ORCID Profile
0000-0003-0421-9264
Current Organisation
University of Oxford
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Publisher: Massachusetts Medical Society
Date: 11-10-2018
Publisher: Elsevier BV
Date: 04-2022
Publisher: Elsevier BV
Date: 03-2017
DOI: 10.1016/J.CMI.2016.10.014
Abstract: Whole genome sequencing (WGS) can help to relate Mycobacterium tuberculosis genomes to one another to assess genetic relatedness and infer the likelihood of transmission between cases. The same sequence data are now increasingly being used to predict drug resistance and susceptibility. Controlling the spread of tuberculosis and providing patients with the correct treatment are central to the World Health Organization's target to 'End TB' by 2035, for which the global prevalence of drug-resistant tuberculosis remains one of the main obstacles to success. So far, WGS has been applied largely to drug-susceptible strains for the purposes of understanding transmission, leaving a number of analytical considerations before transferring what has been learnt from drug-susceptible disease to drug-resistant tuberculosis. We discuss these potential problems here, alongside some of the challenges to characterizing the Mycobacterium tuberculosis 'resistome'-the optimal knowledge-base required for WGS-based assays to successfully direct in idualized treatment regimens through the prediction of drug resistance and susceptibility in the future.
Publisher: Springer Science and Business Media LLC
Date: 17-06-2019
DOI: 10.1038/S41579-019-0214-5
Abstract: Whole genome sequencing (WGS) of Mycobacterium tuberculosis has rapidly progressed from a research tool to a clinical application for the diagnosis and management of tuberculosis and in public health surveillance. This development has been facilitated by drastic drops in cost, advances in technology and concerted efforts to translate sequencing data into actionable information. There is, however, a risk that, in the absence of a consensus and international standards, the widespread use of WGS technology may result in data and processes that lack harmonization, comparability and validation. In this Review, we outline the current landscape of WGS pipelines and applications, and set out best practices for M. tuberculosis WGS, including standards for bioinformatics pipelines, curated repositories of resistance-causing variants, phylogenetic analyses, quality control and standardized reporting.
Publisher: Cold Spring Harbor Laboratory
Date: 08-03-2022
DOI: 10.1101/2022.03.04.22271870
Abstract: Mycobacterium tuberculosis whole-genome sequencing (WGS) using Illumina technology has been widely adopted for genotypic drug susceptibility testing (DST) and outbreak investigation. Oxford Nanopore Technologies is reported to have higher error rates but has not been thoroughly evaluated for these applications. We analyse 151 isolates from Madagascar, South Africa and England with phenotypic DST and matched Illumina and Nanopore data. Using PacBio assemblies, we select Nanopore filters for BCFtools (software) detection of single nucleotide polymorphisms (SNPs). We compare transmission clusters identified by Nanopore and the United Kingdom Health Security Agency Illumina pipeline (COMPASS). We compare Illumina and Nanopore WGS-based DST predictions using Mykrobe (software). Nanopore/BCFtools identifies SNPs with median precision/recall of 99·5/90·2% compared with 99·6/91·9% for Illumina/COMPASS. Using a threshold of 12 SNPs for putative transmission clusters, Illumina identifies 98 isolates as unrelated and 53 as belonging to 19 distinct clusters (size range 2-7). Nanopore reproduces this distribution with addition of 5 singleton isolates to distinct clusters and merging of two cluster pairs. Illumina-based clusters are also replicated using a 5 SNP threshold. Clustering accuracy is maintained using mixed Illumina/Nanopore datasets. Genotyping resistance variants is highly concordant, with 0(4) discordant SNPs (indels) across 151 isolates genotyped at (60,000) SNPs (indels). Illumina and Nanopore sequence data provide comparable cluster-identification and DST results. Academy for Medical Sciences (SGL018\\110), Oxford Wellcome Institutional Strategic Support Fund (ISSF TT17 4). Swiss South Africa Joint Research Award (Swiss national science Foundation and South African national research foundation). Two key types of information can be obtained from laboratory testing of M. tuberculosis isolates to help directly guide public health interventions: drug susceptibility testing (DST) to guide therapy, and bacterial typing to enrich understanding of