1. Module 6 – Laboratory Investigation
Foodborne Disease Outbreak
Investigation Team Training:
Module 6 – Laboratory Investigation
Estimated time: 75 minutes

2. Module Learning Objectives
At the end of this module, you will be able to
Determine the likely causative agent for a foodborne outbreak based on clinical findings and/or suspect food.
List important considerations in the collection and submission of clinical and food specimens to the laboratory.
Interpret results from testing of clinical and food specimens.
Describe three ways subtyping of the causative agent can be used in an outbreak investigation.
> Learning objectives

3. Determining Causative Agent
Determining Causative Agent
Laboratory testing
Clues about causative agent can be gained from:
Clinical findings among ill persons
Suspected food, if known
Determining the causative agent is critical in a foodborne disease outbreak investigation and is used to link cases together to identify an outbreak, exclude unrelated illnesses, link cases with a food vehicle, and determine most effective control measures. Laboratory testing of clinical specimens (and occasionally food) is the best way to determine the causative agent in an outbreak.
[Advance slide.]
However, laboratory testing takes time (and is not always possible). While waiting for the results of laboratory testing, clinical findings and foods eaten by cases can provide insights into the causative agent of an outbreak and often allow investigators to proceed with other investigations in a more timely fashion.
In addition, because laboratory testing of different causative agents requires different specimens and collection, storage, and testing procedures (as we will see later in this module), clues to the likely causative agent also are critical to the laboratory investigations.
So, even though this module is on the “laboratory investigation”, we are going to start by focusing on determining the likely causative agent through methods not using the laboratory (or at least narrowing the list of suspected causative agents).

4. Non-laboratory Methods for Determining Causative Agent
Note to instructor: This is just a transition slide to indicate topics. Move to next slide before beginning lecture.

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
Certain clinical findings differ by causative agent and can provide clues to the general type of causative agent in an outbreak. (In fact, they will sometimes allow you to make a very good guess about the specific causative agent).
Useful clinical findings include
Predominant signs and symptoms of illness displayed by cases (e.g., bloody diarrhea, projectile vomiting, multiple episodes of vomiting), and
Incubation period for the illness (i.e., the time from exposure to the causative agent to onset of illness),
Duration and severity of the illness.
Of course, you might not have all of this information at the outset of an investigation. For instance, if you do not know the exposure that led to illness, you will not know the incubation period. And, if cases are still ill, you will not know the duration of illness. But, often you will know one or two of these things which will be useful in zeroing in on a causative agent.
> Non-laboratory methods

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)
There are many, many agents that can cause foodborne illnesses. For this discussion, it is helpful to divide causative agents into two broad categories:
Illnesses caused by preformed toxins
Illnesses caused by infections/production of toxins within gastrointestinal tract
Let’s look at the clinical findings related to each of these categories separately.
> Non-laboratory methods

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
Illnesses from preformed toxins are caused by ingestion of food already contaminated by toxins. A preformed toxin is ingested; live microorganisms do not need to be consumed to cause illness.
Illness from a preformed toxin manifests more rapidly than does illness from an infection because time for growth and invasion of the intestinal lining is not required. The incubation period for illness resulting from a preformed toxin is short, often in terms of minutes or hours, depending on the toxin.
Signs and symptoms also depend on the toxin ingested but onset is often abrupt and vomiting is common. Other symptoms range from diarrhea and abdominal pain to interference with sensory and motor functions (such as double vision, weakness, respiratory failure, numbness, tingling of the face, and disorientation).
Fever and an elevated white blood cell count are rarely present with preformed toxins.
> Non-laboratory methods

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
Sources of preformed toxin include certain bacteria; toxins found naturally in fish, plants (including algae), or fungi (mushrooms); heavy metals and pesticides; and a myriad of other chemicals. Some toxins are destroyed by heating whereas other are heat stable.
Preformed toxins most often result from bacteria that release toxins into food during growth in the food, such as Staphylococcus aureus, Bacillus cereus, and Clostridium perfringens. Intoxications by these three bacteria are so common that researchers have identified clinical profiles to help recognize these causative agents. (Toxins produced by Staphylococcus aureus and Bacillus cereus have been referred to as “vomiting toxins” because vomiting is a predominant symptom in those intoxications. Toxin produced by Clostridium perfringens has been referred to as “diarrhea toxin” because vomiting is not a common manifestation of intoxication resulting from this causative agent.)
> Non-laboratory methods

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
The other major category of foodborne illnesses are infections. Infections are a consequence of the growth of a microorganism in the body. Illness results from two mechanisms:
Viruses, bacteria, or parasites invade the intestinal mucosa and/or other tissues, multiply, and directly damage surrounding tissues and
Bacteria and certain viruses invade and multiply in the intestinal tract and then release toxins that damage surrounding tissues or interfere with normal organ or tissue function.
The necessary growth of the microorganism (including production and release of toxins) takes time; thus, incubation periods for infections are relatively long, often measured in terms of days as compared to hours or minutes for intoxications.
Symptoms of infection usually include diarrhea, nausea, vomiting, and abdominal cramps. Fever and an elevated peripheral white blood cell count are associated with many infections. Both red and white blood cells can be found in the stool with certain infections.
> Non-laboratory methods

