Foodborne Disease Outbreak Investigation Team Training:

Module 6 – Laboratory Investigation

Laboratory investigations 1

Module Learning Objectives

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. Laboratory investigations > Learning objectives 2

Determining Causative Agent

• Laboratory testing Clues about causative agent can be gained from: • Clinical findings among ill persons • Suspected food, if known  Laboratory investigations 3

Non-laboratory Methods for Determining Causative Agent

Laboratory investigations 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 Laboratory investigations > Non-laboratory methods 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) Laboratory investigations > Non-laboratory methods 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 Laboratory investigations > Non-laboratory methods 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 Laboratory investigations > Non-laboratory methods 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 Laboratory investigations > Non-laboratory methods 9

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 Laboratory investigations > Non-laboratory methods 10

Generalizations by Infection Type

Viruses Bacteria Parasites

Incubation period Less than a day to 4 days Less than a day to a week Possible symptoms Signs Duration 1-4 weeks Vomiting, diarrhea, (rarely bloody) abdominal pain, fever  WBC; fecal WBC Vomiting, diarrhea, (sometimes bloody), abdominal pain, fever  WBC;  fecal WBC 2-5 days Several days to a week Abdominal pain and diarrhea Several weeks Laboratory investigations > Non-laboratory methods 11

Class 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 within 48 hours.

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?

 Laboratory investigations > Non-laboratory methods 12

Class 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?

 Laboratory investigations > Non-laboratory methods 13

Clinical Findings

• Always exceptions to the rules • Unique clinical findings can help pinpoint specific agent • Consult references Laboratory investigations > Non-laboratory methods 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 contamin ation, survival, and proliferation of agent • Common food-illness pairings Laboratory investigations > Non-laboratory methods 15

Foods and Commonly Associated Causative Agents

Food Raw seafood Raw eggs Undercooked meat or poultry Unpasteurized milk or juice Unpasteurized soft cheeses Home-made canned goods Raw hot dogs, deli meats Mussels, clams, scallops Tropical fish Shellfish Mackerel, tuna, bluefish Puffer fish Wild mushrooms Commonly associated causative agent

Vibrio

spp., hepatitis A virus, noroviruses

Salmonella

(particularly serotype Enteritidis)

Salmonella, Campylobacter

, STEC,

Clostridium perfringens Salmonella

,

Campylobacter

,

Yersinia

, STEC

Salmonella

,

Campylobacter

,

Yersinia

,

Listeria

, STEC

Clostridium botulinum Listeria

spp.

Saxitoxin (paralytic shellfish poisoning); Ciguatera poisoning Brevetoxin (neurotoxic shellfish poisoning) Scombrotoxin (histamine fish poisoning) Tetrodotoxin Mushroom poisoning Laboratory investigations > Non-laboratory methods 16

Class Question

Which causative agents are commonly associated with each of these foods?

 Undercooked chicken Unpasteurized milk Raw seafood Raw eggs Deli meats Shiga toxin-producing

E.coli (STEC) Salmonella

Hepatitis A virus

Listeria

Norovirus Laboratory investigations > Non-laboratory methods 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 Laboratory investigations > Non-laboratory methods 18

Laboratory Methods for Determining Causative Agent

Laboratory investigations 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 Laboratory investigations 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 Laboratory investigations > Diagnostic specimens 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) Laboratory investigations > Diagnostic specimens 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

Laboratory investigations > Diagnostic specimens 23

Collection of Stool Specimens (cont’d)

Viruses Suspected Agent Bacteria Parasites

Amount 10 cc bulk stool Bulk stool (rectal swab) 10 cc bulk stool Specimens per patient Preservative One No** One Cary Blair Up to three 10% formalin, PVA Freezing No (only if RNA or antigen testing) Acceptable* No (only if RNA or antigen testing) *Freezing causes die off of

Campylobacter jejuni.

**

Cary Blair acceptable in some laboratories but dilutes stool sample.

Laboratory investigations > Diagnostic specimens 24

Class Question

 Using the references provided, what specimen would you collect if you suspect the following diseases? Are there any special considerations?

Diagnostic Specimen Campylobacteriosis Staphylococcal food poisoning Giardiasis Listeriosis Laboratory investigations > Diagnostic specimens 25

Submission of 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.

Laboratory investigations > Diagnostic specimens 26

Testing of 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 Laboratory investigations > Diagnostic specimens 27

Interpretation of 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 (caused by another agent requiring different testing) – Collected too late in course of illness – Specimen handled improperly Laboratory investigations > Diagnostic specimens 28

Collection of Food Specimens

• Collect as soon as possible and store.

• Follow local policies on collection of foods from 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 collection, consult laboratory about freezing.

Laboratory investigations > Food specimens 29

Submission of 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.

Laboratory investigations > Food specimens 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 Laboratory investigations > Food specimens 31

Testing of 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.

Laboratory investigations > Food specimens 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 Laboratory investigations > Food specimens 33

Class 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.

Laboratory investigations 34

Class Question (cont’d)

Eight of the 10 stool cultures were positive for

Salmonella

. How do you interpret the stool culture results?  Laboratory investigations 35

Class 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?

What information might help you interpret this information properly?

Laboratory investigations 36

Subtyping of Causative Agents

Laboratory investigations 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 Laboratory investigations > Subtyping of causative agent 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 Laboratory investigations > Subtyping of causative agent 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 Laboratory investigations > Subtyping of causative agent 40

PulseNet Laboratory Network

Participating Laboratories PFGE Patterns PulseNet National Database (CDC) • • • • • Standardized testing of

E. coli

O157:H7,

Salmonella Shigella

, ,

Listeria

, and

Campylobacter

Patterns uploaded by testing laboratory • Monitors for similar patterns • Notifies foodborne epidemiologists of clusters • State labs can query Laboratory investigations > Subtyping of causative agent 41

PulseNet Participants

PulseNet headquarters Regional laboratories Local and secondary state laboratories Federal laboratories Laboratory investigations > Subtyping of causative agent 42

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 Laboratory investigations > Subtyping of causative agent 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 Laboratory investigations > Subtyping of causative agent 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 epidemiologic and environmental health studies Laboratory investigations 45

Quick Quiz

Laboratory investigations 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 Laboratory investigations 47

Quick Quiz

2. Most foods can be associated with a variety of causative agents.

A. True B. False Laboratory investigations 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.

Laboratory investigations 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 Laboratory investigations 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 Laboratory investigations 51