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Laurie Post

Retest Analysis: When Original Sample Results and Retest Results Don’t Correlate

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By Laurie Post, Ph.D.
When undesirable microorganisms are detected in a production lot, root cause analysis often includes testing additional samples from the affected lot or related lots of product. However, upon reanalysis, even when a larger sample size is used, the suspect organism cannot be detected in the retest analysis. Correlation differences between original and retest results can occur for several reasons:

  1. Distribution pattern of the microorganisms
  2. Lack of representative sampling
  3. Organism die-off
  4. Level of contamination and probabilities of detection

Distribution
The distribution pattern of microorganisms in a production lot affects the likelihood of detecting the organism in the product. For instance, distribution patterns can be Random or Non-Random.

Random Distribution: Microorganisms are distributed evenly throughout the entire lot. Microorganisms will likely be detected. This type of microbial distribution is not a factor of time since at any point in the sampling process there is an equal opportunity to detect the organism in question (Example A).


Non-random Distribution: Microorganisms are not distributed evenly throughout the entire lot. Microorganisms may not be detected by random sampling. This type of distribution is dependent on time since the microbial contamination is only represented at certain intervals in the production cycle without equal opportunity for detection (Examples B and C)

Example B shows a non-random distribution pattern of organisms that can be present at the startup of production. In this case, the food actually cleans the line, so that over time the contaminant is reduced to undetectable levels. If a retest sample of this lot of production is pulled from containers made later in the day, then the retest results will be negative for the target organism, even though the organism was present.

Example C is another example of non-random microbial distribution that can occur when condensate drips onto exposed product. Condensate began to drip sometime after startup and continues throughout the rest of the day, increasing toward shutdown. If retain samples taken at the beginning of the day were selected for retest analysis, the retest samples would likely be negative for the target organism even
though the organism was present in that lot of production.


Sampling
Microbiological analyses are only as good as the sampling procedure used to procure the sample. Every effort must be made to obtain a RANDOM SAMPLE. “Spot sampling” or taking a single sample will not yield a representative sample. FDA recommends taking 30 random 25g samples during the entire production run for Ready-to-Eat products that will be consumed without a process lethal to microbial pathogens. These include products such as cereals, confectionary products, cheese, dairy, spices – and many others (FDA Bacteriological Analytical Manual). For fluid and powdered products, most manufacturers employ mechanical or auto-samplers. For discrete products, samples are taken on a time production basis (i.e. one candy bar every half hour) and then the analytical unit (the actual amount tested) is made by compositing. Compositing must be performed carefully and thoughtfully, not only to ensure a representative sample, but also to avoid sample contamination.

Organism Die-off
Organisms contaminating a product may not survive at the same level for long periods of time. For example, Escherichia coli (E. coli) is not capable of long-term survival in an inhospitable matrix, such as chocolate, and will eventually die off. Levels may fall below the ability of an assay to detect the organism.


Levels of Contamination and Probability of Detection
The probability of finding a target organism in a retest sample is based on the probability of finding the target organism in the original sample. If 10 samples are collected for analysis and only one tests positive for a pathogen, the level of contamination is one in ten or 10%. Based on a standard statistical analysis, if there is a 10% chance of finding the target organism in the original sample, then the ability of finding the organism a second time by retest sampling is [10% X 10%] or [0.10 X 0.10 = 0.01] or one in a hundred. Similarly, if the level of contamination is 5%, the probability of finding the positive a second time is one in twenty-five and 1% is one in ten thousand. The short hand version of this is to simply square the level of
contamination, as shown here:

Level of Contamination- Probability of Finding the Target Organism in the Original Sample Probability of Finding the Target Organism in a
Retest Sample
One in two (50%)One in four (25%)
One in four (25%)One in sixteen (6.5%)
One in five (20%)One in twenty five (2.5%)
One in ten (10%)One in a hundred (1%)
One in a hundred (1%)One in ten thousand (0.01%)

There are many factors that can result in correlation differences between original and retest results. But, if a retest is desired, pull many random samples and request a retest as soon as possible.

