Microbiological Method Validation: How Do
We Prove that a Method is Fit for Purpose?
Thomas Hammack
Chief
Microbial Methods Development Branch
Division of Microbiology
Office of Regulatory Science
Center for Food Safety and Applied Nutrition
U.S. Food and Drug Administration
Equivalence?
24 h, 35ºC
24 h, 35ºC and 42ºC
•
Why validate methods?
Benefits public health and world trade
False negative results are unacceptable
ISO 17025 lab accreditation demands the use of
validated methods
Validation Programs
•
AOAC Microbiological Guidelines
•
ISO 16140:2003
•
FDA’s Guidelines for the Validation of Analytical
Methods for the Detection of Microbial Pathogens in
Foods
AOAC International
• 1884—Association of Official and Analytical Chemists
– Methods Validation for Fertilizers
– Membership Restricted to Regulatory Chemists
• Supported by USDA
– Dr. Harvey Wiley -“Father of FDA” is also considered the “Father of AOAC”
• 1892—Secretary of AOAC
• 1898—Established AOAC’s Committee on Food Safety
– Housed within FDA
• 1939—Microbiological Sampling of Eggs and Egg products Official Method 939.14 still in use today
• Many AOAC methods Official Methods were developed and validated in FDA Labs
• 1972—FDA published acceptance of AOAC Official Methods in the Federal Register
– Allows FDA to use proprietary rapid methods for the analysis of regulatory samples
• 1979—became an independent non-profit organization no longer tied to FDA, but many FDA employees serve as volunteers
International Organization for
Standardization (ISO)
• International non-governmental organization
– Based in Geneva, Switzerland
– Comprised of 163 national standards institutes including ANSI in the US
• Started in 1926 as the International Federation of National Standardizing Associations (ISA)
– Focused on mechanical engineering
– Disbanded in 1942
– Reorganized as ISO in 1946
– Mission statement is to provide “International Standards for Business, Government and Society”
• 1991 ISO and European Committee for Standardization (CEN) signed the Vienna Agreement
– CEN is comprised of the 31 Member States of the European Union
– Vienna agreement stipulates that CEN and ISO Standards be identical to enable free trade within the EU
• ISO Technical Committee 34 (TC 34) Subcommittee 9 (SC 9)
– ISO exercises its role in the standardization of food microbiological testing methods through TC 34/SC 9
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FDA’s Methods Validation Guidelines
The Science and Research Steering Committee (SRSC), of the Office of Foods and Veterinary Medicine (OFVM), approved guidance to be used for validation of microbiological and chemical methods.Guidelines for the Validation of Analytical Methods for the
Detection of Microbial Pathogens in Foods
http://www.fda.gov/downloads/ScienceResearch/FieldScience/UCM273418.pdf
Guidelines for the Validation of Chemical Methods for the FDA
Foods Program
http://www.fda.gov/downloads/ScienceResearch/FieldScience/UCM298730.pdf
Scope
“These criteria apply to all FDA laboratories that develop and participate in the validation of analytical food methods for Agency-wide implementation in a regulatory capacity. This includes all research
laboratories, and field labs where analytical methods may be developed or expanded for potential regulatory use. These documents will supersede all other intra-agency documents pertaining to
food-related method validation criteria for microbial and chemical analytes. the SRSC will authorize the formation of a Methods Validation Subcommittee (MVS) to serve as the governing body for all method
FDA and Methods Validation
Method validation is a process by which a laboratory confirms by examination, and provides objective evidence, that the particular requirements for specific uses of a method are fulfilled. It serves to
demonstrate that the method can detect and identify an analyte or analytes: • In one or more matrices to be analyzed
• In one or more instruments or platforms
• With a demonstrated sensitivity, specificity, accuracy, trueness, reproducibility, ruggedness
and precision to ensure that results are meaningful and appropriate to make a decision.
• Reliably for its intended purpose. Intended purpose categories include, but may not be
limited to emergency/contingency operations; rapid screening and high throughput testing; and, confirmatory analyses.
• After the method developer has conducted experiments to determine or verify a number of
specific performance characteristics that serve to define and/or quantify method performance.
