AIMS at AOAC Annual Meeting 2023

AOAC INTERNATIONAL Analytical International Methods and Standards Program AOAC Annual Meeting Wednesday, August 30, 2023 1:00pm – 3:00pm

MEETING AGENDA

I. WELCOME, INTRODUCTIONS AND ANNOUNCEMENTS (1:00PM – 1:05PM) Allison Baker, AOAC INTERNATIONAL, will welcome attendees, lead introductions, and highlight the AOAC Micro Community session. II. AIMS OVERVIEW AND SUMMARY (1:05PM – 1:15PM) Erin Crowley (Q Laboratories) and Morgan Wallace will provide an overview of recent AIMS highlights and summation of recent external meetings attended. III. CYCLOSPORA OUTBREAK: BROCCOLI! (1:15PM – 1:25PM) Kalmia Kniel (University of Delaware) will provide an overview of the most recent Cyclospora outbreak in broccoli and how it relates to the importance of the work being done in the Cyclospora WG IV. CYCLOSPORA METHOD DEVELOPMENT AND CHALLENGES (1:22PM – 1:45PM) Mauricio Durigan (FDA) will discuss the current Cyclospora method and review challenges faced as it relates to the AIMS Cyclospora Working Group's development of the SMPR. V. WORKING GROUP ON CYCLOSPORA (1:45PM – 2:20PM) Erin Crowley (Q Laboratories) and Morgan Wallace will present the SMPR draft on the Detection, Identification and Characterization of Cyclospora, and highlight the opening of the public comment period. VI. LEGIONELLA OVERVIEW (2:20PM – 2:35PM) Michael Loewenstein, Q Laboratories, will introduce the challenge of Legionella testing, confirmation of positive results and an overall lack of standards for validating methods. VII. BEYOND CULTURE: CHALLENGES OF LEGIONELLA OUTBREAK RESPONSE FOR PUBLIC HEALTH LABS (2:35PM – 2:55PM) Sarah Price, MilliporeSigma, will provide an overview of state laboratory testing for Legionella, and discuss state outbreak response.

VII. WRAP UP AND ADJOURN (2:55PM – 3:00PM) Next steps and adjourn.

*This agenda is subject to change without notification

Today’s Agenda:  A Year in Review: Cyclospora Working Group  Cyclospora Outbreak: Broccoli  Sampling for Microbial Methods  Working Group on Development of Cyclospora SMPR  Legionella Overview  State Testing and Outbreak Response: Legionella

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Interested in Supporting the Legionella Initiative in AIMS?

Link to Interest Form

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

AIMS Program Meeting Wednesday, August 30 | 1:00PM – 3:00PM CDT

Thank you to our Advisory Panel Members for supporting the current scope of work in the AOAC AIMS!

Scan to sign into the meeting and get access to meeting materials!

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Today’s Agenda:  A Year in Review: Cyclospora Working Group  Cyclospora Outbreak: Broccoli  Sampling for Microbial Methods  Working Group on Development of Cyclospora SMPR  Legionella Overview  State Testing and Outbreak Response: Legionella

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Interested in Supporting the Legionella Initiative in AIMS?

Link to Interest Form

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

After obtaining a PhD in Food Science and Human Nutrition from the University of Florida, Morgan worked as a PI for the USDA Agricultural Research Service in both Pre- and Post- Harvest areas of food microbiology. He worked in the molecular diagnostics industry for more than 20 years in the design and validation of rapid methods for the detection and typing of foodborne pathogens. He is currently an independent consultant specializing in food safety. Erin Crowley, Ph.D. Erin Crowley is the Chief Scientific Officer at Q Laboratories, Inc in Cincinnati, Ohio. Erin and her R&D team serve the industry as an Expert Laboratory with a primary focus on providing high quality method validation for microbiological rapid detection methods. These validations include Independent laboratory evaluations for pathogen detection, qualitative methods and confirmatory assays for AOAC Official Methods of Analysis, AOAC Research Institute Performance Tested Methods Program, MicroVal and AFNOR NF Certification Programs. Erin frequently presents technical symposia globally on Method Validation, Method Verification, Harmonization and ISO standards development often representing the perspective of the third-party laboratory. In addition to being an active member of the International Association of Food Protection (IAFP) and AOAC, Erin currently serves as Immediate Past-President of the AOAC International Board of Directors, Past-Chair of the AOAC Official Methods Board, a member of the MicroVal Technical Committee (MVTC), ISO TC34/SC9/WG 3 Committee on Method Validation and Chair-elect of the JIFSAN Advisory Council. Erin earned a B.S. from the University of Cincinnati and an M.A. from Tufts University. Morgan Wallace, Ph.D.