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
Infections can be broken down a little further: whether they are caused by a virus, bacterium, or parasite. Here are some examples of each. A more complete listing of infectious agents was provided in Module 1.
You will notice a category for “other”. New disease agents continue to be discovered and some fall outside the traditional classifications of living organisms. One example is a newly identified type of infection thought to be transmitted by food and water: prions. Prions are neither bacterial, viral, or fungal in nature and consist only of a misfolded protein, no genetic material. Bovine spongiform encephalopathy (BSE), commonly known as mad-cow disease, is thought to be caused by a prion. You might remember the outbreak of BSE in England in 1986 that affected over 200,000 cattle fed protein supplements that included offal from other animals and that the outbreak was thought to be linked to variant Creutzfeld-Jakob disease in humans. (We won’t say anything more about prions, because they are rare causes of foodborne illness [to the best of our knowledge] but it is a good reminder that new causative agents are being identified.)
Let’s focus on the more common foodborne infectious agents: viruses, bacteria, and parasites and if there are clues that can help you tell them apart.
> Non-laboratory methods

11. Generalizations by Infection Type
Viruses
Bacteria
Parasites
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
Although there is a great deal of overlap in the illnesses caused by the three types of infectious agents (and exceptions among specific pathogens in each group) as well as variability from person to person and outbreak to outbreak, clinical findings often can suggest one kind of infectious agent over the others.
Viruses - Gastrointestinal illnesses caused by viruses are characterized by a relatively short incubation period (e.g., hours for norovirus or rotavirus and hours for astrovirus). Common symptoms include nonbloody diarrhea, vomiting, abdominal cramps, and fever. Vomiting can be pronounced and occur with or without diarrhea. Fecal leukocytes and elevated peripheral white blood cells occur, but are rare. Symptoms usually last for 2-5 days.
Bacteria - Gastrointestinal illness caused by bacteria tends to have incubation periods intermediate between those of viruses and parasites, ranging from less than a day to about a week. Vomiting and diarrhea are prominent symptoms, although the nature of the diarrhea varies with the pathogen. Bloody diarrhea occurs with Campylobacter, enterohemorrhagic and enteroinvasive E. coli, Salmonella, Shigella, and Yersinia enterocolitica. Except for enterohemorrhagic E. coli (E. coli O157:H7), fever is also common. White blood cells are often found in the stools of patients infected with Shigella, Salmonella, and invasive Escherichia coli. Duration of symptoms varies with the pathogen, but typically lasts several days to about a week.
Parasites - In general, incubation periods for parasitic diseases are longer than those for bacteria or viruses, ranging from 1-4 weeks. Abdominal pain and diarrhea tend to predominate. Nausea, vomiting, and fever are less common, although fever is common in symptomatic Trichinella infections and low-grade fever has been noted in Cyclospora infections. Fecal leukocytes are rare. Illness due to parasitic infections tends to be protracted, easily lasting several weeks. Untreated infections with Giardia have been reported to last for years.
> Non-laboratory methods

12. 
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?
The relatively short incubation, vomiting, and lack of diarrhea and fever are suggestive of a preformed toxin.
Of note, norovirus infections can sometimes mimic performed toxins with incubation periods less than a day, a high prevalence of vomiting, and low prevalence of fever.
Answer: Preformed toxin.
> Non-laboratory methods

13. 
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?
The longer incubation period, diarrhea (bloody in some cases), fever, and elevated white blood count suggest an infection, in particular one due to a bacterium.
Answer: Illness likely due to an infection, specifically a bacterium
> Non-laboratory methods

14. Clinical Findings Always exceptions to the rules
Unique clinical findings can help pinpoint specific agent
Consult references
So clinical findings can provide insight into the causative agent of an outbreak and often allow investigators to proceed with other investigations in a more timely fashion. However, investigators need to realize that these categorizations and profiles are gross generalizations. Not all causative agents that fall into a particular category of causative agents follow the rules just provided. For example, although incubation periods for viral diseases tend to be relatively short, hepatitis A virus can be spread through food and has an incubation period closer to a month (not 1-4 days as noted earlier for viruses). And although most parasitic diseases generally do not cause bloody diarrhea, Entamoeba histolytica can cause grossly bloody diarrhea.
Furthermore, unique clinical findings can be helpful in pinpointing the specific agent in an outbreak. For example in the class question about illness among children and staff at a child care center, we decided that the incubation period, signs, and symptoms suggest a “bacterial infection”. Had I mentioned that 10 of the cases (all children) were hospitalized and 8 developed hemolytic uremic syndrome (HUS), might that have helped you to be more specific with the diagnosis”? (Yes, it probably would as Shiga toxin-producing E. coli is the most frequent cause of HUS in children.) And indeed this outbreak was due to E. coli O157:H7.
Investigators should consult the references provided through Epi-Ready for the signs and symptoms associated with specific causative agents to better determine the likely causative agent in an outbreak.
[FOR THE INTERESTED STUDENT: Researchers have examined the usefulness of clinical findings in rapidly categorizing the cause of the outbreak.
Hedberg CW, Palazzi-Churas KL, Radke VJ, et al. The use of clinical profiles in the investigation of foodborne outbreaks in restaurants: United States, 1982–1997; Epidemiol Infect 2008; 136: 65–72. Available at
> Non-laboratory methods

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 contamin-ation, survival, and proliferation of agent
Common food-illness pairings
Certain causative agents are associated with certain foods because the foods
Derive from animal reservoirs of the agent (e.g., Salmonella and Campylobacter in poultry; STEC in cattle) and are frequent contaminants of those foods or products made from those foods.
Derive from plant or animal sources that produce or accumulate toxin without subsequent inactivation (e.g., mussels, clams, scallops that are filter feeders and accumulate saxitoxin)
Provide adequate conditions for contamination, survival, and proliferation of microorganism (e.g., Clostridium botulinum in home-canned goods; E. coli O157:H7 in seed sprouts, Bacillus cereus in fried rice)
As a result, common food-illness pairings have been recognized.
> Non-laboratory methods

16. Foods and Commonly Associated Causative Agents
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
> Non-laboratory methods

