E S C Whole genome sequencing in AST (of bacteria): ESCMID
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Whole genome sequencing in AST (of bacteria):
I D or …What’s new?
C M t h
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Dr Matthew J Ellington
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Principal clinical scientist, AMRHAI, PHE, National Infections Service
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NIHR Health Protection Research Unit in HCAI & AMR, Imperial College London
e: matthew.ellington@phe.gov.uk
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Antibiotic susceptibility testing (AST)
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• Fundamental to diagnostic bacteriology
• Quantitative methods (MIC, mg/L)
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- agar or broth dilution
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- gradient strips (Etests, MICE)
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• Qualitative methods (S/I/R)
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- disc diffusion
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- agar incorporation breakpoint method
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• Automated methods
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WGS in AST: …What’s new?
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EUCAST Sub-committee on the role of whole
genome sequencing (WGS) in AST of bacteria
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• Diverse expertise. Scientific and Clinical.
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Frank M. Aarestrup (Denmark) L
Gunnar Kahlmeter (Sweden)
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Rafael Canton (Spain) Claudio U. Koser (UK)
Michel Doumith (Saudi Arabia) Alasdair MacGowan (UK)
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Oskar Ekelund (Sweden) Mike Mulvey (Canada)
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Matthew J. Ellington (UK, Chair) Thierry Naas (France)
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Christian Giske (Sweden) Tim Peto (UK)
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Henrik Hasman (Denmark) Jean-Marc Rolain (France)
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Katie L. Hopkins (UK) Ørjan Samuelsen (Norway)
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Matt Holden (UK) Neil Woodford (UK)
Jon Iredell (Australia) Dik Mevius (Netherlands)
3 WGS in AST: …What’s new?
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EUCAST Subcommittee on the role of whole
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genome sequencing (WGS) in AST of bacteria
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1. Review literature describing the role of WGS in AST of bacteria
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2. Assess the sensitivity and specificity of WGS vs phenotypic AST
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3. Consider how WGS for AST may be applied in clinical micro labs
4. Consider the epidemiological implications of using WGS
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5. Consider the clinical implications of WGS for the selection of Rx
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6. To describe the drivers and barriers to routine use of WGS
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http://www.clinicalmicrobiologyandinfection.com/article/S1198-743X(16)30568-7/pdf
Clin Microbiol Infect. 2017 Jan;23(1):2-22. doi:10.1016/j.cmi.2016.11.012.
44 WGS in AST: …What’s new?
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Focus on WHO priority organisms
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Organisms Priority resistances
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Enterobacteriaceae E. coli 3GC, FQs
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K. pneumoniae 3GC, carbapenems
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Non-typhoidal Salmonella FQs
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Shigella spp. FQs
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S. aureus - MRSA
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S. pneumoniae - Penicillin
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N. gonorrhoeae - 3GCs
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Plus: P. aeruginosa, Acinetobacter, C. difficile and Mtb
WGS in AST: …What’s new?
Literature at the time n=209
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501 isolates; S. aureus; 5112
AST
143results; 98.8%
isolates;2 WGS 1001 AST
species;
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concordance
results; 96.7% WGS concordance
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388 isolates; 1 species; 1158 AST
results; 88.9% WGS concordance
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E y
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WGS in AST: …What’s new?
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Most appropriate AST comparators
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•What criteria should WGS data be assessed against ?i b
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•clinical breakpoints indicate likelihood of therapeutic success (S) or failure (R) of
antibiotic treatment based on microbiological findings
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•ECOFFs (epidemiological cut-off values) differentiate wild-type (WT) from non-wild-
type (NWT) isolates with an acquired resistance mechanism
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WGS
WGS
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7 ininAST:
AST: …What’s new?
Copyright …What’s new?
© Crown
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Evidence reports – e.g. Enterobacteriaceae
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• Relatively limited number of acquired resistance genes
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and resistance-associated mutations that dominate
epidemiologically in the Enterobacteriaceae
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• High levels of accuracy of genotype-phenotype
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correlation in published studies; means that well-
informed screening approaches can be very accurate.
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• Predicting AST results will be harder for some than for
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others
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• better understanding of the full range of mechanisms is
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required
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• …INCLUDING their interplay
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Complex interplays determine an MIC
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External [drug]
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VEntry + VEfflux
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Periplasmic [drug]
S C u t VHydrolysis
E y a VBinding
It’s a lot more
complex than gene
presence / absence
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WGS in AST: …What’s new?
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Combinatorial resistance: WGS vs. AST
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WGS in AST: …What’s new?
Reuter, Ellington, et al., 2013. JAMA Intern Med 12;173:1397-404r y
Evidence reports – e.g. Enterobacteriaceae
r a
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• Relatively limited number of acquired resistance genes and
L
resistance-associated mutations that dominate
e
epidemiologically in the Enterobacteriaceae
• High levels of accuracy of genotype-phenotype correlation
I D or
in published studies; means that well-informed screening
approaches can be very accurate.
M
• Predicting AST results will be harder for some than for
C
others
u t h
• better understanding of the full range of mechanisms is
S a
required
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• …INCLUDING their interplay
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• Will require more study if improved levels of accuracy across
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large genetically diverse datasets are to be achieved.