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

AIMS- Analytical International Methods & Standards Program

Cyclospora Working Group

Erin S. Crowley, Chief Scientific Officer, Q Laboratories

Morgan Wallace, PhD, Consultant

CYCLOSPORA AGENDA

 I. WELCOME, INTRODUCTIONS AND ANNOUNCEMENTS (1:00PM – 1:05PM)  II. AIMS OVERVIEW AND SUMMARY (1:05PM – 1:15PM)  III. CYCLOSPORA OUTBREAK: BROCCOLI (1:15PM – 1:25PM)  IV. CYCLOSPORA METHOD DEVELOPMENT AND CHALLENGES (1:20PM – 1:40PM)  V. WORKING GROUP ON CYCLOSPORA (1:40PM – 2:20PM)

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In food & agriculture, we set the standard

AOAC CORE SCIENCE PROGRAMS AND INTEGRATED SCIENCE PROGRAMS

Core Science Programs • AOAC science departments/divisions that foundational for all science activities and services provided by AOAC.

Integrated Science Programs & Projects

• Stakeholder Program on Agent Detection Assays (SPADA) • Stakeholder Program on Infant Formula & Adult Nutritionals (SPIFAN) Cannabis Analytical Science Program (CASP) • Food Authenticity Methods Program (FAM) • Gluten & Food Allergens Program (GFA) • Working Group on Folic Acid • Working Group on Acrylamide • Analytical International Methods & Standards Program (AIMS)- Parasites & Viruses

• AOAC Laboratory Proficiency Testing Program

• AOAC Research Institute • Performance Tested Methods SM Program • Reviewed & Recognized Program

• AOAC Standards & Official Methods SM Program

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In food & agriculture, we set the standard

Overview of AIMS program- Cyclospora Working Group

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In food & agriculture, we set the standard

OBJECTIVES OF THE ANALYTICAL INTERNATIONAL METHODS AND STANDARDS PROGRAM (AIMS)  Alternative method development criteria for emerging microbial contaminants (e.g. parasites)

 Validation criteria for the evaluation of alternative (proprietary) methods  Validation where current criteria have challenges  VBNC  Detection without enrichment

 Novel, recently recognized food, feed, and environmental matrices

 Advanced molecular applications, bioinformatics and biotechnology opportunities

 Cutting edge analytical technologies

 Coordinate with other standard setting bodies to avoid duplication of efforts

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In food & agriculture, we set the standard

AIMS

 Determine how we can deal with: Non- culturables, Viable but Not Culturable, Challenging to Culture and Detect Organisms  First organism targeted by AIMS - Cyclospora  Other New and Emerging Contaminants ( Legionella )  Regulatory, Industry (test kit manufacturers and commercial testing labs) and Academia Engagement  Develop SMPR

 Establish definitions  Matrices to cover  Inclusivity/Exclusivity (in silico, using reference materials)  Develop objective performance criteria  Surrogates

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In food & agriculture, we set the standard

CYCLOSPORA AND AIMS

 Industry Presence

 FDA Cyclospora Task Force, April 2023  Hybrid Meeting- OARSA and online  NACMCF Cyclospora Subcommitee  April 2023  Virtual Meeting  IFPA Monthly Town Hall  FDA  AOAC

 IFPA  CPS

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In food & agriculture, we set the standard

CYCLOSPORA AND AIMS

 Working Group

 Currently >30 members  Regular meetings since the 2022 AOAC Annual Meeting  SMPR (Progress to date)  Definitions  Matrices  Exclusivity/Inclusivity  Method Performance Criteria  Surrogates/Positive Control Material

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In food & agriculture, we set the standard

SMPR PROGRESS TO DATE

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In food & agriculture, we set the standard

SMPR PROGRESS TO DATE

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In food & agriculture, we set the standard