17. 
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
Note: Several foods were paired with two different agents (e.g., unpasteurized milk with both Salmonella and STEC and raw seafood with both hepatitis A virus and norovirus). In addition, three different foods (undercooked poultry, unpasteurized milk, and raw eggs) were associated with Salmonella.
Investigators must beware: Most foods can be contaminated with a variety of different causative agents and more than one food can transmit most causative agents.
> Non-laboratory methods

18. New Food Vehicles Bagged spinach White pepper 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
Note to instructor: Do not read this list. It is provided to illustrate the point that although there are common food-causative agent pairings, new food vehicles continue to be recognized, some of which are rather surprising.
Furthermore, new food-causative agent associations are recognized each year. Here is a list of 13 new food vehicles identified through PulseNet, OutbreakNet, and the Foodborne Disease Outbreak Surveillance System from The foods were associated with the following pathogens:
Bagged spinach - E. coli O157:H7
Commercial pasteurized carrot juice - Botulism
Peanut butter - Salmonella
Vegetarian snack food - Salmonella
Dry dog food - Salmonella
Microwaveable pot pies and frozen meals - Salmonella
Dry puffed breakfast cereal - Salmonella
Salami made with contaminated pepper - Salmonella
Canned chili sauce - Botulism
Imported white pepper - Salmonella
Prepackaged cookie dough - E. coli O157:H7
So, care must be taken in inferring the causative agent based on the suspected food item. (But sometimes it can provide a clue.)
> Non-laboratory methods

19. Laboratory Methods for Determining Causative Agent
Note to instructor: This is just a transition slide to indicate topics. Move to next slide before beginning lecture.

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
Now, let us move on to talk about the heart of this module: the laboratory investigation.
Detection of microorganisms in clinical specimens (that is specimens from people who are ill) or from the suspected food vehicle is the best approach to determining the cause of an outbreak. Laboratory testing to identify the causative agent includes culture, gram stain, biochemical analysis, antigen detection, antibody testing, and direct observation (ova and parasites, electron microscopy). Other subsequent tests can be performed to further characterize the pathogen such as serotyping (E. coli and Salmonella) and molecular subtyping. The tests used depend on the specimen and the suspected causative agent.
Although most of the hard work is done by the laboratory, other team members play a critical role in laboratory testing. They are responsible for collecting specimens, packaging them, storing them, transporting them to the laboratory, requesting the proper tests, and providing critical information about the illness, specimen, or food needed to interpret the results.
So let’s focus on what the other team members need to know to collect specimens during an outbreak investigation.

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
Detection of microorganisms in clinical specimens (that is specimens from people who are ill) is the usual approach to diagnosing the cause of the patient’s illness. The appropriate clinical specimen is based on the suspected causative agent. (As discussed earlier, clues to the likely causative agent will include incubation period, signs and symptoms, duration of illness, and suspect food.)
For most infectious agents, stool is the clinical specimen of choice.
For bacteria that cause invasive disease (bacteremia) including Brucella, Listeria, and Yersinia, blood is the specimen of choice.
For Hepatitis A virus and Trichinella spp., the diagnosis is made based on the presence of antibodies in the blood and serum is the specimen of choice.
For heavy metals, urine/hair are the specimens of choice.
For outbreaks involving preformed toxins (e.g., outbreaks due to Staphylococcus aureus or Bacillus cereus, some heavy metals), vomitus or food can be the specimen of choice. However, detection of preformed toxins or toxin-producing microorganisms by the laboratory can be problematic. For some toxins, no test matrices have been developed or clinical tests approved. Furthermore, some toxin-producing microorganisms, such as S. aureus and Clostridium perfringens, are commonly found in the human intestinal tract, so finding them in clinical specimens can be difficult to interpret. As a result, for outbreaks suspected to involve preformed toxins, it is critical to consult with the laboratory before collecting specimens.
(Of note, norovirus often mimics preformed toxins with high rates of vomiting and occasionally incubation periods of less than a day. Since laboratory confirmation of preformed toxins is problematic, some laboratorians suggest testing for and ruling out norovirus before concluding that the causative agent is a preformed toxin.)
> Diagnostic specimens

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; (1): (Table B)
The testing laboratory is the most useful source of information on the appropriate diagnostic specimen. For laboratory staff to provide guidance, you will need to indicate the likely causative agent or provide information on the incubation period, signs, symptoms, illness duration, and/or suspect food.
There also are many good references. CDC’s “Guidelines for Confirmation of Foodborne-Disease Outbreaks” is a good reference. [CDC. Guidelines for Confirmation of Foodborne-Disease Outbreaks. (Table B) MMWR 2000;49(SS01):54-62.]
The other references provided to you in this course are also good. “Diagnosis and Management of Foodborne Illness: A Primer for Physicians and Other Health Care Professional” is particularly easy to use because it lists the causative agents by general category (bacteria, virus, parasite, non-infectious) and then alphabetically by genus and species. (These are the blue booklets.)
> Diagnostic specimens

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
Because stool is the specimen of choice for many foodborne illnesses, let’s talk a little bit more about the collection of stool specimens.
Stool specimens should be collected as soon as possible after onset of illness because patients are most likely to still be shedding the causative agent. (Parasites tend to be shed for a prolonged period by the patient. So the urgency of collecting stool specimens is not as great if that is the suspected agent. But if you are not sure, you should collect them as soon as possible.)
Although confirmation of the diagnosis for all cases might be important for medical treatment, to determine the cause of an outbreak (and save on resources), most public health laboratories request stools from a manageable number of individuals who manifest illness typical of the outbreak and who have not undergone antibiotic treatment. Typically stools are requested from 5-10 patients, but this number varies by laboratory and suspected agent. Check with the testing laboratory to determine the recommended number of stool specimens.
The acceptability of rectal swabs, desired number of specimens per patient, use of preservatives, and acceptability of freezing during storage or transport vary depending on the suspected causative agent and needs to be considered during specimen collection. Some generalizations can be made about viruses, bacteria, and parasites.
Photo source: S. Shiflett
> Diagnostic specimens

