1. FOODBORNE DISEASE OUTBREAK INVESTIGATION TEAM TRAINING MODULE 4 – LABORATORY INVESTIGATION

2. 2 AT THE END OF THIS MODULE, YOU WILL BE ABLE TO 1.Determine the likely causative agent for a foodborne outbreak based on clinical findings and/or suspect food. 2.List important considerations in the collection and submission of clinical and food specimens to the laboratory. 3.Interpret results from testing of clinical and food specimens. 4.Describe three ways subtyping of the causative agent can be used in an outbreak investigation.

3. 3 DETERMINING CAUSATIVE AGENT Laboratory testing Clues about causative agent can be gained from:  Clinical findings among ill persons  Suspected food, if known

4. 4 NON-LABORATORY METHODS FOR DETERMINING CAUSATIVE AGENT

5. 5 USEFUL CLINICAL FINDINGS AMONG ILL PERSONS Predominant signs and symptoms Incubation period (i.e., time from exposure to causative agent to onset of illness) Duration and severity of illness

6. 6 GENERAL CATEGORIES OF FOODBORNE ILLNESS Illnesses caused by preformed toxins Illnesses caused by infections with growth in the gastrointestinal tract with  Direct damage to tissues and/or  Release of toxins in the gut (enterotoxins)

7. 7 ILLNESSES DUE TO PREFORMED TOXINS Ingestion of food already contaminated by toxins Common clinical findings  Short incubation period (minutes or hours)  Abrupt onset  Symptoms depend on toxin −Vomiting common −Fever and elevated white blood cells in peripheral blood rare

8. 8 SOURCES OF PREFORMED TOXINS Bacteria - Staphylococcus aureus, Bacillus cereus, Clostridium perfringens Fish – Scombrotoxin (histamine fish poisoning), tetrodotoxin (puffer fish) Marine algae – Ciguatoxin, saxitoxin (paralytic shellfish poisoning) Fungus – Aflatoxin, mushroom toxins Chemicals – Heavy metals, pesticides

9. 9 ILLNESSES DUE TO INFECTIONS Growth of microorganism in body with direct damage to tissues and/or release of toxins in the gut (enterotoxins) Common clinical findings  Relatively long incubation period  Diarrhea, nausea, vomiting, abdominal pain  Fever, elevated white blood cells in peripheral blood, white blood cells and red blood cells in stool

10. 10 GENERAL TYPES OF INFECTIONS Viruses – Norovirus, hepatitis A virus, rotavirus Bacteria – Salmonella, Shigella, Shiga toxin- producing E. coli Parasites – Cryptosporidium, Cyclospora cayetanensis, Giardia intestinalis, Trichinella Other – prions

11. 11 GENERALIZATIONS BY INFECTION TYPE VirusesBacteriaParasites Incubation period Possible symptoms Signs Duration Less than a day to 4 days Vomiting, diarrhea, (rarely bloody) abdominal pain, fever  WBC; fecal WBC 2-5 days Less than a day to a week Vomiting, diarrhea, (sometimes bloody), abdominal pain, fever  WBC;  fecal WBC Several days to a week Abdominal pain and diarrhea 1-4 weeks Several weeks

12. 12 QUESTION Twenty people became ill after attending a banquet. Onset of illness occurred (on average) about 4 hours after eating at the banquet (range 1-8 hours). All cases reported nausea and vomiting. Two reported diarrhea and none reported fever. No ill persons sought health care. All were well in 48 hrs. Based on the clinical findings reported by cases, is the causative agent likely to be a preformed toxin or infection due to a virus, bacteria, or parasite?  Answer: Preformed toxin.

