Impact, validation and regulatory implication of rapid methods Peter Feng, Research Microbiologist...

Impact, validation and Impact, validation and regulatory implication of regulatory implication of

rapid methods rapid methods

Peter Feng, Research Microbiologist

FDA Center for Food Safety and Applied Nutrition (CFSAN)

Wednesday, May 28, 2008

Impact, validation and regulatory Impact, validation and regulatory implication of rapid methods implication of rapid methods

Peter Feng, Ph.D.Peter Feng, Ph.D.

• Food safety regulations Food safety regulations • impact of food safety laws vs impact of food safety laws vs method developmentmethod development

• Impact of changesImpact of changes• society/lifestyle - new problems, society/lifestyle - new problems, regulations, methodsregulations, methods

• Problems in Food AnalysisProblems in Food Analysis• Rapid Methods - Rapid Methods - definitions and definitions and originorigin• Next generation technologiesNext generation technologies• Advantages and limitationsAdvantages and limitations• ValidationValidation• Impact on regulationsImpact on regulations

Food Safety Law – impact on Food Safety Law – impact on method developmentmethod development

• 1785 - 1785 - State of MA: 1st law on food State of MA: 1st law on food adulterationadulteration

• 1787 – 1787 – U.S. Constitution – Federal U.S. Constitution – Federal

GovernmentGovernment • Society - Agricultural to industrial (mid to Society - Agricultural to industrial (mid to

late 1800’s)late 1800’s)

• 1902 - 1902 - “poison squad” Harvey Wiley, “poison squad” Harvey Wiley, USDAUSDA

• 1906 - 1906 - Food and Drug ActFood and Drug Act, USDA, USDAprohibit manufacture/interstate commerce of prohibit manufacture/interstate commerce of

adulterated andadulterated andmisbranded food, drinks & drugs misbranded food, drinks & drugs (burden of (burden of

proof - Fed Government)proof - Fed Government)

• 1938: 1938: Federal Food, Drug & Cosmetic Federal Food, Drug & Cosmetic ActAct • safety of cosmetic and medical devicessafety of cosmetic and medical devices• tolerance of unavoidable poison in foodtolerance of unavoidable poison in food• standard of product identity, quality and fillstandard of product identity, quality and fill• factory inspection authorityfactory inspection authority• producer must show product safety producer must show product safety

(burden of proof – industry)(burden of proof – industry)

Impact of Changes Impact of Changes

• Population – Population – advances in medicineadvances in medicine• Longevity - > immunosusceptible Longevity - > immunosusceptible

populationpopulation

• Lifestyle – Lifestyle – • Healthier diet – fruits and vegetables; Healthier diet – fruits and vegetables;

> imports> imports• Dual income family – changes in Dual income family – changes in

eating habits. > restaurants, fast food eating habits. > restaurants, fast food & carry out. & carry out.

• ConsequencesConsequences• Convenience industry – RTE; ie: Convenience industry – RTE; ie:

bagged produce.bagged produce.• Mass production & Broad Mass production & Broad

distribution – large outbreaksdistribution – large outbreaks• Bottomline: Bottomline: new food safety new food safety

problems, regs - new methodsproblems, regs - new methods

Microbiological Analysis of Microbiological Analysis of FoodsFoods

• Pathogens – Pathogens – • Presence/absencePresence/absence

• Indicators – Indicators – TVC, coliforms, E. coli,TVC, coliforms, E. coli, enterobacteriaceaeenterobacteriaceae• EnumerationEnumeration - - Direct:Direct: plate counts, membrane plate counts, membrane

filtration (MF)filtration (MF) - - Indirect:Indirect: Most Probably Number Most Probably Number

(MPN); enzymatic activity on specific (MPN); enzymatic activity on specific substrate to estimate cell density substrate to estimate cell density

Pathogen & Toxins in Foods – Pathogen & Toxins in Foods – presence/absencepresence/absence

Solutions Solutions Problems Problems BacteriaBacteria

ToxinsToxins• Complex MatricesComplex Matrices enrichment (dil.)enrichment (dil.)

extractionextraction• Normal floraNormal flora Sel. enrichmentSel. enrichment

little effectlittle effect• Low target levelsLow target levels Post-enrichmentPost-enrichment

concentrationconcentration• Processing (injury)Processing (injury) Pre-enrichmentPre-enrichment

renaturationrenaturation

Indicators in Foods - Indicators in Foods - enumerationenumerationProblems Problems

