AIMS Meeting Book_MYM2023

AOAC INTERNATIONAL Analytical International Methods and Standards Program

AOAC Midyear Meeting Tuesday, March 14, 2023 8:30 AM – 11:30 AM ET Draft Agenda

MEETING AGENDA

I. WELCOME, INTRODUCTIONS AND ANNOUNCEMENTS (8:30AM – 8:35AM) Allison Baker, AOAC INTERNATIONAL, will welcome attendees and lead introductions.

II. PROGRAM OVERVIEW (8:35AM – 8:45AM) Erin Crowley, Q Laboratories and Morgan Wallace will provide a brief overview of recent work done in the AIMS working group. III. THA FDA PERSPECTIVE (8:45AM – 9:00AM) Alexandre da Silva, FDA, will present an overview of the current work being done by the FDA Cyclospora Taskforce, the benefits of AOAC AIMS, and the relevance that AIMS work has to ISO TC34 and FDA. IV. WORKING GROUP SESSION: CYCLOSPORA (9:00AM – 10:00AM) AIMS Co-Chairs, Erin Crowley and Morgan Wallace, will lead the group in a working group meeting that is developing an SMPR for the Detection, Identification, and Characterization of Cyclospora cayetanensis . V. INTRODUCTION TO LEGIONELLA (10:10 AM – 10:25 AM ) Michael Lo e wenstein, Q Laboratories, will introduce the challenge of Legionella testing, confirmation of positive results and an overall lack of standards for validating methods. VI. LEGIONELLA: CHALLENGES AND NEEDS IN THE WATER TREATMENT INDUSTRY (10:25 AM – 10:35 AM ) Dave Christophersen, Dave Christophersen Consulting LLC; Certified Water Technologist, will provide an overview of the challenges of Legionella and the needs of the Water Treatment Industry. VII. LEGIONELLA: THE CONTRACT LABORATORY EXPERIENCE (10:35 AM – 10:45 AM ) Michael Lo e wenstein, Q Laboratories, will provide an overview of the challenges of Legionella from the contract laboratory perspective.

VIII. LEGIONELLA: CHALLENGES FOR THE METHOD DEVELOPER (10:45 AM – 10:55 AM ) Christophe Quiring, Bio-Rad, will provide an overview of the challenges of method development for Legionella.

*This agenda is subject to change without notification Break from 10:00am – 10:10am

IX. ROUNDTABLE DISCUSSION- (10:55 AM – 11:25 AM ) Panelists will participate in a facilitated Q&A discussion about Legionella and respond to questions from the audience.

X. WRAP UP AND ADJOURN (11:25 AM – 11:30 AM )

*This agenda is subject to change without notification Break from 10:00am – 10:10am

Erin Crowley, Chief Scientific Officer, Q Laboratories 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. 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.

Dr. Alexandre da Silva. MSc., Ph.D., FDA Dr. Alexandre da Silva has initiated his career at the Centers for Disease Control and Prevention (CDC) as a Junior Parasitologists in 1992. At CDC he built a parasitology diagnostic program based on molecular and parasitological methods, as well as telediagnosis. Through this program, a robust network for diagnostic response in parasitic diseases was implemented at health departments in the US states and territories. During this period, he developed and implemented several molecular and parasitological methods for

diagnosis of parasitic diseases caused by Microsporidia, Plasmodium (etiologic agent of malaria), Leishmania , the life-threating free-living ameba Naegleria fowleri , Trypanosoma cruzi , Cryptosporidium , Angiostrongylus cantonensis and Cyclospora cayetanensis . He discovered three new species of Cyclospora , isolated from simians originated from Ethiopia and initiated the genomics program on