24. Collection of Stool Specimens (cont’d)
Suspected Agent
Viruses
Bacteria
Parasites
Amount
Specimens per patient
Preservative
Freezing
10 cc bulk stool
Bulk stool (rectal swab)
10 cc bulk stool
One
Up to three
This table highlights some of the differences in the collection and storage of stool specimens when the causative agent is suspected to be a virus, bacterium, or parasite.
If you intend to culture the specimen for bacteria, rectal swabs can be used (since the agent will multiply during the culture process). Some laboratories, however, will not test swabs. If a virus or parasite is the suspected cause, a bulk stool of 10 cc (2 tsp.) must be collected.
Multiple stool cultures per patient are usually not necessary for bacteria or viruses because they are shed continuously for a period after onset of symptoms. If a parasite is suspected, multiple stool samples should be collected at different times because shedding of parasites may be intermittent.
Preservative is necessary to preserve the morphology of protozoal and helminthic parasites which are usually detected through visualization under the microscope. 10% formalin and polyvinyl alcohol preservative (PVA) are the recommended preservatives. Use of these preservatives should be avoided if testing for bacteria (kills bacteria) or viruses (destroys viral morphology). Cary-Blair transport medium, a non-nutritive medium, is used for the preservation of stool samples to be tested for bacteria if there is likely to be a delay between collection and processing of the specimen.
For most bacterial agents, it is acceptable to freeze the stool specimen. Some bacteria, such as Campylobacter jejuni, die off with freezing (and even can die off at room temperature). If C. jejuni is suspected, consult with the laboratory regarding temperatures during storage and transport. Avoid freezing specimens if viral or parasitic agents are the suspected cause of the outbreak because these agents are often diagnosed through visualization. Freezing will destroy the structure of the microorganism. Freezing is acceptable, however, if the specimens are to be tested for RNA (by RT-PCR) or for antigen.
Check with testing laboratory!
No**
Cary Blair
10% formalin, PVA
No (only if RNA or antigen testing)
Acceptable*
*Freezing causes die off of Campylobacter jejuni.
**Cary Blair acceptable in some laboratories but dilutes stool sample.
> Diagnostic specimens

25. 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
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
Note to instructor: Some students will know the answers to these questions without looking them up. (That is not the point of this question.) Encourage groups to look at the references to answer the questions.
Campylobacteriosis - due to a bacterium, Campylobacter jejuni or coli. Stool is the preferred specimen for diagnosis. Campylobacter jejuni, die off with freezing (and even can die off at room temperature). If C. jejuni is suspected, consult with the laboratory regarding temperatures during storage and transport.
Staphylococcus aureus food intoxication – due to preformed toxin. The diagnosis is confirmed by detection of toxin in vomitus or food; however, many labs do not accept vomitus. (Vomitus is only used for toxin screen; it must be neutralized for culture.) [Ask class if the public health laboratory in their state accepts vomitus.]
Giardiasis – due to the parasite, Giardia lamblia. Stool is the preferred specimen. Because Giardia is shed intermittently in the stool, up to three specimens may need to be collected to make the diagnosis. Avoid freezing specimens if viral or parasitic agents are the suspected cause of the outbreak because these agents are often diagnosed through visualization. Freezing will destroy the structure of the microorganism.
Listeriosis – due to Listeria monocytogenes, a bacterium that causes invasive disease. It can be found in the stool of normal individuals. To make the diagnosis of listeriosis, it must be cultured from a sterile body fluid (such as blood or cerebrospinal fluid).
Listeriosis
Blood or cerebrospinal fluid
> Diagnostic specimens

26. 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.
Once the specimen is collected, label the specimen container with the patient’s name or other identifier (so that it can be linked back to the medical chart, a questionnaire, or other data) and a specimen number. According to Clinical Laboratory Improvement Amendments (CLIA) requirements, if a clinical specimen is not clearly labeled, it will be discarded by the laboratory.
Keep a log of the specimens including specimen numbers, date and time collected, and type of sample.
Be sure to provide the necessary information about the illness and the specimen to guide testing by the laboratory and interpretation of results. Provide the laboratory with information on the date of collection, date of onset of symptoms, patient’s signs and symptoms, and requested test(s).
The date of specimen collection allows the laboratory to determine the age of the specimen when they receive/process it (i.e., to determine if it is old and the likelihood that pathogens present have died or multiplied).
The date of onset of symptoms with the date of collection helps the laboratory determine where in the course of the patient’s illness the specimen was collected (and, therefore, the likelihood of isolating a causative agent).
Patient signs and symptoms may give the laboratory ideas about the causative agent and indicate testing in addition to (or instead of) that requested by the investigators. For example, if the patient has bloody diarrhea, the laboratory may suggest the need to culture specifically for E. coli O157:H7 or other Shiga-like toxin producing E. coli.
Don Sharp
#10
12/3/2011
> Diagnostic specimens