the epidemiology and guide interventions to mitigate transmission. DST is typically performed by the “gold standard” culture-based phenotyping method or nucleic acid lification assays targeting specific resistance-conferring mutations. Studies over the last 7 years have shown that prediction of susceptibility profile using Illumina-technology genome sequence data is possible, and can be automated. In a key publication, the CRyPTIC consortium and UK 100,000 Genomes project evaluated the method on over 10,000 genomes including prospectively s led isolates and showed that for first-line tuberculosis (TB) drugs (isoniazid, rif icin, ethambutol, pyrazinamide) a pan-susceptibility profile is accurate enough to be used clinically. The genetic basis of resistance remains imperfectly understood for second-line TB drugs, in particular for new and repurposed drugs (bedaquiline, clofazimine, delamanid, linezolid). Prior work in the field of genotypic DST was heavily based on Illumina technology, which provides short (70-300 base pair) sequence reads of very high quality. Many different softwares (e.g. TBProfiler, Mykrobe, MTBseq, kvarq) have been designed for sequence analysis and genotypic DST. However, the increasingly used Nanopore sequencing platforms yield very different data with much longer sequence reads (frequently over 1kb) and higher error rates including systematic biases. To date, very limited evaluation of Nanopore-based drug susceptibility prediction has been performed using the only two compatible tools (Mykrobe (n=5 independent s les), TBProfiler (n=3 independent s les)). Molecular typing of M. tuberculosis allows lineage identification and detection of putative transmission clusters. In the last decade, multiple M. tuberculosis molecular epidemiology studies have shown how genomic information can complement traditional epidemiology in identifying person-to-person transmission clusters with a high level of resolution. Typically, the number of single nucleotide polymorphism (SNP) disagreements between genomes, or SNP distance, is calculated and single-linkage clustering is performed for genomes falling within retrospectively established transmission thresholds of either 5 or 12 SNPs. Just as with DST, these thresholds were established with Illumina sequencing data. The increased error rate in Nanopore sequencing is believed to lead to inflated SNP distances if standard genome analysis tools are used. Prior to this study it was unknown what impact on isolate-clustering this would incur. Full-scale adoption of genomic sequencing in tuberculosis reference laboratories has so far taken place in a limited number of settings - England, the Netherlands, and New York State - all using Illumina-based sequencing data. Building on current evidence, specific WHO technical guidance and ersification and democratisation of technology, sequencing is expected to be increasingly used in tuberculosis control globally. For the first time, our study offers 4 key deliverables intended to inform adoption of Nanopore technology as an alternative, or a complement, to Illumina. First: a systematic head-to-head comparison of Nanopore and Illumina data for M. tuberculosis drug susceptibility profiling and isolate clustering, including quantitative metrics for cluster precision and recall. Second: an assessment of the impact of mixed Illumina and Nanopore data on clustering which represents an increasingly common challenge. Third: an open-source software pipeline allowing research and reference laboratories to replicate our analytical approach. Fourth: a publicly available curated test set of 151 isolates, including matched Illumina and Nanopore sequence data, and (for a subset of seven isolates) high-quality PacBio assemblies, for method development and validation. Catalogues of drug resistance conferring mutations will keep improving, especially for new and repurposed drugs. Our data confirms that Illumina and Nanopore sequencing technologies can be used to identify those mutations equally accurately in M. tuberculosis . Bacterial molecular typing is constantly shown to support the understanding of disease transmission and tuberculosis control in new settings. The bioinformatics tools and filters we have developed, assessed, and made publicly available allow the use of Nanopore or mixed-technology data to appropriately cluster genetically related isolates. We provide a measure of the expected level of over-clustering associated with Nanopore technology. This study confirms that Illumina and Nanopore sequence data provide comparable DST results and isolate cluster-identification.