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11 WGS in AST: …What’s new?Conclusions from previous report
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• Need for further evidence, could ‘soon’ replace much AST for surveillance
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purposes
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• low impact of the low error rate
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• Need for further evidence, could ‘soon’ reduce need for AST in reference
laboratories unless:
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• to guide treatment
• for agents with poorest genotypic/phenotypic concordance
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• comparative in-vitro activity of new agents
C t h
• ‘longer’ for a paradigm shift to WGS to guide clinical decision making
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• very major errors - gene absence cannot always predict susceptibility
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• robust evidence will be needed
• probably first for TB (for bacteria)
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• surveillance of treatment failure +/- novel resistance mechanisms
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WGS in AST: …What’s new?r y
a
EUCAST Sub-committee on the role of whole
genome sequencing (WGS) in AST of bacteria
i br
• Diverse expertise. Scientific and Clinical.
e
Frank M. Aarestrup (Denmark) L
Gunnar Kahlmeter (Sweden)
I D or
Rafael Canton (Spain) Claudio U. Koser (UK)
Michel Doumith (Saudi Arabia) Alasdair MacGowan (UK)
M h
Oskar Ekelund (Sweden) Mike Mulvey (Canada)
C t
Matthew J. Ellington (UK, Chair) Thierry Naas (France)
S u
Christian Giske (Sweden) Tim Peto (UK)
a
Henrik Hasman (Denmark) Jean-Marc Rolain (France)
E y
Katie L. Hopkins (UK) Ørjan Samuelsen (Norway)
b
Matt Holden (UK) Neil Woodford (UK)
Jon Iredell (Australia) Retired: Dik Mevius (Netherlands)
13 WGS in AST: …What’s new?r y
Committee is reconvened for v2
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• Not a whole new report
• Update of existing sections
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PubMed citations:
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'antimicrobial AND phenotype AND whole genome'
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80
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70
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Citation count
60
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50
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40
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30
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10
0
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Literature scan vs. species n=187
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WGS in AST: …What’s new?r y
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Existing sections – early update findings
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• Not a whole new report
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• Update of existing sections:
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• Good concordances highlighted in previous version for bug /
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drug combinations
• Developed since….
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WGS in AST: …What’s new?r y
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Existing sections – early update findings
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Table adapted from: Su et al., JCM 2019
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• Some species where good evidence base now exists for many key drugs
• Limited extension beyond the previously investigated antimicrobials
17 WGS in AST: …What’s new?r y
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Existing sections – early update findings
• Tb sequencing at PHE for diagnostic testing
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WGS susceptibility predictions vs those of MTBDRplus
and phenotypic testing for isoniazid and rifampin.
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2626 WGS vs pheno results:
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Concordance: 99.2%
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Sensitivity: 94.2%
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Specificity: 99.4%
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WGS in AST: …What’s new?
T. Phuong Quan et al. J. Clin. Microbiol. 2018; doi:10.1128/JCM.01480-17r y
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Systematic sources of error affecting
phenotypic / WGS correlation
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• Incomplete understanding of genotypic basis of phenotypic resistance
• affects sensitivity of WGS prediction (resulting in very major errors)
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• problematic bacteria; problematic antibiotics
• Major gaps in the knowledge base – e.g. mcr-1 & combinatorial Rs
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• Different databases of R loci used – comparisons need to be standardised
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• Flaws with phenotypic AST
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• An inadequate limit of detection of WGS
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• when detection is direct from clinical specimens e.g. TB
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• for most organisms WGS is likely to use cultured (high titre) bacteria
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New sections – AI / machine learning
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I D or Output
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Rules based Model based
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# tested Sens (%) Spec (%) # tested Sens (%) Spec(%) Overall
S a
E. coli Gent 74 100 100 564 87 99
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Mtb Etham 752 100 98.5 3526 91.9 90.3
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S. enterica Ceftriax 648 100 99.8 80, 95%(±1 MIC)
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P. aeruginosa Levo 390 91.9 93.7
Table adapted from: Su et al., JCM 2019
20 WGS in AST: …What’s new?r y
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New sections – AI / machine learning
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AI: Questions arising – listed by Su et al., JCM 2019
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• At what price and turnaround time will WGS-AST replace culture-
based sequencing for routine use in clinical microbiology labs?
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• How do we interpret the presence of an antimicrobial resistance
determinant gene if the susceptibility of the strain is below the MIC?
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• Can genome prediction be used to detect hetero-resistance? Or to
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detect polygenic phenotypes?
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• How important is epistasis in determining the resistance to different
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classes of antibiotics?
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• Can gene amplification as a mechanism of resistance be accurately
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determined from WGS data?
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• How efficiently can WGS-AST prediction software be ported to
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metagenomic-AST
WGS in AST: …What’s new?r y
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New sections …incl’ metagenomes
•
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Metagenomic approaches – Positive early stages for finding AMR, challenges faced
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include: with seq yield, fragment length, locating / localizing mobile resistances
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• New Tech – orthoganol approaches to help with gaps in knowledge base and
delineating heteroresistance
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Press et al., Biorxiv: https://doi.org/10.1101/198713
WGS in AST: …What’s new?
Mulroney et al., SCIENTIFIC REPORTs |
7:1903 | DOI:10.1038/s41598-017-02009-3r y
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WGS based AST - Conclusions – v1
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• Need for further evidence, could ‘soon’ replace much AST for surveillance
L
purposes
• low impact of the low error rate
e
• Need for further evidence, could ‘soon’ reduce need for AST in reference
laboratories unless:
I D or
• to guide treatment
• for agents with poorest genotypic/phenotypic concordance
C M t h
• comparative in-vitro activity of new agents
• ‘longer’ for a paradigm shift to WGS to guide clinical decision making
S u
• very major errors - gene absence cannot always predict susceptibility
a
E
• robust evidence will be needed
y
• probably first for TB (for bacteria)
b
• surveillance of treatment failure +/- novel resistance mechanisms
23
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Still no consensus, but…
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• Reconvened committee to update the report
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– Anticipate general consultation doc’ 2019, Q4
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• Scope extended: AI, (LR-)WGS, metagenomics
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– Mostly early stages and some positives
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• Overall increased evidence base for key
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bug/drugs identified previously
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– Previous per organism/drug patterns persist
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– Limited number of impactful studies
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