Laboratory Investigation

Introduction

It is essential to maintain interagency collaboration in the investigation of outbreaks of foodborne illness.  Communication of the results of each agency’s investigation, including those of the Department of Inspections and Appeals (DIA), Iowa Department Public Health (IDPH), and State Hygienic Laboratory (SHL) is vital to ensuring a thorough multifaceted investigation

Diagnosis of most diseases can be confirmed only if the etiologic agents are isolated and identified from specimens obtained from ill persons.  Specimen collection is then vital to the overall three tier approach of a foodborne illness investigation.  It is important to collect clinical specimens as soon as possible as ill individuals are more willing to provide a specimen during acute illness and are more reluctant to provide a sample when symptoms have subsided.  It is also best to collect these specimens during illness because not all agents are shed after symptoms subside (with some exceptions such as norovirus).  During a suspect foodborne illness outbreak clinical specimens should be sent to SHL for testing.

Environmental samples will either be leftover food suspected of causing illness or environmental surface samples.  These specimens can confirm an etiologic agent and reveal a pathway for transmission of the agent.  In an ideal investigation, the same etiologic agent will be isolated from both the clinical specimen submitted from the patient and the suspect (epidemiologically associated) environmental sample which confirms the source of the outbreak. 

SHL, with locations in Iowa City and Ankeny, has been Iowa’s public health and environmental lab for over 100 years.  In a foodborne outbreak, it has several roles.

• diagnostic testing on patient specimens, cultures foods for pathogens, tests for some toxins causing foodborne diseases, and conducts laboratory surveillance for pathogens in foodborne outbreaks.
• analyzes food specimens to detect, identify, and quantify toxic contaminants (e.g., pesticide residues, heavy metals, volatile organic compounds, and radioactive contamination).
• performs molecular strain typing of pathogens to assist in the epidemiologic investigation.

It is important to notify the Iowa Department of Public Health (IDPH) and SHL immediately when a foodborne outbreak is suspected and provide as much of the following information as possible so the most likely agent can be targeted.

• A summary of signs and symptoms (also helpful to know the worst or predominant symptoms)
• The interval between the consumption of the suspect food and onset of illness (incubation period)
• The duration of symptoms (if known).
• Whether stool specimens were sent to any other lab (provide name of lab)
• The number and type of specimens collected (stool, vomit, or food)

This information will help determine which tests need to be run, and the laboratory can prepare for testing (some tests require special plating media.).  The laboratory can provide sample collection kits with instructions and recommend the type and the number of specimens that should be collected.

Clinical Specimens

The collection of specimens is important in an investigation so an agent can be identified.  Vomit or stool can be collected for foodborne illness (please see instructions on pages 4-11).  It is important to obtain specimens from patients as early in the investigation as possible and preferably while they are still symptomatic.  For example, certain illnesses caused by Bacillus cereus, Clostridium perfringens, and Staphylococcus aureus are of short duration (usually less than 24 hours), so it is critical to collect samples during this period to detect the agent or toxin.  On the other hand, agents like norovirus can be shed up to one week after symptoms subside, so stool specimens may be collected during this period which may still yield positive results for this organism.  Also, as people begin to feel better and their symptoms subside, they may be less motivated to submit samples.

SHL can test specimens for the following agents:

• Enteric bacteria: Culture methods for Salmonella spp., Shigella spp., Yersinia spp., Campylobacter spp., E.coli 0157:H7, Shiga toxin testing and other enteric bacteria as requested.
  • Turn-around time: generally 48-72 hours from receipt.

• Ova and parasite: Identification of intestinal parasites (protozoa, helminths, etc.), including Giardia intestinalis, Cryptosporidium spp. and Cyclospora cayetanensis.
  • Turn-around time: generally 24-48 hours from receipt.

• Norovirus (Norwalk-like virus): RT-PCR for norovirus detection in stools and vomitus.
  • Turn-around time: generally 24-48 hours from receipt.

• Molecular subtyping: Foodborne isolates from patient and food specimens are analyzed by pulsed-field, gel electrophoresis (PFGE) DNA fingerprinting using standardized PulseNet methods.  Patterns are submitted to the CDC’s PulseNet database.
  • Turn-around time: generally 5-7 days from time the PFGE lab receives isolates.

Local public health agency (LPHA) staff should provide, preferably in person, specimen collection kits and directions on collection to patients.  For example, if an outbreak occurs in a school, the agency could take the kits to the school for affected staff and students.  If the outbreak was an event, such as a wedding or a restaurant exposure, agency staff may need to obtain addresses for ill individuals to drop off the kits. Patients who provide specimens for testing should return the kits to the agency for storage, packaging, labeling, and mailing.  Patients should not be responsible for storing, packaging, and mailing their own specimen because it is unlikely they will follow through.  They should, however, be encouraged to label their specimen with their name for correct specimen identification.

