Validation of Microbiological Methods for Use in the Food Industry

1
Validation of Microbiological Methods for Use in
the Food Industry

• Brazilian Association for Food Protection
• 6th International Symposium
• Sao Paulo, Brazil
• June 15th, 2007

2
Introduction

• Hundreds of new methods developed each year
• Pathogenic organisms
• Non-Pathogenic organisms
• Detection
• Identification
• How do you know if you need a new method?
• How do you decide if it is the right method for
  your purpose?

3
Introduction

• Goal of methods evaluation is to find an
  innovative technology that will allow for quick
  and efficient detection and/or quanitation of
  pathogens and spoilage organisms

4
Performance Criteria

• The Three Ss
• Sensitivity
• What is the sensitivity of current method
• What degree of sensitivity is needed
• Specificity
• What is the false positive rate
• What is the false negative rate
• Speed
• What is speed of current method (samples
  processed/day)
• How quickly are results needed

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Performance Criteria

• Costs
• What is cost of current method
• What is cost of instrumentation
• What is cost of disposables/reagents
• What is the cost per test
• Reagents
• Prep time
• Stability
• Availability
• Consistency (Quality Control)

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Performance Criteria

• Versatility
• Product only
• Variety of food matrixes
• Environmental samples only
• Pathogens only
• Microorganisms only
• Bacteria and/or Fungi
• Acceptability of method by scientific community
  and/or Regulators
• AOAC, AOAC-RI, USDA-FSIS, FDA, AFNOR

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Performance Criteria

• Vendor company reputation
• First product on market
• Training
• Vendor provided training on site
• How much, how long
• Technical Service
• Speed of service
• Availability of service (24-7)
• Service contract required

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Technical Evaluation

• Objective
• Justification (benefit of method to company)
• Acceptance Criteria
• Material and Methods
• Test Media/Conditions
• Microorganisms
• Genus, species, source
• Inoculum preparation
• Inoculation Procedure
• Statistical Analysis
• Results
• Next Steps

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Case Study 1 Dichloran-Rose Bengal Agar Yeast
and Mold Method Evaluation
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Dichloran-Rose Bengal Agar Yeast and Mold Method
Evaluation

• Objective Determine validity of a 2 day yeast
  and mold method using DRB agar incubated at 30C
  or 35C
• Justification Reduced product holding time,
  resulting in significant cost savings to the
  plant
• Acceptance Criteria Recovery efficiencies must
  be equivalent to the current 5 day PDA method

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Dichloran-Rose Bengal Agar Yeast and Mold Method
Evaluation

• Microorganisms
• Mold Cultures
• A.niger, Penicillium spp., and Paecilomyces spp.
• Yeast Cultures
• Z.ballii, S.cerevisiae, and a plant isolate
• Inoculum Preparation
• Organisms were harvested from aPDA plates by
  washing with sterile water
• 1ml from each individual mold or yeast suspension
  was added to 20 mls DI water
• Molds serially diluted
• Yeast adjusted to a spec reading of 1.00, then
  serially diluted

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Dichloran-Rose Bengal Agar Yeast and Mold Method
Evaluation

• Material and Methods
• product was inoculated with 100 cfu/g of target
  organisms
• 0.1ml of inoculated product surface plated onto
  each media (aPDA, DRBA)
• aPDA incubated at 25C
• Counted at 3 and 5 days
• DRBA incubated at 30C and 35C
• Counted at 2, 3, 4, and 5 days

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Dichloran-Rose Bengal Agar Yeast and Mold Method
Evaluation

• Statistical AnalysisAn analysis of variance
  (AOV) was done to test if the total counts for
  DRB at 2 and 5 days was significantly different
  from aPDA at 5 days

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Dichloran-Rose Bengal Agar Yeast and Mold Method
Evaluation

• Results
• DRB at 2 days-30C was statistically equivalent to
  aPDA at 5 days for mold recovery
• Molds were pale in color Penicillium spp. was
  white on DRB (green on aPDA). The other 2 test
  molds were pale yellow
• Yeast counts on DRB at 30C were significantly
  lower than counts on aPDA at 2 and 5 days
• Mold and Yeast counts were significantly lower on
  DRB at 35C vs. aPDA

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Dichloran-Rose Bengal Agar Yeast and Mold Method
Evaluation

• Conclusion
• Due to overall decreased recovery of yeast and
  mold, and the mold visual observations the
  Dichloran-Rose Bengal Agar Yeast and Mold
  recovery medium is not recommended.