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RESEARCH
VALIDATION
Micro
Method
Validation
Sub-group
The Office of Foods and Veterinary Medicine & the SRSC
“Roadmap
for Microbiological Method Development and Validation”
IMPLEMENTATION
Organizational
Partnerships
•BAM Council •FERN MCC •IFSH •ORA “Micronauts” VERIFICATION10
ESTABLISHES
validation needs and priorities in consultation with theSRSC-Micro Super-group, FDA Bacteriological Analytical Manual Council (BAM Council), FERN Method Coordinating Committee, ORA “micronauts” inter-center working groups and others as appropriate
ADOPTS
procedures to govern all administrative processes needed for emergency and non-emergency method validation proposals and studies.PROVIDES
planning, guidance, oversight, and resources to participating laboratories during the method development and validation process; will be the responsible authority for recommendations, evaluations and final approval of all validation studies from planning through field implementation.CONSULTS
with other governmental, and independent (commercial, and international) validation bodies to harmonize validation standards where possible and practices11
BROAD REPRESENTATION
from CFSAN, ORA, CVM, and NCTR with additional
expertise from biostatisticians and QA/QC managers
CURRENT MICRO MVS COMPOSITION:
ORA
Palmer Orlandi (co-Chair), Cathy Burns
CFSAN Thomas Hammack (co-Chair), William Burkhardt, Darcy Hanes
CVM
Beilei Ge
NCTR
Steven Foley
FERN
Don Burr
NCFST
(CFSAN Moffett)Ravinder Reddy
The Method Validation Sub-Group
Qualitative assays
Analyte
Bacteriological, e.g.
Salmonella spp.
Pathogenic Escherichia coli
Listeria monocytogenes
Shigella spp
Vibrio spp
Campylobacter spp
Microbial toxins
Viral pathogens, e.g.
Hepatitis A virus
Norovirus
Enterovirus
Parasitic protozoan pathogens, e.g.
Cryptosporidium
Cyclospora cayetanensis
Bioengineered analytes, e.g.
Genetically-modified foods (GMOs)
Applications
Pre- and selective enrichment
Microbial analyte recovery and concentration
Screening, high-throughput, confirmation
Procedures
Phenotypic, e.g.
Biochemical characterization
Antibiotic resistance traits Antigenic characterization
Genetic, e.g.
Nucleic acid isolation/concentration
Polymerase Chain Reaction
Conventional, Real-time
Reverse transcription
Sequencing, e.g.
Whole genome
Selective sequencing
Single nucleotide polymorphism (SNP) analysis
Strain-typing applications
METHOD VALIDATION SCOPE OF RESPONSIBILITY
Pathogens, Genetic Material, Toxins and Antigens
1. Submission of a new or original method, OR,
2. Any significant modification of a method that may alter its performance specifications or changes to the fundamental science of an existing method. Categories include:
• Substitutions of reagents/apparatus
• Expansion of the scope of an existing method to include additional analytes.
• Changes in intended use i.e. screening or confirmatory.
• Platform extensions or significant parameter changes e.g. adaptation to another real-time PCR thermal cycler.
• Matrix extensions.
• Changes to time/temperature incubation periods, or enrichment media.
• In cases where the sample preparation and/or the extraction procedure/analytical method is modified from the existing test procedure and protocol, i.e the new method should demonstrate that the modifications do not adversely affect the precision and accuracy or bias of the data obtained.
• Modification of a method’s performance range e.g. specificity, sensitivity beyond previously validated levels.
Levels of Validation
Four levels of performance are defined. The hierarchy of
scrutiny will provide general characteristics on the method’s
utility and insights for its intended use, the assessed risk, and
the food-borne illness potential for an analyte-matrix pairing.
Not all methods will or should be validated to meet the requirements of a Level 4: full collaborative study.
Method Validation Criteria
Level One
The lowest level of validation, with all the work done by one lab. Inclusivity and exclusivity testing has been tested, but by a limited number of strains. The analyte was tested at a level based on the intended use of the method, with just normal background flora. There is no aging of the artificially-inoculated samples and no comparison to an existing reference culture
method. The expectation would be for the originating lab to continue to conduct further testing to eventually elevate the method to a higher level of validation.
