Micro Community Meeting Book
AOAC INTERNATIONAL Micro Community Meeting
AOAC Annual Meeting New Orleans, Louisiana Sunday, August 27, 2023 | 4:30PM – 5:30PM CDT Draft Agenda Moderator: Allison Baker (AOAC INTERNATIONAL)
I. WELCOME AND OVERVIEW OF MICROBIOLOGY ACROSS AOAC INTERNATIONAL (4:30PM – 4:40PM) Allison Baker and invited speakers will give an overview of the current micro projects within AOAC, highlight upcoming initiatives, and introduce hot topics for discussion. II. THE ROLE OF AOAC INTERNATIONAL COMMUNITIES & MICRO COMMUNITY (4:40PM – 4:45PM) Allison Baker will review the role of the AOAC Micro Community and provide ways in which Community Members can be engaged in AOAC INTERNATIONAL. III. MICRO COMMUNITY SURVEY RESULTS (4:45PM – 4:50PM) Allison Baker will review community survey results. IV. TOPIC OF INTEREST: METAGENOMICS (4:50PM – 5:10PM) Mike Sussman (US Department of Agriculture) will provide an overview of metagenomics with respect to big data, data driven life science and smart farming. He will review the future of this data and artificial intelligence. V. MICRO COMMUNITY TOPICS OF INTEREST (5:10PM – 5:25PM) Erin Crowley (Q Laboratories) and Mike Clark (Bio-Rad) will moderate discussions on additional topics of interest:
• Alignment of Matrix Categories • Alignment of Test Portion Sizes • Alignment of Validation Guidance • Consensus Validation Schemes & Revisions to Appendix J
VI.
NEXT STEPS AND ADJOURNMENT (5:25PM – 5:30PM) Allison Baker will discuss future meetings of the Micro Community and discuss avenues for communication for community members.
5:30pm – 6:30pm Community Mixer
This agenda is subject to change without notice.
Get Involved in AOAC
Color Additives from Natural Sources: Microbial Contaminants Subgroup Interest Form
AIMS WG and Advisory Panel Interest Form
CASP Microbial Contaminants WG Sign-up Form
https://form.jotform.c om/231104883953054
https://form.jotform.c om/91005746932154
https://form.jotform.co m/232343816410044
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
AOAC Micro Community Meeting
Please scan to sign into the meeting!
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
Today’s Agenda Overview of Current Micro Projects and Programs in AOAC
The Role of the Micro Community
Overview of Micro Community Survey Results
Featured Presentation: Metagenomics
Topics of Interest: Open Discussion
Next Steps
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
Microbiology at AOAC
Core Programs • Analytical Solutions Forum • Standards Development • Official Methods of Analysis o OMAs • Proficiency Testing Program o Micro Testing Programs • Research Institute o PTMs Certifications
Integrated Science Programs
• AIMS • CASP • Color Additives from Natural Sources • SPADA
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
Proficiency Testing: Microbiology Programs • Standard Microbiology Program • Pathogen-Free Microbiology Program • Meat Microbiology Program • Listeria Swab Program • Salmonella in Liquid Egg Program • Cannabis and Hemp Program Want to learn more? Contact Shane Flynn at sflynn@aoac.org
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
Official Methods of Analysis SM Active ERPs Microbiology Guidance Used Method Types
Methods Adopted Food Microbiology >160 methods CASP Microbial Contaminants 3 methods Environmental & Biothreat agent 5 methods
OMA, Appendix J
Qualitative
Methods for Food & Environmental Surfaces
OMA, Appendix I (BTAM Guidelines)
Quantitative
CASP Microbial Contaminants
CASP Microbial Contaminants (in progress)
Identification
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
Performance Tested Methods SM Source of Volunteer Experts ERP for Microbiology Methods for Food & Environmental Surfaces Guidance Used OMA, Appendix J Method Types Qualitative
Methods Certified
Food Microbiology >290 methods
OMA, Appendix I (BTAM Guidelines)
Quantitative
ERP for Cannabis Microbial Contaminants
CASP Microbial Contaminants 30 methods
Cannabis Microbial Contaminants (in progress)
Identification
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
Current Microbiology in Integrated Science Programs
Analytical International Methods and Standards (AIMS) Program
Cannabis Analytical Science Program (CASP)
Color Additives fromNatural Sources (CANS)
Stakeholder Program for Agent Detection Assays (SPADA)
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
Analytical International Methods and Standards Program (AIMS)
Link to Form
Link to Form
Currently drafting an SMPR for Cyclospora Funding currently underway for new WG: Legionella
AIMS Working Group Interest Form
AIMS Advisory Panel Interest Form
AIMS Meeting Wednesday, August 30 | 1:00 – 3:00PM
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
Cannabis Analytical Science Program (CASP)
Link to WG Sign Up Link to Interest Form
Currently drafting SMPR for Detection and Enumeration of Listeria spp. in cannabis infused edibles Micro Validation Guidelines
Join a CASP Working Group!