SolutionsSolutions • Complex MatricesComplex Matrices pre-pre-filtration, dilutionsfiltration, dilutions• High Cell densityHigh Cell density serial serial dilutiondilution

Conventional microbiological Conventional microbiological analysis of foodsanalysis of foods

FoFoodod

++

BuffBufferer

IncuIncubatebate

Pre-enrichmentPre-enrichment

resuscitationresuscitation

Sub cultureSub culture

Selective Selective mediamedia

Selective Selective enrichmentenrichment

selectionselection

growth growth mediamedia

Non-Non-selective selective

mediamedia

IncubateIncubate

Sub cultureSub culture

Post-enrichmentPost-enrichment

> cell #> cell #Differential/Differential/

Selective agarSelective agar mediummedium

IncubateIncubate

PresumPresumptive ptive identificidentificationation

BiocheBiochemical mical

testingtesting

IdentificationIdentification

SerotypingSerotypingand/orand/orPFGEPFGE

CharacterizationCharacterization

plateplate

isolaisolatestes

Pick coloniesPick colonies

pathogenspathogenstoxinstoxins

extractextractConc.Conc.

TestTest

indicatorsindicatorsSerial dilutionsSerial dilutions

EnumerationEnumerationTVC, MPN, MFTVC, MPN, MF

Commercial kits (rapid methods) – Commercial kits (rapid methods) – food borne pathogensfood borne pathogens

TargetTarget AbAb AbAb DNADNAAbAb DNADNA

CampylobacterCampylobacter 00 33 33 1717 4 4

C. botulinum C. botulinum toxintoxin 0 0 11 00 5 5 1 1

E. sakazakiiE. sakazakii 00 00 00 1 1 1 1

EHEC EHEC (non-O157)(non-O157) 0 0 0 0 00 16 16 0 0

O157O157 00 1515 11 4141 9 9

Shiga toxinShiga toxin 0 0 33 00 10 10 0 0

Listeria & LmListeria & Lm 11 88 33 22 22 19 19

SalmonellaSalmonella 44 2121 33 4242 1010

S. enteritidisS. enteritidis 00 00 00 4 4 0 0

S. aureusS. aureus 00 33 11 9 9 4 4

enterotoxinenterotoxin 22 55 00 1111 0 0

19981998 2006200619901990

““Rapid”Rapid” MethodsMethods

• DefinitionDefinition• ““Rapid”?Rapid”?

• Origin? Origin?

•““..determines the presence of ..determines the presence of dangerous strain of E. coli in 5 to 10 dangerous strain of E. coli in 5 to 10 min instead of 48 hr…” min instead of 48 hr…” WA Post WA Post (1997)(1997)

19401940 198019801960196019501950 19701970 19901990 20002000

pre-APIpre-API ““rapid method”rapid method”

biotechnologybiotechnology

History of History of BiotechnBiotechnologyology

DNADNA

Genetic EngineeringGenetic Engineering

Monoclonal AntibodyMonoclonal Antibody

Tissue Culture TechnologyTissue Culture Technology

Fermentation TechnologyFermentation Technology

Human & animal health care productsHuman & animal health care products

Custom-design of raw agricultural Custom-design of raw agricultural

commoditiescommodities

Engineered enzymes & proteins Biosensor Engineered enzymes & proteins Biosensor

applicationsapplications

Natural ingredients & chemicals by Natural ingredients & chemicals by

fermentation or cell culturefermentation or cell culture

DNA probe & monoclonal antibody-based DNA probe & monoclonal antibody-based

detection systemsdetection systems

Bioreactor design, down-stream processing Bioreactor design, down-stream processing

& product purification& product purification

FundameFundamental ntal

InformatiInformationon

1950’s1950’s

1960’s1960’s

1970’s1970’s

1980’s1980’s

1990’s 1990’s and and

beyonbeyondd

Basic Basic Technology Technology

DevelopmentDevelopment

FutureFutureApplicaApplicationstions

Harlander (1989)Harlander (1989)

Delphi Forecast on Rapid Delphi Forecast on Rapid Methods Methods (1981)(1981)

