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
College Park, MD 20740
 Equivalence?

24 h, 35ºC

Does or =

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
http://www.fda.gov/downloads/ScienceResearch/FieldScience/UCM273418.pdf
 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
– TC 34/SC 9 started standardizing “horizontal” reference methods for bacteria in foods in the 1970s
 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 18
formation of a Methods Validation Subcommittee (MVS) to serve as the governing body for all method
validation concerns.”
 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.
 The Office of Foods and Veterinary Medicine & the SRSC
“Roadmap for Microbiological Method Development and Validation”

RESEARCH
Micro
Method
Organizational
Partnerships
Validation
•BAM Council
•FERN MCC
VALIDATION Sub-group
•IFSH
•ORA “Micronauts”
VERIFICATION

IMPLEMENTATION
9
The Method Validation Sub-Group (Microbiology)

ESTABLISHES validation needs and priorities in consultation with the

SRSC-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 practices

10
 The Method Validation Sub-Group
(Microbiology)
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

11
METHOD VALIDATION SCOPE OF RESPONSIBILITY
Pathogens, Genetic Material, Toxins and Antigens
 Qualitative assays  Applications
 Analyte  Pre- and selective enrichment
 Bacteriological, e.g.  Microbial analyte recovery and concentration
 Salmonella spp.  Screening, high-throughput, confirmation
 Pathogenic Escherichia coli  Procedures
 Listeria monocytogenes  Phenotypic, e.g.
 Shigella spp  Biochemical characterization
 Vibrio spp  Antibiotic resistance traits Antigenic characterization
 Campylobacter spp Genetic, e.g.
 Microbial toxins  Nucleic acid isolation/concentration
 Viral pathogens, e.g.  Polymerase Chain Reaction
 Hepatitis A virus  Conventional, Real-time
 Norovirus  Reverse transcription

 Enterovirus  Sequencing, e.g.

 Whole genome
 Parasitic protozoan pathogens, e.g.
 Selective sequencing
 Cryptosporidium
 Single nucleotide polymorphism (SNP) analysis
 Cyclospora cayetanensis
 Strain-typing applications
 Bioengineered analytes, e.g.
 Genetically-modified foods (GMOs)
 Method Validation is Required for…
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 further
validation, procedures outline in this document will be followed.
 Method 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.
 Method Validation Criteria
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.
Microbiology Validation Category Examples
I. General Qualitative Guidelines for Microbial Analytes
Table 1. ORIGINATING Laboratory Requirements
Originating Laboratory Level One: Urgent Level Two: Independent lab Level Three: Multiple Level Four: Full †AOAC

Criteria usage validation lab collaborative collaborative study Collaborative Study

# of target organism 10 (20 for 50 (unless 50 aren't 50 (unless 50 aren't 50 (unless 50 aren't
50a,b
(inclusivity) Salmonella) available)a,b available)a,b available)a,b

# of non-target organism
10 strains 30 strainsc 30 strainsc 30 strainsc 30 strainsc
(exclusivity)

# of foods 1 or mored 1 or mored 1 or mored 1 or mored Up to 20 foodse

set level based on One inoculated levelf

One inoculated levelf and One inoculated levelf One inoculated levelf
# of analyte levels/food matrixf intended use and and uninoculated
uninoculated level and uninoculated level and uninoculated level
negative control level

Replicates per food at each

20 20 20 20 20
level tested

Aging of inoculated samples

No Yes Yesg Yesg Yesg
prior to testing

In 1 food at +1 In 1 food at +1 In 1 food at +1

Normal background In 1 food at +1 log>analyte at log>analyte at log>analyte at log>analyte at
Addition of competitor strain h
flora fractional positivef analyte level fractional positivef fractional positivef fractional positivef
analyte level analyte level analyte level

Comparison to recognized
No Yes, if available Yes, if available Yes, if available Yes, if available
method
 Microbiology Validation Category Examples, continued
Table 2 - General Qualitative Guidelines for Microbial Analytes-Collaborating
Laboratory Requirements

Level Two: Independent lab Level Three: Multiple lab Level Four: Full
Collaborating Laboratory Criteria AOAC Collaborative Study
validation collaborative 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

