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Division of Foodborne, Waterborne, and Enviromental DiseasesNational Center for Emerging and Zoonotic Infectious Diseases
Antisera QC and IQCP and Associated Challenges
Patt i FieldsEnteric Diseases Laboratory Branch (EDLB)
CDC
2017 APHL Annual MeetingProvidence, Rhode IslandJune 14, 2017
For more information please contact Centers for Disease Control and Prevention
1600 Clifton Road NE, Atlanta, GA 30333Telephone, 1-800-CDC-INFO (232-4636)/TTY: 1-888-232-6348E-mail: [email protected] Web: www.cdc.gov
Thank you !Patt i [email protected]
National Center for Emerging and Zoonotic Infectious DiseasesDivision of Foodborne, Waterborne, and Environmental Diseases
The findingsand conclusions in this report are those of the author and do not necessarily represent the official position of the Centers for Disease Control and Prevention.
Serotyping is a subtyping method Characterizat ion of strains based on immunologic
react ivity of surface structures, commonly: Lipopolysaccharide (O antigen) Flagellin protein (H antigen)
In Salmonella, it is a bit more complicated than other enteric organisms because: Includes species and subspecies identification because
isolates of different subspecies can have the same O and H antigens
Most Enterobacteriaceaehave only one H antigen; Salmonella commonly has two different H antigens
2500+ serotypes have been described (and counting)
Background: Serotyping
Phenotypic methods Detect/differentiate expressed antigens using antibodies Hyper-immune rabbit antisera or monoclonal antibodies Can be a challenged to maintain antisera required
• 250+ reagents needed for all Salmonella serotypes• 180 O groups described for E. coli
Genetic methods Target genes responsible for serotype: rfb region, fliC, fljB
• Bonus: Replicates the phenotypic serotyping scheme Target surrogate markers, e.g. , MLST
• Drawback: Can’t type things you’ve never seen before Variety of methods can be used: PCR, PCR-probe, WGS
CDC plans to maintain phenotypic serotyping “Reference Center for Traditional Microbiological Methods”
Methods for serotyping
Why is serotyping important?Why do we want to keep it?
An internat ional “ language” The US has 50+ years of Salmonella surveillance data
based on serotype Many serotypes have unique niches and/or epidemiology Many do not (or has not been described)
No longer the definit ive subtyping method We now have genetic subtyping methods that provide a
LOT more discrimination than serotyping can Some of the antigenic detail in Kauffmann-White Scheme
no longer necessary Can we simplify the serotyping scheme?
Salmonella Serotyping Reagents Many specificit ies are available from CDC
free-of-charge for the purpose of nat ional surveillance
CDC had funding to replenish ant isera supply in late 1990s- early 2000s APHL was a huge partner. Thanks! Contracted with the state of California to
produce more that 150 regents A “10 year supply” has lasted 15+ years, but
some specificities are running out
Salmonella Serotyping ReagentsQC
Expirat ion dates CDC reagents: re-evaluate RTD and extend
expiration dates when needed
Antisera QC under CLIA Reagents need to be QC’d every 6 months Not a problem for commonly used reagents
• QC performed when working dilution is made, used up within 6 months
What about rarely used reagents? QC at time of use (a LOT of work)
Another solution: IQCP
What is IQCP? Individualized Quality Control Plan The alternative CLIA quality control (QC) option. Provides for equivalent quality testing to meet the
CLIA regulations for non-waived tests. Considers the entire testing process: pre-analytic,
analytic and post analytic Consider the risks in each of these phases and
applicable regulatory requirements Develop a plan to mitigate risks
https://www.cms.gov/regulat ions-and-guidance/legislat ion/clia/downloads/cliabrochure11.pdf
CDC’s approach to IQCP for serotyping CDC QMS team led us through the process Developed one IQCP for all serotyping we do:
“Quality control plan for phenotypic determination of serotype in Campylobacter, Escherichia coli, Salmonella, Shigella, Vibrio cholerae, and Yersinia enterocolitica”
Basic organization Purpose and scope Overview of workflow List of risks QC plan to address risks QA plan Supporting documentation
Flow chart
Risks (1 of 3 pages)
QC Plan
Support ing documentat ion for 5-year QC interval for ant isera
QC records for:155 Salmonella reagents (thanks PHLs!)
20 E. coli reagents5-20 reagents for each of the other organisms
Stability of E. coli O and H ant isera
Antisera produced between 1981 and 1991 No change in RTD in 24-34 years (and count ing…)
Stability of Salmonella serotyping reagents
Did not t rack when a lot was depleted Shorter “# years lasted” could be due to missing QC
records or a lot being depleted Oldest Salmonella reagent on inventory is 36 years old Reagents made by the state of California are 13 to 20
years old (or depleted).
Category#
reagents
# years lasted, range
Reagents that failed QC 3Reagents that never failed QC (yet) Monoclonal antibody reagents, H antigens 3 14-15 Monoclonal antibody reagents, O antigens 25 5-18 Absorbed rabbit antiserum reagents, H antigens 28 2-20 Rabbit antiserum reagents, H antigens 53 6-31 Rabbit antiserum reagents, O antigens 41 4-36 Rabbit antiserum reagents, Vi antigen 2 11-27
Total 155 2-36% of reagents that never failed QC (yet) 98.1%
Summary by reagent type
# years lasted
# reagents
% of reagents
1-5 12 8%6-10 26 17%11-15 41 27%16-20 45 30%21-25 11 7%>25 17 11%
Total 152 100%
Summary by number of years reagent lasted
Salmonella serotyping reagents that failed
ReagentYear of
Manufacture Year of last
QC pass# years lasted before failure What happened?
Ha, rabbit
antisera1979 2002 23
4/2009: CDC was alerted by a SHD that the reagent failed their QC. Result confirmed at CDC the next day. Lot removed from inventory and other states notified via Enteric Bacteriology Listserv.
H2, absorbed
rabbit antisera
1998 2004 6
12/2006: Routine testing at CDC indicated titer had fallen and cross reactions seen at revised RTD. Lot removed from inventory and states notified via Enteric Bacteriology Listserv.
O10, mouse
monoclonal antibody
2000 na na
8/2010: Reagent failed QC while testing bulk in advance of dispensing. Reagent is a blend of two monoclonal antibodies and it appears one died.
CDC is happy to assist in any way we can with QC and QA issues
CDC Enteric Bacteriology Discussion [email protected] Contact Blake Dinsmore at [email protected] if you
would like to be added to the list.
Salmonella Lab Inbox [email protected] Team Inbox [email protected]
Patt i [email protected] 639 1848
For more information please contact Centers for Disease Control and Prevention
1600 Clifton Road NE, Atlanta, GA 30333Telephone, 1-800-CDC-INFO (232-4636)/TTY: 1-888-232-6348E-mail: [email protected] Web: www.cdc.gov
Thank you !Patt i Fields
National Center for Emerging and Zoonotic Infectious DiseasesDivision of Foodborne, Waterborne, and Environmental Diseases
The findingsand 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.
Salmonella Serotyping ReagentsCDC Inventory
Category Reagent Status SolutionH monospecific 2 out of stockH monospecific 7 out of stockH monospecific v out of stockH monospecific z24 out of stockH monospecific z28 out of stockO grouping 28 out of stockO monospecific 7 low bulk available for dispensingO monospecific 8 low bulk available for dispensingO monospecific 9 lowH typing y low bulk available for dispensingH monospecific z51 low