CASP Advisory Panel Interest Form
CASP Meeting Tuesday, August 29 | 1:00 – 3:00PM
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
Color Additives from Natural Sources
Subgroup drafting SMPR for Microbial Contaminants in Color Additives from Natural Sources
Link to Form
Color Additives from Natural Sources Meeting Monday, August 28 | 3:30 – 5:00PM
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
Stakeholder Program on Agent Detection Assays (SPADA)
AOAC SPADA Biothreat Agents
Completed
Guidance Documents
Current Working Group and Scope: Standards for NGS Biothreat Agent Detectors Working Group Development of SMPR for Detection of Multiple Biothreat Organism by Amplicon Sequencing In Publications Amplicon Sequencing Minimal Information (ASqMI): Quality and Reporting Guidelines for Actionable Calls in Biodefense Applications [featured article on JAOAC webpage] Standard Requirements for Nucleotide Sequences used in Biothreat Agent Detection, Identification, and Quantification: Verified Next Generation Sequences (VNGS) [new OMA Appendix]
SMPRs
OMA OMA, Appendix I, BTAM Validation Guidelines X
Public Safety
B. anthracis F. tularensis
X X X X X X X X X X X X X
OMA, Appendix O, Environmental Factors for Validating BTAM Assays OMA, Appendix P, Guidance for Soil Collection, Characterization and Application for BTAM and Site Evaluations OMA, Appendix Q, Recommendations for Developing Molecular Assays for Microbial Pathogen Detection Using Moder In Silico Approaches OMA, Appendix R, Guidelines for Verifying and Documenting the Relationships Between Microbial Cultures
Y. pestis B. mallei
B. pseudomallei
Variola virus
Field Safety B. anthracis
F. tularensis
Y. pestis
B. pseudomallei Brucella suis Variola virus Staphylococcal Enterotoxin B Venezuelan Equine Encephalitis Virus
X
Coxiella burnetti
X
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
Roles of AOAC Microbiology Community
How the community can engage with AOAC Weigh in on AOAC standards and methods Provide expertise on new technologies Identify needs for standards, methods, and relevant guidance Help AOAC align globally Identify laboratories willing to participate in AOAC collaborative studies Provide expertise and updates regarding newly conducted research Develop and validate methods
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
Micro Community
Survey Results
Part 1: Member Status
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
Micro Community Survey Results Part 2: AOAC INTERNATIONAL Meeting Attendance
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
Micro Community Survey Results
Part 3 : Topic Areas of Interest
Topic at ASF: Metagenomics!
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
Featured Speaker: Michael Sussman on Metagenomics Michael Sussman received his BS from Tulane University, MS in molecular
microbiology from the University of Illinois at Chicago and Ph.D. in molecular biology and biochemistry in biomedical science from the University of Connecticut. He trained in two molecular virology post-doctoral fellowships at Plum Island Animal Disease Center and Michigan State University. Until 2003, Dr. Sussman was employed as a research scientist in animal vaccine development. From 2003 to 2009, Michael led the US Department of Agriculture’s biotechnology testing program. From 2009 to 2013 he served as the Director of the USDA, Agricultural Marketing Service Laboratory Division. Since 2013, Dr Sussman is currently serving as a senior research scientist for the US Department of Agriculture, Agricultural Marketing Service’s Agricultural Analytics Division in Washington, DC. Dr. Sussman was the Chairperson for ISO TC 34/SC 16 from 2008 until 2018. He became the current committee manager of ISO/Technical committee 34 “Food products”/SC 16 “Horizontal methods for molecular biomarker analysis” in 2017 and also serves as an expert in a number of ISO, AOAC, and federal policy committees. He most recently has served the Chair for the SPADA working group to develop a SMPR for NGS reference sequences.
