Principles of Microbiological Testing: Methodological Concepts ...
-
Upload
truonghanh -
Category
Documents
-
view
223 -
download
2
Transcript of Principles of Microbiological Testing: Methodological Concepts ...
1
Principles of Microbiological Testing:Methodological Concepts , Classes and
ConsiderationsRussell S. Flowers
Silliker Group Corp.
Relating Microbiological Testing and Microbiological Criteria toRelating Microbiological Testing and Microbiological Criteria toPublic Health GoalsPublic Health Goals
October 31October 31-- November 1, 2005November 1, 2005
Gallaudet University Kellogg Conference CenterGallaudet University Kellogg Conference Center
Washington, DCWashington, DC
Basic Principles of Microbiological Basic Principles of Microbiological Testing of FoodTesting of Food
Collect sample(s) of the food Collect sample(s) of the food Prepare homogenates Prepare homogenates Analyze homogenatesAnalyze homogenates
Direct MicroscopyDirect MicroscopyDetection/Enumeration Detection/Enumeration
•• Quantitative; Colony Counts, MPNQuantitative; Colony Counts, MPN•• Qualitative; enrichment followed by detection Qualitative; enrichment followed by detection
technique and isolation of target organismtechnique and isolation of target organism•• Evidence of growth may be detected by various Evidence of growth may be detected by various
means; visual, biochemical, immunological, genetic means; visual, biochemical, immunological, genetic
2
Detection TechnologiesDetection TechnologiesEnzyme immunoassayEnzyme immunoassay
ImmunoImmuno--capturecaptureImmunoImmuno--precipitationprecipitation
Nucleic acid hybridizationNucleic acid hybridizationDNA/RNADNA/RNAPCR & other nucleic amplification methodsPCR & other nucleic amplification methods
ElectrochemicalElectrochemicalEnzymatic amplificationEnzymatic amplificationElectrical separationElectrical separationChromatographic separationChromatographic separation
Quantitative Microbial AnalysesQuantitative Microbial AnalysesLower Limit "Rules of Thumb"Lower Limit "Rules of Thumb"
<10 <10 -- 100 cfu/g MPN100 cfu/g MPN>10 >10 -- 100 cfu/g Viable Counts100 cfu/g Viable Counts>10>1033 -- 101044 cfu/g cfu/g DEFTDEFT>10E>10E44 -- 101055 cfu/g ELISA, Flow Cytometry,cfu/g ELISA, Flow Cytometry,
Quantitative PCRQuantitative PCR>10>1055 -- 101066 cfu/g Direct microscopy, cfu/g Direct microscopy,
SpectrophotometrySpectrophotometry
Sensitivity may be increased by methods Sensitivity may be increased by methods through enrichment of multiple samples at through enrichment of multiple samples at the limit of detection (calculated MPN)the limit of detection (calculated MPN)
3
Precision of Colony CountsPrecision of Colony CountsFor Poisson distribution, Variance (s2 ) = Mean
S D xR S D (% ) = 1 0 0 = 1 0 0m e a n x
( )x
SD RSD (%)
± ±400 20.0 5.0 360 - 440200 14.1 7.1 172 - 228100 10.0 10.0 80 - 12050 7.1 14.1 36 - 6425 5.0 20.0 15 - 3510 3.2 31.6 4 - 16
Mean Colony Count
95% Limit
Values
Qualitative DetectionQualitative DetectionEnrichment(s) followed by detection, Enrichment(s) followed by detection, and confirmation (if required)and confirmation (if required)
Confirmation Confirmation •• differential isolation, differential isolation, •• Identification of target organism Identification of target organism
Limit of Detection is dependent on;Limit of Detection is dependent on;Number of samplesNumber of samplesSize of samples Size of samples
4
TwoTwo--class Plans (c = 0): class Plans (c = 0): Probabilities of AcceptanceProbabilities of Acceptance
ICMSF, 1986. Microorganisms in Foods, Sampling for microbiological analysis: Principles and applications, University of Toronto Press, Toronto.