Microbial enumeration:Microbial enumeration:• automated colony automated colony

counting counting (1991)(1991)• by microbial metabolites by microbial metabolites

(2001)(2001)• by microbial activity by microbial activity

(2001)(2001)

Microbial identification:Microbial identification:• miniaturized miniaturized

biochemical biochemical (1986)(1986)

• selective selective media/automation media/automation (1997)(1997)

• specific specific substrates/metabolites substrates/metabolites (1996)(1996)

Adapted from Gutteridge & Arnott (1989)Adapted from Gutteridge & Arnott (1989)

Rapid Methods - Rapid Methods - Foodborne PathogensFoodborne Pathogens

1. Miniaturized Biochemical 1. Miniaturized Biochemical IdentificationIdentification(API 20E, MicroID, Crystal, RapidID (API 20E, MicroID, Crystal, RapidID

32E, etc)32E, etc)• pure culturespure cultures; incubations ; incubations

2. Modification - Conventional 2. Modification - Conventional methodsmethods(ATP & Chromogenic, fluorogenic (ATP & Chromogenic, fluorogenic

substrates, etc.)substrates, etc.)• ATP – fast; total counts only – ATP – fast; total counts only – no IDno ID• substrates – incubations & substrates – incubations &

presumptive data, presumptive data, costcost

3. Automated systems3. Automated systems (various technol(various technol: eg. biochemical, FA, eg. biochemical, FA,

C oxidation)C oxidation)• identification – identification – pure culturepure culture• detection – few manipulation; detection – few manipulation; need need

enrichmentenrichment

Rapid Methods – Food Rapid Methods – Food borne Pathogensborne Pathogens

4. Antibody-based Assays4. Antibody-based Assays• Latex Agglutination:Latex Agglutination: low sensitivity low sensitivity

(log 7); (log 7); pure culturepure culture• ELISA:ELISA: manipulations: manipulations: need need

enrichmentenrichment. . • IMS:IMS: = = selective selective enrichmentenrichment; binding ; binding

efficiency?; not all foods; culture efficiency?; not all foods; culture not pure; not pure; notnot stand alonestand alone..

• Immunoprecipitation:Immunoprecipitation: fast (min), but fast (min), but need enrichmentneed enrichment

5. Nucleic acid-based Assays5. Nucleic acid-based Assays• DNA probes:DNA probes: manipulations; manipulations; need need

enrichmentenrichment• Phage:Phage: luxlux or or inaina; few on market; ; few on market;

need enrichmentneed enrichment • PCR:PCR: inhibitors in foods; inhibitors in foods; need need

enrichmentenrichment

““Ideal” Ideal” Rapid Rapid

MethodMethodss

ConventiConventional onal

MethodsMethods

-- antibody assaysantibody assays -- DNA assaysDNA assays -- Automated assaysAutomated assays -- Miniaturized Miniaturized biochemical IDbiochemical ID

00 1 1 dayday

2 2 daysdays

existing rapid methodsexisting rapid methods

Food Analysis - timelineFood Analysis - timeline

<< 8 hr 8 hr

3 days3 days

Enrichment/Enrichment/isolationisolation

confirmationconfirmation

key problemskey problems in food testing – in food testing – food complexity; speedfood complexity; speed

History of History of BiotechnolBiotechnology.2ogy.2

DDNNAA

Genetic EngineeringGenetic EngineeringMonoclonal AntibodyMonoclonal Antibody

Fermentation & Tissue Culture Fermentation & Tissue Culture Technology, PCRTechnology, PCR

Health care productsHealth care products

Engineered enzymes & proteinsEngineered enzymes & proteins

DNA probe & monoclonal antibody DNA probe & monoclonal antibody

assaysassays

Custom-designed agricultural Custom-designed agricultural

commoditiescommodities

bioreactors, down-stream processing & bioreactors, down-stream processing &

purificationpurification

ingredients & chemicals by ingredients & chemicals by

fermentation or cell culturefermentation or cell culture

Basic Basic informationinformation

1950’s & 1950’s & 60’s60’s

Basic Basic Technology Technology DevelopmeDevelopme

ntnt1970’s & 1970’s &

80’s80’s

ApplicatioApplicationsns

1990’s1990’s

Modified fromModified from Feng (2001)Feng (2001)

Tech. Dev.Tech. Dev.2000’s2000’s

Automated sequencing, bioinfomatics (BLAST), microfluidics,Automated sequencing, bioinfomatics (BLAST), microfluidics,MALDI-TOF, nanotechnology,MALDI-TOF, nanotechnology,

rt-time PCR, DNA and phenotypic microarrays, biosensors. rt-time PCR, DNA and phenotypic microarrays, biosensors.