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Case Study 2 Rapid Check Salmonella Test Kit
Evaluation
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Rapid Check Salmonella Test Kit Evaluation

• Objective Determine validity of the Strategic
  Diagnostics Inc. Rapid Check antibody lateral
  flow method for the detection of Salmonella in
  comparison to the BAX PCR test method
• Justification Reduce testing cost, false
  positives rate and technician time

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Rapid Check Salmonella Test Kit Evaluation

• Acceptance Criteria
• Speed shorter time to results vs. PCR?
• Sensitivity greater or equivalent to PCR?
• Specificity greater or equivalent to PCR
• Cost
• Less than or equal to BAX PCR system
• Cost per test
• Versatility food products only, environmental
  samples only, or both?

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Rapid Check Salmonella Test Kit Evaluation

• Organisms and Inoculum Preparation
• A cocktail of 5 Salmonella spp.
• A cocktail of 7 non-Salmonella spp.
• E.coli (2), Citrobacter, Bacillus, Klebsiella,
  Enterobacter (2)
• Individual cultures grown overnight in BHI at 35C
• Salmonella strains pooled, diluted to 100cfu/ml
• Non-Salmonella strains pooled, diluted to 1,000
  cfu/ml

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Rapid Check Salmonella Test Kit Evaluation

• Methods
• Inoculation of samples
• With Salmonella
• With non-Salmonella strains
• With both
• Pre-enrichment of samples
• Traditional medium Lactose for 24 hours
• SDI medium for 5 hours
• Secondary enrichment
• Tetrathionate for 24 hours

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Rapid Check Salmonella Test Kit Evaluation

• Methods (cont)
• BAX PCR analysis
• 3 hour re-growth
• Cell lysis
• 4-8 hour PCR cycle
• SDI lateral flow assay
• Load 150ul onto SDI cartridge
• Develop for 10 minutes

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Rapid Check Salmonella Test Kit Evaluation

• Results
• Sensitivity
• Results were more consistent with SDI when
  recovering at the threshold level (1000 cfu/ml in
  the TT broth)
• Equivalent results with both methods above the
  threshold level
• SDI 5 hour pre-incubation media did not
  consistently support growth above the threshold
  level (acceptance criteria)
• Specificity
• No cross reactivity with non-Salmonella organisms
  with either method

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Rapid Check Salmonella Test Kit Evaluation

• Results Speed

BAX-PCR SDI w/ 5 hour medium SDI
Enrichment 24 hours 5 hours 24 hours
Secondary 18 hours 18 hours 18 hours
Re-growth 3 hours 0 hours 0 hours
Cell lysis 0.5 hours 0 hours 0 hours
Analysis time 8 hours 10 minutes 10 minutes
Total 53.5 hours 23 hours, 10 minutes 42 hours, 10 minutes
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Rapid Check Salmonella Test Kit Evaluation

• Conclusions
• SDI shown to be as sensitive as BAX-PCR
• 5 hour medium not recommended
• No cross reactivity observed with SDI
• SDI gave results sooner than PCR
• PCR has more steps, more prone to technician
  error
• Some degree of subjectivity with SDI
• SDI easier to use 1 step inoculation of 1 single
  cartridge

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Rapid Check Salmonella Test Kit Evaluation

• Conclusions
• SDI can be successfully used for food and
  environmental samples
• No additional equipment needed (heat blocks,
  thermal cycler)
• Cost per test of SDI less than BAX-PCR
• SDI approved for use in place of PCR
• Appropriate for use by labs analyzing a smaller
  number of samples

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Value of Method Validation

• Need to validate method on your intended product
  rule out matrix interference
• Determine minimum regulatory requirements (AOAC,
  AFNOR, etc)
• Determine what is the right method for your lab
  based on volume of testing and number of
  technicians
• Base selection of methodology on need
• Sensitivity
• Specificity
• Speed
• Cost
• Lab space