27. 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
You will need to indicate to the laboratory the test(s) you wish to be run which again will be based on the suspected causative agent.
In most instances, stool collected from cases who are thought to be infected with a bacterium will be cultured. But the desired tests/cultures must be stated because in most laboratories, “routine stool cultures” are limited to screening for Salmonella and Shigella species and Campylobacter jejuni/coli. Some laboratories now routinely test for Shiga toxin-producing E. coli (STEC).
Cultures for Vibrio and Yersinia species, and Campylobacter species other than jejuni/coli require additional media or incubation conditions and advance notification and communication with the laboratory.
Viruses are often diagnosed through the detection of viral RNA in the stool using reverse transcription polymerase chain reaction (RT-PCR). Other methods for diagnosis include direct and immune electron microscopy of fecal specimens, detection of a fourfold increase of specific antibodies in acute- and convalescent-phase blood samples, and observation of viral particles by electron microscopy.
Most parasites are diagnosed through microscopic examination of the stool looking for eggs (ova) and parasites, called O&P exam. However, Cyclospora cayetanensis and Cryptosporidium spp. require special staining or other techniques.
> Diagnostic specimens

28. Interpretation of 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 (caused by another agent requiring different testing)
Collected too late in course of illness
Specimen handled improperly
Note to instructor: Before advancing the slide, ask students to suggest different reasons for both positive and negative results of stool testing.
[Advance slide.]
Finally, once you receive the results of stool testing, you need to interpret the results and consider all possible interpretations regardless of whether the results are positive or negative for the suspected causative agent.
For example, a positive clinical specimen may suggest that the patient’s illness was caused by that pathogen. But it might also result from contamination during collection or processing or colonization of the patient (i.e., the causative agent can be present but not cause illness [e.g., Listeria in stool specimens]). Test results that are not consistent with the patient’s signs and symptoms might suggest specimen contamination or colonization of the patient.
Similarly, a negative clinical specimen may have alternative explanations beyond the obvious (i.e., the patient’s illness is not due to the causative agent tested for). The specimen may have been collected too late in the course of the illness, when the patient was no longer excreting the pathogen in adequate numbers for detection. The specimen may have been improperly collected or mishandled during storage, transport, or processing leading to the death of any microorganisms present.
In interpreting the results, be sure to consider the characteristics of the test itself (e.g., detection thresholds, sensitivity and specificity) and the experience of the testing laboratory.
> Diagnostic specimens

29. 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.
Let’s move on to talking about the collection and testing of food samples.
Because testing of food samples takes significant time and resources, generally foods are tested only after implication by epidemiologic or environmental investigations. But foods can grow old or be discarded. (The food service establishment might even intentionally discard foods to get rid of the evidence!) Therefore, if the presumed exposure involves food, collect and store—but do not test—the food to which cases may have been exposed until evidence suggests it might be the source of the outbreak.
Because of the possibility of food tampering for private gain and the inability to determine the chain of custody, some jurisdictions do not routinely collect food samples/leftovers from private homes. Determine and follow local policies regarding collection of food samples from private homes.
In collecting food samples, you will want to collect samples of foods consumed by cases. Check storage areas or discarded foods in the trash. If no samples remain from the food consumed by cases, you might decide to collect ingredients or raw items used in the suspect food or intact (unopened) packages or other containers bearing the same code number. If no specific food has come under suspicion, you will likely collect samples of any potentially hazardous foods left from the suspect meal.
Consult with the testing laboratory on collection techniques and storage. Specific techniques are used to collect different food types. In general, food samples that are frozen when collected should remain frozen until examined. Perishable foods should be refrigerated. The acceptable length of storage is food dependent. If analysis of perishable foods is unlikely to occur within 48 hours of collection, consult with the laboratory about freezing the sample (–40 to –80C). But realize certain bacteria (e.g., Campylobacter jejuni) die when frozen, affecting laboratory results.
> Food specimens

30. 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.
For each food sample, label the container with a code for the establishment and the specimen number.
Maintain a log of specimens and record the following information:
Code and specimen number
Name and type of product
Brand of product
Product manufacturer and code or lot number
Name of person collecting the sample
Date, time, and place of collection
Establishment name
Condition of packaging (e.g., open, intact)
Some investigators will photograph the label of the food.
Be sure to provide the laboratory with information that will be useful in interpreting test results. Because the presence and quantity of contaminants in food are in a dynamic state, their presence and quantity will change with time. Samples collected during an investigation may not be representative of the food ingested when the problem occurred. The investigator should note when the food was served, the date the sample was collected, and handling/treatment of the food since that time (e.g., temperatures at which food was held or contact with human hands or environmental surfaces since serving), and share that information with the testing laboratory.
> Food specimens

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
For some investigations, laboratory specimens will need to be collected following the proper rules of chain of custody.
Chain of custody requires maintenance of a chronological written record that identifies who had control over the specimen during what time (including who obtained the specimen, who transported/delivered it, and who disposed of it).
Each person signing the form is responsible for the care and preservation of the sample while it is in their possession. Those signing may be called to testify in a criminal proceeding.
Every agency has its own form and is responsible for retaining records regarding the custody of a particular sample. If the sample is transferred to a second agency, that agency will initiate another chain of custody form.
Information collected through the chain of custody process is considered confidential/classified. It should be safe-guarded and maintained in a secure location.
> Food specimens

32. 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.
Testing of food specimens is challenging for a number of reasons. Physical and chemical components of a food product sometimes interfere with culture methods, producing negative results. Because food is not a sterile commodity, resident competing flora, whether normal or spoilage, can interfere with isolation of a suspected causative agent. Furthermore, different methods need to be used for different foods and different causative agents and generally accepted procedures are not available for all foodborne causative agents. (Testing of food for viruses is almost nonexistent. FDA does testing for norovirus in seafood.)
Because of these challenges, not all laboratories do food testing. Sometimes the public health laboratory tests foods and sometimes the state agricultural agency tests foods. Sometimes foods need to be submitted to some other reference laboratory. It will be important to identify the appropriate test site BEFORE collecting and submitting the specimen and to consult with that laboratory about testing.
> Food specimens