“…“…7 technologies with 7 technologies with greatest impact on life science greatest impact on life science

research…”research…” (Scientist 19:6, 2005)(Scientist 19:6, 2005)

11. . Automated DNA sequencingAutomated DNA sequencing2. Basic Local Alignment Search 2. Basic Local Alignment Search

Tool (BLAST)Tool (BLAST)3. DNA microarray (DNA Chip)3. DNA microarray (DNA Chip)4. Yeast two-hybrid assay – 4. Yeast two-hybrid assay – novel novel

protein-protein interactionsprotein-protein interactions

5. MALDI-TOF - 5. MALDI-TOF - matrix-assisted laser matrix-assisted laser desorption ionization-time of flightdesorption ionization-time of flight

6. Microfluidics6. Microfluidics7. Optical trap/tweezers – 7. Optical trap/tweezers – use of laser to use of laser to

move cells, organellemove cells, organelle, , atoms without cell atoms without cell disruptiondisruption

Next Generation Next Generation TechnologiesTechnologies

• DNA Microarrays (DNA Chips)DNA Microarrays (DNA Chips)• Simultaneous detection/characterization of Simultaneous detection/characterization of

multiple genotypemultiple genotype• Capable of 400,000 tests per chipCapable of 400,000 tests per chip• Ie: MWG; Panorama – K-12 genomic array (> Ie: MWG; Panorama – K-12 genomic array (>

4000 ORF)4000 ORF)• Phenotypic Microarray (phenotypes)Phenotypic Microarray (phenotypes)

• Simultaneous detection/characterization of Simultaneous detection/characterization of multiple phenotype multiple phenotype

• Ie: Omnilog – Phenotype microarray (2000 Ie: Omnilog – Phenotype microarray (2000 phenotypic growth curves)phenotypic growth curves)

• real-time PCR real-time PCR • Rapid amplification & real-time results Rapid amplification & real-time results

(SYBRgreen, Taqman, FRET hybridization (SYBRgreen, Taqman, FRET hybridization probes, molecular beacons)probes, molecular beacons)

Ie: LightCycler & iQ-Check – Ie: LightCycler & iQ-Check – SalmonellaSalmonella and and L. monocytogenesL. monocytogenes

• Biosensors – Biosensors – • Biological component (antibodies, ligands, Biological component (antibodies, ligands,

etc) – specificity. etc) – specificity. • Physical component (fluorescence, light, Physical component (fluorescence, light,

electromagnetic wave, etc) – detection. eg: electromagnetic wave, etc) – detection. eg: flow cytometry, SPR, etc. flow cytometry, SPR, etc.

• Rapid detection in minutes. in-line Rapid detection in minutes. in-line monitoring?monitoring?

rt-PCR and biosensor assays for food borne pathogensrt-PCR and biosensor assays for food borne pathogens

PathogenPathogen rt-PCRrt-PCR biosensorsbiosensors

B. cereusB. cereus 11 00CampylobacterCampylobacter 22 33C. bot./perf.C. bot./perf. 22 00E. coli O157:H7E. coli O157:H7 66 55E. sakazakiiE. sakazakii 11 00Listeria spp.Listeria spp. 44 44L. monocytogenesL. monocytogenes 66 22SalmonellaSalmonella 66 44ShigellaShigella 22 00S. aureusS. aureus 22 00

Logistics to considerLogistics to consider

• Practicality – Practicality – • Technology - fascination or Technology - fascination or

publicity? publicity? • Many “solutions looking for Many “solutions looking for

problems”problems”• Applicability – Applicability – ample formats to meet ample formats to meet

all needsall needs• User’s needs – characterization or User’s needs – characterization or

screeningscreening• Screening - amount of Screening - amount of

data/discrimination vs cost?data/discrimination vs cost?• volume of testing - volume of testing - instrument instrument

capacity; disposible; costcapacity; disposible; cost• Advantages and LimitationsAdvantages and Limitations

• Fast – DNA chips, biosensor, rt-PCR Fast – DNA chips, biosensor, rt-PCR - < 1hr assay time- < 1hr assay time

• Food matrix interference? Food matrix interference? • Validation?Validation?