Instructions for clinical specimen collection

In foodborne outbreaks in which gastrointestinal illness is predominant, stool samples, rectal swabs and vomitus can be collected.  The following instructions are for collecting samples for bacteria, parasites and viruses.

How to collect and ship

Instructions for stool collection:

Pass the stool into a clean, dry, plastic disposable container (margarine, cottage cheese, etc) or place a large plastic bag, kitchen wrap, or newspaper over the back portion of the toilet seat to catch the specimen.

DO NOT MIX URINE OR WATER WITH THE STOOL SPECIMEN.

Instructions for enteric bacteria kit (culture):

Kits are available from SHL and test request forms are on SHL’s web page. Use the stick provided to pick up a portion of the stool (equivalent to the size of a navy bean) and MIX it into the semi-solid medium in the bottle.  It is best to select portions that are bloody or covered in mucous if present.  Discard the stick.

• For liquid stool, add approximately ½ to 1 teaspoon of stool to the bottle and MIX with the stick.
  • Rectal swabs are acceptable for children and the elderly if they are unable to pass a stool. Collect two rectal swabs and insert them into the transport medium. Break or cut the sticks so that the bottle cap may be securely closed.
• TIGHTEN the cap and place the bottle in the Whirl-pak or zip-lock bag.
• Label the bottle with the patient’s name and date of collection.
• Complete all information on the report form. Place it and the bottle into the mailing kit.
• Use carrier service or first class postage to mail the kit to the SHL, or hand deliver

Instructions for parasite kit (ova and parasite detection):

Kits are available from SHL and test request forms are on SHL web page.

Use one of the sticks in the kit to place a portion of the stool (about the size of a navy bean or walnut) into the formalin vial. Place any blood or mucus present with the stool into the vials.

• For watery stool, pour the stool into the vial up to its neck.
• For hard, formed stools, take samples from small areas from the inside and outside of the stool.
• Use the stick to mix the formalin and the stool thoroughly.
• Use the second stick to mix another sample of stool into the P.V.A vial.
• Discard both sticks.
• Tighten the caps on both vials and clean the outside of the vials with rubbing alcohol or soap and water if they are soiled.
• Label both vials with the patient’s name and date of collection.
• Place vials into the Whirl-Pak.
• Wash your hands thoroughly. If liquid from either vial gets on the skin, flush it with running water.
• Complete all information on other side of the form. Place it and both vials into the mailer provided and mail it to the SHL, using first class postage.

Instructions for norovirus (only) detection by PCR:

Criteria for specimen collection

• The best specimen is a pea-sized (or greater) portion of stool  in a sterile container.
• SHL Enteric kit Cary-Blair transport media can also be used if other enteric pathogens are suspected but this is not the best transport method for detection of norovirus. Note: if both bacteria and norovirus are suspected, ideally collect both transport types: stool in sterile container and in Carey-Blair media.
• Stool samples should be stored at 4°C .

Transport

• Ship specimens to SHL ASAP on ice packs.
• Ship Monday through Thursday to guarantee arrival at the laboratory before the weekend.
• Contact the lab at 319-335-4500 for further questions or instructions for delivery if specimens are to be shipped Friday through Sunday.

Vomit sample for bacteria or norovirus 

• Collect the vomitus in a clean, dry plastic container with a tightly sealed lid. Note: vomit is not the best specimen for norovirus and will only be tested if no other specimens are available.
• Transport (follow instructions above for norovirus)

Clinical specimen kits available from SHL

The kits that pertain to this section of the manual are the following:

• Stool collection for ova and parasites (O & P) kit
  • SHL test request form
  • Collection instructions 
• Stool (enteric) culture kit
  • SHL test request form
  • Colection instructions
• Biological defense kit
  • (form not available on web site at this time but included in each kit)

Environmental specimens

Environmental specimens are either samples of the food from an event or swabs from surface areas and equipment where food was prepared that are collected by the environmental health specialist.  Although many food samples may be collected, the laboratory may hold the samples and wait to begin testing until epidemiological evidence suggests a specific food or foods.  Remember that leftover food from the outbreak should not be served again until after food has been cleared by health department or inspector, or it should be discarded after a sample is collected.

SHL can test environmental specimens collected for the following agents:

Culture methods for all the major foodborne pathogens including, Staphylococcus aureus, Bacillus cereus, Salmonella spp., Shigella spp., E.coli 0157:H7, Campylobacter spp.,  Clostridium perfringens and indicator bacteria (total and fecal coliform and aerobic plate count);

• PCR methods for Salmonella spp., E.coli 0157:H7, Listeria spp and Campylobacter spp.;
• Enterotoxin assay for Staphylococcus aureus (enterotoxins A-E) and Bacillus cereus (diarrheal toxin only).
• Turn-around time: generally 48-72 hours from receipt.