Composition of Lot Number of sample units tested % acceptable % defective 5 10 20 60 100
98 2 .90 .82 .67 .30 .13 95 5 .77 .60 .36 .05 .01 90 10 .59 .35 .12 < < 80 20 .17 .11 .01 70 30 .03 .03 < 50 50 .01 < 40 60 < 30 70
Applications of Microbiological Testing at Applications of Microbiological Testing at Various LevelsVarious Levels
Nationally and InternationallyNationally and InternationallyEpidemiological data; e.g., outbreaks, recalls, etc.Epidemiological data; e.g., outbreaks, recalls, etc.Baseline studiesBaseline studiesInternational Trade (SPS)International Trade (SPS)
Industry specificIndustry specificTrade association studiesTrade association studiesRetail surveysRetail surveys
CompanyCompanyAcross different production facilities and linesAcross different production facilities and linesCustomer/supplier (purchase specifications)Customer/supplier (purchase specifications)
Facility/product specificFacility/product specificHACCP HACCP Prerequisite programs (GMP, GHP)Prerequisite programs (GMP, GHP)
5
Applications of Microbiological Applications of Microbiological Testing in Food Safety ProgramsTesting in Food Safety Programs
Microbiological testing plays an Microbiological testing plays an essential role in HACCP essential role in HACCP
Hazard analysisHazard analysisProcess validationProcess validationMonitoring of critical ingredients Monitoring of critical ingredients and high risk finished productsand high risk finished productsVerification of CCPs and the Verification of CCPs and the overall process overall process
Applications of Microbiological Applications of Microbiological Testing in Food Safety ProgramsTesting in Food Safety Programs
GMP/GHP Programs GMP/GHP Programs Determine potential for post Determine potential for post process contaminationprocess contaminationEstablish adequacy and frequency Establish adequacy and frequency of cleaning and sanitationof cleaning and sanitationDetect difficult areas to clean and Detect difficult areas to clean and sanitizesanitize
6
Applications of Microbiological Applications of Microbiological Testing in Food Safety ProgramsTesting in Food Safety Programs
Compliance testingCompliance testingMandatory regulatory programsMandatory regulatory programsPurchase specificationsPurchase specificationsDocumentation in case of litigationDocumentation in case of litigation
Problem solvingProblem solvingOften microbiological data exists but is Often microbiological data exists but is only being used for acceptance on a given only being used for acceptance on a given unit of production (batch, lot, day)unit of production (batch, lot, day)Trend analysis of this data often identifies Trend analysis of this data often identifies the most likely source of a problem, or pin the most likely source of a problem, or pin points areas for further investigation points areas for further investigation
Microbiological Criteria (Codex)Microbiological Criteria (Codex)
A microbiological criterion defines the acceptability A microbiological criterion defines the acceptability of a product or a food lot, based on the absence or of a product or a food lot, based on the absence or presence, or number of microorganisms including presence, or number of microorganisms including parasites, and/or quantity of theirparasites, and/or quantity of theirtoxins/metabolites, per unit(s) of mass, volume, toxins/metabolites, per unit(s) of mass, volume, area, or lot .area, or lot .
7
the food , and the point in the food the food , and the point in the food chain to which it applies chain to which it applies any actions to be taken when the any actions to be taken when the Criterion is not metCriterion is not met
Microbiological Criteria should Microbiological Criteria should specify (CODEX)specify (CODEX)
Microorganisms and reasons for concernMicroorganisms and reasons for concern
Analytical methods to be usedAnalytical methods to be used
Sampling plan and size of analytical unitsSampling plan and size of analytical units
Microbiological limitsMicrobiological limits
Numbers of units to be in conformity Numbers of units to be in conformity
Microbiological Criteria Components
Establishment and Application- CAC / GL 21 - 1997
8
Uses of Uses of Microbiological Criteria Microbiological Criteria Assess tAssess the safety of foodhe safety of foodVerifVerifyy//validavalidatete procedures in HACCPprocedures in HACCPDemonstrate aDemonstrate adherencedherence to GMP/GHPto GMP/GHPDemonstrate tDemonstrate the utility (suitability) of a food or he utility (suitability) of a food or ingredient for a particular purposeingredient for a particular purposeEstablish tEstablish the keeping quality (shelfhe keeping quality (shelf--life) of certain life) of certain perishable foodsperishable foodsAs a regulatory tool to drive industry As a regulatory tool to drive industry improvementimprovementAs verification that a Performance Objective or As verification that a Performance Objective or FSO has be FSO has be acheivedacheived
• Standard—a mandatory criterion that is part of a law or ordinance.
• Guideline—an advisory criterion issued by a control authority, industry association, or food producer to indicate what might be expected when best practices are applied.
• Specification—Part of a purchasing agreement between a buyer and supplier of a food; such criteria may be mandatory or advisory according to use.