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
How SPADA got us to metagenomics
Mike Sussman Senior Research Scientist US Department of Agriculture, Marketing and Regulatory Programs
ASqMi and VNGS are progressive developments
A Marine company has just arrived at its rally point. As they discuss the plan of attack, a sergeant notices the plants are changing colors around him. He immediately yells “gas, gas, gas” and the Marines quickly put on gas mask and protective suits. Plants changing colors to alarm a company that toxins are present, it can not be true. It is true, and this is one of many capabilities that “deoxyribonucleic acid (DNA) computing” can provide the Department of Defense (DOD). Submitted by Captain J.F. Hudson Jr to Major Stophel, CG 3 22 Februry 2008
ASqMi and VNGS are progressive developments
• Leonard Adleman made the first DNA computation in 1994 by using his DNA computer to solve the “’traveling salesman’ mathematical problem -- how a salesman can visit a given number of cities without passing through any city twice.” • At that time, sequencing was slow and expensive. Now it is a little different • Since that time, DNA has been used to mimic known computer logic circuits with amazing success. • If we can envision and enable synthetic DNA as a computing device, are we missing something in the potential for DNA to already be computing in living cells. • Is there a living computing system involved or is it just magical kingdom randomly operating in time and space? • Does extend to communication between and among organisms? • Is this a role for metagenomics? • Can metagenomics that includes the sequence of all of the bacteria, all of the phages and all of the hosts in a community or an ecosystem help us to understand the interactions that permit pathogens to flourish under certain conditions?
Broadening the scope of SPADA
• The US DOD sponsors the work of SPADA • Fortunately, and according to the economics of scientific research private business and military research are usually the most successful at moving the needle of progress • In fact, we made some progress and attracted you all to the table • Whole genome sequencing technology has been on a rise for a while and does not appear to slowing down • Meta (Pytorch), Google (Tensor) and Nividia (Samba) are expanding neural networks and AI, so we have alot to look forward to in the future.
Amplicon Sequencing Minimal Information (AsqMI): Quality and Reporting Guidelines for Actionable Calls in Biodefense Applications
• Amplicon sequencing (AS) is a good biosurveillance tool compared to quantitative polymerase chain reaction for routine biosurveillance of biothreat agents. • The added specificity provide by sequencing the amplicon leads to fewer false negative and false positive calls. • Appropriate controls and workflow reporting are necessary to address difference in sequencing platforms, the approaches used to amplify and sequence the target and the algorithms associated with base calling, i.e., Fourier transform or neural network (AI).
Use for analysis of aerosol collection dry filter unit (DFU) filters as part of concept of operation-based routine testing to make actionable calls. Standard Method Performance Requirements (SMPRs ) for Detection of Multiple Biothreat Agent Organisms in Environmental Samples by Amplicon Sequencing Simultaneous detection of Bacillus anthracis , Francisella tularensis , Yersinia pestis , Burkholderia mallei , Burkholderia pseudomallei , Brucella abortus , Coxiella burnetii , and Variola virus from DFU filters or polytetrafluoroethylene (PTFE) filters.
A suitable method can target some or all of these agents as appropriate for the specific biodefense program or concept of operations.