Cyclospora cayetanensis at CDC with funds from the Advanced Molecular Detection initiative. This program was funded at $500,000/year for 5 years. In September of 2014, Alex joined the U.S. Food and Drug Administration (FDA) to build and lead a program focused on foodborne parasitology at CFSAN. He currently leads the FDA Cyclospora task force to address knowledge gaps, develop prevention-based approaches and enhance the FDA’s response to Cyclospora issues. He is the elected Convener Support Team for the ISO Technical Committee 34, Subcommittee 9 (ISO TC34/SC9) /WG6 (Foodborne Parasites) for a term of 3 years starting in 2021 and the lead of an ISO project to develop an international standard for the detection of C. cayetanensis in produce under ISO TC34/SC9/WG6. Alex has authored/co-authored more than 130 publications and book chapters in biological studies, detection of parasites, surveillance, and outbreak investigations of parasitic diseases. He serves as associated editor on editorial boards of reputable scientific journals such as Journal of Clinical Microbiology, Applied and Environmental Microbiology and Journal of Food Protection.

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).

Dave Christophersen, CWT (Certified Water Technologist), Dave Christophersen Consulting LLC Knowledgeable in wastewater treatment, boiler water treatment and pretreatment, cooling water, and membrane technologies. Worked in many industries including oilseed, steel, manufacturing, power, chemical, mining, commercial & institutional, and others. Functions included business management, field management, technical support, sales and marketing, chemical product development and product formulation of water treatment chemicals, and training of field and site personnel.

45 years of experience in the water treatment industry including managing water treatment programs for industrial sites for boilers, cooling systems, wastewater systems, and membranes. Operated many field pilots for various chemical and equipment applications along with performing laboratory treatability studies. Conducted trainings on many topics of water treatment including water treatment chemistry, boiler and cooling systems and treatment, wastewater treatment and jar testing, membranes systems and cleaning procedures, plant operations, and industrial services.

Christophe Quiring, Bio-Rad Christophe has a master’s degree in applied microbiology and biological engineering. He began his career in 1994 as a scientist with the French company Solabia- Biokar Diagnostics. In this position, he developed culture media and methods for a wide range of pathogens and quality indicators for food and water testing. Christophe joined Bio-Rad’s Food Science Division in 2004 as R&D Manager. He

then broadened his field of action to molecular technologies applied to food diagnostic. He currently holds the position of Business Unit R&D Manager and leads a team in charge of the design of new products based on both cultural techniques and PCR and ddPCR. During his career, Christophe has developed many methods which have been validated according to the ISO 16140 standard and AOAC guidelines. Christophe is actively Involved ISO and CEN working groups regarding Salmonella, Listeria and enrichments steps. He is also member of the AOAC Expert Review Panel for Microbiology Methods for Food & Environmental Surfaces.

Analytical International Methods and Standards (AIMS) Program Session Tuesday, March 14 | 8:30AM – 11:30AM ET

Thank you to our Advisory Panel who support the current scope of work in AIMS!

Scan to sign into the meeting and access meeting materials!

AOAC INTERNATIONAL Midyear Meeting March 13 –17, 2023 | Gaithersburg, MD

AOAC INTERNATIONAL HOUSEKEEPING ITEMS

This mee t ing will be conduct ed in accordance wit h AOAC Policies & Procedures

AOAC INTERNATIONAL Midyear Meeting March 13 –17, 2023 | Gaithersburg, MD

AOAC INTERNATIONAL HOUSEKEEPING ITEMS

This mee t ing is be ing recorded

Please mut e your lines and unmut e only when speaking

AOAC INTERNATIONAL Midyear Meeting March 13 –17, 2023 | Gaithersburg, MD

AIMS- Analytical International Methods & Standards P rogram Erin Crowley, Q Laboratories Morgan Wallace, Retired

Co-Chairs

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 o 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

AOAC INTERNATIONAL Midyear Meeting March 13 –17, 2023 | Gaithersburg, MD

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Additional Challenges in Validation of New Microbiology Technologies • Establish validation criteria (SMPRs and guidance) for novel methods • Tests for microbes that can’t be enriched o Viruses o Parasites • Microbial enumeration methods • How to standardize production of spiked samples for validation studies • Methods with more than one purpose (ddPCR, detection plus typing, risk testing) • Indicator organism testing • Strain typing methods

AOAC INTERNATIONAL Midyear Meeting March 13 –17, 2023 | Gaithersburg, MD

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Cyclospora Background • Cyclospora cayetanensis - coccidian protozoan parasite