Types of Acceptance Criteria
9
Sampling PlansSampling Plans
Define the probability of Define the probability of detecting a microorganisms or detecting a microorganisms or other hazards in a lotother hazards in a lotNone can ensure the absence None can ensure the absence of a particular hazardof a particular hazardShould be administratively and Should be administratively and economically feasibleeconomically feasible
Types of Microbiological Types of Microbiological Sampling PlansSampling Plans
Attributes plans:
Qualitative analytical results (presence/absence) orquantitative results that have been grouped(e.g. <10 cfu/g, 10 to 100 cfu/g, >100 cfu/g)
Variables plans:
Non-grouped quantitative analytical results
Require distributional assumptions be made
10
IndicatorsIndicatorsShould indicate something :Should indicate something :
ContaminationContaminationSurvivalSurvivalRecontaminationRecontaminationGrowthGrowth
Should be easy to determineShould be easy to determineShould behave as pathogen Should behave as pathogen
(growth, survival) when(growth, survival) when used used instead of testing for pathogeninstead of testing for pathogen
Cannot be relied upon as Cannot be relied upon as "proof" that pathogen of "proof" that pathogen of concern is absent concern is absent
Pathogen not measurablePathogen not measurable
Example : < 1 Example : < 1 SalmonellaSalmonella / 10 kg of / 10 kg of dried eggdried egg--productproduct
Enterobacteriaceae are good indicators of Enterobacteriaceae are good indicators of adequate pasteurisation andadequate pasteurisation andcontrol of recontaminationcontrol of recontamination
11
Salmonella criterion for dried egg productsSalmonella criterion for dried egg productscase 11 : n = 10 c = 0, 25g case 11 : n = 10 c = 0, 25g
samplessamples
lots containing 1 S. per 83 glots containing 1 S. per 83 gwill be rejected with 95% will be rejected with 95%
probabilityprobabilitylots containing < 1 S. per 7.7 kg
will be accepted with 95% probability
A producer would need to test 565 end-productsto verify that he would meet this criterion
Indicators are measurableIndicators are measurable
Example: Absence of EnterobacteriaceaeExample: Absence of Enterobacteriaceaein 1 g of eggin 1 g of egg--productproduct
a) case 7 : n = 5, c = 2 * (use : biscuit)a) case 7 : n = 5, c = 2 * (use : biscuit)b) case 8 : n = 5, c = 1 (dried egg)b) case 8 : n = 5, c = 1 (dried egg)c) case 9 : n = 10, c = 1 (use : tiramisu)c) case 9 : n = 10, c = 1 (use : tiramisu)
* if adequate heating is assured, no testing is necessary
12
Utility and Indicator TestingUtility and Indicator Testing
ExamplesExamplesAerobic plate countAerobic plate countColiforms or Enterobacteriaceae (EB)Coliforms or Enterobacteriaceae (EB)Escherichia coli Escherichia coli Yeast and mold countYeast and mold count
Species or groups or microorganisms whose presence may indicate the potential for the presence of pathogens or the extent to which good manufacturing and hygiene practiceswere adhered to during manufacture
Salmonella Incidence in Relationship Salmonella Incidence in Relationship to to E.coliE.coli Most Probable Number (MPN) Most Probable Number (MPN)
in Raw Prein Raw Pre--formed Meat Pattiesformed Meat PattiesEE.. ccoollii MMPPNN//gg SSaammpplleess
WWiitthhiinn MMPPNNRRaannggee
SSaammpplleess PPoossiittiivveeFFoorr SSaallmmoonneellllaa
WWiitthhiinn MMPPNN RRaannggee
%% PPoossiittiivveeWWiitthhiinn MMPPNN
RRaannggee
<<33 227700 22 00..7733--55 440066 2200 44..99
5511--110000 5544 33 55..66110011--224400 9966 44 44..11
224411--11,,110000 6655 33 44..6611,,110011--1111,,000000 5566 99 1166..11
>>1111,,000000 2255 55 2200..00
13
No indicators availableNo indicators availableExample : <1 C. botulinum in 1000 ton of Example : <1 C. botulinum in 1000 ton of
lowlow--acid canned meat productacid canned meat product
Reliance on Reliance on Process Criteria (Process Criteria (botbot cook)cook)
and GMP and GMP
No Microbiological Criteria
To Test or Not to TestTo Test or Not to Test ??Severity of the hazard(s)Severity of the hazard(s)New information linking the food to illnessNew information linking the food to illnessWhether the food isWhether the food is
Commonly involved in diseaseCommonly involved in diseasePrimarily destined for a sensitive populationPrimarily destined for a sensitive populationFrom a country with endemic disease of importance to From a country with endemic disease of importance to
food safetyfood safetyHistory of consistency and compliance History of consistency and compliance Distribution of contaminant(s)Distribution of contaminant(s)
Homogenous, heterogeneous, stratifiedHomogenous, heterogeneous, stratifiedAbility to sampleAbility to sample
Sufficient numbersSufficient numbersRandom samplingRandom sampling
14
Depending on the application the Depending on the application the requirement for confidence in the data requirement for confidence in the data
will be different will be different Method performance must be appropriate for the Method performance must be appropriate for the ““purpose intendedpurpose intended”” e.g.e.g.