Intended Use: Emergency needs.
A method developed for the detection of an analyte, or a matrix not previously recognized or identified as a threat to food safety or public health. As the first level in the development of any method designed for regulatory use; performance of the method at this level of scrutiny will determine, in part, whether further validation is useful or warranted.NOTE:
Under emergency situations where the rapid development and deployment of a method is needed to immediately address an outbreak event, Level 1 criteria should be followed as closely as the situation will allow. Representatives of the MVC and Agency subject matter experts (SMEs) should be in close consultation with the originating laboratory. Once the crisis has past and it has been determined that there is a need for furtherMethod Validation Criteria
Level Two
This is a more robust study, with the possibility of regulatory strength depending on the circumstances. The originating lab has done a more comprehensive initial study, with
inclusivity/exclusivity levels at the AOAC Collaborative Study level. If possible, a comparison has been done to an existing reference culture method. One other independent laboratory has participated in the collaborative study. Some of the criteria of the study are at a lower level than the full AOAC study, but still appropriate for the developing method at this stage.
Intended Use: Emergency needs.
Slightly higher false-positive rates are acceptable as all samples analyzed with methods validated to this level will require confirmatory testing.Level Three
This is a validation level that should have full regulatory strength. Most of the criteria followed by the originating lab are at the AOAC level, including inclusivity/exclusivity, analyte levels, competitor strains, aging, and comparison to existing method when available. The additional collaborating labs follow many of the criteria of an AOAC collaborative study.
Intended Use: All methods validated to this level of scrutiny are acceptable for use in any and all circumstances e.g. confirmatory analyses; regulatory sampling, and
compliance support.
Level Four
This validation level has criteria equivalent to the AOAC Collaborative Study. Any
method reaching this level of validation should be able to be submitted for adoption by the AOAC as a fully collaborated method.
Originating Laboratory Criteria
Level One: Urgent usage
Level Two: Independent lab validation
Level Three: Multiple lab collaborative
Level Four: Full collaborative study †AOAC Collaborative Study # of target organism (inclusivity) 10 (20 for Salmonella) 50 (unless 50 aren't available)a,b 50 (unless 50 aren't available)a,b 50 (unless 50 aren't available)a,b 50 a,b # of non-target organism
(exclusivity) 10 strains 30 strainsc 30 strainsc 30 strainsc 30 strainsc
# of foods 1 or mored 1 or mored 1 or mored 1 or mored Up to 20 foodse
# of analyte levels/food matrixf
set level based on intended use and
negative control
One inoculated levelfand
uninoculated level
One inoculated levelf
and uninoculated level
One inoculated levelf
and uninoculated level
One inoculated levelf
and uninoculated level Replicates per food at each
level tested 20 20 20 20 20
Aging of inoculated samples
prior to testing No Yes Yesg Yesg Yesg
Addition of competitor strainh Normal background
flora
In 1 food at +1 log>analyte at
fractional positivef analyte level
In 1 food at +1 log>analyte at fractional positivef analyte level In 1 food at +1 log>analyte at fractional positivef analyte level In 1 food at +1 log>analyte at fractional positivef analyte level Comparison to recognized
method No Yes, if available Yes, if available Yes, if available Yes, if available
Table 1. ORIGINATING Laboratory Requirements
I.