• Newly recognized – described 1993-1994 as a human gastrointestinal pathogen

• Produces oocysts that are resistant to harsh environmental/chemical conditions (infectious). May take days to weeks to become sporulated and infectious • Among the Cyclospora species, only Cyclospora cayetanensis is known to infect humans; other species are associated with infections in other animals • Cyclospora has a complex life cycle and can only multiply within the infected hosts

AOAC INTERNATIONAL Midyear Meeting March 13 –17, 2023 | Gaithersburg, MD

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Life Cycle

AOAC INTERNATIONAL Midyear Meeting March 13 –17, 2023 | Gaithersburg, MD

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Cyclospora Background • Previously considered to be a pathogen acquired during childhood in developing nations • In the U.S. and Europe, cyclosporiasis associated with travel or consumption of contaminated imported foods. • However, in recent years, the U.S. has seen an increase in cases and positive samples associated with domestic produce o Contaminated berries, fresh cilantro, basil and, more recently, ready-to-eat bagged salads.

AOAC INTERNATIONAL Midyear Meeting March 13 –17, 2023 | Gaithersburg, MD

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Cyclospora-Public Health Impact • In the last three years cases have increased approximately 300%

• More Prevalent? • Better Detection? • Outbreaks of Cyclosporiasis generally occur during the warmer months of May – September for the northern hemisphere and November – March for the southern hemisphere.

AOAC INTERNATIONAL Midyear Meeting March 13 –17, 2023 | Gaithersburg, MD

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AIMS • Determine how we can deal with: Non- culturables, Viable but Not Culturable, Challenging to Culture and Detect Organisms o First organism targeted by AIMS - Cyclospora

o Other New and Emerging Contaminants ( Legionella ) o Regulatory, Industry (test kit manufacturers and commercial testing labs) and Academia Engagement o Develop SMPR – Establish definitions – Matrices to cover – Inclusivity/Exclusivity (in silico, using reference materials) – Develop objective performance criteria – Surrogates

AOAC INTERNATIONAL Midyear Meeting March 13 –17, 2023 | Gaithersburg, MD

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Testing Landscape • Early laboratory detection methods were limited to direct microscopy and staining methods • This is not a perfect method, since there is no way to increase the concentration of the parasite in the sample to “detectable levels”

• These methods lack the required sensitivity for the detection of low concentration of oocysts in food matrices

• It can be challenging to differentiate Cyclospora from other Coccidian parasites in a sample

• Molecular and other advanced techniques for detecting and typing Cyclospora are critical for understanding and containing its spread

AOAC INTERNATIONAL Midyear Meeting March 13 –17, 2023 | Gaithersburg, MD

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Testing Landscape • Several efforts have been made to develop molecular detection methods for both food and environmental process water • Still several significant knowledge and data gaps that hamper the implementation of effective measures to prevent the contamination of produce with the oocysts of this parasite • These methods have been used almost exclusively for to assist epidemiological investigations and surveys to estimate the prevalence of C. cayetanensis in commodities and growing regions.

• A gap exists in commercially available methodology and reference materials (stable oocysts)

AOAC INTERNATIONAL Midyear Meeting March 13 –17, 2023 | Gaithersburg, MD

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FDA Bacteriological Analytical Manual Revised in 2016; Recently updated as of 4/22 1. Produce wash procedure 2. Isolation of DNA from produce wash

3. Identification by PCR amplification 4. Custom primers, probes and DNA 5. Currently validated for leafy greens such as lettuces, cilantro, and basil; soft fruit such as raspberries, blackberries, or strawberries; and whole vegetables such as beans or peas. Extended to blueberries (4/22)

AOAC INTERNATIONAL Midyear Meeting March 13 –17, 2023 | Gaithersburg, MD

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FDA Bacteriological Analytical Manual

AOAC INTERNATIONAL Midyear Meeting March 13 –17, 2023 | Gaithersburg, MD

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Cyclospora and AIMS o First Order of Business – Develop SMPR  Establish definitions  Matrices to cover  Inclusivity/Exclusivity • in silico • using reference materials)  Develop objective performance criteria  Are surrogates and/or controls needed?

