AOAC Annual Meeting_AIMS

AOAC Annual Meeting Sunday, August 28, 2022 | 8:00AM – 11:00AM MT

MEETING AGENDA

I. WELCOME, INTRODUCTIONS AND ANNOUNCEMENTS (8:00AM – 8:05AM) Palmer Orlandi, Deputy Executive Director and Chief Science Officer, AOAC INTERNATIONAL

II. PROGRAM REVIEW & PROCESS OVERVIEW (8:05AM – 8:20AM) Erin Crowley, Q Laboratories and Morgan Wallace, Rheonix

III. GUEST SPEAKER: GENETIC DIVERSITY OF HAV AND NOROVIRUS (8:20AM – 8:40AM) Dr. Jacquelina Woods, FDA GCSL

IV. GUEST SPEAKER: TAXONOMY AND PHYLOGENETICS OF CYCLOSPORA (8:40AM – 9:00AM) Dr. Alexandre da Silva, US FDA-Center for Food Safety and Applied Nutrition

V. WORKING GROUP SESSION (9:00AM – 10:50AM) Led by AIMS Co-Chairs, Erin Crowley, Q Laboratories and Morgan Wallace, Rheonix

VI. WRAP UP AND ADJOURN (10:50AM – 11:00AM) Palmer Orlandi, Deputy Executive Director and Chief Science Officer, AOAC INTERNATIONAL

06/27/22 Version 1 – Subject to Change Without Notice *Item requires a live vote

AIMS Advisory Panel Members

Forms the AOACAIMS AdvisoryPanel which advises on programdirection and priorities

SUPPORTFORCURRENT SCOPEOFWORKIN AIMS IS MADEPOSSIBLE BYTHEFOLLOWING:

In Food & Agriculture, We Set the Standard

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, Scientific Director of Applied Markets, Rheonix Morgan Wallace is the Scientific Director of Applied Markets at Rheonix where he is a co-developer of the Listeria PatternAlert assay which enables rapid identification of recurring Listeria in food facilities. He also supports the Rheonix Beer SpoilerAlert assay for identifying spoilage organisms in beer. Throughout his career, Morgan has worked extensively with industry, validating organizations, and governments developing and supporting rapid methods in food and beverage safety, quality, and methods for pathogen strain typing. Prior to working for Rheonix, Morgan

managed the validations group of DuPont Qualicon and was a PI for the USDA Agricultural Research Service in both Pre- and Post-Harvest areas of food microbiology. Morgan earned a BA in Political Science, a BS in Microbiology and Cell Science, and a Ph.D. in Food Science and Human Nutrition with an emphasis on food microbiology, all from the University of Florida. He also is a former United States Army Medic.

Jacquelina Woods, Ph.D., CFSAN GCSL Jacquelina Woods is a research microbiologist for CFSAN’s Gulf Coast Seafood Laboratory with over 20 years’ experience in the fields of virology and microbiology. Dr. Woods is a recognized expert in the areas of enteric virus extraction, detection, characterization, and enumeration in food and environmental samples. She has developed and validated sensitive and effective virological methods, which are currently being used in foodborne outbreak investigations and surveillance.

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.

AIMS- Analytical International Methods & Standards Program

Erin Crowley, Q Laboratories Morgan Wallace, Rheonix

Co-Chairs

AOAC Annual Meeting Sunday, August 28, 2022 | 8:00AM – 11:00AM MT

MEETING AGENDA

WELCOME, INTRODUCTIONS AND ANNOUNCEMENTS (8:00AM – 8:05AM) Palmer Orlandi, Deputy Executive Director and Chief Science Officer, AOAC INTERNATIONAL PROGRAM REVIEW & PROCESS OVERVIEW (8:05AM – 8:20AM) Erin Crowley, Q Laboratories and Morgan Wallace, Rheonix GUEST SPEAKER: GENETIC DIVERSITY OF HAV AND NOROVIRUS) (8:20AM – 8:40AM) Dr. Jacquelina Woods, FDA GCSL GUEST SPEAKER: TAXONOMY AND PHYLOGENETICS OF CYCLOSPORA (8:40AM – 9:00AM) Dr. Alexandre da Silva, US FDA-Center for Food Safety and Applied Nutrition WORKING GROUP SESSION (9:00AM – 10:50AM) Led by AIMS Co-Chairs, Erin Crowley, Q Laboratories and Morgan Wallace, Rheonix WRAP UP AND ADJOURN (10:50AM – 11:00AM) Palmer Orlandi, Deputy Executive Director and Chief Science Officer, AOAC INTERNATIONAL