Food Safety Considerations in the Production of Traditional Fermented Products: A Review Article

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By Laurie Post, Ph.D.
A recent article in the Journal of Food Safety by authors at the Federation University in Australia reviewed the processes used in the production of Japanese rice koji and miso and developed a hazard assessment for these fermented foods.

Miso is a traditional fermented soybean paste made for thousands of years. The most common type of miso is made from rice koji, salt, and soybeans using a two stage fermentation process. The first step is the production of koji where rice is fermented with a mold such as Aspergillus oryzae at 30°C for 48 h. The koji and salt are then added to boiled soybeans and fermented by yeasts and bacteria for 2 to 24 months. Koji and miso are increasing in popularity in western countries where contemporary miso varieties are not pasteurized as consumer demand is for more natural products containing live microorganisms. While correctly prepared fermented foods are rarely associated with foodborne illness outbreaks, there have been several reports of illness. These were primarily due to pathogenic microorganisms introduced into the products from external sources such as raw materials or the processing environment. Hygiene and fermentation conditions must be carefully monitored to ensure food safety.


The authors point out that many of the production steps in the manufacture of koji and miso do not fit into contemporary food safety guidelines. Although a reduction in pH is a typical food safety hurdle for fermented foods, this does not apply to non-acidic foods such as koji or miso. Use of a certified safe mold as the starter culture and monitoring appropriate temperatures to ensure prolific growth of the mold (and to minimize growth of unwanted bacteria) are requirements for the safe production of rice koji. An optimized salt content and reduced water activity (Aw) is a food safety hurdle in the production of miso. Focusing on the quality of ingredients and hygienic practices along with the development of standardized
fermentation conditions, would also assist in safe production of these fermented foods. The authors conclude that research on unpasteurized koji and miso is needed to determine optimized temperatures, pH and Aw levels, and salt concentrations during the fermentation process that would assure these fermented foods comply with food safety requirements.

Regulatory Watch Out!

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Use of Unapproved Food Additives – FDA’s Public Inventory

Any substance used or intended for use in food must be authorized by the FDA for use as a food additive under the Federal Food, Drug, and Cosmetic (FD&C) Act, unless the use of that substance is generally recognized as safe (GRAS) by qualified experts or qualifies for an exemption.


As part of its on-going compliance activities, FDA conducts post-market activities to monitor the food supply for chemical contaminants. FDA identifies foods that contain a substance for which there is no authorization as a food additive and then reviews the regulatory status of this substance. FDA scientists analyze whether there is a basis to conclude that the intended use of the substance is GRAS or meets a
listed exception to the food additive definition. When FDA scientists determine that a substance is an unapproved food additive because it is not GRAS for its intended use (and does not meet a listed exception), they deem the additive to be unsafe and any food that contains the additive is adulterated.

On July 12, 2023, FDA released a public inventory of certain food ingredients that do not have GRAS status as determined by the agency and are therefore deemed unsafe. The inventory is a useful tool to assure the use of authorized ingredients.


The FDA Concludes Voluntary Pilot Program to Evaluate Alignment of Third-Party Food Safety Standards
with FSMA Rules


Food manufacturers may find that they are required to comply with one of the Global Food Safety Initiative standards in addition to the FDA’s Preventive Controls for Human Food or Produce Safety Rules. The FDA recently concluded a voluntary pilot program to evaluate alignment of private third-party food safety audit standards including BRC, FSSC22000, SQF and Global G.A.P. with applicable FDA regulations.


The pilot program was launched to help both FDA and industry gain a better understanding of whether these standards align with FDA regulations. Alignment between these standards may allow a company to structure their food safety plan to eliminate redundancy in their documentation and auditing programs.

Buyers and others in the food supply-chain often use third-party audits to assess the quality and safety of a product. Buyers, such as importers and receiving facilities, might stipulate an audit as part of a purchase agreement. In addition, three FSMA regulations – the Preventive Controls for Human Food rule, Preventive Controls for Animal Food (PC Animal Food) rule, and Foreign Supplier Verification Programs (FSVP) rule – allow for third-party audits to be used as supplier verification activities. Points of alignment would provide confidence that, in general, the third-party standards used to audit suppliers adequately address applicable FDA food safety requirements.


