les from 573 patients from the GAinS sepsis cohort and CSF s les from 243 patients from the ChiMES meningitis cohort that had been evaluated using standard clinical microbiology methods, identifying relevant pathogens in many cases where no pathogen had previously been detected. Castanet is intended for use in defining the distribution of pathogens in s les, diseases and populations, for large-scale clinical studies and for verifying the performance of routine testing regimens. By providing sequence as output, Castanet combines pathogen identification directly with subtyping and phylo-epidemiology.
Publisher: Elsevier BV
Date: 2019
Publisher: Microbiology Society
Date: 19-01-2016
Publisher: American Association for the Advancement of Science (AAAS)
Date: 16-04-2021
Abstract: A year into the severe acute respiratory syndrome coronavirus 2 pandemic, we are experiencing waves of new variants emerging. Some of these variants have worrying functional implications, such as increased transmissibility or antibody treatment escape. Lythgoe et al. have undertaken in-depth sequencing of more than 1000 hospital patients' isolates to find out how the virus is mutating within in iduals. Overall, there seem to be consistent and reproducible patterns of within-host virus ersity. The authors observed only one or two variants in most s les, but a few carried many variants. Although the evidence indicates strong purifying selection, including in the spike protein responsible for viral entry, the authors also saw evidence for transmission clusters associated with households and other possible superspreader events. After transmission, most variants fizzled out, but occasionally some initiated ongoing transmission and wider dissemination. Science , this issue p. eabg0821
Publisher: Springer Science and Business Media LLC
Date: 20-10-2021
DOI: 10.1038/S41467-021-25649-6
Abstract: Persistent hepatitis C virus (HCV) infection is a major cause of chronic liver disease, worldwide. With the development of direct-acting antivirals, treatment of chronically infected patients has become highly effective, although a subset of patients responds less well to therapy. Sofosbuvir is a common component of current de novo or salvage combination therapies, that targets the HCV NS5B polymerase. We use pre-treatment whole-genome sequences of HCV from 507 patients infected with HCV subtype 3a and treated with sofosbuvir containing regimens to detect viral polymorphisms associated with response to treatment. We find three common polymorphisms in non-targeted HCV NS2 and NS3 proteins are associated with reduced treatment response. These polymorphisms are enriched in post-treatment HCV sequences of patients unresponsive to treatment. They are also associated with lower reductions in viral load in the first week of therapy. Using in vitro short-term dose-response assays, these polymorphisms do not cause any reduction in sofosbuvir potency, suggesting an indirect mechanism of action in decreasing sofosbuvir efficacy. The identification of polymorphisms in NS2 and NS3 proteins associated with poor treatment outcomes emphasises the value of systematic genome-wide analyses of viruses in uncovering clinically relevant polymorphisms that impact treatment.
Publisher: Microbiology Society
Date: 03-2020
DOI: 10.1099/JGV.0.001387
Publisher: F1000 Research Ltd
Date: 13-10-2015
DOI: 10.12688/F1000RESEARCH.7111.1
Abstract: The routine availability of high-depth virus sequence data would allow the sensitive detection of resistance-associated variants that can jeopardize HIV or hepatitis C virus (HCV) treatment. We introduce ve-SEQ, a high-throughput method for sequence-specific enrichment and characterization of whole-virus genomes at up to 20% ergence from a reference sequence and 1,000-fold greater sensitivity than direct sequencing. The extreme genetic ersity of HCV led us to implement an algorithm for the efficient design of panels of oligonucleotide probes to capture any sequence among a defined set of targets without detectable bias. ve-SEQ enables efficient detection and sequencing of any HCV genome, including mixtures and intra-host variants, in a single experiment, with greater tolerance of sequence ersity than standard lification methods and greater sensitivity than metagenomic sequencing, features that are directly applicable to other pathogens or arbitrary groups of target organisms, allowing the combination of sensitive detection with sequencing in many settings.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 09-2018
DOI: 10.1002/HEP.29877
Abstract: New directly acting antivirals (DAAs) provide very high cure rates in most patients infected by hepatitis C virus (HCV). However, some patient groups have been relatively harder to treat, including those with cirrhosis or infected with HCV genotype 3. In the recent BOSON trial, genotype 3, patients with cirrhosis receiving a 16‐week course of sofosbuvir and ribavirin had a sustained virological response (SVR) rate of around 50%. In patients with cirrhosis, interferon lambda 4 ( IFNL4 ) CC genotype was significantly associated with SVR. This genotype was also associated with a lower interferon‐stimulated gene (ISG) signature in peripheral blood and in liver at baseline. Unexpectedly, patients with the CC genotype showed a dynamic increase in ISG expression between weeks 4 and 16 of DAA therapy, whereas the reverse was true for non‐CC patients. Conclusion: These data provide an important dynamic link between host genotype and phenotype in HCV therapy also potentially relevant to naturally acquired infection. (H epatology 2018 00:000‐000).