AOAC INTERNATIONAL 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

Overview • ISPAM History- How did we get here? • First of many- Non-culturables, Viable but Not Culturable o New and Emerging contaminants o First will be Cyclospora • Industry and Regulatory Engagement o CDC Typing Method o FDA BAM Chapter 19B o NACMCF • Technology Applications- DDPCR, V- DDPCR • Objectives of WG o Validation Criteria o Suitable Reference Material

AOAC INTERNATIONAL 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

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ISPAM History – International Stakeholder Panel on Alternative Methods • Formed in 2011 o Initial charter was to: – develop harmonized, internationally accepted standard validation guidelines for alternative (rapid) chemical and microbiological methods

– avoid duplication of efforts for the validation of proprietary methods

o Charter expanded to address a variety of other high-profile needs through the formation of working groups

• Sunset in 2019 after many accomplishments

AOAC INTERNATIONAL 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

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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 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

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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 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

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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 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

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Life Cycle

AOAC INTERNATIONAL 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

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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 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

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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 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

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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

• Techniques for detecting and typing critical for understanding and containing spread

AOAC INTERNATIONAL 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

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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 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

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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 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

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FDA Bacteriological Analytical Manual

AOAC INTERNATIONAL 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

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CDC- Advanced Molecular Detection and Prototype Typing

AOAC INTERNATIONAL 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

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2021-2023 National Advisory Committee on Microbiological Criteria For Foods (NACMCF)

AOAC INTERNATIONAL 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

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International Fresh Produce Association- Virtual Town Hall • Dr. Max Teplitski, Chief Science Officer, IFPA • Key Points: 1. We need to confirm the routes of human fecal contamination

2. Establish mitigation strategies

3. Develop better analytical methods

AOAC INTERNATIONAL 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

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Progress To Date • Currently 27 members of the WG

• Meetings every other Friday for 90 minutes- 5 meetings to date

• Formed a Definitions Subgroup o Identified definitions to include in SMPR (in meeting book) o Harmonized definitions-ISO and FDA • Submission of joint symposia with FDA, AOAC and IAFP – Virus and Parasites PDG for IAFP 2023

AOAC INTERNATIONAL 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

Thank you!

AOAC INTERNATIONAL 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

Jacquelina Woods, PhD FDA CFSAN OFS DSST AOAC Annual Meeting August 28, 2022

Genetic Diversity of Hepatitis A Virus and Norovirus

Enteric Viruses

• Associated With Gastroenteritis • Caliciviruses

• Viruses associated with systemic infections • Enteroviruses • Parechoviruses • Viruses associated viral hepatitis • Hepatitis A virus

• Noroviruses • Sapoviruses

• Astroviruses • Adenoviruses 40, 41

Hepatitis A Virus

Why Hepatitis A Virus (HAV)

• Much of the world--HAV infections are endemic • Infected in early childhood and virtually all adults are immune • In developed countries, few persons are infected in early childhood- most adults remain susceptible to HAV-unless vaccinated • Virus shedding starts 10-14 days before the onset of symptoms

Hepatitis A Virus • WHO worldwide 2010 • 14 million cases 30,000 deaths • Acute infection of liver • Asymptomatic or subclinical in children under 5 • Symptomatic in older children and adults • Incubation 14-20 days • Prolonged or relapsing symptoms • Acute liver failure • 0.015-0.15% cases

Hepatitis A Virus

• Transmitted via fecal oral route • 4 infection types

• Asymptomatic • Symptomatic

• Cholestatic • Fulminant • Stable in the environment • Infects hepatocytes • How virus reaches the liver from the gut is not well understood

Hepatitis A Virus • Member of Family Picornaviridae Genus Hepatovirus • + strand RNA virus with poly A tail • 7.5 kb and low GC content compared to Picornaviruses • Single ORF • Slow replication and non-cytopathic

Hussain et al. 2011

Hepatitis A Virus

• Human strains

• 3 genotypes I, II, III • 7 sub genotypes • Classification based on VP1-2A coding region • IA, IB, IC, IIB, IIIA, IIIB • IA– 60% infections, IB– 14% infections, IIIB– 21% infections • IA, IB, IIIB– responsible for 30, 30, and 41% of fulminating hepatitis

Aboubakr and Goyal 2019

Hepatitis A Virus

• Antigenic conservation • Antibody response to antigens of a virus • Antibody response to HAV is conserved among the different strains of HAV, Some antigenic conservation of HAV capsid extends to distantly related Hepatovirus identified in bats • Which bind monoclonal antibodies in bats • Vaccine development • Mid 1990s

HAV genotypes

LU38 FH3 LY6

AH3 FH1GBM F.G.