• Extraction • Detection  Standardized reference materials

AOAC INTERNATIONAL Midyear Meeting March 13 –17, 2023 | Gaithersburg, MD

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Cyclospora and AIMS • Working Group will continue today • Currently >30 members • Regular meetings since the 2022 AOAC Annual Meeting • SMPR (Progress to date) o Definitions o Matrices o Exclusivity/Inclusivity o Method Performance Criteria o Surrogates/Positive Control Material

AOAC INTERNATIONAL Midyear Meeting March 13 –17, 2023 | Gaithersburg, MD

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Thank you!

AOAC INTERNATIONAL Midyear Meeting March 13 –17, 2023 | Gaithersburg, MD

SARS COV-2

• Inclusivity o There are 15,764 accessions. Additional genome sequences may be included if desired. 3.1.1.1 Report inclusivity for the primers and for the probe as the number of genomes with a perfect match sequence out of the total number analyzed and report the genomes with mismatches. For example, “of the 15,764 target genomes analyzed, 15,702 were perfect matches for the forward primer. Those with mismatches were… The nature of the mismatches were…”

AOAC INTERNATIONAL Midyear Meeting March 13 –17, 2023 | Gaithersburg, MD

SARS-COV-2

• Exclusivity o Report exclusivity as the number of genomes with no matching sequences out of the number of near neighbor genomes analyzed and report close matches where binding could occur. For example, “of the X near neighbor genomes analyzed against the primer sequence, Y showed no matching sequences. Those showing potential for binding by the primer included…”

AOAC INTERNATIONAL Midyear Meeting March 13 –17, 2023 | Gaithersburg, MD

SARS-COV-2

AOAC INTERNATIONAL Midyear Meeting March 13 –17, 2023 | Gaithersburg, MD

Variola Virus SMPR 2014.006

AOAC INTERNATIONAL Midyear Meeting March 13 –17, 2023 | Gaithersburg, MD

Variola Virus SMPR 2014.006

AOAC INTERNATIONAL Midyear Meeting March 13 –17, 2023 | Gaithersburg, MD

AOAC INTERNATIONAL Midyear Meeting March 13 –17, 2023 | Gaithersburg, MD

AOAC INTERNATIONAL Midyear Meeting March 13 –17, 2023 | Gaithersburg, MD

AOAC INTERNATIONAL Midyear Meeting March 13 –17, 2023 | Gaithersburg, MD

FDA Cyclospora Taskforce, the benefits of AOAC AIMS, and the relevance that AIMS work has to ISO TC34 SC9 and FDA

Alexandre J. da Silva, MSc, PhD SBRBPAS Expert, Senior Parasitology Advisor FDA- CFSAN- Office of Food Safety-Division of Produce Safety March 14 th , 2023

Cyclosporiasis • Cyclosporiasis outbreaks have been associated with the consumption of fresh fruits and vegetables around the world, including in the U.S. • According to CDC there were 39 cyclosporiasis outbreaks from 2000 to 2017. A food vehicle of infection was identified (or suspected) for 17 of the 39 outbreaks. • According to the Centers for Disease Control and Prevention (CDC), there have been roughly 4,600 reported domestically-acquired cases of cyclosporiasis from 2018 to 2021. • During the spring and summer months there is often an increase in cyclosporiasis acquired in the United States (i.e., “domestically acquired”). • The exact timing and duration of these seasonal increases in domestically acquired cyclosporiasis can vary, but reports of illnesses tend to increase starting in May. • The number of cases have peaked between June and July in past years. https://www.fda.gov/food/foodborne-pathogens/cyclosporiasis-and-fresh-produce Cyclospora Prevention, Response and Research Action Plan | FDA Cyclosporiasis Outbreak Investigations — United States, 2021 (cdc.gov)

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C. cayetanensis

• While there are species of Cyclospora that infect animals, however C. cayetanensis only infects humans. • Cyclosporiasis is also endemic, or commonly found among, certain populations around the world, and was previously thought to occur only in produce grown in those regions. • The parasite must be shed by an infected person (1). It must remain in the environment for 1-2 weeks before becoming infective to other humans (2). Once infective, people can become sick by ingesting contaminated food or water and continue the cycle (3). •

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2

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To date, there are 21 valid species of Cyclospora.