Publisher: Oxford University Press (OUP)
Date: 13-03-2020
DOI: 10.1093/CID/CIAA254
Abstract: Meta-analysis of patients with isoniazid-resistant tuberculosis (TB) given standard first-line anti-TB treatment indicated an increased risk of multidrug-resistant TB (MDR-TB) emerging (8%), compared to drug-sensitive TB (0.3%). Here we use whole genome sequencing (WGS) to investigate whether treatment of patients with preexisting isoniazid-resistant disease with first-line anti-TB therapy risks selecting for rif icin resistance, and hence MDR-TB. Patients with isoniazid-resistant pulmonary TB were recruited and followed up for 24 months. Drug susceptibility testing was performed by microscopic observation drug susceptibility assay, mycobacterial growth indicator tube, and by WGS on isolates at first presentation and in the case of re-presentation. Where MDR-TB was diagnosed, WGS was used to determine the genomic relatedness between initial and subsequent isolates. De novo emergence of MDR-TB was assumed where the genomic distance was 5 or fewer single-nucleotide polymorphisms (SNPs), whereas reinfection with a different MDR-TB strain was assumed where the distance was 10 or more SNPs. Two hundred thirty-nine patients with isoniazid-resistant pulmonary TB were recruited. Fourteen (14/239 [5.9%]) patients were diagnosed with a second episode of TB that was multidrug resistant. Six (6/239 [2.5%]) were identified as having evolved MDR-TB de novo and 6 as having been reinfected with a different strain. In 2 cases, the genomic distance was between 5 and 10 SNPs and therefore indeterminate. In isoniazid-resistant TB, de novo emergence and reinfection of MDR-TB strains equally contributed to MDR development. Early diagnosis and optimal treatment of isoniazid-resistant TB are urgently needed to avert the de novo emergence of MDR-TB during treatment.
Publisher: Elsevier BV
Date: 11-2017
Publisher: Elsevier BV
Date: 12-2020
Publisher: Springer Science and Business Media LLC
Date: 20-04-2016
DOI: 10.1038/NCOMMS11465
Abstract: Nature Communications 6: Article number: 10063 (2015) Published: 21 December 2015 Updated: 20 April 2016 In Supplementary Data 3 of this Article, one of the Staphylococcus aureus accession codes is incorrect, as follows: SRR2101499 should be ERR1197981.
Publisher: F1000 Research Ltd
Date: 12-10-2022
DOI: 10.12688/WELLCOMEOPENRES.18026.1
Abstract: Patients with severe COVID-19 disease require monitoring with pulse oximetry as a minimal requirement. In many low- and middle- income countries, this has been challenging due to lack of staff and equipment. Wearable pulse oximeters potentially offer an attractive means to address this need, due to their low cost, battery operability and capacity for remote monitoring. Between July and October 2021, Ho Chi Minh City experienced its first major wave of SARS-CoV-2 infection, leading to an unprecedented demand for monitoring in hospitalized patients. We assess the feasibility of a continuous remote monitoring system for patients with COVID-19 under these circumstances as we implemented 2 different systems using wearable pulse oximeter devices in a stepwise manner across 4 departments.
Publisher: Springer Science and Business Media LLC
Date: 21-12-2015
DOI: 10.1038/NCOMMS10063
Abstract: The rise of antibiotic-resistant bacteria has led to an urgent need for rapid detection of drug resistance in clinical s les, and improvements in global surveillance. Here we show how de Bruijn graph representation of bacterial ersity can be used to identify species and resistance profiles of clinical isolates. We implement this method for Staphylococcus aureus and Mycobacterium tuberculosis in a software package (‘Mykrobe predictor’) that takes raw sequence data as input, and generates a clinician-friendly report within 3 minutes on a laptop. For S . aureus , the error rates of our method are comparable to gold-standard phenotypic methods, with sensitivity/specificity of 99.1%/99.6% across 12 antibiotics (using an independent validation set, n =470). For M . tuberculosis , our method predicts resistance with sensitivity/specificity of 82.6%/98.5% (independent validation set, n =1,609) sensitivity is lower here, probably because of limited understanding of the underlying genetic mechanisms. We give evidence that minor alleles improve detection of extremely drug-resistant strains, and demonstrate feasibility of the use of emerging single-molecule nanopore sequencing techniques for these purposes.
