Investigating Legionella: PCR Screening and Whole-Genome Sequencing
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1 Investigating Legionella: PCR Screening and Whole-Genome Sequencing June 13, 2017 Kimberlee Musser, PhD Chief, Bacterial Diseases Wadsworth Center
2 1976 Philadelphia Legionnaires' disease outbreak 2 July 21-24, 1976 Bellevue-Stratford Hotel The total number of cases reached 221, 34 had died.
3 June 13, International Symposium on Legionella, ASM Press, 1993
4 4 Reported Cases of Legionellosis by year New York # Cases NYS Legionellosis NYC Legionellosis Total Year
5 Legionella Culture Archive 2945 archived ( ) L. pneumophila L. pneumophila L. pneumophila L. pneumophila L. anisa 84 L. pneumophila 3 50 L. micdadei 42 L. pneumophila L. pneumophila 4/5 24 L. feelei 20 L. bozemanii 133 Legionella species 77 L. pneumophila no serotype *55 % Lp1 80 % Lp
6 June 13, PFGE Analysis 1990 implemented (27 yrs exp.) Lab notebooks, reports 2003 Bionumerics Database entries 415 patterns
7 Investigating Outbreaks ( ) 7 Total of 668 investigation testing requests Average 19 requests per year 128 total facilities/locations Spanning 1 time testing to multiyear sampling (2-34 years)
8 Historic Outbreaks with Sources Identified 8 Hospital Nursing home/assisted living Category Potable water 20 Potable water /Cooling tower 1 Cooling tower 2 Potable water 10 Cooling tower 3 Private home Potable water 2 Hotel Potable water 1 Correctional Facility Potable water 2 Residential Complex Potable water 2 Cooling Tower 1 School Potable water 1 Cooling tower = 7, Potable water = 39 *36% success rate for determining source 46/128 Number
9 9 Real-time PCR testing Started testing in 1998 (19 yrs exp.) Validations for clinical testing, NYS approvals Side by side testing comparisons Screen samples, confirm identification of isolates and detect in clinical specimens L. pneumophila serogroup 1 L. pneumophila (1-15) Legionella spp. Inhibition control L. anisa L. micdadei
10 10 PCR +/Cult - PCR -/Cult +
11 Legionella Outbreak Communication 11 Assessment of facility Interaction with facility Review of maintenance records Sampling recommendations Environmental Health Legionella Case Reports Interviews Medical record reviews Interaction with facility, physicians Community or HAI Request cultures, specimens HAI Epi BCDC Epi Discussing testing volumes, types Preparing ordering media, labelling Testing timelines Shipping, Testing info Lab Regional Epi
12 Can whole-genome sequencing (WGS) provide additional resolution to outbreak investigations? 12
13 Ongoing pilot project with the CDC Legionella Lab AMD funding-spring Build an analytic bioinformatics pipeline for WGS SNPbased detection Retrospectively sequence characterized Legionella pneumophila outbreaks Determined the ability of WGS at correctly separating unrelated outbreak clusters and have a baseline of cluster definition for future usage
14 Results SNPs Maximum parsimony tree, ref seq based on 24,424 Core SNP matrix
15 NYS Facility Investigation 15 4 patients diagnosed in Aug/Sept 2015 Water restrictions and remediation occurred November 2015 facility letter final culture January th patient diagnosed No evidence that the patient spent any time in areas of facility where Legionella pneumophila serogroup 4 (Lp4) has been recovered from potable water and no distinct water exposure could be identified. Patient s Lp4 isolate was indistinguishable by PFGE from other patients at facility and two environmental cultures from the potable water system WGS initiated
16 PFGE and serotyping were performed on 4 patient isolates and 3 environmental samples 16 5 th patient All isolates were determined to be a serogroup 4* PFGE results indicated the isolates were all the same strain
17 5 th patient WGS SNP Analysis 17 5 th patient The 5 th patient was actually a closer match to Patient B, Patient A, and environmental sources than Patient C was. All patient and environmental samples in this investigation were 1-13 SNPs different when compared to the 5 th patient and >23,000 SNPs different from any other of the >320 Legionella we have sequenced to date.
18 18 The Challenge of interpretation of SNP differences 1 SNP difference is meaningful <10 SNPs, <5 SNPs indicate the same source? Heterogeneity in sources Reference genome- when is this critical for interpretation of an outbreak?
19 June 13, Legionella Persistence in Facilities 20 SNPs different
20 Legionella Whole-genome Sequencing Legionella to date
21 WGS activities 21 Outbreak Investigations Facility Persistence CT Heterogeneity Serogroup switching Primary specimens CDC AMD wgmlst
22 June 13, BACTERIOLOGY LABORATORY Liz Nazarian Deb Baker Dianna Bopp Nellie Dumas Amy Saylors BIODEFENSE LABORATORY Christina Egan, Michael Perry Lisa Mingle Kara Mitchell Teresa Passaretti Danielle Wroblewski William Lainhart Bioinformatics Core- Pascal Lapierre APPLIED GENOMIC TECHNOLOGIES CORE Matt Shudt, Patrick Van Roey Anna Kidney Yan Zhu Tanya Halse Lisa Thompson Acknowledgements Tissue and Media Core, Security, Director s Office, DLO, CLIMs group Jill Taylor, Vicky Derbyshire, Ron Limberger, Colleen Walsh NYC PHL- Jen Rakeman, Scott Hughes, Tony Tran, John Kornblum, Laura Kornstein, Ying Lin NYC Epidemiology- Don Weiss, Robert Fitzhenry, Isaac Benowitz NYSDOH NYS CEH/NYC CEH CDC Pneumonia Response and Surveillance Laboratory (NCIRD/DBD/RDB)- Brian Raphael, Natalia A. Kozak-Muiznieks, Shatavia S. Morrison, Claressa E. Lucas, Jeffrey W. Mercante, Jonas M. Winchell CDC AMD Funding
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