Cyclospora species

Host type Host species

References

C. cayetanensis

Human Simian Simian Simian Eastern mole Eastern mole Simian

Homo sapiens Papio anubis Colobus guereza

J Parasitol. 1994;80:625–9

C. papionis

Emerg Infect Dis. 1999 :651-658 Emerg Infect Dis. 1999 :651-658 Emerg Infect Dis. 1999 :651-658

C. colobi

C. cercopitheci

Cercopithecus aethiops

C. duszynskii

Scalopus aquaticus

Syst Parasitol. 2018;95(2-3):271-279

C. yatesi

Scalopus aquaticus

Syst Parasitol. 2018;95(2-3):271-279

C. macacae C. schneideri

Macaca mulatta

Parasitol Res. 2015;114(5):1811-1816 Mem Inst Oswaldo Cruz. 2005 2 :103-10

Snake

Anilius scytale scytale

C. Angimurinensis C. parascalopi C. ashtabulensis C. megacephali

Rodent

Chaetodipus hispidus Parascalops breweri Parascalops breweri Scalopus aquaticus

J Parasitol 1990 76: 325-331 Parasitology 1989 75: 508-513 Parasitology 1989 75: 508-513 J Protozool 1988 35: 223-226

Mole Mole Mole Mole

C. talpae

Talpa europaea

Folia Parasitol (Praha) 1968 15: 275-277

Lizard

Ninia sebae sebae

Ann Trop Med Parasitol 1965 59: 159- 163 Bull Soc Path Exot 1933 26: 415-420

C. niniae

C. viperae

Snakes

Vipera aspis, Coluber scalaris, Coronella austriaca, Natrix viperinus

C. scinci

Snake

Scincus officinalis

Bull Mus Natn Hist Nat Paris 1924 30: 100-101 Bull Soc Path Exot 1924 17: 871-873 Bull Soc Path Exot 1924 17: 868- 871 Bull Mus Natn Hist Nat Paris 1924 30: 501-502 Enteritis des Maulwurfs. Arb K Gesundheitsamte 1902 18: 378- 416 Arch Zool Exp Gen 1881 9: 387-404

C. tropidonoti

Snakes

Natrix natrix, Natrix stolata

C. babaulti C. zamenis

Snake Snake

Vipera berus

Coluber viridiflavus viridiflavus

C. caryolytica

Mole

Talpa europaea

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millipede

Glomeris sp.

C. glomericola

Oocysts, Sporocysts, Sporozoites

Cyclospora

Eimeria

Cryptosporidium

Isospora

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Progress Updates Entered in CPTTS (FDA Cyclospora Prevention, Response and Research Action Plan)

Reports Generated to CFSAN (Center and Offices)

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FDA Cyclospora Prevention, Response and Research Action Plan

Priority area A - Prevention Action

Priority area B - Response Action

Expand the capacity of FDA, states, foreign governments, and academia to sample and test for C. cayetanensis (on hold until BAM method is updated) Develop and provide training and updates for FDA, state and foreign regulators on Cyclospora. Provide technical support for Cyclospora- related investigations. Continue to engage with the states regarding consistent control strategies for C. cayetanensis .

Develop and deliver education and outreach on prevention measures for growers in collaboration with industry, academia, regulators and other partners (several webinars were delivered in 2022). Engage with industry, academia, and test kit companies to develop rapid tests kits to detect C. cayetanensis , and to develop industry best practices that can be used to test for C. cayetanensis. Work with industry to research and identify practical ways of controlling C. cayetanensis in the environment and on produce.

