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Sampling Protocol for Foodborne Illness Outbreak

ByMasoud Alebouyeh

PhD of Medical BacteriologyFoodborne and Waterborne Diseases Research Center,

Shahid Behehsti University of Medical Sciences,Tehran, Iran

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Primary Mode of Transmission

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• Food – Select if the initial transmission of an enteric illness was associated with ingestion of a common, potentially contaminated food or beverage.

• A foodborne disease outbreak is defined as an incident in which two or more persons experience a similar illness resulting from the ingestion of a common food.

• Water – Select if the initial transmission of any type of illness (enteric and otherwise) was associated with exposure via ingestion, inhalation, contact, or another route to treated or untreated recreational water, drinking water (including bottled water), water not intended for drinking or water of unknown intent.

• A waterborne disease outbreak is defined as two or more cases of a similar illness that are epidemiologically linked to a common water exposure. The source case should not be considered part of the first cluster of illness. See Appendix A on page 39 for examples on how to choose a mode of transmission for certain beverages and foods/drinks containing ice.

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• Animal contact Select if the initial transmission of an enteric illness was associated with exposure (physical contact) to farm animals, reptiles, or any other animals potentially infected with pathogens causing gastrointestinal illness in humans. • Person-to-person –Select if the initial transmission of an enteric illness was associated with direct contact with an infected person. These outbreaks are typically defined by two or more cases of illness in a common setting of exposure, such as a nursing home or a school. Although environmental contamination is oftentimes a factor in person-to-person outbreaks, if most of the cases had known direct contact or likely had the opportunity for direct contact with one another, consider the primary mode of transmission to be person-to-person.

. Environmental contamination other than food/water Select if the initial transmission of an enteric illness was associated with exposure to a contaminated environment (i.e., contaminated environmental surface or fomite). For example, the source case vomits in a public restroom and the following day people become sick after visiting the same restroom. This would not be considered a person-to-person outbreak because those who became sick did not come into direct contact with the source case.

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Epi Kits An Epi Kit is a collection of all the supplies you will need to do a field investigation. It should be stored in a place that is readily accessible to all personnel who may need to use it. This includes access to the kit on the weekends and in the evening. A checklist should be used prior to using the epi kit to ensure the necessary items are included. It is also a good idea to do this after the investigation is over to see what needs to be replaced. The following Epi Kit materials list can be used as a checklist. The minimal Epi Kit will contain the following items: • A large cooler • Ice packs• Sterile containers (e.g., WhirlPak bags) • Sterile sampling utensils • Non-cotton swabs (resin in the cotton swab, it may affect the growth of microbes) or swab test kits. • At least 15 sterile bags. • At least 15 sterile spoons. • Six sterile specimen collection containers or devices (e.g. WhirlPak bags). • Non-sterile self-sealing plastic bags (e.g. unused Zip-Lock® bags) • Properly calibrated temperature-measuring devices. • Sterilizing equipment. • One of each item listed under supporting equipment.

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Epi kit (continued)Sterile tubes Sterilizing & Disinfecting Agents . Ethyl alcohol 95% solution • Propane torch • Sodium or calcium hypochlorite Refrigerants • Ice • Blue ice packs • Rubber or plastic bags which can be filled with water and frozen • Heavy-duty plastic bags for ice Media Media• Tubes of transport media • Pre-enrichment or enrichment broth as appropriate

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• Supporting Equipment • Foodborne outbreak form (Copies can be printed off the CDC

Website.) • Ballpoint pen with waterproof ink • Fine point waterproof permanent marker such as a Sharpie • Roll of adhesive or masking tape • Labels and waterproof tags with eyelet and wire ties • Tamper-evident seals* • Matches • Buffered distilled water or 0.1% peptone water (5 ml in screw capped

tubes) • Test tube rack • Investigational forms *Tamper-evident seals and labels can be obtained from laboratory supply companies or by searching for “tamper-evident seals (or labels)” online.

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INSTRUCTIONS FOR COLLECTING BULK STOOL SAMPLES

1. Make sure the patient information section on the side of the vial is completed.

• 2. Collect stool onto a newspaper or into a clean container, such as a specimen cup or a clean plastic tub.

• For best results, select areas of the stool that appear bloody or watery. If the stool is formed (hard), sample small amounts from each end and the middle.

• 3. Using a plastic spoon or tongue depressor, put about 3-4 tablespoons of stool in the specimen container.

• 4. Put lid on the specimen container and label container with patient’s name. • 5. Put the specimen container in a zip lock plastic bag. • 6. Keep the specimen container with the stool sample cool, either in a

refrigerator or on a freeze pack in a cooler. DO NOT FREEZE. • 7. Contact the Health Department at ____________ to make arrangements

for pick-up or delivery of both stool samples.

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In the cases of parasite related outbreaks:

• Since parasites are shed intermittently, 3 samples, collected 24-48 hours apart within a 10 day period, are required to adequately test for parasites. Each sample is collected into two tubes (one pink and one blue): one for the cyst stage and one for the trophozoite stage.

• Stool specimens in the Ova & Parasite solutions are shipped to Labs without refrigerant.

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RECOMMENDATIONS FOR COLLECTION of CLINICAL SPECIMENS FOR VIRUSES DIAGNOSIS (Stool Samples)

• Submit 2-5 bulk stool specimens (25-30 g/10-20 ml). Acute diarrheal stool is best.

• Timing. Specimen collection for viral testing should begin on day 1 of the epidemiologic investigation. Any delays to await testing results for bacterial or parasitic agents could preclude establishing a viral diagnosis.

• Ideally, specimens should be obtained during the acute phase of illness (i.e., within 48 72 hours after onset) while the stools are still ‐‐ liquid or semisolid because the amount of viral excretion is greatest.

• With the introduction of realtime RT PCR assays, the ability to detect ‐viruses (norovirus, sapovirus) in specimens collected later in the illness has been improved. In specific cases, specimens might be collected later during the illness (i.e., 7 10 days after onset), if the testing is necessary ‐‐for either determining the etiology of the outbreak or for epidemiologic purposes (e.g., a specimen obtained from an ill foodhandler who might be the source of infection).

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RECOMMENDATIONS FOR COLLECTION of CLINICAL SPECIMENS FOR VIRUSES DIAGNOSIS (Stool Samples) continued

• Number and Quantity. Ideally, specimens from at least 5 ill persons should be collected during the acute phase of illness. Bulk samples (i.e., 10 50 ml of stool placed in a stool cup ‐‐or urine container) are preferred, as are acute diarrhea specimens that are loose enough to assume the shape of their containers. Serial specimens from persons with acute, frequent, high volume diarrhea are ‐ useful as reference material for the development of assays.

• The smaller the specimen and the more formed the stool, the lower the diagnostic yield.

• Rectal swabs are of limited or no value because they usually contain insufficient quantity for typing of the strains.

• Storage and Transport. Stool specimens should be kept refrigerated at 4°C. At this temperature, specimens can be stored without compromising diagnostic yield for 2 3 ‐‐weeks, during which time testing for other pathogens can be completed. If the specimens have to be transported to a laboratory for testing, they should be bagged and sealed and kept on ice or frozen refrigerant packs in an insulated, waterproof container. If facilities for testing specimens within 2 3 weeks are not available, specimens can be frozen for antigen ‐‐or PCR testing.

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Patient must DO NOT:

• i) Pass the stool directly into the vial. • ii) Urinate on the stool or into the vial. • iii) Pass the stool into a toilet. • iv) Overfill the collection vial.

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Vomitus and serum• Vomitus Vomiting is a characteristic, but not unique symptom of virus illness, and vomitus can be collected to supplement the diagnostic yield from stool specimens during an investigation. • Recommendations for collection, storage, and shipment of vomitus specimens are the same as those for stool

specimens. Briefly, Let the patient vomit directly into a specimen container that has been thoroughly cleaned and boiled in water. Take the specimen directly to the laboratory. If this is not possible refrigerate (but do not freeze) the specimen.

. Serum Timing. If feasible, acute and convalescent phase serum ‐ ‐ specimens should be obtained to test for a diagnostic >4 fold ‐rise in IgG titer to viruses. Acute phase specimens should be obtained ‐ during the first 5 days of symptoms, and the convalescent phase specimen should be collected from the ‐ third to sixth week after resolution of symptoms.

Number and Quantity. Ideally, 10 pairs of specimens from ill persons (i.e., the same persons submitting stool specimens) and 10 pairs from well persons (controls) should be obtained. Adults should provide 15 ml of blood, and children should provide 3 4 ml. ‐‐

• Storage. Specimens should be collected in tubes containing no anticoagulant, and the sera should be spun off and frozen. If a centrifuge is not available, a clot should be allowed to form, and the serum should be decanted and frozen. If this step cannot be accomplished, the whole blood should be refrigerated but not frozen. For antibody studies the specimens need not be refrigerated during the day of the collection (unless the weather is extremely hot) but should be kept out of direct sunlight. Centrifuge the blood and send only the serum for analysis. If no centrifuge is available, store the blood specimens in a refrigerator until a clot has formed; then remove the serum and pipette it into an empty sterile tube. Refrigerate the tubes of spun or unspun serum and ship them refrigerated.

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Environmental Specimens for viral outbreaks 

• Current realtime RT PCR methods allow ‐detection of viruses in water, food, and environmental specimens.

• If a food or water item is strongly suspected as the source of an outbreak, then a sample should be obtained as early as possible and stored at 4°C .

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Urine samples for chemicals

• Urine• Urine is the preferred specimen if the suspected toxicant is

an inorganic chemical (e.g. lead, arsenic, mercury). Urine should also be collected if the toxicant is unknown. Freeze promptly.

• Procedure: Clean the area around the urethral orifice with a pad that has been pre-moistened with a 4% tincture of iodine or other appropriate antiseptic. Begin to urinate into the toilet and collect 30ml of midstream urine. The specimen should be refrigerated but not frozen.

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Samples for chemical outbreaks

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Food samples• Do not freeze food samples because certain foodborne bacteria (such as gram-negative

bacteria and Clostridium perfringens) die off rapidly during frozen storage. All samples that are not frozen should be stored and shipped at 4 ºC.

• Solid Food or Mixture of Two or More Food Items:• 1) Cut or separate portions of food with a sterile knife or other sterile implement. For a solid

uniform food (e.g. roast), whenever possible collect at least four samples of 0.875 ounces (for a total of 3.5 ounces) each from the center and other representative locations throughout the food item.

• 2) For food mixtures, collect at least four samples of 25-50 grams/ml for a total of 100-200 grams/ml each from the center and other representative locations throughout the food item.

• 3) Transfer the sample to a sterile primary container. • 4) Label the primary container, taking care to identify food from different batches. • 5) Seal the primary container with tamper-evident tape. • 6) Place the primary container in a secondary self-closing plastic bag such as an unused

Ziploc® bag. 7) Pack the secondary container in an insulated container (e.g. a cooler) with cold packs or other refrigerant around the sample containers. Do not freeze the sample or use dry ice in the cooler.

• 8) Take or ship all samples to the reference labs as quickly as possible.

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Food samples (continued)• Liquid Food or Beverages This category includes all beverages and liquid food such as gravy, soup, sauce, etc. Stir or thoroughly shake the item to be sampled and collect the sample in one of the following ways: 1) Pour or ladle, with sterile utensil, at least 200 ml of the liquid into a sterile primary container;

OR Put a long sterile tube into the liquid and then cover the top with a gloved finger, transferring a 200 ml sample to a sterile primary container.

• Dip a Moore swab in the liquid or into the pipe so that liquid circulates around it. Leave in place for several hours, if possible. Transfer swab to a jar containing enrichment broth. Refrigeration is not usually necessary.

• If the liquid is not too thick, pour 1 to 2 litres through a membrane filter. Transfer the• filter pad aseptically to a jar containing enrichment broth. Refrigeration is not usually

necessary.2) Label the primary container. 3) Seal the primary container with tamper-evident tape. 4) Place the primary container in a secondary self-closing plastic bag such as an unused Ziploc® bag. 5) Pack in an insulated container with cold packs or refrigerant around the sample container. Do not

freeze or use dry ice. 6) Take or ship all samples to the reference labs as quickly as possible.

