1. The Role of the Laboratory in Foodborne Outbreak Investigations Dubai, February 17, 2014 Cheryl Bopp, M.S. Unit Lead, Epidemic Investigations Laboratory Enteric Diseases Laboratory Branch 404-639-1798 cbopp@cdc.gov

2. Epidemic Investigations Laboratory CDC ♦A unit of the Enteric Diseases Laboratory Branch ♦Responsibility: To conduct laboratory investigations of outbreaks of foodborne and waterborne bacterial diseases 2

3. Enteric Diseases Laboratory Branch: Foodborne Bacteria and Toxins ♦Salmonella, Shigella, diarrheagenic Escherichia coli, Yersinia enterocolitica, and Cronobacter sakazakii in infants ♦Campylobacter ♦Listeria ♦Vibrio cholerae, Vibrio parahaemolyticus, Vibrio spp. ♦Foodborne toxins –Clostridium botulinum and Clostridium perfringens –Bacillus cereus –Staphylococcus aureus

4. Outline ♦Why investigate foodborne disease outbreaks? ♦Foodborne outbreak investigations in the United States –Role of clinical laboratories –Role of local and state public health laboratories ♦ Example of a foodborne outbreak investigation

5. Why Investigate Foodborne Disease Outbreaks? ♦Identify the etiologic agent and food vehicle(s) ♦Contain the outbreak by removing the contaminated food from distribution ♦Share information with food regulatory agencies and the food industry so that in the future similar outbreaks may be prevented ♦Learn about newly emerging pathogens and their modes of transmission

6. Role of Clinical Laboratories in Foodborne Outbreak Investigations ♦Responsibility of clinical microbiology laboratories: –Test clinical specimens –Report the isolation of foodborne pathogens to the physician ♦In the US, clinical laboratories are crucial in detecting foodborne outbreaks because they: –Report suspected outbreaks to public health authorities –Forward isolates from suspected foodborne illness to local or state public health laboratories

7. Role of Local and State Public Health Laboratories in Outbreak Investigations 1.Provide advice on specimen collection 2.Receive food, environmental specimens for testing 3.Receive clinical specimens for testing 4.Receive isolates from clinical laboratories for identification, serotyping, and DNA fingerprinting by PFGE 5.Perform PFGE and submit DNA fingerprints to PulseNet (or send isolates to another public health lab for PFGE) 6.Interpret and report laboratory results to the outbreak investigation team

8. Role of the Public Health Laboratory in Foodborne Outbreak Investigations 1.Advise the outbreak investigation team on specimen collection -What type of specimens to collect -How many specimens and the volume of each specimen for optimal culture results -How to store and transport specimens to the public health laboratory

9. What Types of Specimens to Collect ♦Clinical specimens –Fecal –Blood cultures –Serum –Vomitus, etc. ♦Food specimens ♦Water and environmental specimens ♦Animal Specimens

10. CDC Guidelines for Collection of Fecal Specimens http://www.cdc.gov/foodsafety/outbreaks/investigating-outbreaks/specimen-collection.html

11. Food, Environmental, and Animal Specimens 2. Receive food, environmental, animal specimens –To conserve laboratory resources, nonclinical specimens are tested only for the implicated pathogen –Decide on optimal detection methods for the outbreak pathogen –Culture methods –Molecular methods (immunoassay, PCR, etc) ♦Serotype and PFGE all isolates of the outbreak pathogen

12. Limitations of Food Testing for Confirmation of Outbreak Vehicle ♦In many outbreak investigations, food testing is negative for the etiologic agent. Reasons include: –The food item was incorrectly identified as the vehicle –The food vehicle was not available for collection and other non-epidemiologically relevant foods were tested instead –The food was contaminated during preparation and not before (unprepared food not contaminated) –Food not evenly contaminated with the pathogen so sampling and testing of a portion of the vehicle may produce false negative results –The laboratory methods were inadequate

13. Collection of Water Samples in Foodborne Outbreaks ♦Collect water if investigation suggests –cross contamination of foods by water –outbreak is waterborne not foodborne ♦Well water and surface waters (streams, rivers, ponds, lakes) may be contaminated with Salmonella, Campylobacter, or E. coli O157:H7 in rural or agricultural areas –PFGE of patient and water isolates may be crucial to implicate the water source

14. Collection of Environmental Samples ♦Surfaces ♦Food preparation equipment –Slicers, blenders, etc. ♦Other samples –Soil –Sawdust