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FDA Cyclospora Prevention, Response and Research Action Plan

Priority area C - Filling Knowledge Gaps Action Action Evaluate trends and distribution of

Advance the development of genotyping methods for C. cayetanensis in clinical, food and environmental samples.

cyclosporiasis cases in the US (a proposal for a prevalence study was submitted to CDC NHANES; data from NORS was compiled in a manuscript) Work with the National Advisory Committee on Microbiological Criteria for Foods (NACMCF) to address knowledge gaps related to Cyclospora (ongoing and NACMCF should provide feedback in 2023). Collaborate with industry, academia, states, local and foreign partners to promote research to better understand the prevalence of C. cayetanensis in agricultural water

Convene a Joint Institute for Food Safety and Nutrition (JIFSAN) Research Meeting on Cyclospora (replaced with a new objective)

Explore the role of wastewater in contributing to C. cayetanensis contamination in produce.

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New Objectives for The FDA Cyclospora Project starting in FY 2023 • Fostering conversations with Cyclospora cayetanensis experts outside the FDA: Explore future collaborations, share data and define research priorities.

• Repository of C. cayetanensis oocysts to perform high priority Agency activities.

• Fostering studies towards in vitro propagation, viability and infectivity of C. cayetanensis.

• Strengthening Root Cause Analysis of Cyclosporiasis Outbreaks.

• Science-based approaches to prevent contamination of produce with C. cayetanensis oocysts.

• Annual Prioritization of For-Cause Inspections.

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Advancements

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NP ISO 9811 ( C. cayetanensis ) • The current draft of NWIP 9811 is based on the FDA BAM 19b method and include a qPCR targeting the C. cayetanensis 18srRNA gene. • The protocol will require modifications in the qPCR step. • The consensus among members of WG6 was to replace the current 18SrRNA-based qPCR to increase specificity. • The modified draft will be submitted to TC34/SC9 for approval. • A interlaboratory study will be planned to validate the new C. cayetanensis ISO standard.

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Thank you

Alexandre J. da Silva, MSc, PhD SBRBPAS Expert-Senior Parasitology Advisor-FDA/CFSAN/OFS Alexandre.dasilva@fda.hhs.gov

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What is Legionnaires Disease?

 Caused by Legionella spp.

 More deadly presentation of legionellosis characterized by pneumonia. • Distinct from Pontiac Fever which is typically self limiting, non-pneumonia presentation

 Named after the first known outbreak discovered after a convention meeting of Legionnaires at the Bellevue-Stratford Hotel in Philadelphia in 1976.

 Initial outbreak species identified as/named Legionella pneumophila (serogroup 1).

 Now over 50 known species, over half have been implicated in infection/disease

Legionnaires’ Disease Quick Facts (ref. CDC)

 Legionnaires’ Disease is underreported. A study by Collier et al estimates that the true number of Legionnaires Disease cases is 1.8-2.7 times higher than currently reported.

 In general, Legionnaires’ Disease is not spread person to person. It is spread by inhaling contaminated water droplets.

 Approximately 10% of people diagnosed with Legionnaires’ Disease will die.

 People at increased risk include: • Being over 50 years old • Current or former smokers • People with chronic lung disease • Being immunocompromised • People with cancer • People with other chronic health issues (e.g., diabetes, kidney failure, liver failure, etc.)

Legionnaires Disease is on the Rise

Source: CDC Surveillance Report - Crude incidence rates of reported confirmed cases of Legionnaires’ disease by year—NNDSS United States, 2000–2017.

Facilities Encouraged to Test for Legionella spp.

 Very little regulation in the US (more in the EU/UK)  Various standards/guidance documents recommend having a Water Management Program (WMP). • Utilizes HACCP principles to identify critical control points, and risk mitigation measures • Say that measures should be “validated”, but typically stop short of saying how that is to be accomplished · Virtually everyone agrees, testing for Legionella spp. is the only way to properly validate

Challenges with Legionella spp. Testing

 Cultural method: ISO 11731

• 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. • CDC: “There is no ‘safe’ level or type of Legionella”

Challenges Facing Alternative Methods

 Acceptability of Alternative Methods?