General Qualitative Guidelines for Microbial Analytes
Collaborating Laboratory Criteria Level Two: Independent lab validation
Level Three: Multiple lab collaborative
Level Four: Full
collaborative study AOAC Collaborative Study
# of laboratories providing usable data 2 5 10 10
# of foods 1 or morea 1 or morea 1 or morea 1 to 6 foodsb
# of strains of organism 1 per food 1 per food 1 per food 1 per food
# of analyte levels/food matrixc 2 levels: One inoculated levelc
and uninoculated level
3 levels: One inoculated levelc
one at 1 log higher and uninoculated level
3 levels: One inoculated levelc, one at 1 log higher and
uninoculated level
3 levels: One inoculated levelc
one at 1 log higher and uninoculated level
# of replicate samples/food 8 per analyte level 8 per analyte level 8 per analyte level 6 per analyte level †
Aging of inoculated samples prior to testing Noe Yesd Yesd Yesd
Comparison to Recognized Method Yes, if available Yes, if available Yes, if available Yes, if available
Microbiology Validation Category Examples,
continued
Table 2 - General Qualitative Guidelines for Microbial Analytes-Collaborating Laboratory Requirements
Originating Laboratory Criteria Category One: Urgent usage Category Two: Independent lab validation Category Three: Multiple lab collaborativ e Category Four: Full collaborativ e study
Replicates per strain 3 3 8 8
Comparison to recognized methoda No Yes, if available Yes, if available Yes, if available Collaborating Laboratory Criteria Level One: Urgent usage Level Two: Independent lab validation Level Three: Multiple lab collaborative
Level Four: Full collaborative study
# of laboratories
providing usable datab n/a 2 5 10
Replicates per strain n/a 3 8 6
Comparison to
Recognized Methoda n/a Yes, if available
Yes, if
available Yes, if available
II. General Qualitative Guidelines for Food-borne Microbial Pathogens
That Present Unique Isolation and/or Enrichment Challenges
†Microbiology Validation Category Examples,
continuedTable 1. ORIGINATING Laboratory Requirements
III. CRITERIA AND GUIDANCE FOR THE VALIDATION OF FDA-
DEVELOPED MOLECULAR-BASED ASSAYS
•Inclusivity and exclusivity
•Target gene(s) and controls (positive and negative).
•Comparison to the Reference Method
IV. FOOD MATRIX EXTENSION FOR VALIDATED MICROBIOLOGY
METHODS
The validation of a method for a food matrix not previously included in a validation study is necessary to assure that the new matrix will produce neither high false positive (matrix is free from cross reactive substances) no high false negative rates (matrix is free of inhibitory substances)
•
Guidance to Support Field Laboratory Expedience
•
Guidance for the Formal Expansion of Validated Food Matrices
Microbiology Validation Category Examples,
continuedVI. CRITERIA AND GUIDANCE FOR THE VERIFICATION AND VALIDATION
OF COMMERCIALLY- AVAILABLE MICROBIOLOGICAL DIAGNOSTIC KITS AND PLATFORMS
1. For commercially-available microbiological diagnostic kits whose performance parameters have been fully validated in a collaborative study monitored and evaluated by an independent accrediting body e.g. AOAC-OMA, AFNOR, etc. 2. For commercially-available microbiological diagnostic kits whose performance parameters are supported by data
obtained through an abbreviated validation protocol and evaluated by an independent accrediting body e.g. AOAC-RI.
Validation of an Alternative method: Demonstration that adequate confidence is provided when the results obtained by the alternative method i.e. the commercially-available kit, are comparable to or exceed those obtained using the reference method using the statistical criteria contained in the approved validation protocol.
Verification: The confirmation by examination and the provision of objective evidence that specified requirements have been fulfilled.
Criteria
For Kits Fully Validated in a Collaborative Study Monitored by an Independent Accrediting Body
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Current Micro MVS Priorities
I. Hepatitis A Virusa. Real-time RT-qPCR assay to detect Hepatitis A virus b. In a food matrix (green onions)
Status: Final report nearing completion
2. †Non-O157:H7 STEC
Screening method to detect non-O157:H7 STECs using the BioPlex
Status: Multi-lab collaborative study has been completed..
3. ‡Salmonella Enteritidis
Isolation and Detection of Salmonella Enteritidis (SE) from Whole Shell Eggs-Cultural and Molecular Applications
4.