New sequencing tools can provide a metagenomic non-targeted approach, but crically removing false positives will be more important
SPADA VNGS ( Stakeholder Panel on Agent Detection and Analysis Verified Next Generation Sequence ) The sequence is findable, accessible, interoperable and reusable (FAIR). a) has a unique URI b) sequence data and metadata is version controlled c) sequencer provider is the responsible party d) sequence is in a stable format – provenance etc. maintained – FASTQ, FAST5, pod5 e) Use of standard DNA sequence ontologies is encouraged a) sample name (sample ID) b) raw reads c) Assemblies and alignments - d) B or A g M anism name, e) strain name f) identification method g) sample type h) Host i) Isolation provider name j) isolation acquisition identity k) taxonomic identification l) contact name m) clinical or environmental sample. Minimum Annotation Information – metadata BED, WGG, GFF3, VCF, GTF, SBOL, etc.
SPADA VNGS Sequence Instrument Quality Metrics (1) Base quality score .— Statistical algorithms used for base calling shall be known, verified and converted to a Q score. Average base quality score Q>20. Single base quality score for the targeted region Q>30. (2) Artefacts.— No artefacts found in final sequence. (3) Sequencing platform specific error profiles . — All platform associated errors should be resolved (4) Variation in quality scores across the sequence read. —Sequence reads shall have an overall resolved Q score >20 (5) Biases in sequence data driven by base composition. — GC-rich sequence bias shall be anticipated and resolved, based on species specificity or nucleic acid repair (6) Departure from suboptimal library fragment sizes . — If possible, average library fragment size shall be provided as metadata
SPADA VNGS Sequence Instrument Quality Metrics
(7) Contamination from known and unknown species other than the sequencing target . — Contaminating species sequences shall be removed (8) Insert size .— Insert size and type of library preparation should be provided. (9) Number of reads.— The minimum read depth is 20X. (10) Base calling.— Base calling protocol should be provided. (11) Sequence length distribution.— Library quality should be recorded. (12) Length of longest contig.— Length of longest contig can be provided. (13) N50 .— N50 should be given in annotations. (14) NG50 .— NG50 should be given in annotations.
(15) Number of contigs.— Number of contigs should be given in annotations. (16) Base composition .— The proportions of the four bases (adenine, cytosine, guanine, and thymine or uracil) present in DNA or RNA expressed as the percentage (mol %) of G plus C should be given in annotations. (17) Coverage .— At the run level, minimum 20X. At the sample level, it depends on the application. (18) Breadth of coverage .— Coverage for 95% of the genome should be at the minimum level or higher depending on the expected application: 100% of target sequences should be at the minimum coverage or higher. (19) Cluster density .— The total length of all contigs or scaffolds should approximate the known genome size of the target organism. SPADA VNGS Sequence Instrument Quality Metrics
Thank you
Microbiology Community: Topics of Interest Erin Crowley and Mike Clark
Moderators
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
Micro Community Topics of Interest • Alignment of Matrix Categories • Alignment of Test Portion Sizes • Alignment of Validation Guidance • Consensus Validation Schemes & Revisions to Appendix J
What does this mean to Community members?
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 | New Orleans, LA •
The State of Things…
Validation Schemes
ISO 16140-2
AOAC Research Institute (RI)
AFNOR French National Organization for Standardization
AOAC Performance Tested Method (PTM)
Microval European certification body for microbiology methods NordVal International Nordic certification body: Denma rk , Finland, Iceland, Norway, Sweden
AOAC Official Method of Analysis (OMA)
Validation Scope Food categories i.e Raw meat and ready to cook meat products
Validation Scope Very specific i. e Ground beef (20% fat)
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
ISPAM – Microbiology Method Harmonization
International Stakeholder Panel on Alternative Methods = ISPAM
Working Group (WG) on Microbiology Guidelines
• Compare validation guidelines • Identified areas of difference • Promote harmonization
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
Five Priority Areas Identified
1. Reference Methods
2. Number of Levels/Samples/Fractional Positives
3. Selection of Food/Category (sample matrix)
4. Results analysis & Criteria/Statistical Analysis
5. Data Sets for Collaborative Study/Sample Size
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
AOAC ISPAM