33. 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
Note to instructor: Before advancing the slide, ask students to suggest different reasons for both positive and negative results of food testing.
[Advance slide.]
As with clinical specimens, results of food testing must be interpreted carefully. Interpretation of food testing results can be particularly challenging because samples collected during an investigation may not be representative of the food ingested when the problem occurred.
Investigators should consider all possible interpretations of the results provided. A positive result could suggest that the food is contaminated with the causative agent and is the source of the outbreak. However, the mere presence of microorganisms in food does not mean the food was the source of the illness. Food is usually not sterile; microorganisms can be isolated from samples but they may not be responsible for the illness under investigation. With some causative agents, it is necessary that large numbers of the organisms be present in a food for it to be hazardous. Examples of these agents include: Bacillus cereus, Clostridium perfringens and Staphylococcus aureus. Usually 106 organisms per gram of food are necessary before there is a danger of food poisoning from these agents. Similarly, the presence of spores of Clostridium botulinum (the causative agent for botulism) alone is insufficient to make the diagnosis of botulism since spores are prevalent in soil and marine sediments and often found on the surfaces of fruits, vegetables, and in seafoods; demonstration of the toxin is required to make the diagnosis. Furthermore, food can become contaminated after the outbreak exposure (sometimes by the cases themselves) or bacteria initially present in low numbers can multiply.
Similarly, negative findings can have multiple interpretations. It could mean the food is not the source of the outbreak. It could result from subsequent handling or processing of the food that caused the death of microorganisms present. If contamination of the food was not uniform, a negative result could mean that the sample missed the contaminated portion. In addition, as noted on the previous slide, the properties of some food components interfere with culture methods.
So be careful when interpreting laboratory results. Don’t jump to conclusions. Consider the circumstances around the food specimens, the quality of the samples, test limitations, and capacity of the testing laboratory. And always consult with the laboratory testing the specimen for an interpretation of the results and recommendations for follow-up testing.
> Food specimens

34. 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.
Note to instructor: Read scenario to students.

35. Class Question (cont’d)
Class 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. Class Question (cont’d)
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?
Answer: The chicken could be the source of the outbreak but raw chicken is commonly contam-inated 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 epidemiologic studies, findings from an environmental health assessment of the chicken preparation, subtyping of isolates from cases and the chicken

37. Subtyping of Causative Agents
Note to instructor: This is just a transition slide to indicate topics. Move to next slide before beginning lecture.

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
Subtyping is the identification of strains or groupings of microorganisms below the species level. Methods of subtyping are based on phenotypic and genetic characteristics that tend to differ between different strains of the same species but are the same among isolates that arise from the same parent (that is those that have a common ancestry).
As a result, subtyping of microorganisms isolated from foods, production environments, and cases in an outbreak can facilitate conclusions about contamination patterns, transmission routes, and sources of an outbreak.
A variety of subtyping methods exist including serotyping, phage typing (which is based on the pattern of bacteriophages to which a bacterium is susceptible), antimicrobial susceptibility, pulsed field gel electrophoresis (PFGE) , restriction-fragment length polymorphism (RFLP) analysis, ribotyping, multiple-locus variable number tandem repeat analysis (MLVA). Not all subtyping methods are available for all microorganisms.
Some subtyping methods are more difficult to perform than others or take more time or are less reproducible. Some are more expensive. Some discriminate between strains better than others. The most appropriate method will depend on the particular setting and causative agent involved.
> Subtyping of causative agent

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
Uses of subtyping:
Because outbreaks of foodborne diseases are typically caused by exposure to a single subtype of the causative agent, subtyping can help link together cases of a particular disease to suggest an outbreak. Sometimes this is necessary to recognize an outbreak due to a widespread low level disease.
Since foods are not sterile and can be contaminated with potential disease-causing agents without causing disease, subtyping can be used to determine if a food item that is contaminated with the causative agent is related to a particular outbreak such as the example given earlier of raw chicken contaminated with Salmonella.
Subtyping results can be used as part of the case definition in an epidemiologic investigation allowing investigators to exclude cases of disease that are not related to an outbreak. Increased specificity of the case definition decreases misclassification of cases improving the chances that a food vehicle will be linked to illness. This is particularly critical for more common causative agents.
Subtyping can link together outbreaks in different locations and better characterize the scope of an outbreak (and where the outbreak is (or is not) occurring). Knowing the geographic extent of an outbreak lets investigators focus their efforts. Furthermore, it is not uncommon that until we examine this larger pattern of disease that the source of the outbreak becomes apparent. For example in an investigation of an outbreak of E. coli O157:H7 linked to alfalfa sprouts in Michigan, the source of the contaminated sprouts could not be determined (because a number of different producers were possible) until an outbreak of the same PFGE pattern in Virginia was linked to sprouts and the source of the sprouts in Virginia was more limited in scope.
> Subtyping of causative agent

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
Let’s focus on one particular method of molecular subtyping that has received (rightfully) a lot of attention with regards to foodborne outbreak detection and investigation in recent years: pulsed field gel electrophoresis (PFGE). In PFGE, DNA from an isolate is broken up into fragments using certain enzymes and the fragments from the isolate are separated across a gel matrix using a pulsing electrical field. The DNA fragments migrate according to their molecular weight. The smaller pieces move through the gel more quickly and the larger pieces move more slowly resulting in bands. These bands are concentrations of different sized pieces of DNA.
The banding pattern (which looks like a barcode) will be unique for an isolate (and particular testing parameters). Different DNA composition will result in different PFGE banding patterns. But bacteria descended from the same original parent (which is usually the case in an outbreak) will have the same DNA composition and their DNA fingerprints will be indistinguishable when the same methods are used.
This image shows an example of PFGE patterns for individual patients. Each horizontal line is an isolate from a patient.
[Advance slide] The red box indicates a cluster of indistinguishable patterns. These patterns would be called an “outbreak strain”.
PFGE subtyping is so good at linking together related isolates of the same pathogen that a national pathogen-specific surveillance system (mentioned earlier) has been built around it: PulseNet.
Cluster of indistinguishable patterns
> Subtyping of causative agent

41. PulseNet Laboratory Network
Participating Laboratories
PulseNet National Database (CDC)
PFGE Patterns
Standardized testing of
E. coli O157:H7,
Salmonella,
Shigella,
Listeria, and
Campylobacter
PulseNet is a national network of public health and food regulatory agency laboratories coordinated by the Centers for Disease Control and Prevention (CDC). The network consists of over 80 public health and regulatory laboratories including those from state health departments, local health departments, and federal public health agencies (CDC, USDA/FSIS, FDA).
PulseNet participants perform standardized molecular subtyping (or “fingerprinting”) of foodborne disease-causing bacteria by PFGE including Escherichia coli O157:H7, Salmonella, Shigella, Listeria, and Campylobacter. Participating laboratories upload PFGE patterns and isolate information electronically to a server located at CDC.
PulseNet monitors the patterns for collections of similar patterns in the past 2-4 months. When a cluster of similar patterns is identified, PulseNet notifies foodborne epidemiologists.
Participating laboratories can query the PulseNet database for specific PFGE patterns they have isolated and can download PFGE patterns from other participating laboratories to their local databases for comparison. These databases are frequently updated and are available on- demand to participants, allowing for rapid comparison of the patterns and detection of clusters.
Monitors for similar patterns
Notifies foodborne epidemiologists of clusters
State labs can query
Patterns uploaded by testing laboratory
> Subtyping of causative agent

42. PulseNet Participants
PulseNet's national laboratory network is made up of 87 laboratories−at least one in each state and many states include federal and local laboratories.
PulseNet-triggered outbreak investigations have resulted in the recall of well over one half billion pounds of contaminated food. More importantly, these investigations have highlighted unrecognized problems in food production and distribution industries, such as the beef, produce, tree nut, peanut, egg, and spice industries, ultimately resulting in major change to industrial processes and improved food safety.
PulseNet headquarters
Local and secondary state laboratories
Regional laboratories
Federal laboratories
> Subtyping of causative agent

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
In late December 2010, PulseNet detected a suspicious cluster of E. coli O157 infections in the Midwest of only 8 cases. Staff from the Wisconsin and Minnesota departments of health immediately interviewed cases and determined that all cases had eaten in-shell hazelnuts (also known as filberts). Most of the in-shell hazelnuts were purchased from bulk bins at retail food stores.
Tests on the hazelnuts recovered in a patient's home identified a E. coli O157:H7 strain that matched patient isolates by DNA analysis. Source tracing identified a common distributor for the hazelnuts consumed by ill persons. Laboratory testing conducted on different packaging of in-shell hazelnuts from this source identified E. coli O157:H7 matching the outbreak strain by DNA analysis. (Laboratory testing conducted by the Minnesota Department of Agriculture on in-shell hazelnuts, the California Department of Public Health on mixed nuts containing in-shell hazelnuts, and the Wisconsin Department of Agriculture, Trade and Consumer Protection and Wisconsin Department of Health Services on mixed nuts containing in-shell hazelnuts identified E. coli O157:H7 matching the outbreak strain by DNA analysis.)
The company recalled all of its products in March 2011, containing the outbreak. In less than 3 months, and with only 8 illnesses, the outbreak and food source were identified, the product recalled, and no one died. CDC and its food safety partners also discovered a new food vehicle for E. coli O157:H7 infections—hazelnuts. The results of this investigation also helped nut producers focus their efforts and re-examine the safety of their production practices.
> Subtyping of causative agent

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
Subtyping of isolates from clinical and food specimens has been extremely helpful in the detection of outbreaks and successful identification of the source. However, several issues with regards to subtyping should be mentioned:
Matching of subtypes should not be considered proof of a common exposure among cases, merely that the isolates share a common ancestry. (Some microorganisms are very stable resulting in limited variability in subtypes. As a result, subtypes can match, but the cases do not result from exposure to the same source.) An epidemiologic investigation is necessary to demonstrate that there is a common source of exposure.
Identification of non-matching subtypes of an organism does not mean that cases of a particular disease are not related. Outbreaks can be caused by more than one organism or subtype if the food or other vehicle was contaminated with multiple organisms/subtypes. An example is the nationwide outbreak of salmonellosis associated with eating salami. Initially, Salmonella Montevideo with a particular PFGE pattern was isolated from case patients. Testing of retail salami from the implicated producer identified two different Salmonella serotypes: Senftenberg and Montevideo so the case definition was expanded. The outbreak was ultimately linked to red and black pepper that was found to be contaminated by both strains of Salmonella.
Investigators need to explore subtyping results while patients can still recall exposures and while vehicles of transmission (e.g., food items) are still available for testing. Therefore, subtyping needs to be performed on a routine basis and as rapidly as possible, with concurrent serotyping and PFGE analysis, where possible.
Pathogens have different degrees of genetic variability or heterogeneity and current methodologies do not always differentiate between strains in a meaningful way. Sometimes use of more than one subtyping method (e.g., MLVA and PFGE) will help. However, more discriminating subtyping methods are needed that can be standardized and used outside the research setting.
Rapid diagnostic methods based on polymerase chain reaction, enzyme immunoassay (EIA), and similar procedures are becoming available for some foodborne illnesses. These tests (similar to “quick pregnancy tests”) are often referred to as culture-independent diagnostics and allow quick identification of a pathogen and rapid initiation of treatment. However, some of these tests lack the sensitivity and specificity of other diagnostic methods and usually do not result in a culture that can be forwarded to the public health laboratory for further characterization. This will inhibit subtyping of isolates and interfere with the detection and investigation of possible outbreaks.
> Subtyping of causative agent