Genotype IA

AH2

AH1 FH2

NCACG

HM-175

L-A-1 HAF-203

1000

Genotype IB

1000

PA21 GA76

MBB

Genotype IIIA

P27

1000

1000

NOR21

612

1000 989

Genotype IIA

CF53

Genotype IV simian strain

Genotype IIB

SLF88

CY-145

Genotype V simian strain

AGM27

Kathrine Stene-Johansen NIPH, 2017

0.1

Hepatitis A Virus

• Attenuation in HAV associated with loss of immunogenicity • HM175 Melbourne Australia • 735 nt 5’ UTR (10% of the genome) • 63 nt 3’ UTR • Both have significant °2 structure • HM175/18f • Cell culture adapted variants

Hepatitis A Virus • Dual Types • Naked • Quasi enveloped virion • Exosome like vesicles • Can contain 1-3 virion • Quasi enveloped (eHAV) • eHAV sera • Naked virus • Shed in feces

Feng 2020

Hepatitis A Virus

• Capsid • 60 copies of 4 mature capsid proteins • VP4, VP3, VP2, VP1 • Pseudo T=3 symmetry • IRES • Internal Ribosome Entry Site • Ribosome bind for translation

https://viralzone.expasy.org/809?outline=all_by_species

Hepatitis A Virus

Hepatitis A Virus

• Severity of infection dependent upon mutations in IRES and/or the VP1/2A region • VP1-2A

• ↑mutations results in ↓severity • ↓mutations results in ↑severity

• IRES • Mutation associated with virulence with different genotypes • ↑IRES variability, decrease in severity and in some cases with other genotypes ↓IRES variability resulted in decrease severity

Norovirus

Norovirus • Family Caliciviridae • Genus norovirus • 49 Genotypes

• 9 GI, 37 GII, 3 GIII, 2 GIV, 2 GV, 2 GVI, 1 GVII (dog), 1 GVIII (human), 1 GIX (human reclassified from GII.15), 1 GX (bat ) • 2 new proposed genogroups GNA1 (Porpoise) and GNA2 (sea lion) • Classified GI-GX • Unclassified noroviruses • Bats, sea lions, porpoise • Mammalian host species • Humans, dogs, cats, pigs, mice, sheep, cattle

Norovirus

• Leading cause of gastroenteritis worldwide • Over 5 million cases in US annually • Norovirus non-enveloped viruses • + ss RNA genome 7.5 kb • 5’ end covalently linked to VPg and 3’ poly A tail • 3 ORFs and murine norovirus has 4 ORFs • Murine norovirus has complex genome

Norovirus Genogroups

Chhabra et al 2019

Norovirus GI

Chhabra et al 2019

Norovirus GII

Chhabra et al 2019

Norovirus 3 ORFS • ORF1

• Polymerase region • Nonstructural proteins • Capsid region • Major structural proteins • VP1

• ORF2

Campillay-Veliz et al, 2020

• Shell (S) and protruding (P) domain P1 and P2 • P2 domain highly variable and interacts with HBGAs

• ORF 3

• Minor structural proteins • VP2

Norovirus • Current classification

• Complete VP1 AA sequences (Capsid) and ORF1 NS region (encodes for the RNA dependent RNA polymerase--RdRp) • Based on RdRp region • 60 P types • 14 GI, 37 GII, 2 GIII, 1 GIV, 2 GV, 2 GVI, 1 GVII, 1 GX • GII.4 variants • Phylogenetic clustering • Endemic in 2 geographically diverse locations

Norovirus

• Inconsistent Classification • Limited data mid 1990

• Divided into genogroups and genotypes based on partial RdRp sequences • Later classification based on 5’ end of VP1 region (capsid) • Region C

• 2013 NCWG (Norovirus Classification Working Group) • Proposed nomenclature change • GI, GII, GII, GIX– human • GII.11, GII.18, GII.19– swine • GIV.2– cats and dogs • VP1 complete AA sequence • Capsid region and ORF2

Cavicchio et al, 2022

Norovirus • Recombination

• ORF1/2 junction • Increase norovirus diversity • Dual typing

• Partial RdRp and partial VP1 region • P type and Genotype • Region B and region C

• 14 GI P types • 27 GII P types

• 9 GI capsid genotypes • 22 GII capsid genotypes

Norovirus

• Limitations of current classification system • P-type • 762 nucleotide from RdRp region

• But 85% of the sequenced use are from the whole genome • 14 GI P types • 27 GI P types

