PulseNet Updates: Transitioning to WGS for Reference Testing and Surveillance - APHL
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National Center for Emerging and Zoonotic Infectious Diseases PulseNet Updates: Transitioning to WGS for Reference Testing and Surveillance Heather Carleton, PhD, MPH; Steven Stroika; Kelley Hise, MPH Enteric Diseases Laboratory Branch Division of Foodborne, Waterborne and Environmental Diseases 50-State Call, Foodborne Epidemiologists October 12, 2018
EDLB Vision
REPLACE all enteric workflows: With ONE cost-efficient and
• Identification precise method:
• Serotyping • All of this information
• Virulence profiling can be derived from the
• Antimicrobial susceptibility genome sequence
• Subtyping for surveillance and
outbreak investigations
WGS
PulseNet Isolates by PFGE and WGS
1996-2017
80000
70000
60000
50000
40000
30000
20000
10000
0
1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017
PFGE Isolates minus WGS WGS
PulseNet Transition Timeline to WGS Surveillance at
States
Nov 2nd, 2018 January 15th, 2019
Transition period
• Stopped PFGE for Listeria • Update databases to BioNumerics • PulseNet state and local PHLs:
and Campylobacter 7 and use for PFGE analysis • Submit state sequence data through BN
• Receive analysis certification in for analysis; upload analyzed sequence
WGS from PulseNet data to PN national database; upload
• Develop and implement sequence reads to NCBI with limited
validation plan for identification, metadata
serotyping, virulence and • Perform local cluster detection using
resistance gene detection if cgMLST and allele codes; compare to
necessary under CLIA or CAP national database
Considerations for WGS Transition Timeline Time of double workloads for PHL microbiologists – performing both PFGE and WGS Funding concerns with double workload – cannot afford to fully implement WGS while still performing PFGE When BN7 analysis tools would be validated for sharing with states Time needed for fully transitioning to BN7 and new WGS analysis tools Feedback from state partners about feasibility of timelines
PulseNet Prioritization for WGS
Conduct WGS on as many isolates using the following priority schedule: (1)
Listeria monocytogenes, (2) STEC, (3) Salmonella, (4) Other species
– If 100% of Salmonella cannot be sequenced, utilize a random sequencing
approach (i.e. 1 of every 3 Salmonella received in laboratory) or as agreed to
by lab and epi
– Other organisms may be further prioritized if funded to do so (i.e.
Campylobacter in FoodNet sites)
WGS Genotyping Tools Available in BioNumerics
Listeria Salmonella Escherichia Campylobacter
(O157/Non-O157/Shigella)
Resistance Resistance Resistance Resistance
Plasmid Plasmid Plasmid Plasmid
Virulence (genes Virulence (stx/eae/etc.
for pathovars) used to determine
Pathotype)
Lineage Antigenic formula Serotype
and serotype Serotype Serotype_wgs Pathotype
Example of output from BioNumerics
WGS Analysis for Surveillance and Outbreak Detection in BioNumerics at States Tool Listeria Salmonella Escherichia Campy Core genome MLST Yes Yes Yes Yes (cgMLST) – primary cluster detection method Allele code Yes Available Jan Available Jan Available nomenclature- primary 2019 2019 March 2019 cluster detection method Whole genome MLST Yes Yes Yes Yes (wgMLST) – as further analysis needed SNP analysis – as further Yes Yes Yes Yes analysis needed
Cluster detection with cgMLST: comparing to entire
database
• Select 1 or more entries and
find matches in local (or
national) database based on
allele differences
• Generate a line list of isolates
within 20 allele differences
• Can export line list and further
analyze potential cluster by
generating a tree51 Alleles 19 Alleles 0 Alleles
71 Alleles 36 Alleles 7 Alleles
Cluster detection: Allele Codes
LMO1.0 - 5 . 1 . 1 . 2 . 5 . 1
Organism version Allele Code
Nomenclature is organism specific with different thresholds for the digits
When sequences have partial names, it means they are singletons in clusters below their
last digit.
The sequences below are approximately within 36 and 19 alleles of each other.
Sequence A LMO1.0 - 5 . 1 . 2
Sequence B LMO1.0 - 5 . 1 . 2 . 2 . 5 . 1Database Cluster Detection
Default settings include allele code matches up to the
5th digit (~7 allele difference) and all human entries in
the last 120 days
Found clusters are displayed below with Allele code
and number of cases
Defaults can be changed to match on any digit in the
allele code (1st, 2nd, 3rd, 4th, 5th, or 6th), historical
cases, non-human, cluster size, and/or number of
days to check
Selecting clusters and choosing OK opens a
comparison of those entries in BioNumerics
Further refine clusters in a dendrogram by cgMLST or
wgMLST allele differencesDatabase Cluster Detection
Your PHL can view
dendrograms by different
allele schemes (cgMLST,
wgMLST, MLST)
Your PHL can label nodes by
right-clicking node on tree
and adding tree labels
denoting allele differences
at that node by scheme
chosenWorkflows and Turn-Around-Times
Day 0 Day 1-2 Day 3-4 Day 5-6
DNA extraction and DNA sequencing
Isolate received or Pure culture
growth Library preparation
recovered at PHL
Day 6-7
*TAT is measured at
time of upload to
National Database PulseNet
National
Databases Organism-specific Reference ID
Database Database
Genotyping and Sequence data QC
surveillance analysis and identificationTurn-Around-Times
Starting January TAT will be calculated from the date the isolate was received (or
recovered) in the PHL to the date of upload to the national database
– Should be 7 working days or less
Tentative Transition to use of wg/cgMLST by January 15, 2019
– All PHL’s migrated to BioNumerics v7.6
– Laboratorians certified for WGS analysis
– Testing and validating WGS nomenclature (Allele Codes)Upcoming trainings PulseNet Webinars (coming early December) Food Safety COE WGS training (BioNumerics and WGS interpretation) webinars PulseNet/OutbreakNet Regional meetings CSTE/AMD Molecular Epi training – includes more than foodborne MiSeq/BioNumerics training in February
Questions?
For more information, contact CDC
1-800-CDC-INFO (232-4636)
TTY: 1-888-232-6348 www.cdc.gov
#PulseNet
The findings and conclusions in this report are those of the authors and do not necessarily represent the
official position of the Centers for Disease Control and Prevention.
Telephone: 404-639-4558
E-mail: PulseNet@cdc.gov Web: www.cdc.gov/pulsenet
#PulseNetNational Center for Emerging and Zoonotic Infectious Diseases WGS Allele Codes in SEDRIC
WGS Allele Codes are Already in SEDRIC! They were incorporated for Listeria a few months ago and can be found in: – PulseNet Outbreak Line List Editor – Object Explorer – Browser – Search Bar
Line List Editor
Object Explorer
Browser
Search Bar
For more information, contact:
Lyndsay
For moreBottichio (xmm8@cdc.gov)
information, contact CDC
404-639-0570 (232-4636)
1-800-CDC-INFO
https://sedric.cdc.gov
TTY: 1-888-232-6348 www.cdc.gov
The findings and conclusions in this report are those of the authors and do not necessarily represent the
official position of the Centers for Disease Control and Prevention.You can also read