15. Collection of Animal Specimens ♦Animals are often colonized with Salmonella, Campylobacter, or E. coli O157:H7, etc. – cattle and other ruminants such as goats, sheep – poultry – household pets (dogs, cats, reptiles, frogs)

16. Role of the Public Health Laboratory in Foodborne Outbreak Investigations 3. Receive clinical specimens for culture ♦If clinical laboratory testing did not determine the pathogen, the public health laboratory tests for additional pathogens –An important role of public health laboratories is to maintain capacity to test for all important foodborne pathogens and toxins Examples: norovirus, Cl. perfringens, Staph enterotoxin –Or the public health laboratory may forward specimens to another laboratory

17. Top 5 US Foodborne Pathogens 2000-2008; http://www.cdc.gov/foodborneburden ♦Top 5 US foodborne pathogens (estimated percent of all foodborne disease) –Norovirus (58%), Nontyphoidal Salmonella (11%), Clostridium perfringens (10%), Campylobacter spp. (9%), Staphylococcus aureus (3%) ♦In the US, clinical laboratories often test stools only for Salmonella, Shigella, Campylobacter ♦Public health laboratory testing essential to detect outbreaks of norovirus, C. perfringens, Staph enterotoxin

18. CDC Guidelines for Confirmation of Foodborne Disease Outbreaks ♦http://www.cdc.gov/outbreaknet/references_resources/ guide_confirming_diagnosis.htmlhttp://www.cdc.gov/outbreaknet/references_resources/ guide_confirming_diagnosis.html ♦Information on incubation period, clinical syndrome, and confirmatory testing for: –Bacterial agents –Chemical agents –Viral agents –Parasitic agents

19. Table B-1. Guidelines for confirmation of foodborne-disease outbreaks (Bacterial) Etiologic agentIncubation periodClinical syndromeConfirmation Campylobacter jejuni/coli 2-10 days; usually 2-5 days Diarrhea (often bloody), abdominal pain, fever Isolation of organism from clinical specimens from two or more ill persons OR Isolation of organism from epidemiologically implicated food Clostridium perfringens 6-24 hrsDiarrhea, abdominal cramps; vomiting and fever uncommon Isolation of 10 6 organisms/g from stool of two or more ill persons, provided specimen is properly handled. OR Demonstration of enterotoxin in the stool of two or more ill persons OR Isolation of 10 5 organisms/g from epidemiologically implicated food, provided specimen is properly handled Nontyphoidal Salmonella 6 hrs-10 days; usually 6-48 hrs Diarrhea, often with fever and abdominal cramps Isolation of organism of same serotype from clinical specimens from two or more ill persons OR Isolation of organism from epidemiologically implicated food Staphylococcus aureus 30 min-8 hrs; usually 2-4 hrs Vomiting, diarrheaIsolation of organism of same phage type from stool or vomitus of two or more ill persons OR Detection of enterotoxin in epidemiologically implicated food OR Isolation of 10 5 organisms/g from epidemiologically implicated food, provided specimen is properly handled Yersinia enterocolitica1-10 days; usually 4-6 daysDiarrhea, abdominal pain (often severe)Isolation of organism from clinical specimen from two or more ill persons OR Isolation of pathogenic strain of organism from epidemiologically implicated food Guidelines for Confirmation of Foodborne Disease Outbreaks http://www.cdc.gov/outbreaknet/references_resources/guide_confirming_diagnosis.html http://www.cdc.gov/outbreaknet/references_resources/guide_confirming_diagnosis.html Etiologic agent Incubation period Clinical syndrome Confirmation of Etiology

20. Isolation of Pathogens from Diarrheal Outbreaks of Unknown Etiology CDC Algorithm fecal specimen XLD or HEK MAC TCBS TET or SEL Broth CVACIN or SS Shigella Salmonella Shigella Salmonella E. coli Vibrio cholerae Vibrio parahaemolyticus HEK Campylobacter Jejuni/coli Yersinia enterocolitica Salmonella CTSMAC E. coli O157:H7 diarrheagenic E. coli: STEC, ETEC, EPEC, EIEC Sub 37 o C TET to new TET, Incubate at 42 o C HEK 25oC 2 days