HEPATITIS A VIRUS SEAFOOD AND PRODUCE ASSOCIATED OUTBREAKS 1997-2018

*

*

*

*

*

* *

*

*

*

*

not detected not detected

detected

detected and characterized

detected and characterized

*Imported Product *Imported Product

NOROVIRUS SEAFOOD AND PRODUCE ASSOCIATED OUTBREAKS 2016-2017

*

*

*

*

detected and characterized

*Imported Product

www.fda.gov

Norovirus Foodborne Outbreak Investigations 2016-2018

Hepatitis A Virus Foodborne Outbreak Investigations 2013-2017

Foodborne Viruses • Reported prevalence is increasing • Approx 2000 cases probable to everyone reported • Highly infectious • There are asymptomatic carriers of virus. • It is generally a reflection of poor handling/sanitary conditions

QUESTIONS?

Taxonomic Aspects of the Genus Cyclospora : 21 species and counting…

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

Phylum Apicomplexan A large group of protozoa including approximately 4000 named species.

They produce oocysts which are excreted by the definitive host and can persist in the environment.

In some apicomplexan species the excreted oocysts undergo a process called sporulation to become infectious (e.g., Cyclospora , Eimeria, Toxoplasma ).

Cryptosporidium oocysts do not sporulate.

2

Oocysts, Sporocysts, Sporozoites

Cyclospora

Eimeria

Cryptosporidium

Isospora

3

Morphologic differentiation based on sporulation –Taxonomy

4

5

To date, there are currently 21 valid species of Cyclospora. The molecular tools applied for detection and characterization of C. cayetanensis be thoroughly evaluated for specificity Cyclospora species Host type Host species References C. cayetanensis Human Homo sapiens J Parasitol. 1994;80:625–9 C. papionis Simian Papio anubis Emerg Infect Dis. 1999 :651-658 C. colobi Simian Colobus guereza Emerg Infect Dis. 1999 :651-658 C. cercopitheci Simian Cercopithecus aethiops Emerg Infect Dis. 1999 :651-658

C. duszynskii

Eastern mole Eastern mole Simian

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

6

millipede

Glomeris sp.

C. glomericola

New Species -2018

7

C. Cayetanensis

E. nieschulzi

E. mitis

E. tenella

8

Diversity of Cyclospora sp.

These Cyclospora spp. are

morphologically identical to C. cayetanensis

9

www.fda.gov

Cyclospora sp. in monkeys

10

11

Phylogenetic relationships ( C. cayetanensis and Eimeria )

Mitochondrial genome

18SrRNA

Apicoplast genome

12

Conclusions • The genus Cyclospora encompass 21 valid species and most of them cause infections in animals. • A large number of Eimeria species (over 130) has been described as pathogens of mammals, birds, reptiles and fish. • C. cayetanensis is the only species in this genus known to infect humans. • Despite the limitations the genetic databases contain enough information to allow reproducible phylogenetic analysis using genes such as 18SrRNA (sequences are not curated). • The phylogenetic relationship between the genus Cyclospora and Eimeria was established based on analysis of 18SrRNA gene as well as mitochondrial and apicoplast genomes. • It is expected that new species of the genus Cyclospora and Eimeria be described in the future causing direct implications on approaches used to detect C. cayetanensis in environmental samples.

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AIMS- Analytical International Methods & Standards Program – Working Group

Erin Crowley, Q Laboratories Morgan Wallace, Rheonix

Co-Chairs

AIMS Objectives • First Area of Study - How do we perform a validation study for a non-culturable organism? (e.g., Cyclospora or Norovirus) • What does an Inclusivity/Exclusivity Study look like? o Can in silico analysis be used? • How to produce Standard Reference Materials? • How to spike samples for testing? • Can we confirm that a sample that is presumptive positive is a true positive? • Will methods produce a quantitative result? If so, how do we validate?

AOAC INTERNATIONAL 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

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Cyclospora – AIMS Objectives and Possible Extensions

• Working Group Established • Arrive at an applicability statement • Produce consensus on: o Definitions (draft produced by sub-group) o Matrices to address – Produce (types) – Agricultural Waters?