13. 13 QUESTION 51 children and staff from a childcare center developed gastroenteritis following a field trip to a dairy farm. Symptoms included diarrhea (100%), bloody diarrhea (27%), fever (45%), and vomiting (45%). White blood cell counts were elevated for the 10 patients tested. The average incubation period among cases was 3.5 days (range: 2-10 d). Based on the clinical findings reported by cases, is the causative agent likely to be a preformed toxin or infection due to a virus, bacteria, or parasite?  Answer: Illness likely due to an infection, specifically a bacterium

14. 14 CLINICAL FINDINGS Always exceptions to the rules Unique clinical findings can help agent Consult references

15. 15 SUSPECT FOODS Certain causative agents are associated with certain foods because the foods  Derive from animal reservoirs of agent  Derive from plants/animals that produce or accumulate toxin  Provide adequate conditions for contamination, survival, and proliferation of agent Common food-illness pairings

16. 16 FOODS AND COMMONLY ASSOCIATED CAUSATIVE AGENTS FoodCommonly associated causative agent Raw seafoodVibrio spp., hepatitis A virus, noroviruses Raw eggsSalmonella (particularly serotype Enteritidis) Undercooked meat or poultry Salmonella, Campylobacter, STEC, Clostridium perfringens Unpasteurized milk or juiceSalmonella, Campylobacter, Yersinia, STEC Unpasteurized soft cheesesSalmonella, Campylobacter, Yersinia, Listeria, STEC Home-made canned goodsClostridium botulinum Raw hot dogs, deli meatsListeria spp. Mussels, clams, scallopsSaxitoxin (paralytic shellfish poisoning); Tropical fishCiguatera poisoning ShellfishBrevetoxin (neurotoxic shellfish poisoning) Mackerel, tuna, bluefishScombrotoxin (histamine fish poisoning) Puffer fishTetrodotoxin Wild mushroomsMushroom poisoning

17. 17 QUESTION Which causative agents are commonly associated with each of these foods? Shiga toxin-producing E.coli (STEC) Salmonella Hepatitis A virus Listeria Norovirus Undercooked chicken Unpasteurized milk Raw seafood Raw eggs Deli meats 

18. 18  Bagged spinach  Carrot juice  Peanut butter  Dog food  Pot pies  Broccoli powder on snack food  Canned chili sauce  Hot peppers  White pepper  Raw cookie dough  Whole, raw papaya  Hazelnuts  Pine nuts NEW FOOD VEHICLES

19. 19 LABORATORY METHODS FOR DETERMINING CAUSATIVE AGENT

20. 20 LABORATORY TESTING “Laboratory responsibility” Important role of other team members  Collect specimens  Package and store them  Transport them to the laboratory  Request proper test(s)  Provide information about illness and specimen

21. 21 DIAGNOSTIC SPECIMENS Need to have suspicion of likely causative agent because appropriate specimen differs by agent  Stool – Most infectious foodborne agents  Blood – Bacteria that cause invasive disease (bacteremia)  Serum – Hepatitis A virus and Trichinella  Urine/hair – Heavy metals  Vomitus, stool, suspected food – Preformed toxins

22. 22 GUIDANCE ON DIAGNOSTIC SPECIMENS Testing laboratory “Diagnosis and Management of Foodborne Illness: A Primer for Physicians” CDC. “Guidelines for Confirmation of Foodborne- Disease Outbreaks.” MMWR 2000; 49 (1): 54- 62. (Table B)

23. 23 COLLECTION OF STOOL SPECIMENS Collect and submit as soon as possible Typically from 5-10 cases not treated with antibiotics with illness characteristic of outbreak Considerations that depend on suspected agent  Acceptability of rectal swabs  If more than one specimen is needed per patient  Use of preservative  Acceptability of freezing Photo source: S. Shiflett

24. 24 COLLECTION OF STOOL SPECIMENS Suspected Agent VirusesBacteriaParasites Amount Specimens per patient Preservative Freezing *Freezing causes die off of Campylobacter jejuni. **Cary Blair acceptable in some laboratories but dilutes stool sample. 10 cc bulk stool Bulk stool (rectal swab) 10 cc bulk stool One Up to three No** Cary Blair10% formalin, PVA No (only if RNA or antigen testing) Acceptable*