3 levels: One inoculated levelc 3 levels: One inoculated 3 levels: One inoculated levelc
2 levels: One inoculated levelc
# of analyte levels/food matrixc one at 1 log higher and levelc, one at 1 log higher and one at 1 log higher and
and uninoculated level
uninoculated level uninoculated level 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
II. General Qualitative Guidelines for Food-borne Microbial Pathogens
That Present Unique Isolation and/or Enrichment Challenges†
Table 1. ORIGINATING Laboratory Requirements
Category Category
Category
Category Two: Three:
Originating Laboratory Four: Full
One: Urgent Independent Multiple lab
Criteria collaborativ
usage lab collaborativ
e study
validation e
Replicates per strain 3 3 8 8
Comparison to Yes, if Yes, if Yes, if
No
recognized methoda available available available

Table 2. COLLABORATING Laboratory Requirements

Level Two: Level Three:
Collaborating Level One: Level Four: Full
Independent lab Multiple lab
Laboratory Criteria Urgent usage collaborative study
validation collaborative
# of laboratories
n/a 2 5 10
providing usable datab
Replicates per strain n/a 3 8 6
Comparison to Yes, if
n/a Yes, if available Yes, if available
Recognized Methoda available
Microbiology Validation Category Examples, continued
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
V. PLATFORM EXTENSION
 Microbiology Validation Category Examples, continued
VI. CRITERIA AND GUIDANCE FOR THE VERIFICATION AND VALIDATION
OF COMMERCIALLY- AVAILABLE MICROBIOLOGICAL DIAGNOSTIC KITS AND PLATFORMS

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
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.
 Current Micro MVS Priorities
I. Hepatitis A Virus
a. 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. Norovirus
a. Real-time RT-qPCR assay to detect Noroviruses
b. In a food matrix (molluscan shellfish)
Status: planning phase underway in collaboration with the ORA-CFSAN virology working group

23
Validation of an Identification Method for Shiga-
toxigenic E. coli Somatic (O) Groups using the
BioPlex Suspension Array System
Method Authors
Andrew Lin
Teresa Lee
Laurie Clotilde
Julie A. Kase
Insook Son
J. Mark Carter
Carol R Lauzon

24
Validation of a STEC Molecular Serotyping Method
• 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

25
Validation of a STEC Molecular Serotyping Method
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

26
Validation of a STEC Molecular Serotyping Method

Method Status
• In-House Validation Study Completed
– Paper
– LIB

• 5 Lab Collaborative Study Successfully Completed

• Approved by the BAM Council

27
Validation of a STEC Molecular Serotyping Method
In-house Validation Results Inclusivity Panel

28
Validation of a STEC Molecular Serotyping method
Exclusivity Panel

40
 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
30
Validation of a STEC Molecular Serotyping method
BioPlex Results

31
 Validation of a Method for
Salmonella in Whole Shell Eggs

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

32
 Validation of a Method for Salmonella in
Whole Shell Eggs
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
33
 Validation of a Method for Salmonella in
Whole Shell Eggs
Clean surface Clean surface
Surface disinfection
Surface disinfection

20 eggs to a container
Hand homogenize sample 20 eggs + 2L TSB
24 h at 35oC
Hold at room Temp (20-24oC)
for 96 h
1 ml to 10 ml TT 0.1 ml to 10 ml RV
24 h at 35oC 24 h at 42oC
25 ml eggs to 225 ml TSB + ferrous
sulfate; 24 h at 35oC
Streak on XLD, BS, HE Streak on XLD, BS, HE
1 ml to 10 ml TT 0.1 ml to 10 ml RV 24 h at 35oC 24 h at 35oC
24 h at 35oC 24 h at 42oC

Streak on XLD, BS, HE Streak on XLD, BS, HE TSI, LIA TSI, LIA
24 h at 35oC 24 h at 42oC
24 h at 35oC 24 h at 35oC
TSI, LIA TSI, LIA
24 h at 35oC 24 h at 35oC
Serological Test Serological Test
Serological Test Serological Test
34
CURRENT BAM METHOD PROPOSED BAM METHOD
Validation of a Method for Salmonella in
Whole Shell Eggs

35
Validation of a Method for Salmonella in
Whole Shell Eggs

36
Validation of a Method for Salmonella in
Whole Shell Eggs

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

37
Validation of a Method for Salmonella in
Whole Shell Eggs
Collaborative Studies

Tentative Schedule

•Culture Method Alone

Fall 2012

•Culture Method Plus qPCR Method

Spring 2013

38