– Drinking Water? – Other matrices?

o Address challenges with quantitation o Address viable but non-culturable – DD PCR as a possible solution o Strain Typing

AOAC INTERNATIONAL 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

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Cyclospora – Nuts and Bolts of the WG

• Deliverable, completed in draft form - o Determine what terms need definitions o Determine which definitions are currently available – FDA/USDA/CDC – ISO – Others o Arrive at consensus on definitions • Next Deliverables - o Arrive at consensus on matrices o SMPRs

AOAC INTERNATIONAL 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

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AIMS Document Workflow- Qualitative

AOAC INTERNATIONAL 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

AIMS Document Workflow- Quantitative

AOAC INTERNATIONAL 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

AIMS Document Workflow- Confirmatory Methods

AOAC INTERNATIONAL 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

Bioinformatic Workflow

AOAC INTERNATIONAL 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

AIMS Possible Extensions • What does a validation study for a typing method entail? Is this different for a non-culturable organism? • Do we need a reference method? • What current screening method parameters are relevant? o False Positive/False Negative? o Inclusivity/Exclusivity? o Limit of Detection? • Do we need new descriptors for typing method performance? o Discriminatory Power? o Performance in samples with more than one strain?

AOAC INTERNATIONAL 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

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Item to Address – Working Group as a Whole • Reach consensus on the purpose of the working group so that sub-groups can begin to produce deliverables • Identify most urgently needed sub-groups and SMEs

AOAC INTERNATIONAL 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

Items to Address – Possible Breakout Groups 1. Begin to build consensus on how to perform an

4. Begin to build consensus on whether confirmation of a presumptive positive result is needed and if so, how is this performed? 5. Begin to build consensus on whether enumeration performance requirements are needed and if so, how are these methods validated?

Inclusivity/Exclusivity Study - In silico? On available strains? 2. Determine matrices which SMPR(s) will address 3. Begin to build consensus on how to spike samples for testing or obtain naturally occurring positive materials

AOAC INTERNATIONAL 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

Thank you!

AOAC INTERNATIONAL 2022 Annual Meeting & Exposition August 26 – September 1, 2022 | Scottsdale, AZ

Definitions

From Cryptosporidium/Giardia ISO Standard 18744-Microbiology of the food chain — Detection and enumeration of Cryptosporidium and Giardia in fresh leafy green vegetables and berry fruits Cyclospora - Single-celled protozoan parasites and are classified as obligate intracellular coccidian parasites in the phylum Apicomplexa. (In introduction?) Species of Cyclospora develop in the gastrointestinal tract of vertebrates throughout their entire live cycle. Immature (unsporulated) oocysts are then shed in feces. Cyclospora oocysts must mature in the environment (outside the host), under favorable conditions, to become infective to someone else. Cyclospora cayetanensis – A species of the genus Cyclospora . It appears that all human cases are caused by this species; no animal reservoirs for C. cayetanensis have been identified. Oocyst - A hardy, thick-walled stage of the life cycle of coccidian parasites. This is the stage that is shed in the feces of people infected with parasites such as Cyclospora and Cryptosporidium . Protozoan oocysts may vary in size and shape. Cryptosporidium oocysts are spherical in shape and ~4-5 um in diameter. Cyclospora oocysts are spherical in shape and 8-10 um in diameter. Related species of Eimeria oocysts can be 10-30 um in length and width and are more ovoid in shape. (In introduction?) The process by which immature (noninfective) coccidian oocysts develop into the mature, infective form. (Also see “coccidian” and “oocyst.”) The conditions and setting in which sporulation occurs varies among coccidian parasites. For example, Cryptosporidium oocysts are shed in the feces in an already sporulated state. In contrast, Cyclospora oocysts must mature in the environment (outside the host), under favorable conditions, to become infective to someone else. Fresh Leafy green vegetable - Plant leaves eaten as a vegetable, which have not been subjected to any process, except perhaps cutting and washing. Fresh berry fruits - Small, round or oblong, fleshy and juicy fruit, which has not been subjected to any process except perhaps cutting and washing. Internal extraction control - (Oo)cysts that may be added in defined numbers to the sample prior to processing to assure that the method is operating properly. Internal Amplification Control – A DNA segment added to the master mix for PCR from which a segment is amplified to determine if the PCR reaction was successful. Positive PCR control – A DNA segment, possibly a synthetic version of the target DNA, added to a PCR master mix without sample to determine that the PCR reaction worked successfully. Negative PCR control – A sample known to be negative for the target and run with unknown samples to provide evidence that large scale cross contamination did not occur. This sample is not run through the extraction process. Sporulated oocyst - The mature, infective form of a coccidian oocyst. Herbs(?) -

Negative Process control – A known negative sample run through the extraction process and then tested by PCR to provide evidence that large scale cross contamination did not occur. Positive Process control – A sample known to be positive run through the extraction process and then tested by PCR to demonstrate that all phases of the method were preformed successfully. Surrogate Organism – A non- Cyclospora cayetanensis protozoan parasite used as an indicator that the extraction and detection method worked successfully. It can also be used as a tool to validate a method. Consideration should be given if this organism is genetically or physiologically similar to C. cayetanensis .