Publisher: Springer Science and Business Media LLC
Date: 17-06-2020
DOI: 10.1038/S41598-020-66713-3
Abstract: Epstein-Barr virus (EBV) reactivation is common in sepsis patients but the extent and nature of this remains unresolved. We sought to determine the incidence and correlates of EBV-positivity in a large sepsis cohort. We also hypothesised that EBV reactivation would be increased in patients in whom relative immunosuppression was the major feature of their sepsis response. To identify such patients we aimed to use knowledge of sepsis response subphenotypes based on transcriptomic studies of circulating leukocytes, specifically patients with a Sepsis Response Signature endotype (SRS1) that we have previously shown to be associated with increased mortality and features of immunosuppression. We assayed EBV from the plasma of intensive care unit (ICU) patients with sepsis due to community-acquired pneumonia. In total 730 patients were evaluated by targeted metagenomics (n = 573 patients), digital droplet PCR (n = 565), or both (n = 408). We had previously analysed gene expression in peripheral blood leukocytes for a subset of in iduals (n = 390). We observed a 37% incidence of EBV-positivity. EBV reactivation was associated with longer ICU stay (12.9 vs 9.2 days p = 0.004) and increased organ failure (day 1 SOFA score 6.9 vs 5.9 p = 0.00011). EBV reactivation was associated with the relatively immunosuppressed SRS1 endotype (p = 0.014) and differential expression of a small number of biologically relevant genes. These findings are consistent with the hypothesis that viral reactivation in sepsis is a consequence of immune compromise and is associated with increasing severity of illness although further mechanistic studies are required to definitively illustrate cause and effect.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 27-04-2018
DOI: 10.1002/HEP.29837
Abstract: Hepatitis C virus (HCV) genotype (gt) 3 is highly prevalent globally, with non‐gt3a subtypes common in Southeast Asia. Resistance‐associated substitutions (RASs) have been shown to play a role in treatment failure. However, the role of RASs in gt3 is not well understood. We report the prevalence of RASs in a cohort of direct‐acting antiviral treatment‐naive, gt3‐infected patients, including those with rarer subtypes, and evaluate the effect of these RASs on direct‐acting antivirals in vitro . Baseline s les from 496 gt3 patients enrolled in the BOSON clinical trial were analyzed by next‐generation sequencing after probe‐based enrichment for HCV. Whole viral genomes were analyzed for the presence of RASs to approved direct‐acting antivirals. The resistance phenotype of RASs in combination with daclatasvir, velpatasvir, pibrentasvir, elbasvir, and sofosbuvir was measured using the S52 ΔN gt3a replicon model. The nonstructural protein 5A A30K and Y93H substitutions were the most common at 8.9% (n = 44) and 12.3% (n = 61), respectively, and showed a 10‐fold and 11‐fold increase in 50% effect concentration for daclatasvir compared to the unmodified replicon. Paired RASs (A30K + L31M and A30K + Y93H) were identified in 18 patients (9 of each pair) these combinations were shown to be highly resistant to daclatasvir, velpatasvir, elbasvir, and pibrentasvir. The A30K + L31M combination was found in all gt3b and gt3g s les. Conclusion: Our study reveals high frequencies of RASs to nonstructural protein 5A inhibitors in gt3 HCV the paired A30K + L31M substitutions occur in all patients with gt3b and gt3g virus, and in vitro analysis suggests that these subtypes may be inherently resistant to all approved nonstructural protein 5A inhibitors for gt3 HCV. (H epatology 2018).
Publisher: Springer Science and Business Media LLC
Date: 2012
Publisher: Elsevier BV
Date: 06-2018
Publisher: Elsevier BV
Date: 2022
Publisher: Elsevier BV
Date: 11-2021
Publisher: Oxford University Press (OUP)
Date: 23-07-2020
Abstract: Targeted metagenomics using strand-specific libraries with target enrichment is a sensitive, generalized approach to pathogen sequencing and transcriptome profiling. Using this method, we recovered 13 (76%) complete human respiratory syncytial virus (RSV) genomes from 17 clinical respiratory s les, reconstructed the phylogeny of the infecting viruses, and detected differential gene expression between 2 RSV subgroups, specifically, a lower expression of the P gene and a higher expression of the M2 gene in RSV-A than in RSV-B. This methodology can help to relate viral genetics to clinical phenotype and facilitate ongoing population-level RSV surveillance and vaccine development. Clinical Trials Registration. NCT03627572 and NCT03756766.
Publisher: American Society for Microbiology
Date: 22-09-2020
DOI: 10.1128/JCM.00382-20
Abstract: Viral genetic sequencing can be used to monitor the spread of HIV drug resistance, identify appropriate antiretroviral regimes, and characterize transmission dynamics. Despite decreasing costs, next-generation sequencing (NGS) is still prohibitively costly for routine use in generalized HIV epidemics in low- and middle-income countries. Here, we present veSEQ-HIV, a high-throughput, cost-effective NGS sequencing method and computational pipeline tailored specifically to HIV, which can be performed using leftover blood drawn for routine CD4 cell count testing.
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for M. Azim Ansari.