SMPR PROGRESS TO DATE

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In food & agriculture, we set the standard

SMPR PROGRESS TO DATE

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In food & agriculture, we set the standard

SMPR PROGRESS TO DATE

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In food & agriculture, we set the standard

SMPR PROGRESS TO DATE

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In food & agriculture, we set the standard

SMPR PROGRESS TO DATE

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In food & agriculture, we set the standard

SMPR PROGRESS TO DATE

• Proposed use of G Block DNA of target sequence(s). • Method developers will likely use the same target regions so can pool resources. • Target DNA will be added to extracted matrix free of naturally occurring Cyclospora . • Traditional number of fractionally positive samples can be used for this portion of the study (not true for spiked matrix study). • Results will be determined either by an arbitrary C t cutoff (e.g. 38 C t or lower for a positive result) or by a commercial method’s algorithm.

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In food & agriculture, we set the standard

SMPR PROGRESS TO DATE

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In food & agriculture, we set the standard

SMPR PROGRESS TO DATE

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In food & agriculture, we set the standard

SMPR ISSUES UNDER DISCUSSION

 Objective Criteria  What are the minimum sensitivities that would make a method useful (X number of oocysts in Y amount of each matrix)?  Theoretical and experimentally derived LOD  How to address sample preparation methods that concentrate a sample (e.g. BAM 19c)?  Benchmarks to judge method performance

 Pure cloned targets or G Blocks without matrix present  Oocysts in centrifuged pellets from real-world matrices  Should 19b serve as the reference method?

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In food & agriculture, we set the standard

SMPR ISSUES UNDER DISCUSSION

 Surrogates  Use to demonstrate sample preparation efficacy?

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In food & agriculture, we set the standard

SMPR ISSUES UNDER DISCUSSION

 Other interested parties  NACMCF  CFIA  ISO

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In food & agriculture, we set the standard

THANK YOU!

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In food & agriculture, we set the standard

Kali Kniel, University of Delaware

Dr. Kniel’s laboratory explores issues of food safety and public health that involve transmission of viruses, protozoa, and pathogenic bacteria. Few laboratories work with this extent of different microorganisms. Specific projects may address survival or transmission routes through various complex food systems. In light of needs related to the FDA FSMA Produce Safety Rule, research includes assessing risks associated with pre-harvest contamination of raw agricultural commodities through water and soil amendments. Research projects focus on survival and persistence in water and poultry litter and subsequent transfer onto the edible portion of the crop. Investigations into novel detection methods as well as biocontrol are also ongoing. Efforts are made to correlate behaviors of different microorganisms, including viral surrogates, with climate or other environmental metadata in our assessments. We also work with nearby government agencies and connect our research to the needs of policy makers. We have contributed data critical to addressing contemporary food safety questions; specifically, prevalence of pathogens in manure and time needed between application of manure and time to harvest. Students on all levels assist with this research.

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

Mauricio Durigan, OARSA FDA/CSAN

Dr. Mauricio Durigan joined FDA/CFSAN in 2016 with the Division of Virulence Assessment (DVA) in the Office of Applied Research and Safety Assessment (OARSA). Dr. Durigan has over 15 years of experience in Molecular Diagnostics and Epidemiology of Parasitic Diseases. Since Dr. Durigan joined FDA, he has been working with the development of streamlined methods to detect C. cayetanensis and the validation of regulatory methods for molecular epidemiology tools. The major project aims the validation of a sensitive method to detect C. Cayetanensis in environmental sources like water matrices. This is a very challenging study that represents major food safety and regulatory issues that should be addressed by the agency. His specialties include Genetics and Molecular Biology, Next-Generation Sequencing, Foodborne Parasitology, Molecular Parasitology, Epidemiology of Parasitic diseases, and Environmental Microbiology.

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

Michael Loewenstein, Q Laboratories

Michael Loewenstein is the current Vice President of Scientific Consulting at Q Labs LLC in Cincinnati, OH. Mr. Loewenstein earned his B.S. in Microbiology, with a research focus in Molecular Genetics and Cellular Physiology from The Ohio State University. After graduation in 2011, Michael obtained a position in Corporate R&D Microbiology at Procter & Gamble before joining Q Labs in January 2019. Throughout the course of his career, Michael has developed a substantial depth of expertise in “end-to-end” Microbiology - from product development and formulation through microbiological control of manufacturing, and he has published several articles on this subject. Michael currently serves in an active volunteer capacity for several professional associations, including: the AOAC CASP Microbial Contaminants Expert Review Panel, the Microbiology Committee and founding member of the Manufacturing Hygiene Task Force of the Personal Care Products Council (PCPC), and the cooling water subcommittee of the Association of Water Technologies (AWT).