 Industry is hesitant due to lack of data.

 CDC Guidance: • “(Alternative Methods)…should be validated against a standard method by a third party…”

• Need a framework for the most modern methods

 Challenges of alternative methods: • Drawing relationships between alternative method result units to CFU for Water Treatment Industry

• Differentiation between viable and non-viable cells

• Determining appropriate validation scheme when cultural reference method may not be consistently, quantitatively accurate.

Dave Christophersen

Water Management Plan Water Safety Plan HACCP H azard A nalysis

C ritical C ontrol P oints

Should I Test for Legionella?  Expert opinion on routine Legionella testing are moving towards more testing  Although not required, testing is highly recommended to verify control practices  Random testing in the absence of an assessment and management program is unwise, and annual “spot-checks” are rarely if ever meaningful  Legionella testing must be combined with other monitoring strategies  Perform testing within the context of a water management plan with control limits and a define contingency plan  Rapid methods improve water safety, especially in remediation

AOAC Can Help Industries Current Problem Culture  Culture is accepted, difficult to run, slow, and the known standard  Culture deficiencies not well known  Colony Forming Units (CFU’s) well established  CFU’s used to qualify testing labs qPCR  Modern qPCR capabilities not well known  Analysis is on genomic units (GU’s)  Lacking certification standards  Lacking Alert and Action guidelines based upon GU’s

 Culture has testing certification standards  Alert and Action somewhat established based upon CFU’s

Quickly create a certification for qPCR testing (months not years) Water treater needs a fast method as good or better than the ISO 11731 culture method Water treaters desire options on test methods, and one that is AOAC certified

Independent Lab Perspective Validation of Cultural and Molecular Alternatives

Why is the need so critical?

environmental organisms difficult to culture

ISO 11731 CDC (01/2005)

employs selective agars and treatment steps

method, as written, is highly subjective

Why are alternative methods not more widely used?

 Industry relies heavily on external guidance • Governmental bodies (e.g., CDC, DOH, etc.) • Independent laboratories

• professional organizations (e.g., ASHRAE, ASHE, AWT, etc.)  Independent method validation is new to the industry.  ISO is currently the only option, and gaps exist in current validation standards. • Detection/quantification vs confirmation/typing  AOAC brings a breadth and depth of experience to meet a pressing industry need.

Click to edit Master title style

Challenges for Legionella PCR implementation AOAC MID-YEAR MEETING – March 2023 Christophe Quiring

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Click to edit Master title style ISO 11731 culture media v PCR technologies

Traditional method (Microbiological culture)

q PCR

dPCR

Speci ficiy

Sensi tivi ty

Time to negative resul ts

3 days

4 hours

7 hours

Time to posi tive resul ts

14 days

4 hours

7 hours

No effect of accompanying flora

User experience

VBNC detection

Dead cel l s no detection

*

*

Expression of the resul ts

CFU

GU

GU

National Regulations Recogni tion

*with v-PCR

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Click to edit Master title style PCR Legionella Implementation – Technical Challenges

Dead cells: v-PCR available, using of an easy, fast and safe reagent

For quantification: need of a Primary Standard. Stability in time ? Slighest variation in time How to confirm PCR results ? Compared to a non-optimized culture method

Technical Challenges

Dead cells

Viable but cannot form colonies on agar plates (VBNC)

Viable and can form colonies on agar plates

Culture ISO 11731

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Click to edit Master title style PCR Legionella Implementation – Regulatory Challenges

No correlation between CFU(culture) & GU (PCR) Comparison between the international ‘gold’ standard, the culture method, the ISO 11731 (but media non optimized) and a more specific, sensitive method, the PCR Easier to determine it for Legionella pneumophila than Legionella spp. Need of Primary Standard to calibrate the Quantification Standards New technology, digital PCR, can overcome those challenges : absolute quantification (no use of Quantification Standards) Recognition by national regulations as an official tool for routine analysis: slow-move but, with the increase, year after year of Legionella outbreaks and the global increase of temperature and humidity, awareness is rising.

Regulatory Challenges

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