25. 25 QUESTION Using the references provided, what specimen would you collect if you suspect the following diseases? Are there any special considerations?  Stool, do not freeze Blood or cerebrospinal fluid Vomitus or food, not all labs accept vomitus Stool, do not freeze, multiple specimens might be necessary Campylobacteriosis Staphylococcal food poisoning Giardiasis Listeriosis Diagnostic Specimen

26. 26 STOOL SPECIMENS Label specimen clearly and maintain log. Complete necessary laboratory forms. Provide patient information:  Date of collection,  Date of onset, and  Signs and symptoms.

27. 27 TESTING STOOL SPECIMENS Routine stool culture  Salmonella, Shigella, STEC, Campylobacter jejuni/coli Special requests  Vibrio, Yersinia, E. coli O157:H7*, other Campylobacter species  Viruses – detection of viral RNA by RT-PCR or visualization of virus under electron microscope  Most parasites – visualization by ova and parasite exam; Cryptosporidium or Cyclospora require special techniques *Most public health laboratories routinely test for E. coli O157:H7

28. 28 INTERPRETING STOOL SPECIMEN TESTING Positive for certain agent Negative for certain agent  Patient’s illness caused by agent  Patient colonized with agent but illness not caused by agent  Contamination of specimen  Patient’s illness not caused by agent  Collected too late in course of illness  Specimen handled improperly 

29. 29 FOOD SPECIMENS Collect as soon as possible and store. Follow local policies on food collection private homes. Test when food implicated by other studies. Check with laboratory on collection and storage.  Keep frozen foods frozen.  Refrigerate perishable foods.  If testing >48 hours after collecting, consult lab about freezing.

30. 30 SUBMITTING FOOD SPECIMENS Label container and maintain log. Complete necessary laboratory forms. Provide sample information:  Date of collection,  When originally served, and  Handling since time of ingestion that caused illness.

31. 31 CHAIN OF CUSTODY Chronological written record that identifies who had control over specimen during what time Includes all persons handling sample Persons signing form  Are responsible for sample while in their possession  May be called to testify in criminal proceeding Each agency has own form

32. 32 TESTING FOOD SPECIMENS Challenging because  Food components can interfere with culture.  Food is not sterile.  Different methods are needed for different foods and causative agents.  Generally accepted procedures are not available. Not all public health laboratories do food testing → May need to submit to reference laboratory Consult with testing laboratory.

33. 33 INTERPRETATION OF FOOD SPECIMEN TESTING Positive for certain agent Negative for certain agent   Agent cause of outbreak (i.e., food is source)  Agent present but not the cause of the outbreak (e.g., contaminated after the fact)  Agent not present (i.e., food not source)  Non-uniform contamination  Agent inactivated/killed during handling  Complexity of food product and testing procedures precluded detection of agent

34. 34 QUESTION An outbreak of gastroenteritis occurs following a wedding dinner reception. Ill persons reported diarrhea (100%), bloody diarrhea (25%), vomiting (80%), and fever (50%). The average incubation period for development of illness was 2 days. Stool specimens were collected from 10 ill persons. The dinner was catered by a local business. No foods from the dinner were available except raw chicken from a package used in the chicken Cordon Bleu. A sample was collected and submitted to the laboratory.

35. 35 QUESTION (CONT’D) Eight of the 10 stool cultures were positive for Salmonella. How do you interpret the stool culture results?  Answer: Symptoms among patients (i.e., diarrhea some of which is bloody, vomiting, and fever) and the incubation period (average of 2 days) are consistent with salmonellosis. A large number of stools are positive. Salmonella is likely to be the causative agent for this outbreak.