Publisher: F1000 Research Ltd
Date: 20-06-2023
DOI: 10.12688/WELLCOMEOPENRES.18026.2
Abstract: Patients with severe COVID-19 disease require monitoring with pulse oximetry as a minimal requirement. In many low- and middle- income countries, this has been challenging due to lack of staff and equipment. Wearable pulse oximeters potentially offer an attractive means to address this need, due to their low cost, battery operability and capacity for remote monitoring. Between July and October 2021, Ho Chi Minh City experienced its first major wave of SARS-CoV-2 infection, leading to an unprecedented demand for monitoring in hospitalized patients. We assess the feasibility of a continuous remote monitoring system for patients with COVID-19 under these circumstances as we implemented 2 different systems using wearable pulse oximeter devices in a stepwise manner across 4 departments.
Publisher: Elsevier BV
Date: 04-2014
Publisher: Elsevier BV
Date: 04-2017
Publisher: F1000 Research Ltd
Date: 02-12-2019
DOI: 10.12688/WELLCOMEOPENRES.15603.1
Abstract: Two billion people are infected with Mycobacterium tuberculosis , leading to 10 million new cases of active tuberculosis and 1.5 million deaths annually. Universal access to drug susceptibility testing (DST) has become a World Health Organization priority. We previously developed a software tool, Mykrobe predictor , which provided offline species identification and drug resistance predictions for M. tuberculosis from whole genome sequencing (WGS) data. Performance was insufficient to support the use of WGS as an alternative to conventional phenotype-based DST, due to mutation catalogue limitations. Here we present a new tool, Mykrobe , which provides the same functionality based on a new software implementation. Improvements include i) an updated mutation catalogue giving greater sensitivity to detect pyrazinamide resistance, ii) support for user-defined resistance catalogues, iii) improved identification of non-tuberculous mycobacterial species, and iv) an updated statistical model for Oxford Nanopore Technologies sequencing data. Mykrobe is released under MIT license at ykrobe-tools/mykrobe. We incorporate mutation catalogues from the CRyPTIC consortium et al. (2018) and from Walker et al. (2015), and make improvements based on performance on an initial set of 3206 and an independent set of 5845 M. tuberculosis Illumina sequences. To give estimates of error rates, we use a prospectively collected dataset of 4362 M. tuberculosis isolates . Using culture based DST as the reference, we estimate Mykrobe to be 100%, 95%, 82%, 99% sensitive and 99%, 100%, 99%, 99% specific for rif icin, isoniazid, pyrazinamide and ethambutol resistance prediction respectively. We benchmark against four other tools on 10207 (=5845+4362) s les, and also show that Mykrobe gives concordant results with nanopore data. We measure the ability of Mykrobe -based DST to guide personalized therapeutic regimen design in the context of complex drug susceptibility profiles, showing 94% concordance of implied regimen with that driven by phenotypic DST, higher than all other benchmarked tools.
Publisher: Public Library of Science (PLoS)
Date: 31-03-2023
DOI: 10.1371/JOURNAL.PGPH.0001754
Abstract: There were approximately 10 million tuberculosis (TB) cases in 2020, of which 500,000 were drug-resistant. Only one third of drug-resistant TB cases were diagnosed and enrolled on appropriate treatment, an issue partly driven by a lack of rapid, accurate drug-susceptibility testing (DST) tools deployable in peripheral settings. In 2014, World Health Organization (WHO) published target product profiles (TPPs) which detailed minimal and optimal criteria to address high-priority TB diagnostic needs, including DST. Since then, the TB community’s needs have evolved new treatment regimens, changes in TB definitions, further emergence of drug resistance, technological advances, and changing end-users requirements have necessitated an update. The DST TPP’s revision was therefore undertaken by WHO with the Stop TB Partnership New Diagnostics Working Group. We describe the process of updating the TPP for next-generation TB DST for use at peripheral centres, highlight key updates, and discuss guidance regarding technical and operational specifications.
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Timothy Walker.