The results of the pilot program can be found in The FDA Concludes Voluntary Pilot Program to Evaluate Alignment of Third-Party Food Safety Standards with FSMA Rules. The FDA’s statements regarding alignment of the standards are referenced in the table below and apply only to the specified audit standards and addenda listed.


The reviews listed in the table focused on assessing third-party food safety standards and not the overall quality of the audit programs or qualifications of auditors. The FDA’s review and the findings from this pilot do not constitute an endorsement of any one food safety audit standard, or audits conducted under such standards. The FDA also adds a qualifying statement that third-party audits are not a substitute for FDA or state regulatory inspections for compliance with FDA regulations, including the Preventive Controls for Human Food Rule or the Produce Safety Rule.

Third-Party Food Safety Audit Standard and Applicable
Addendum		Scope Name
BRC Global Standard Food Safety plus the Global Standard Food Safety,
Issue 9, Interpretation Guideline		Preventive Controls for Human Food (PCHF)
FSSC 22000 Scheme 5.1 for Food Manufacturing plus the FSSC 22000, Version 3 FSMA PCHF Report Addendum
Preventive Controls for Human Food (PCHF
SQF Food Safety Code: Food Manufacturing, Edition 9 plus the SQF
Addendum for the Preventive Controls for Human Food		Preventive Controls for Human Food (PCHF)
GLOBALG.A.P. Integrated Farm Assurance – All Farm Base Crops Base – Fruit and Vegetables Checklist. Version 5.4-1- GFS plus the GLOBALG.A.P. Food Safety Modernization Act Produce Safety Rule Add-on Module Version 1.3Produce Safety Rule (PSR)
*Did not include Subpart E, related to Agricultural Water (as applicable to non-sprout produce) or Subpart M, related to sprouts

IAFP 2023 – Symposium Discusses the Investigation of Ambiguous Outbreaks

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A symposium at IAFP 2023 (S64), “Investigating Ambiguous Outbreaks and Adverse Events” sponsored by the Institute for the Advancement of Food and Nutrition Science (IAFNS) Food Microbiology Committee discussed the topic of “ambiguous outbreaks,” those outbreaks of foodborne illness for which definitive sources and/or pathogens targets are not known.

The FDA will issue a public health advisory for outbreak investigations that result in specific, actionable steps for consumers to take to protect themselves. However, some outbreak and adverse event investigations may not conclusively identify a source or reveal any contributing factors. Actionable steps for consumers are not available. FDA maintains a list of outbreak and adverse event (self-reported) investigations managed by their CORE Response teams. Although an investigation may reveal a particular product or product category as a possible source of illness, the FDA will not publicly name a specific product until there is sufficient epidemiological evidence to implicate that product as a cause of illness or an adverse event.

Many investigations do not identify a pathogen or food vehicle responsible for an outbreak or self-reported illnesses in an adverse event despite extensive testing for numerous potential microbial and chemical adulterants. What learnings can be taken from these investigations that could improve resolution and contribute to an improved prevention strategy? This symposium delved into the question. A recording of the symposium will be available from IAFP.

Speakers discussed improved investigational approaches and tools, the use of Root Cause investigation and analysis to uncover factors leading to attribution, improved outbreak communication strategies for reaching consumers and industry, and new areas of research.

Ambiguous outbreaks can be grouped into one or more of the following categories: outbreaks with a known source and unknown agent, a known agent and unknown source, or an unknown agent and unknown source. Dr. Craig Hedberg, University of Minnesota, discussed improved investigational tools to detect and identify a causative agent such as culture independent diagnostic tests (CIDT) and whole genome sequencing. He also reviewed advances in epidemiological and environmental assessments that improve the chances of making connections between an illness and the source when studying outbreaks for which definitive sources and/or pathogens targets are not known.

Dr. Tim Jackson, FDA, reviewed reasons why the root cause of an outbreak is often ambiguous. Investigations into outbreaks are usually retrospective, analytical testing is statistically limited and often inconclusive, and some foods with short shelf lives may not be available for analysis. Additionally, there may be limited tools for analysis of some causative agents. With multi-component or assembled products, processes and supply chains may be complex with a variety of growers/suppliers, manufacturers, retailers, and distributors. Accurate and precise traceability programs are essential to the evaluation of underlying causes. Dr. Jackson discussed the tools available for root cause investigation and analysis and provided examples of how they have been used in the resolution of outbreaks.