Industrial process monitoring.Industrial process monitoring.Regulatory screening of foods.Regulatory screening of foods.Industrial process verification.Industrial process verification.Regulatory compliance with criteria.Regulatory compliance with criteria.Forensic investigation.Forensic investigation.
Greater Accuracy &Precisionrequired
AOAC Method Validations Protocols AOAC Method Validations Protocols Suggested for Various ApplicationsSuggested for Various Applications
Process & Product Process & Product monitoringmonitoring
Raw materialsRaw materialsInIn--process testingprocess testing
Process Validation & Process Validation & VerificationVerification
Process validationProcess validationHACCP verificationHACCP verification
Regulatory TestingRegulatory Testing
Single Lab (SLV)Single Lab (SLV)1010--20 samples per 20 samples per matrix/level matrix/level
MultiMulti--Lab Validation Lab Validation (MLV)(MLV)
Similar to SLV but Similar to SLV but with multiple labs with multiple labs and more samplesand more samples
Harmonized Harmonized Collaborative Collaborative Validation (HCV)Validation (HCV)
Validation protocolsValidation protocolsApplicationsApplications
15
Method Validation CriteriaMethod Validation CriteriaRuggednessRuggednessInclusivityInclusivityExclusivityExclusivityMatricesMatricesComparison to Comparison to standardstandard
CorrelationCorrelationAgreementAgreement
PerformancePerformanceFalse neg.False neg.False pos.False pos.RepeatabilityRepeatabilityReproducibilityReproducibility
Validation Level # Labs
# Rep Samples/
Lab
Total # Rep
SamplesSensitivity
(% +ve) 95% CISLV 1 20 20 50% 27.6 - 72.4%MLV 2 20 40 50% 34.2 - 65.8%HCV 10 6 60 50% 37.1 - 62.9%
Comparison of Confidence Intervals for 3 Levels of Comparison of Confidence Intervals for 3 Levels of Validation for a Qualitative Method, assuming the Validation for a Qualitative Method, assuming the same performance characteristics in each same performance characteristics in each
Note: CI of HCV would normally be greater than SLV or MLV due to inter-laboratory variance (repeatability), not included in above analysis.
16
Factors affecting the Confidence Factors affecting the Confidence (Uncertainty) in Microbiological Data (Uncertainty) in Microbiological Data
Sampling plan, method and sizeSampling plan, method and sizeMethod performance characteristics Method performance characteristics
SensitivitySensitivityRepeatabilityRepeatabilityRuggednessRuggedness
Laboratory performance Laboratory performance Error ratesError ratesReproducibilityReproducibility
Uncertainty in Microbiological Uncertainty in Microbiological AnalysesAnalyses
Types of ErrorTypes of ErrorInherent Inherent -- part of the procedure or method part of the procedure or method Performance Performance -- error associated with laboratory and/or error associated with laboratory and/or analystanalyst
Sources of ErrorSources of ErrorDistribution of microorganisms in matrix Distribution of microorganisms in matrix State of the microorganismsState of the microorganismsSampling planSampling planSampling handlingSampling handlingAnalysisAnalysisReportingReporting
17
Distribution of microorganismsDistribution of microorganisms
HomogenousHomogenousHeterogeneousHeterogeneousRandomRandomNonNon--randomrandomStratified Stratified
Example AIncidence = 6 pos. per 100 x 1 lb.
samplesLevel = 6 cells per 100 lbs.
Example BIncidence = 6 pos. per 100 x 1 lb.
samplesLevel = 600 cells per 100 lbs.