Instructions for food specimen collection

How to collect and ship

1. Always wash hands thoroughly (with soap and warm water) before beginning sampling. Plastic gloves can be used also.
2. Obtain samples of any suspect leftover foods as soon as possible.
3. Collect 100-400 grams (1/2-1 lb, ½-1 pint) of any indicated food or if less product is available, all of it.  If the volume is greater than 200 grams, sampling should be representative, i.e. taken from throughout the sample, not just one portion.
4. Use sterile containers and sample implements to collect it, using aseptic technique.
5. Collect packaged foods in their original containers if possible.  If the sample is a fresh or canned commercially prepared food, note the name of the manufacturer or processor, code or lot number and other identifying characteristics.  If possible, collect an unopened container of the same lot number. Obtain the original commercial container or sample if it is available, even if it is in the trash (Note this appropriately. It can be useful in the investigation.).  When the sample must be placed in containers other than the original container, sterile whirl-paks and ziploc bags (provided), sterile Mason jars (boiled in water for 10 minutes), and new aluminum foil are acceptable.
6. Label each container or bag with date/time of collection, type of sample and name of establishment. Use labels and the waterproof pen provided.
7. Do not freeze samples unless they are collected in a frozen state. If they are frozen, keep them frozen.
8. Place each Whirl-pak sample in a Ziploc bag to avoid leakage and cross-contamination from other samples.
9. Complete SHL chain-of-custody form found in the kit and place in plastic sleeve.
10. Place frozen ice packs, samples, and chain-of-custody form in cooler. With bubble-wrap or newspaper, make sure samples are protected from direct contact with ice packs. Samples should be packed to prevent breakage, spillage, or change in temperature.
11. Samples should be delivered to the laboratory as soon as possible. Hand deliver or ship overnight (return address label provided).  If shipping on a Friday, you must specify “Saturday delivery” with arrival before noon.
12. Notify appropriate SHL personnel of the type and number of samples being shipped and description of the outbreak (e.g. time of onset and description of symptoms are helpful to determine causative agent, and approximate time of arrival) by calling 800 421-4692 and asking for a member of SHL’s foodborne outbreak team.

Instructions for sampling equipment or surfaces

• Swab contact method (qualitative):
  1. To sample equipment or surfaces for suspected bacterial pathogens, open the sterile swab container, grasp the end of the stick and remove the swab aseptically.
  2. Hold the swab handle to make an angle contact with the surface (pencil-to-paper angle). 
  3. Rub the swab head slowly and thoroughly over the surface. Place swab back into sleeve.
  4. Label and ship to laboratory according to the package directions.

For quantitative surface analysis, contact the laboratory and request SHL’s (meat carcass) sample kit that includes sterile sponge, diluent, template, sterile gloves, and instructions.

Laboratory result interpretation

A. Pathogen guidelines

[table]

B. Food indicator bacteria results

• Aerobic Plate Count: high aerobic colony counts indicate that one of three situations has occurred.  The first is that raw food or ingredients contained high populations of microorganisms and that it received no or insufficient heating and no other potentially lethal treatment to sufficiently decrease the population.  The second is that the food was held at temperatures conducive to bacterial growth long enough to allow surviving spores to germinate and the resulting cells to multiply. The third is that contamination occurred after the food had been cooked.
• Fecal coliform bacteria: indicates a fecal contamination source, likely to be found on raw foods of animal origin.  Their presence in heated foods; however, suggests post-processing fecal contamination.

[table]

C. Molecular subtyping results

Isolates with the same PFGE pattern as the outbreak pattern may be interpreted as indistinguishable from the outbreak stain.  Isolates that have indistinguishable DNA fingerprint patterns are more likely to have originated from a common source; while isolates that have different DNA fingerprint patterns are less likely to have originated from a common source.

Environmental sampling kits available from SHL

The kits that pertain to this section of the manual are (forms are not available on website at this time but included in each kit):

• Food sample collection kit
• Biological defense kit

Important notes on laboratory result interpretation

1. Laboratory results are only as good as the specimens that are collected.
2. A positive result or a confirmation from an epidemiologically implicated person or environmental specimen strongly suggests that the person or environmental specimen was the source or vehicle of infection. However, a positive result from a person or environmental specimen without an epidemiological association does not suggest or prove that the person or environmental specimen was or was not the source or vehicle.
3. A negative or absent result should not rule out an association; the only interpretation is that the pathogen was not found in the specimen.

References

1.  Center for Disease Control and Prevention, 2000, Appendix B, Guidelines for Confirmation of Foodborne-disease Outbreaks. CDC Surveillance Summaries, March 17, 2000. MMWR 49:52-64 (http://www.cdc.gov/mmwr/preview/mmwrhtml/ss4901a3.htm).

2.   International Association for Food Protection, 1999. Procedures to Investigate Foodborne Illness, fifth edition.