Michael Vasser, CDC, examined the impact of reoccurring, emerging, and persisting (REP) strains on ambiguous outbreaks. Some enteric bacterial strains cause acute outbreaks linked to specific sources. Other strains that are closely related by whole genome sequencing, referred to as REP strains, can reoccur and periodically cause acute outbreaks. They can also emerge and increase in frequency or persist and cause illnesses over periods of months or years, despite investigation and prevention efforts. They do not present as typical acute outbreaks. While illnesses caused by REP strains may be associated with a common reservoir or source, the linkage is often inferred but not proven. Mr. Vasser commented that, in fact, most illnesses reported through PulseNet are not linked to a source (90% of isolates do not have an assigned cluster code). REP strains could actually be the cause of a larger fraction of illnesses than
typical outbreaks. Investigation of REP strains will create opportunities to characterize new sources of enteric disease and develop novel prevention approaches.


Mitzi Baum, STOP Foodborne Illness, discussed the critical need for improved communication channels to the public and industry when outbreaks occur. Stop Foodborne Illness is a non-profit public health organization in the United States dedicated to the prevention of illness and death from foodborne pathogens. The organization was founded in 1993 following an E. coli O157:H7 outbreak in California and the Pacific Northwest. While ambiguity in investigations does occur, it is essential that public health officials provide relatable and actionable information to consumers in these circumstances.



The Eight Most Important Recommendations for Controlling Listeria monocytogenes in Retail Delicatessens

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The USDA Food Safety and Inspection Service (FSIS) recently published a guidance document that provides recommendations that retailers can use in delicatessens to control L. monocytogenes contamination of ready-to-eat (RTE) meat and poultry products. This 2023 document is a revised version of the 2015 FSIS Best Practices Guidance for Controlling Listeria monocytogenes (Lm) in Retail Delicatessens. It reflects the FSIS’s current thinking on this topic and is not a regulation. While focused on small firms, the recommendations made will be useful for all meat and poultry retail firms.


Recommendations are based on the joint FSIS, Food and Drug Administration (FDA), and Centers for Disease Control and Prevention (CDC) 2013 Listeria monocytogenes in Retail Delicatessens Interagency Risk Assessment. The assessment summarizes the risks posed by L. monocytogenes from the consumption of RTE foods commonly prepared and sold in retail delis.

The guidance addresses:

  • Actions retailers can take in the delicatessen (deli) area to decrease the potential for
    L. monocytogenes growth and cross-contamination.
  • Steps retailers can take to help ensure that deli products are maintained under sanitary conditions
    that do not allow L. monocytogenes adulteration of the product.
  • Information from the U.S. Food and Drug Administration’s (FDA) Food Code, scientific literature,
    other guidance documents, and lessons learned from meat and poultry establishments that
    retailers can use to control L. monocytogenes.
  • Helpful tools that retail firms can use to identify potential gaps in current best practice procedures.
  • The eight most important recommendations for retail deli operations.

Retailers can use the best practices identified in this guidance to help ensure that RTE meat and poultry products in the deli area are handled under sanitary conditions and are not adulterated, as defined in the Federal Meat Inspection Act (FMIA) and Poultry Products Inspection Act (PPIA).

While these practices are designed to control L. monocytogenes specifically, they also may help control other foodborne pathogens that may be introduced into the retail deli environment and other facilities where consumers take possession of food. No single action or practice will control L. monocytogenes contamination. Therefore, by following the best practices in this guidance and the Food Code, retailers can help ensure that RTE products are not adulterated with L. monocytogenes, and that the potential for listeriosis is decreased.

A Deli Self-Assessment Tool is provided for deli operators in Appendix A of this guidance to help them identify the best practices they are using and to assess whether they need to adopt others.

A Comprehensive Approach: The Eight Most Important Retail Deli Recommendations to Prevent
Conditions That May Cause Adulteration (Source: USDA FSIS

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