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Limitations in Legionella Testing Michael Loewenstein Vice President, Scientific Consulting Q Labs LLC

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Brief Legionella History • Pneumonia outbreak in 1976 at Bellevue-Stratford Hotel • Etiologic agent identified as new waterborne pathogen o Legionella pneumophila serogroup 1 o Now over 50 named Legionella spp. (about half known to cause disease) • Method developed over next few decades to isolate L. pneumophila . o ISO 11731; Water Quality – Enumeration of Legionella

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Legionnaires’ Disease • ~10,000 cases in 2018. • Mortality estimated ~10% • Those at higher risk include: • >50 years old • Current or former smoker • Estimated burden is 1.8-2.7x

• Chronic lung disease (e.g., COPD) • Immunocompromised • Underlying illness (e.g., diabetes, renal or hepatic failure) • Recent healthcare stay

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Is Legionella Testing Required? • Increasingly expected in recent years as part of a WMP.

• A few jurisdictions require testing – most notably New York City and New York State

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Status of Legionella Testing • Generally accepted standard is solid culture o ISO 11731:2017; Water Quality – Enumeration of Legionella • Molecular methods not well understood o Some guidance docs mention potential as a “negative screen” • Water Treatment Industry, broadly speaking, not familiar with standards development/method validation

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Limitations of Solid Culture

environmental organisms difficult to culture

ISO 11731 CDC (01/2005)

employs selective agars/treatment steps

method, as written, is highly subjective

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

High Backround

• Selective pre-treatment (acid/heat)

• Antibiotics

• Literature estimates range up to 50%+ initial Legionella population may be lost

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Challenges with Legionella spp. Testing • Long time to result (7-14+ days) • Concerns about consistent quantitative accuracy

• Concerns about inclusivity • Concerns about VBNC

• Infectious dose is unknown. Setting acceptable limits is up to the Water Management Team. o CDC: “There is no ‘safe’ level or type of Legionella”

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

ISO 11731 has poor qualitative detection efficiency

0% 20% 40% 60% 80% 100%

CDC recommends action (potable)

system is poorly controlled (10-fold increase) system uncontrolled (100-fold increase)

CDC recommends action (non-potable)

0.2

0.77

1.4

4.8

111

114

1300

7400

Inoculum (CFU/mL)

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Culture often misses Legionella spp.

80%

68%

60%

52%

40%

33%

20%

16%

16%

15%

0%

Not Detected

Culture

qPCR

Legionella spp.

L. pneumophila SG1

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Challenges Facing Alternative Methods • Industry is hesitant due to lack of data/regulatory support • CDC Guidance: “(Alternative Methods)…should be validated against a standard method by a third party…” • Challenges of alternative methods: o CFU comparison to alternative method units o Differentiation between viable and non-viable cells o Determining appropriate validation scheme given reference method challenges

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 •

Questions?

Sarah (Sally) Powell Price, MilliporeSigma

Joined MilliporeSigma (Merck KGaA Darmstadt) in 2020 as a Regulatory Consulting Expert for Public Health with a focus on food, water, and environmental pathogen testing. Previously, she served as Director of Lab Operations at Puretech Health, and Lab Supervisor at the New York City Department of Health Public Health Laboratory supporting food and legionella outbreak response testing. She has a Bachelor's degree in Biology from Hamilton College, MSc in Microbiology & Immunology from James Cook University in Australia and did continuing coursework in public health at Harvard TH Chan School of Public Health. She co-chairs the International and Governmental Relations Committee for the Association of Food and Drug Officials (AFDO), is a member of the Association of Public Health Laboratories (APHL), WHO Food Safety Communities of Practice, and currently sits as an Advisory Panel member for several working groups with AOAC International

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 •

Interested in Supporting the Legionella Initiative in AIMS?

Link to Interest Form

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA