Best Practices in Pathogen and Allergen Testing E. coli O157 Testing in the Beef Industry: Sampling, Detection, COA, and its Consequences Ross Ullman American Foods Group 2018
Regulatory Requirements ➢ USDA declared E. coli O157 as an adulterant in raw ground beef after the Jack in the Box outbreak of 1992. ➢ FSIS published Notice 44-02 on Oct 7, 2002, stating that, FSIS considered E. coli O157:H7 a Hazard reasonably likely to occur at all stages of handling raw beef products. ➢ FSIS Notice 05-09 required that grinding establishment must receive COA with each lot of boneless beef trimmings.
FSIS Assessment of E. coli O157 as a Hazard Reasonably Likely to Occur ➢ FSIS notice 44-02 stating that, E. coli O157:H7 is a hazard reasonably likely to occur at all stages of handling raw beef products. ➢ FSIS required all establishments producing raw beef products to reassess their HACCP plans ➢ Establishments to determine adequacy of pre- requisite interventions ➢ To incorporate one or more Critical Control Points (CCPs) to prevent contamination ➢ Beef grinders to use raw material testing a part of purchase specifications as an acceptable CCP ➢ Efficacy of CCPs were to be evaluated by on-going verification activities. FSIS recommends that verification activities include microbial testing
E. coli O157 testing of trim ➢ Wide spread sampling and testing in effect as of December 2002 ➢ No standard or guidelines for a sampling plan or testing methods by USDA ➢ Cultural method (Gold Standard) the only available confirmatory method used by FSIS Lab ➢ Industry needed rapid, accurate and reliable method. ➢ Cannot afford to wait for confirmation ➢ Make the decision for the disposition based on initial screening results ➢ Test results have regulatory consequences
FSIS Position on Presumptive Positives ➢ From: FSIS Directive 10,010.1 “Microbiological Testing Program and Other Verification Activities for Escherichia coli O157:H7 in Raw Ground Beef Products and Raw Ground Beef Components and Raw Ground Beef Patty Components”, Revision 1, 3/3/04 ➢ Definition: “A sample is presumptive positive when analytical steps of microbiological analysis indicate the strong possibility that E. coli O157:H7 is present, but additional steps are needed to confirm the presence or absence of the organism.” ➢ Policy: “FSIS points out, however, that if an establishment finds a sample of one of these [beef] products to be presumptive positive for E. coli O157:H7, that product would only be allowed to move off site under appropriate controls for proper disposition at official establishments, landfill operations, or rendering. If the establishment’s confirmation testing finds the sample negative for the pathogen, that product may be shipped in commerce under normal procedures .
Why does Beef Industry test trimmings? ➢ Beef Industry’s commitment to Food Safety ➢ To comply with Regulatory Requirements ➢ To comply with customer’s demand ➢ Prevent recalls, lawsuits and to protect the Brand Name
Elements of E. coli O157 Trim Testing Program 1. Lot Definition 2. Sampling Plan, and Sampling Method 3. Laboratory and Testing Methodology 4. COA 5. Product Disposition Decision Tree & Using Test Results in Statistical Process Control
1. Lot Definition ➢ Combo Bin: A unit of production, 2000 lb ➢ In a 5-combo lotting system a combo is a sub-lot ➢ Lot: Usually 5 combo bins – 10,000 lb. ➢ AFG uses 1 combo bin as one lot ➢ One test represent one lot, provided robust sampling has been applied
2. Sampling Plan ➢ Perhaps the most important part of the program ➢ Trim sampling plan requires N=60 sampling ➢ In Single Combo as a lot, 60 pieces are collected from a single combo (2000 lb) as opposed to 12 pieces from each combo in 5- combo lot (12x5= 60) ➢ More samples, more confidence in negative results and less product devaluation ➢ The smaller the lot size the more robust the plan is
2. Sampling Method ➢ USDA prescribed excision sampling is labor intense and increases the chances of cross contamination, and employee safety issues ➢ AFG uses semi-automated sampling device called N60 Plus sampling method. ➢ The N60 Plus method takes away most of the operator related errors, and collects far more microbial load per sample than the N=60 excision method
326 2.2 2.2 1. F IGURE 1. M EAN L OG TPC, TCC AND ECC/ SAMPLE OBTAINED FROM BEEF TRIM SAMPLES COLLECTED USING THE IEH N60 P LUS S AMPLER ™ OR SURFACE EXCISION .
Antigen / Antibody
3. Testing: Choice of Analytical Method ➢ Two classes of screening platforms: Immunoassay or Lateral Flow Device (LFD) based, and DNA based (PCR) methods ➢ Both methods consist of two modules: enrichment and detection ➢ Enrichment: E. coli O157 level in the sample have to increase from <10 cfu/375 g to detectable levels/ml of the enrichment broth: ➢ 10,000-100,000 cfu/ml for LFD methods ➢ 1000-10,000 cfu/ml for PCR methods
E. coli O157:H7 VIRULENCE TARGETS O157/O26/O45/O103/O111/O121/O145/ O…. RFB uid STX1 Ph EAE STX2 Ph H1-50
Some Commercial Kit Virulence Targets O-157 PCR 1 RFB UID STX1 Ph EAE STX2 Ph H7 PCR 2
LFD: Antigen-Antibody ➢ LFDs are generally less sensitive than PCR based methods by a factor of one log ➢ LFDs give a higher rate of false positives and false negatives than PCR ➢ False positives: cross reacting organisms ➢ Non-toxigenic E. coli O157s ➢ False negatives: Improper enrichment to result in minimum detection limits
3-Genetic DNA based identification • Polymerase Chain Reaction (PCR) Single target • Multiplex PCR (multi-targets) • Real Time PCR (RTPCR)
PCR Test System Targeting all Virulent Factors O-157 UID RFB STX1 Ph EAE STX2 Ph H7
PCR 2 AND LFD1 PCR1 IMS Initial Reactive LFD 2 With AND Without IMS IMS Presumptive Positive Presumptive Positive
4. Certificate of Analysis (COA) ➢ Components of raw ground beef cannot be sold without a certificate of analysis for E.coli O157 ➢ The COA at the minimum should convey the following information: Date, Establishment #, PO, customer ID, Lot #, boning date, Name of the Lab, ISO Certification Number, representation regarding sampling and sample weight, test method, and test result
5. Product Disposition Decision Tree & Statistical Process Control ➢ Need a clear, well-defined plan of action when there is positive product including disposition of the product ➢ Event period or “hot” day ➢ Statistical Process Control is used for determining the prevalence of positive product and what is product is affected
Ground Beef Testing for E. coli O157 ➢ Not required by USDA, conducted as a verification to raw material sampling by facility, or customer requested ➢ Grab samples, 375g samples ➢ Time intervals & Lotting (supported by validation study) ➢ Isolating positive product by intensified sampling – product disposition
Allergen Testing ➢ When to test ▪ Product change over ▪ After clean-up or sanitation ➢ Method ▪ Test Kits (LFD) ▪ Cannot be detected by PCR ➢ What can be detected ▪ Milk, Egg, Soy, Peanuts, Gluten, Mustard, Sesame, and others… ➢ Decisions made from results
Conclusion Important elements in a pathogen verification test program in order of significance: ➢ 1) Establishment of statistically based sampling program ➢ 2) Selection of the Analytical Method for testing and the competency of the Laboratory services ➢ 3) Generation of accurate COA; managing the product disposition is based on information presented in COA
Conclusion ➢ 4) The higher the number of samples, and the smaller the lot size, the higher confidence in the negative COA ➢ 5)To hold foreign supplier of beef to the same exact standards of frequency and verification testing as domestic producers Important factors in Allergen testing: ➢ Determine when testing should occur ➢ Have established protocol for positive results ➢ Use results to evaluate effectiveness of cleaning
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