MODULE 4 LABORATORY INVESTIGATION FOODBORNE DISEASE OUTBREAK INVESTIGATION

MODULE 4 – LABORATORY INVESTIGATION FOODBORNE DISEASE OUTBREAK INVESTIGATION TEAM TRAINING

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. 2

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

NON-LABORATORY METHODS FOR DETERMINING CAUSATIVE AGENT 4

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 5

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) 6

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 7

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 8

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 9

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

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

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. 12

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 13

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

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 15

FOODS AND COMMONLY ASSOCIATED CAUSATIVE AGENTS Food Commonly associated causative agent Raw seafood Vibrio spp. , hepatitis A virus, noroviruses Raw eggs Salmonella (particularly serotype Enteritidis) Undercooked meat or poultry Salmonella, Campylobacter, STEC, Clostridium perfringens Unpasteurized milk or juice Salmonella, Campylobacter, Yersinia, STEC Unpasteurized soft cheeses Salmonella, Campylobacter, Yersinia, Listeria, STEC Home-made canned goods Clostridium botulinum Raw hot dogs, deli meats Listeria spp. Mussels, clams, scallops Saxitoxin (paralytic shellfish poisoning); Tropical fish Ciguatera poisoning Shellfish Brevetoxin (neurotoxic shellfish poisoning) Mackerel, tuna, bluefish Scombrotoxin (histamine fish poisoning) Puffer fish Tetrodotoxin Wild mushrooms Mushroom poisoning 16

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

NEW FOOD VEHICLES § 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 18

LABORATORY METHODS FOR DETERMINING CAUSATIVE AGENT 19

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 20

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 21

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) 22

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 23

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

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

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. 26

TESTING STOOL SPECIMENS Routine stool culture § Salmonella, Shigella, STEC, Campylobacter jejuni/coli Special requests § Vibrio, Yersinia, E. coli O 157: H 7*, 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 O 157: H 7 27

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

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. 29

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. 30

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 31

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. 32

INTERPRETATION OF FOOD SPECIMEN TESTING Positive 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) Negative for certain agent § 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 33

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. 34

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. 35

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 36

SUBTYPING OF CAUSATIVE AGENTS 37

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 38

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 39

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 40

PULSENET LABORATORY NETWORK Participating Laboratories Standardized testing of • E. coli O 157: H 7, • Salmonella, • Shigella, • Listeria, and • Campylobacter PFGE Patterns uploaded by testing laboratory Pulse. Net National Database (CDC) • Monitors for similar patterns • Notifies foodborne epidemiologists of clusters • State labs can query 41

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

E. COLI O 157: H 7 AND HAZELNUTS December 2010, cluster of 8 cases of E. coli O 157: H 7 identified through Pulse. Net 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 43

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 44

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 45

QUICK QUIZ 46

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 47

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

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. 49

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 50

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 51