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Food samples (continued)• Raw/Cooked Meat or Poultry • There are several methods of collecting these samples depending on the type of

sample being taken. Use ONE of the following methods: • 1) Using a sterile utensil or sterile gloved hand, put at least 200 grams of the

chicken, poultry part or large cut of meat into a large sterile primary container; OR For large cuts of meat, a sterile sponge should be wiped over a large area of the meat. Then put the sponge into a primary sterile container; OR Using a sterile utensil(s), cut four 25-50 gram portions of meat or skin from different areas of the carcass or cut of meat and put it into a sterile container; OR Moisten a swab in buffered distilled water or 0.1% peptone water. Wipe the swab over a large section of the carcass or piece of meat. Place swab in enrichment broth.

2) Label the primary container. 3) Seal the primary container with tamper-evident tape. 4) Place the primary container in a secondary self-closing plastic bag such as an unused Ziploc® bag.5) Pack in an insulated container with cold packs or refrigerant around the sample container. Do not freeze or use dry ice. 6) Take or ship all samples to the laboratory as quickly as possible.

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Food samples (continued)• Frozen Foods Use one of the following methods: 1) Place unopened packaged frozen food items in a sterile plastic bag (primary container). Call

the reference labs for instructions if the sample is greater than 800 gram; OR Use sterile utensils to chip unwrapped frozen material and transfer at least 25 grams of chips taken from each of four different locations for a total of 100-200 grams of the frozen food item or from each food item (e.g., all foods in a frozen dinner) into a sterile primary container.

2) Label the primary container.

3) Seal the primary container with tamper-evident tape.

4) Place the primary container in a secondary self-closing plastic bag such as an unused Ziploc® bag.

5) Pack in an insulated container with cold packs or refrigerant around the sample container and ship or store on dry ice to maintain frozen state.

6) Take or ship all samples to the reference labs as quickly as possible.

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Environmental samples• 1) A sterile non-cotton swab moistened with sterile 0.1% peptone water or buffered

distilled water

• 2) Swab the food contact surfaces of the equipment or environmental surfaces. Put swab in a sterile container with enrichment broth.

• 3) Label the primary container (container with swab) with the following: date and time sampled equipment or sample source and location sampled, name of sampler, and name of county in which the investigation is being conducted.

• 4) Seal the primary container with tamper-evident tape

• 5) Complete a foodborne illness Form and place with the primary container in a secondary selfclosing plastic bag such as an unused Ziploc® bag.

• 6) Pack in an insulated container with cold packs or refrigerant around the sample container. Do not freeze or use dry ice.

• 7) Take or ship all samples to the SLPH as quickly as possible.

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Water samples

• Collect water from suspected areas, including from bottles in refrigerators, ice cubes, basins, etc. When taking water from a tap, let the water run for 10 seconds before collecting the sample. To sample water that has not been standing in proximal pipes, let water run for 5 minutes. Place sterile jar under running water and let it fill to 2.5 cm from the top. Collect 5-10 litres.

• Alternatively, membrane filters can be used. Moore swabs may be used to collect water samples from streams or plumbing; they should be left in place for up to 48 hours and then transferred to sterile jars containing enrichment broth.

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Guidelines for Laboratory Identification of Clostridium botulinum

• Specimen A. Acceptable specimens 1. Clinical specimens 2. 2. Postmortem specimens 3. 3. Culture/isolate 4. 4. Food samples, solid or liquid 5. 5. Environmental samples

B. Rejection criteria 6. Incomplete documentation: All specimens must include the sender's name and telephone number to

contact for the preliminary report and additional information. 7. Improper packaging/shipping 8. Do not ship specimens to higher-level LRN laboratories without prior approval. IV. Materials A. Media: Anaerobic media (chopped meat or equivalent); follow standard laboratory procedures. B. Supplies C. 1. Port-A-Cul vials (Becton Dickinson; catalog #4321609) or equivalent D. 2. Leakproof containers (i.e., sealed plastic bags, and other plastic containers) E. 3. Petroleum jelly, or petrolatum (Fisher Scientific; catalog #P661LB), or equivalent (e.g., Vaseline) F. 4. Sterile, nonbacteriostatic water .