Rapid Methods – Pros and Rapid Methods – Pros and Cons Cons

Reliance on cultural Reliance on cultural enrichment enrichment • Assay in min, but enrichment takes Assay in min, but enrichment takes

daysdays• limits speed but offers other benefitslimits speed but offers other benefits

Screening applicationScreening application• (-) accepted; (+) Is presumptive, need to (-) accepted; (+) Is presumptive, need to

be confirmedbe confirmed Food dependant performance Food dependant performance

and interferenceand interference• Need for comparative analysisNeed for comparative analysis

DNA assays DNA assays • not detect gene expressionnot detect gene expression• Can’t detect pre-formed toxinsCan’t detect pre-formed toxins

Single target formatSingle target format• Ideal for screening applicationIdeal for screening application

Over abundance of assaysOver abundance of assays• Lack of comparative evaluation or Lack of comparative evaluation or

validationvalidation

Sample Analysis Time of Sample Analysis Time of Various AssaysVarious Assays

ELISA:ELISA: 45 45 minmin - 2 - 2 hhenrichment:enrichment: 16 - 72 16 - 72 hh

Immunoprecipitation:Immunoprecipitation: 10 10 minminenrichment:enrichment: 18 - 24 18 - 24 hh

DNA Probes:DNA Probes: 2 - 2.5 2 - 2.5 hhenrichment:enrichment: 48 48 hh

PCR:PCR: 1 - 4 1 - 4 hhenrichment:enrichment: 16 - 72 16 - 72 hh

Bacteriophage:Bacteriophage: 3 3 hh enrichment:enrichment: 22 22 hh




Screening applicationScreening application• (-) accepted; (+) is presumptive, need to (-) accepted; (+) is presumptive, need to


and interferenceand interference• Varying sensitivitiesVarying sensitivities• Need for comparative analysisNeed for comparative analysis



Over abundance of assaysOver abundance of assays• Lack of comparative evaluation or Lack of comparative evaluation or


Detection Sensitivity of Detection Sensitivity of Assays Assays

AssayAssay BacteriaBacteria (cfu/g)(cfu/g) Toxins Toxins (ng/ml)(ng/ml)

Adenosine triphosphate (ATP)Adenosine triphosphate (ATP) 10104 4 NANALatex AgglutinationLatex Agglutination 10107 7 NANAReverse Passive LAReverse Passive LA NANA 0.5 - 0.5 -

4.0 4.0 ELISAELISA 10104 4 - 10- 1055

0.01 - 10.01 - 1Immunomagnetic SeparationImmunomagnetic Separation < 10< 1033 NANAImmunodiffusion (1-2 Test)Immunodiffusion (1-2 Test) 10105 5 - 10- 1066

NANAImmunoprecipitationImmunoprecipitation 10104 4 - 10- 1055 NANA

for stxfor stx 10107 7 - 10- 1088 6 - 6 - 20 20

DNA ProbeDNA Probe 10104 4 - 10- 1066 NANA

phage (INA or bioluminescence)phage (INA or bioluminescence) 25 - 100 25 - 100 NANA

PCRPCR 10101 1 - 10- 1022 NANA

BiosensorBiosensor 10101 1 - 10- 1022 NANA

NA - information not available or applicableNA - information not available or applicable




Screening applicationScreening application• (-) accepted; (+) Is presumptive, need to (-) accepted; (+) Is presumptive, need to


and interferenceand interference• Varying sensitivitiesVarying sensitivities• Need for comparative analysisNeed for comparative analysis



Over abundance of assaysOver abundance of assays• Lack of comparative evaluationLack of comparative evaluation or or


Rapid Methods in food Testing – Rapid Methods in food Testing – validations claimsvalidations claims

• “…“….our assay is approved by the FDA .our assay is approved by the FDA for testing X pathogen in foods…..”for testing X pathogen in foods…..”