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 epidemiologic and environmental health studies
One final comment with regards to the laboratory investigation. Although some of the most convincing evidence in a foodborne outbreak investigation is isolation of the causative agent from the suspected food source, the causative agent is not always isolated from the suspected vehicle. In fact, that is the case in the majority of investigations.
An analysis of foodborne outbreaks reported to the National Outbreak Reporting System (from ) shows that among outbreaks with a confirmed etiology, the agent was isolated from a food item in only 14%. Similarly, in a review of published reports from 54 outbreaks by Craig Hedberg, it was noted that in 27 (50%) outbreaks the agent was recovered from the implicated food vehicle.
So relying solely on laboratory evidence to take action during an outbreak would result in inaction in the majority of outbreaks. If control measures are not undertaken as quickly as possible, it is a sure thing that more people will become ill. So other information – results of environmental health studies, epidemiologic studies, and supporting laboratory studies (e.g., food safety research) – are critical!

46. Quick Quiz
These “quick quizzes” are meant to be a rapid self-assessment so that participants can see if they understood the most important concepts in this module.
The instructor will read the question aloud.
Think about the answer for a moment.
When asked, please indicate your answer by raising the appropriate color-coded card.
If you do not get an answer right, please make note of it and revisit the module during the break or over lunch (or talk to the instructor) to see if you can determine the correct answer. This is meant to be quick. We will not discuss the answers at any length.
[NOTE: Instructors should make sure everyone is aware of the correct answer and briefly explain why it is the answer, but should not go into great detail. The detailed explanations with each question are provided to you to make sure you agree with the answer. If students have questions they should ask the instructor during the break or over lunch.]

47. Quick Quiz
Which of the following causative agents tend to have the longest incubation period?
Preformed toxins
Viruses
Bacteria
Parasites
ANSWER: D. Parasites.
Illnesses due to preformed toxins do not require growth of a microorganism in the body after ingestion for illness to occur and have short incubation periods, often measured in terms of minutes or hours.
Illnesses due to infections require growth of the microorganism (including production and release of toxins) and take time; thus, incubation periods for infections are relatively long, often measured in terms of days as compared to hours or minutes for intoxications.
Gastrointestinal illnesses caused by viruses are characterized by a relatively short incubation period (e.g., hours for norovirus or rotavirus and hours for astrovirus). Gastrointestinal illnesses caused by bacteria tend to have incubation periods intermediate between those of viruses and parasites, ranging from less than a day to about a week. Gastrointestinal illness due to parasites, in general, have the longest incubation periods, ranging from 1-4 weeks.

48. Quick Quiz
Most foods can be associated with a variety of causative agents.
True
False
ANSWER: A. True.
Certain causative agents are associated with certain foods because the foods provide adequate conditions for contamination, survival, and proliferation of the microorganism. As a result, common disease-causative agent pairings have been observed.
Most foods, however, can be contaminated with a variety of different causative agents and more than one food can transmit most causative agents.

49. Quick Quiz
The following are true statements about stool specimens collected during a foodborne disease outbreak EXCEPT
Stool is the specimen of choice for most causative agents.
Routine stool cultures cover the most common foodborne disease causative agents.
The method of collection and handling depend on the suspected causative agent.
Freezing of stool specimens can interfere with detection of some causative agents.
ANSWER: B. Routine stool cultures cover the most common foodborne disease causative agents.
In most laboratories, “routine stool cultures” are limited to screening for Salmonella and Shigella species and Campylobacter jejuni/coli. Some laboratories now routinely test for Shiga toxin-producing E. coli (STEC). Routine stool cultures do not cover viruses (the most common cause of outbreaks of foodborne illness), selected bacteria, or parasites.
The remaining statements are true:
Stool is the specimen of choice for most foodborne illness causative agents.
The method of collection and handling depends on the suspected causative agent.
Although freezing of stool specimens is acceptable for most bacterial agents, some bacteria such as Campylobacter jejuni, die off with freezing. Because viral or parasitic agents are often diagnosed through visualization and freezing destroys the structure of the microorganism, freezing should be avoided if these agents are suspected. (Freezing is acceptable if the specimens are to be tested for RNA or for antigen.)

50. Quick Quiz
Which of the following is a possible interpretation of a negative result on a routine stool culture?
The patient’s illness was not caused by agents detected through routine cultures.
The specimen was collected too late in the course of illness.
The specimen was handled improperly, killing any causative agents present.
All of the above
ANSWER: D. All of the above.
A negative result on a routine stool culture may have alternative explanations beyond the obvious (i.e., the patient’s illness is not due to the causative agents tested for). The specimen may have been collected too late in the course of the illness, when the patient was no longer excreting the pathogen in adequate numbers for detection. In addition, the specimen may have been improperly collected or mishandled during storage, transport, or processing leading to the death of any microorganisms present.

51. Quick Quiz
Subtyping of isolates from cases of the same disease can be used for all of the following EXCEPT
Link cases together
Link outbreaks in different geographic locations
Link foods with cases
As sole proof of an outbreak
ANSWER: D. As sole proof of an outbreak.
In a foodborne outbreak, subtyping of isolates from cases and food can be used to
Link cases together
Link outbreaks in different geographic locations
Link foods with outbreaks
Refine the case definition decreasing misclassification in epidemiologic studies
Matching of subtypes, however, should not be considered proof of a common exposure among cases, merely that the isolates share a common ancestry. An epidemiologic investigation is necessary to demonstrate that there is a common source of exposure.