Norovirusa. Real-time RT-qPCR assay to detect Noroviruses
b. In a food matrix (molluscan shellfish)
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Method Authors
Andrew Lin
Teresa Lee
Laurie Clotilde
Julie A. Kase
Insook Son
J. Mark Carter
Carol R Lauzon
Validation of an Identification Method for
Shiga-toxigenic
E. coli
Somatic (O) Groups using the
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•
STECs are a significant public health concern
–
Non-O157 STECs are responsible for over 60% of STEC
infections or an estimated 112,000 illnesses in the U.S. each year
– Over 74.2% of STEC infections in the U.S. are caused by serogroups O26
(23.9%), O45 (7.8%), O91 (2.3%), O103 (16.7%), O111 (12.6%), O121 (7.5%), and O145 (3.4%)
– O26, O103, O111, O121, and O145 are known to cause HC and HUS, and
O45 is associated with HC
– Other serogroups that may cause HC and HUS, but are less commonly
isolated, are O91, O113, and O128
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BioPlex Assay Overview
• Must be performed on Pure Cultures
• Bead-based assay that can be multiplexed
– Conventional and real-time PCR assays for STEC O serogroup - limited by resolution of band sizes
on a gel, or the # of fluorescent channels
• 96-well plates
• Targets = nucleic acid, proteins – Ab, antigens, cytokines
• Each bead = a separate assay
• 100 different color-coded beads (magnetic
or polystyrene)
– Unique color comes from different ratios of two distinct flourophores
• Two lasers, fluidics, optics, detectors
• Automated, High-Throughput, Fast, Expandable
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Method Status
•
In-House Validation Study Completed
–
Paper
–
LIB
•
5 Lab Collaborative Study Successfully Completed
•
Approved by the BAM Council
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In-house Validation Results Inclusivity Panel
40
Exclusivity Panel
BioPlex Results
•
Fluorescence signals are quantified for each micro
bead
•
Signal to background ratios are calculated, where
background is measured using all no template
reactions
•
Samples were considered to be positive when
signal to background ratio was greater than 5.0
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BioPlex Results
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Development and Validation of a Method for the
Detection and Isolation of Salmonella Enteritidis (SE)
from Whole Shell Eggs
Guodong Zhang
Eve Thau
Eric W. Brown
Thomas Hammack
Validation of a Method for
33
Background
•
SE is the second most commonly isolated Salmonella serotype
in the United States
–
14.18% (1968-1998)
•
SE is most commonly associated with whole shell eggs and
egg products
•
FDA’s egg rule (74 FR 33030) recommends various preventive
control measures including sampling and sample analysis
•
FDA’s BAM reference culture method for the detection of SE in
whole shell eggs takes 9 days to complete
•
There are no AOAC Official Methods of Analysis methods for
the detection of SE in whole shell eggs
Validation of a Method for Salmonella in
Whole Shell Eggs
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Clean surface Surface disinfection
20 eggs to a container Hand homogenize sample
Hold at room Temp (20-24oC)
for 96 h 25 ml eggs to 225 ml TSB + ferrous sulfate; 24 h at 35oC 1 ml to 10 ml TT 24 h at 35oC Streak on XLD, BS, HE 24 h at 35oC TSI, LIA 24 h at 35oC Serological Test 0.1 ml to 10 ml RV 24 h at 42oC Streak on XLD, BS, HE 24 h at 42oC TSI, LIA 24 h at 35oC Serological Test
CURRENT BAM METHOD
Clean surface
Surface disinfection
20 eggs + 2L TSB
24 h at 35
oC
1 ml to 10 ml TT
24 h at 35
oC
Streak on XLD, BS, HE
24 h at 35
oC
TSI, LIA
24 h at 35
oC
Serological Test
0.1 ml to 10 ml RV
24 h at 42
oC
Streak on XLD, BS, HE
24 h at 35
oC
TSI, LIA
24 h at 35
oC
Serological Test
PROPOSED BAM METHOD
Validation of a Method for Salmonella in
35
Validation of a Method for Salmonella in
Whole Shell Eggs
36
Validation of a Method for Salmonella in
Whole Shell Eggs
37
Method Status
•
In-House Validation Study of Culture Method
Complete
–
Manuscript in preparation
–
Report to BAM Council in preparation
•
In-House Validation Study of qPCR Methods in
Progress
–
ABI SE qPCR Method Promising
–
Evaluation of an internal qPCR method in progress
Validation of a Method for Salmonella in
Whole Shell Eggs
38