Harmonized approaches for • Number of levels/samples/fractional positives • Results analysis/criteria/statistical analysis • # data sets for collaborative study/sample size • Food Classification Table and replaced & “all foods” with “ broad range of foods ” • Reference method
Updated Validation Protocols • AOAC Guidelines (2012) • ISO 16140-2:2016 (2016)
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
Food Categories — ISO and AOAC INTERNATIONAL
Table A1: Classification of categories and suggested target combinations for verification studies
Ready-to-eat, ready-to- reheat meat products
Raw poultry and ready-to- cook poultry products
Raw meat and ready-to-cook meat products (except poultry)
Ready-to-eat, ready-to-reheat meat, poultry products
Heat-processed milk and dairy products
Raw milk and dairy products
Raw and ready-to-cook fish & seafoods (unprocessed)
Ready-to-eat, ready-to-reheat fishery products
Fresh produce and fruits
Processed fruits and vegetables
Dried cereals, fruits, nuts, seeds and vegetables
Eggs and egg products (derivatives)
Cannabis and cannabis- derived matrices
Environmental samples (food or feed production)
Chocolate, bakery products & confectionary
Multi-component foods or meal components
Primary production samples (PPS)
Infant formula and infant cereals
Pet food and animal feed
Non-food categories
AOAC categories
Food categories
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA TECHNICAL BULLETIN: TB02MAY2016: Acceptable Validation Claims for Proprietary/Commercial Microbiology Methods for Foods and Environmental Surfaces Level of Claim Acceptable Validation Claims for Proprietary /Commercial Microbiology Methods for Foods & Environmental Surfaces Acceptable Multiple Matrix Claims Claim Number of Matrices Number of Categories/Groups 1 Broad Range of Foods 15 (3 foods/categories) 5 Variety of Foods ≥ 10 5 Selected Foods ≥ 5 2 Food Category/Group ≥ 5 1 Environmental Surfaces 7 NA Selected Surfaces 2–6 NA Acceptable Environmental Surfaces Air Filter Material Cast Iron Coated to prevent rusting Sealed Concrete Plastic Polyethylene, Polypropylene, Polycarbonate Rubber Ceramic Glazed earthen material or glass Stainless Steel
Test Portion Size and Dilution Scheme- AOAC Policy • If there is any change to the dilution ratio, the changes must be validated. Note* Methods cannot claim a different test portion to enrichment broth ration of a given matrix than that of the ratio of the approved validation. Any such changes must be validated. A smaller test portion of the same matrix may be claimed if the portion to broth ratio is the same as the larger portion that was validated. • If a method has an approved validation for a certain test portion size, then the claim for that method may include portions up to that test portion size. To claim a test portion larger than the approved validated test portion size, then validation is required. • AOAC reserves the right to require validation data to support any deviation in test portion size that is not equal to the test portion size in the approved validation. The smallest acceptable test portion size claimed must be greater than or equal to the smallest portion of the reference method used as part of the approved validation study.
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
Basic AOAC Standards Development Activity Framework
Works with AOAC to define scope of work to be launched, including development of new programs and/or new working groups (WGs) Advisory Panel Meeting
WG begin work and draft consensus documents via web conference and reach general consensus on draft standard between AOAC meetings WG Meetings
WG chair(s) present final draft standard along with reconciled comments for deliberation and consensus. Community Consensus
Approval of Standard follows consideration of all input and with oversight by Official Methods Board (OMB). Publication of Standard is in the OMA Approval/Publication of Standard
Draft standard is posted for public comments. Comment period is ≥ 30 days Virtual public comment session via webconference Draft Standard Public Comment Period
At AOAC meetings, launch WG effort by refining a preset scope of work into a basic applicability statement from which the WG to began drafting the standard (e.g.,
SMPR, guidelines, etc) Launching New WG Activities
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
What Do We Need? • Call for Microbiology Community Working Groups • Align with AOAC Standards Development Framework • Align with other Communities
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
Next Steps • Get involved! • Meeting Frequency:
o Proposed: Community Meetings Quarterly
• Next Meetings:
o Proposed: January 2023 o Proposed: AOAC Midyear Meeting (March 2024)
• Communication for the Community:
o AOAC Teams/SharePoint Sites o LinkedIn Group
AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA
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