21. Role of the Public Health Laboratory in Foodborne Outbreak Investigations 4. Receive isolates from clinical laboratories –Confirmation of identification of pathogen –Serotyping of pathogen Salmonella and E. coli serotyping important –Toxin testing (C. perfringens, E. coli) –DNA fingerprinting by PFGE

22. Role of the Public Health Laboratory in Foodborne Outbreak Investigations 5.Perform PFGE and submit DNA fingerprints to the PulseNet Database –If laboratory does not have PFGE capacity, isolates are sent to another public health lab for PFGE testing

23. Why does CDC use PFGE instead of other DNA fingerprinting methods? ♦PFGE is the most epidemiologically relevant method for E. coli O157:H7 and Salmonella –A 1993 investigation of an E. coli O157:H7 outbreak in the western United States first demonstrated the utility of PFGE as a DNA fingerprinting method ♦Because of PulseNet highly standardized methods and Bionumerics software, PFGE fingerprints from different laboratories can be compared

24. 19201940196019802000 Serotyping MEE REA PFGE RAPD MLST Phage typing Bacteriocin typing AFLP * Microarray-based multi-target sequencing Evolution of Subtyping for Bacteria MLVA Ribotyping MBMS* Plasmid profiles The future of subtyping is whole genome sequencing

25. Role of the Public Health Laboratory in Foodborne Outbreak Investigations ♦Report and interpret laboratory results for the outbreak investigation team –Provide written reports of laboratory results to the investigation team leader and interpret the significance of the results –Provide timely updates of ongoing testing Speed versus accuracy: If presumptive results are reported, the laboratory must clearly state that the results have not been confirmed

26. An example of an outbreak investigation ♦Cereal-associated outbreak of Salmonella serotype Agona infections in multiple states, 1998 –Role of the clinical laboratories –Role of the public health laboratories

27. Clinical laboratories isolated Salmonella enterica serotype Agona from 23 cases in Illinois and 9 cases in Pennsylvania ♦Clinical laboratories isolated Salmonella ♦Forwarded isolates to state public health laboratories for serotyping

28. Public health laboratories in Illinois, Pennsylvania, and other states reported a large increase in Salmonella serotype Agona isolates

29. Preliminary results by state outbreak investigation teams implicated one brand of toasted oats cereal from one grocery store chain ♦Interviewed 11 patients: all mentioned grocery store chain A as their regular grocery store ♦10/11 mentioned purchasing plain toasted oats cereal from the store ♦No other common food item or social event identified ♦Contacted grocery store chain A and producer of plain toasted oats cereal ♦All states with cases had grocery store chain A

30. Public health laboratory testing: all Salmonella Agona isolates from cases who ate the implicated cereal had the same PFGE DNA “fingerprint” ♦In addition, Salmonella Agona isolates from persons who had not consumed the implicated brand of cereal had different PFGE “fingerprints”

31. State outbreak investigation teams conducted a case-control study: ill persons were 20 times more likely to have eaten the implicated cereal ♦74 ill persons in 10 states enrolled; 151 healthy household members used as controls ♦Phone interview –Shopping habits –Cereal consumption –Other food items associated with salmonellosis ♦Ill persons 20 times more likely to have eaten plain toasted oats cereal produced by Company B ♦No other food item implicated

32. State public health laboratories and the CDC isolated Salmonella Agona from the implicated cereal ♦Toasted oats obtained from case homes (open packages) tested at state public health laboratory and CDC by multiple methods ♦S. Agona isolated from toasted oats cereal by state public health laboratories and CDC ♦Unopened packages from the same lot obtained from Company B sent to federal regulatory agency laboratory for testing ♦Regulatory agency isolates S. Agona from cereal in intact packages

33. Public health laboratories performed PFGE on Salmonella Agona isolates from cereal and DNA fingerprints match human isolates CC

34. Outcomes of the Outbreak Investigation ♦The cereal product was recalled by the manufacturer. ♦CDC recommended that consumers not eat the cereal product. ♦Additional infections were prevented.

35. Lessons from this outbreak investigation ♦Laboratories were critical to outbreak recognition –Clinical laboratories isolated Salmonella and forwarded isolates to public health laboratories –Public health laboratories serotyped and PFGE fingerprinted clinical isolates –Public health laboratories isolated the outbreak strain of Salmonella serotype Agona from the implicated food ♦Timely communication between laboratorians, epidemiologists and regulatory officials enabled rapid identification of the food vehicle

36. Thank you!