ca. 100 cells /clump
Incidence vs. LevelIncidence vs. Level
18
Distribution of MicroorganismsDistribution of MicroorganismsHom ogeneous d is trib u tion
A
B
C
Non-hom ogeneous d is tribu tion
Sampling plan Sampling plan (others will cover in detail)(others will cover in detail)
Sample selectionSample selectionrandomrandomstratifiedstratifiedspecified frequencyspecified frequency
Sampling methodSampling methodNumber of samplesNumber of samplesSize of samples (analytical unit)Size of samples (analytical unit)
Note: Statistical considerations of sampling plans generally assume that the method ection is error free
19
AnalysisAnalysisMethod Selection and PerformanceMethod Selection and Performance
inherent accuracy and precision of the inherent accuracy and precision of the method assuming all procedures are done method assuming all procedures are done perfectly perfectly
Analyst PerformanceAnalyst Performancetrainingtrainingproficiencyproficiency
Laboratory PerformanceLaboratory Performancequality systemsquality systemsproficiencyproficiency
Method validation and proficiency Method validation and proficiency testing are essential components of a testing are essential components of a
laboratorylaboratory’’s quality systems quality systemand are necessary to determine and are necessary to determine
UncertaintyUncertainty of a microbiological data of a microbiological data resultresult
20
Method ValidationMethod Validation
WhyWhyIt sets the performance parameters It sets the performance parameters of the method and demonstrates of the method and demonstrates laboratory competencylaboratory competency
WhenWhenUpon introduction of a new method, Upon introduction of a new method, the revision of a method, or addition the revision of a method, or addition of a new test matrixof a new test matrix
Proficiency TestingProficiency Testing
Laboratory proficiency measures a Laboratory proficiency measures a laboratorylaboratory’’s ability to generate data that is s ability to generate data that is comparable to other laboratories for comparable to other laboratories for identical samples.identical samples.
Analyst proficiency measures a analystAnalyst proficiency measures a analyst’’s s ability to generate data that is comparable ability to generate data that is comparable to other analysts for identical samples.to other analysts for identical samples.
21
There are 240 private and hundreds more in-plant and corporate food testing labs in the U.S. Approximately 100 labs participate in the AOAC Split Sample Program. This program allows them to benchmark their proficiency against other labs. The Silliker lab data represents 22% of the test volume, and shows significantly better accuracy than the rest of the population.
0
0.5
1
1.5
2
2.5
3
3.5
% Errors
Salmonella Listeria E. Coli 0157:H7 Total
SillikerOthers
Pathogen Testing ErrorsPathogen Testing ErrorsAOAC Split Sample Program AOAC Split Sample Program (Jan 01 (Jan 01 -- Oct 03)Oct 03)
Quality RequirementsQuality RequirementsSummary and ConclusionsSummary and Conclusions
Microbiology is not an exact science Microbiology is not an exact science Many sources of variance not related to mistakesMany sources of variance not related to mistakesMany manual steps subject to human errorMany manual steps subject to human errorInterpretation of data must consider this variationInterpretation of data must consider this variationProduct specifications and process guidelines should Product specifications and process guidelines should be based on sufficient data to know the coefficient of be based on sufficient data to know the coefficient of variationvariationUncertainty of the analytical result must be considered Uncertainty of the analytical result must be considered when establishing microbiological criteria, including when establishing microbiological criteria, including variance associated with the sampling plan, method of variance associated with the sampling plan, method of analysis and laboratory performance.analysis and laboratory performance.
22
Summary and ConclusionsSummary and ConclusionsLimitations of Microbiological TestingLimitations of Microbiological Testing
Often it is not practical to test a sufficient Often it is not practical to test a sufficient number of samples for confidence in lot number of samples for confidence in lot acceptanceacceptanceNonNon--random sampling may cause random sampling may cause incorrect conclusions to be drawnincorrect conclusions to be drawnFinished product testing determines Finished product testing determines outcomes, not causes or controlsoutcomes, not causes or controlsNo feasible sampling plan can ensure No feasible sampling plan can ensure absence of a pathogenabsence of a pathogen
Summary and ConclusionsSummary and ConclusionsUses of Microbiological TestingUses of Microbiological Testing
Establish baseline dataEstablish baseline dataControl ingredientsControl ingredientsIdentify highly contaminated lotsIdentify highly contaminated lotsAssessing control of the environmentAssessing control of the environmentVerify compliance of PO and FSO (within Verify compliance of PO and FSO (within limits of sampling and testing)limits of sampling and testing)Verify control of within HACCP/GHP systemsVerify control of within HACCP/GHP systemsValidate a HACCP/GHP system provides a Validate a HACCP/GHP system provides a desired level of controldesired level of control
23
Summary and ConclusionsSummary and ConclusionsIf FSOs or POs are within a range allowing If FSOs or POs are within a range allowing practical application of sampling plans and practical application of sampling plans and testing protocols, then testing protocols, then
Microbiological criteria may be useful to verify Microbiological criteria may be useful to verify acceptability of the lot relative to PO and FSO acceptability of the lot relative to PO and FSO
Across Lot/Batch testing may be useful to Across Lot/Batch testing may be useful to verify that a food safety system or process is verify that a food safety system or process is continuing to function as intended continuing to function as intended