• ““..FDA-approved method X quickly ..FDA-approved method X quickly determines the disease causing toxins determines the disease causing toxins produced by EHEC in foods, especially produced by EHEC in foods, especially ground beef and milk.”ground beef and milk.”

• ““.. Company X received FDA clearance .. Company X received FDA clearance to market a rapid test for to market a rapid test for E. coliE. coli O157:H7, O157:H7, which is often transmitted via ground which is often transmitted via ground beef”beef”

• “…“…the FDA is currently using this assay the FDA is currently using this assay in their labs.”in their labs.”

Test Kit Validations in the Test Kit Validations in the U.S.U.S.

• Clinical: Clinical: FDAFDA (Center for Devices and (Center for Devices and Radiological Health)Radiological Health)

• Food: Food:

• U.S. USDA U.S. USDA Microbiology Microbiology Laboratory GuidebookLaboratory Guidebook Food Safety Inspection ServiceFood Safety Inspection Service (internal validation)(internal validation)

• U.S. FDA U.S. FDA Bacteriological Analytical Bacteriological Analytical ManualManualCenter for Food Safety & Applied Center for Food Safety & Applied NutritionNutrition (internal validation)(internal validation)

• AOAC Int. AOAC Int. - - Official Official Methods Methods (Collaborative Study)(Collaborative Study)

- Peer-Verified Methods - Peer-Verified Methods (non-(non-propriety) propriety)

AOAC-RI -AOAC-RI - Test Kit Performance Test Kit Performance Tested Methods Tested Methods (proprietary) (proprietary) (AOAC Research Institute)(AOAC Research Institute)

AOAC: Official vs AOAC: Official vs Performance Tested Performance Tested

• Administrator:Administrator: AOACAOAC

• Application fee: Application fee: 35K35K• Reviewers:Reviewers: General General

Referee, Methods Referee, Methods Committee (volunteers)Committee (volunteers)

• Studies:Studies: in-house, in-house, precollab, collabprecollab, collab

• Lab Requirements:Lab Requirements: 15 15 (academia, industry, (academia, industry, government)government)

• Sample Sample Requirements:Requirements: 5 5 typestypes

• Seed Levels:Seed Levels: uninoculated, lowuninoculated, low high (5 high (5 replicates each).replicates each).

• Status:Status: Official Official Standard MethodStandard Method• Official 1st action - 2 Official 1st action - 2

yrs yrs • Official Final action - Official Final action -

indefiniteindefinite

• Administrator:Administrator: AOAC RI AOAC RI

• Application fee:Application fee: 21K21K• Reviewers:Reviewers: 2 Experts 2 Experts

selected by AOAC RI selected by AOAC RI (retainer)(retainer)

• Studies:Studies: proposed proposed protocol protocol

• Lab Requirements:Lab Requirements: 2 (Sponsor & RI 2 (Sponsor & RI contractor) contractor)

• Sample Sample Requirements:Requirements: nonenone

• Seed Levels: Seed Levels: specified by study specified by study protocolprotocol

• Status:Status: Not officialNot official• Validated status - Validated status -

renewed annually + renewed annually + 10K10K

OfficialOfficial -- AOAC Official Method AOAC Official Method 996.09996.09

Research Institute -Research Institute -PERFORMANCE TESTEDPERFORMANCE TESTED

AOAAOACCRESEARCH INSTITUTERESEARCH INSTITUTELICENSE NUMBER 930706LICENSE NUMBER 930706

AOAC Method DesignationsAOAC Method Designations

AOAC – eCAM Validation AOAC – eCAM Validation Program Program

• REG – Regulatory MethodsREG – Regulatory Methods• Specified in regs by National & Specified in regs by National &

International regulatory agenciesInternational regulatory agencies• HCV – Harmonized Collaboratively HCV – Harmonized Collaboratively

Validated MethodsValidated Methods• Meets ISO and AOAC standards – full Meets ISO and AOAC standards – full

collaborativecollaborative• MLV – Multi-Laboratory Validated MLV – Multi-Laboratory Validated

MethodsMethods• Same as above, but only 2-8 lab Same as above, but only 2-8 lab

collaborativecollaborative• SLV – Single Laboratory Validated SLV – Single Laboratory Validated

MethodsMethods• Single lab validationSingle lab validation

• DNV – Developmental Non-DNV – Developmental Non-Validated MethodsValidated Methods• Provide information only on Provide information only on

potentially useful methods; not potentially useful methods; not optimized or validatedoptimized or validated

Validation Programs - Key Validation Programs - Key PointsPoints

• FDA (CDRH) - reviews and FDA (CDRH) - reviews and approves assays used in clinical approves assays used in clinical testing.testing.