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Sample collection for botulism1. Feces: Place into sterile unbreakable container and label carefully. Confirmatory evidence of botulism may be obtained

from 10-50 g quantities (English walnut size); botulism has been confirmed in infants with only "pea-sized" stool samples. Taken from CDC Laboratory Response Network (LRN) I. General: Laboratory Response Network (LRN) laboratories.

2. Enema: Place ~20 ml into sterile u tainer and label carefully. If an enema must be given because of constipation, a minimal amount of fluid (pre specimen so th

3. Gastric aspirate or vomitus: Place ~20 ml into sterile unbreakable container and4. Serum: Use red top or separator type tubes to obtain serum (no anticoagulant). 0 ml of serum for mouse toxicity tests

(usually 20 ml whole blood); serum volumes less than 3 ml will provide inconclusive results. Whole blood should not be sent as it typically undergoes exessive hemolysis during transit.

5. Tissue or exudates: Place into sterile unbreakable container and label carefully. imens should be placed in Port-A-Cul vials (Mena, 1978) and sent to the y, preferably without refrigeration, for attempted isolation ecimens of intestinal contents from different levels of intestines. Place ~10 g per specimen into sterile unbreakable Obtain gastric content, serum, and tissue iate (refer to A. 3, 4, and 5 above). uspicious isolates anaerobically (overlay liquid media with 2-inch re). 8. ens: Foods should be left in their original containers if possible, or containers and labeled carefully. Place containers contamination during shipment. Empty containers with remnants of suspected s can be examined. t medium (e.g., Port-A-Cul tubes) (Mena, 1978). Environmental swabs which spores may be isolated) may be sent in plastic containers without ed. n. B. hipping: Refer to Shipping Procedure; complete and attach appropriate gerant (sealed ice packs, cold packs), labeled "MEDICAL EMERGENCY, BIOLOGICAL HAZARD, y the most rapid means available. Most of the major airlines have a special package handling service for expedited shipments. Do not send by U.S. Postal Service; ship as hazardous materials. unavoidable delay of several days is anticipated, the specimens (serum or th dry vides boratory confirmation of botulism, priority should be given to nbreakable con ferably sterile, nonbacteriostatic water) should be used to obtain the at the toxin will not be unnecessarily diluted. label carefully. Samples should be obtained as soon as possible after the onset of symptoms and before antitoxin is given. Enough blood should be collected to provide at least 1 be sent as it typically undergoes excessive hemolysis during transit. Spec appropriate laborator of C. botulinum.

6. Postmortem: Obtain sp small and large container and label carefully. specimens if/as appropr 7. Culture: Ship s sterile petroleum jelly or petrolatum; melt/temper prior to overlaying cultu Cultures may be shipped at

room temperature or refrigerated. 8. Food specim placed in sterile unbreakable individually in leakproof containers (i.e., sealed plastic bags) to prevent

crossfood 9. Swab samples (environmental or clinical): Send clinical swabs in an anaerobic transpor (from any medium. Swabs may be

shipped at room temperature or refrigerat Collect 3-4 swabs from each potential site.10. Environmental samples: Collect a sample in the size indicated for each possible locatio (1) Soil (50-100 g) (2) Water (=100

ml)

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Suggested specimens based on form of botulism

1. Foodborne a. Clinical material: Serum, gastric contents, vomitus, stool, return from sterile water or saline enema b. Autopsy samples: Intestinal contents and gastric contents (serum if availab c. Food samples 2. Infant a. Feces b. Return from sterile water or saline enema c. Serum: Although circulating toxin may be detected in infants with botulism, it is rare. Shipment of other specimens

should not be delayed while waiting for serum collection. d. Postmortem samples: Intestinal contents from different levels of small and large intestine e. Food and environmental

samples as appropriate for the investigation 3. Woundd. Serum e. Exudate, tissue, or swab samples of wound transported in an anaerobic transport medium f. Feces or return from sterile water enema (wound may not be source) g. An isolate of suspected C. botulinum (maintain under anaerobic conditions)

4. Intentional toxin release (inhalational or ingested) h. Serum i. Feces or return from sterile water enema j. Food, solid or k. Environmental or nasal swabs l. Gastric aspirate

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