36. 36 QUESTION (CONT’D) The raw chicken was also positive for Salmonella. How do you interpret the food test results, given the results of the stool cultures?  Answer: The chicken could be the source of the outbreak but raw chicken is commonly contaminated with Salmonella. If chicken is properly prepared, Salmonella present will be killed. What information might help you interpret this information properly? Answer: Results from analytic epi studies, findings from an EH assessment of the chicken preparation, subtyping of isolates from cases and the chicken

37. 37 SUBTYPING OF CAUSATIVE AGENTS

38. 38 SUBTYPING OF CAUSATIVE AGENT Characterization of microorganisms below the species level using characteristics that  Differ between strains  Are same among isolates with common origin Variety of subtyping methods (e.g., serotyping, phage typing, antibiotic susceptibility, pulsed field gel electrophoresis [PFGE], multiple-locus variable number tandem repeat analysis [MLVA]), not all of which are available for all organisms Not all methods equally discriminatory

39. 39 USES OF SUBTYPING IN OUTBREAK INVESTIGATION Usefulness in outbreaks based on presumption  Isolates in an outbreak have a common origin  Single strain will be the culprit in most outbreaks Uses  Link cases together  Link foods with outbreaks  Refine case definition decreasing misclassification in epidemiologic studies  Link outbreaks in different locations

40. 40 PULSED FIELD GEL ELECTROPHORESIS (PFGE) Separation of DNA fragments in a gel using a pulsing electric field Creates visual banding pattern unique for isolate Different DNA composition  different PFGE Indistinguishable patterns suggest similar origin of isolates  Cluster of indistinguishable patterns

41. 41 PULSENET LABORATORY NETWORK Participating Laboratories PFGE Patterns PulseNet National Database (CDC) Monitors for similar patterns Notifies foodborne epidemiologists of clusters State labs can query Standardized testing of E. coli O157:H7, Salmonella, Shigella, Listeria, and Campylobacter Patterns uploaded by testing laboratory

42. 42 PULSENET PARTICIPANTS PulseNet headquarters Regional laboratories Local and secondary state laboratories Federal laboratories

43. 43 E. COLI O157:H7 AND HAZELNUTS December 2010, cluster of 8 cases of E. coli O157:H7 identified through PulseNet Follow-up interviews revealed in-shell hazelnuts consumed by all cases Outbreak strain isolated from hazelnuts Traceback led to common distributor Product recalled March 2011

44. 44 SUBTYPING ISSUES  Matching of subtypes not proof of common exposure  Association of multiple subtypes with one outbreak  Need for routine subtyping (in real time)  Limited ability of available methods to distinguish between strains  Patient isolate not available if rapid diagnostic testing used

45. 45 NO SMOKING GUN? Most convincing evidence about the source of an outbreak is isolation of causative agent from suspected vehicle (i.e., food) Causative agent isolated from food vehicle in only 14% of outbreaks with a confirmed etiology Importance of epi and EH studies

46. 46 QUICK QUIZ

47. 47 QUICK QUIZ 1.Which of the following causative agents tend to have the longest incubation period? A.Preformed toxins B.Viruses C.Bacteria D.Parasites

48. 48 QUICK QUIZ 2.Most foods can be associated with a variety of causative agents. A.True B.False

49. 49 QUICK QUIZ 3.The following are true statements about stool specimens collected during a foodborne disease outbreak EXCEPT A.Stool is the specimen of choice for most causative agents. B.Routine stool cultures cover the most common foodborne disease causative agents. C.The method of collection and handling depend on the suspected causative agent. D.Freezing of stool specimens can interfere with detection of some causative agents.

50. 50 QUICK QUIZ 4.Which of the following is a possible interpretation of a negative result on a routine stool culture? A.The patient’s illness was not caused by agents detected through routine cultures. B.The specimen was collected too late in the course of illness. C.The specimen was handled improperly, killing any causative agents present. D.All of the above

51. 51 QUICK QUIZ 5.Subtyping of isolates from cases of the same disease can be used for all of the following EXCEPT A.Link cases together B.Link outbreaks in different geographic locations C.Link foods with cases D.As sole proof of an outbreak