• FDA (CFSAN) - does FDA (CFSAN) - does notnot review or review or approve assays used for approve assays used for pathogens & toxin testing in foodspathogens & toxin testing in foods

• Performance Tested Methods by Performance Tested Methods by AOAC Research InstituteAOAC Research Institute - - not not officialofficial

• Collaborative Study Methods of Collaborative Study Methods of AOAC InternationalAOAC International - - Official Official (Standard) Methods (Standard) Methods (must be performed (must be performed as specified)as specified)

Rapid Methods in food Rapid Methods in food Testing – claimsTesting – claims

• ““..determines the presence of dangerous ..determines the presence of dangerous strain of E. coli in 5 to 10 min instead of strain of E. coli in 5 to 10 min instead of 48 hr…” 48 hr…” NewspaperNewspaper

• “…“….our assay is approved by the FDA for .our assay is approved by the FDA for testing pathogen X in foods…..” testing pathogen X in foods…..” kit kit Company Sales RepsCompany Sales Reps

• ““..FDA-approved X method quickly ..FDA-approved X method quickly determines the disease causing toxins determines the disease causing toxins produced by EHEC in foods, especially produced by EHEC in foods, especially ground beef and milk.” ground beef and milk.” kit Company kit Company NewsletterNewsletter

• ““.. Company X received FDA clearance to .. Company X received FDA clearance to market a rapid test for market a rapid test for E. coliE. coli O157:H7, O157:H7, which is often transmitted via ground which is often transmitted via ground beef.” beef.” kit Company Press Releasekit Company Press Release

• “…“…the FDA is currently using this assay the FDA is currently using this assay in their labs” in their labs” kitkit Company BrochureCompany Brochure

Validation – Misconceptions?Validation – Misconceptions?• Validated methods are good?Validated methods are good?

• Compared Compared vsvs standard or official standard or official methodmethod

• Tested with seeded foodsTested with seeded foods• Validated methods are applicable to Validated methods are applicable to

all foods?all foods?• Exotic importsExotic imports

• complexity of foodscomplexity of foods• food-dependant assay food-dependant assay efficiencyefficiency• different sensitivitiesdifferent sensitivities• abundance of assaysabundance of assays• regulatory testingregulatory testing

Importance of ValidationImportance of Validation

Rapid Methods - Impact on Rapid Methods - Impact on RegulationRegulation

Increased Sensitivity Increased Sensitivity • ““zero tolerance” pathogens zero tolerance” pathogens • Health Risk?Health Risk?• Upgrade in Quality Control Upgrade in Quality Control

Programs? Programs? • Cost? Cost?

Assay-dependent Assay-dependent Regulations?Regulations?

False-negative reactions?False-negative reactions? Applicability: Applicability: ie: All Foods?ie: All Foods?

Endorsement/conflict of Endorsement/conflict of Interest?Interest?

Food Microbiology: Fundamentals & Food Microbiology: Fundamentals & FrontiersFrontiers, , 22ndnd ed. ed. 20012001

ASM Press, Washington DCASM Press, Washington DC

Feng, P. Chap. 38. Development and Impact Feng, P. Chap. 38. Development and Impact of Rapid Methods for Detection of of Rapid Methods for Detection of Foodborne Pathogens, pp.775-796.Foodborne Pathogens, pp.775-796.

Food Microbiology: Fundamentals & Food Microbiology: Fundamentals &

FrontiersFrontiers, , 33rdrd ed. 2007 ed. 2007ASM Press, Washington DC.ASM Press, Washington DC.

Feng, P. Chap. 43. Rapid Methods for the Feng, P. Chap. 43. Rapid Methods for the Detection of Foodborne Detection of Foodborne

Pathogens: Current and next-generation Pathogens: Current and next-generation technologies, pp.911-934.technologies, pp.911-934.

[email protected]@fda.hhs.gov

Impact, validation and regulatory implication of rapid methods Peter Feng, Research Microbiologist...

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