AOAC 2022 Midyear Meeting - AIMS Meeting Book


AOAC MIDYEAR MEETING Wednesday, March 16, 2022 1:00pm ET – 3:00pm ET Draft Agenda Moderator: Palmer Orlandi (AOAC INTERNATIONAL)

I. WELCOME & INTRODUCTIONS (Orlandi) (1:00pm – 1:05pm) Palmer Orlandi (AOAC) will welcome attendees and lead introductions.

II. AIMS PROGRAM LAUNCH (Crowley & Wallace) (1:05pm – 2:10pm) AIMS Working Group Co-Chairs, Erin Crowley (Q Laboratories) and Morgan Wallace (Rheonix), will lead the launch of the AIMS Program. a. OVERVIEW, EXPECTATION, BACKGROUND (1:05pm – 1:10pm) b. AOAC STANDARDS DEVELOPMENT PROCESS OVERVIEW (McKenzie) (1:10pm – 1:35pm) Deborah McKenzie (AOAC) will provide an overview of the AOAC standards process and Working Group processes and responsibilities a. LAUNCH OF AIMS PROGRAM (Crowley and Wallace) (1:35pm – 2:05pm)

Erin Crowley and Morgan Wallace will do a launch presentation for the program providing information on the background, current challenges, and propose scope for the consensus standards to be developed

III. CONCEPT AND APPLICATIONS OF VIABILITY DROPLET DIGITAL PCR (v-ddPCR) (Kiefer) (2:05pm – 2:35pm) Anthony Kiefer (International Flavors and Fragrances, Inc., IFF) will provide insights on the applications of viability droplet digital PCR (v-ddPCR).





Items and times on this agenda are subject to change without notice.



AOAC INTERNATIONAL Policies and Procedures • AOAC INTERNATIONAL Policy on Antitrust • AOAC INTERNATIONAL Policy on Use of Association Name, Initials, Identifying Insignia, Letterhead and Business Cards • AOAC INTERNATIONAL Policy on Volunteer Conflict of Interest


AOAC DUE PROCESS FOR DEVELOPMENT OF NON ‐ METHOD CONSENSUS STANDARDS AND DOCUMENTS Policy for Due Process in the Development of AOAC Standards and Consensus Documents

AOAC INTERNATIONAL (AOAC) develops and publishes voluntary consensus standards and documents in support of method development, method modification, and method use or implementation. AOAC establishes voluntary consensus standards and documents in accordance to national and international principles for ensuring due process. AOAC’s consensus and standards development activities include participation from diverse perspectives across a given analytical industry or community area, discipline or issue. Standards are developed upon consensus of stakeholders. Approval of standards is based upon confirmation that the standard has been developed in accordance with AOAC’s processes 1 and principles, and as outline in Figure 1. Examples of AOAC consensus documents and standards include voluntary consensus standards; performance standards; methods of analysis; guidance documents; concepts of operation; best practice recommendation documents, and sampling approaches. POLICY STATEMENT AOAC develops national and international analytical non ‐ method consensus and standards documents according to the national and international principles for ensuring due process in the development of consensus documents and standards: openness, transparency, lack of dominance, balance, due consideration, coordination, consensus, and appeals. The AOAC Official Methods Board oversees the implementation of scientific and technical policies and procedures established by the AOAC Board of Directors including those for ensuring due process in establishing standards and consensus documents as outlined in Figure 1. The rules of engagement and parliamentary procedures for AOAC standards developing bodies follow Robert’s Rules of Order (latest edition) in accordance with AOAC bylaws and policies.

Figure 1 ‐ Overview Flow for Consensus & Standard Development

1 AOAC has documentation on the details of the processes used to develop its standards and consensus documents. These supporting documentation include terminology, process details in text with an accompanying flowchart, and a document stage handout.

Approved by AOAC Board of Directors on June 10, 2020.


March 16, 2022

Deborah McKenzie Senior Director, Standards & Official Methods SM AOAC INTERNATIONAL

In Food & Agriculture, We Set the Standard


Integrated Science Programs & Projects • Community thematic program; ongoing deliverables; Incorporate >2 AOAC Core Programs & Services • Programs with AOAC brand identity as panels - replaces “Program” “Panel” • 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)

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

• AOAC Laboratory Proficiency Testing Program

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

• AOAC Standards & Official Methods SM Program

In Food & Agriculture, We Set the Standard


A concerted standards development process

Adherence to Standards Principles

Leverage global membership, partnerships and body of science to form collaboration consensus volunteer bodies

Scientific and Performance Based

Forms Working Groups as needed

Integrated Science Programs

In Food & Agriculture, We Set the Standard


1. New Program/Project Formed

2. Program/Project Scope Defined for Period of Time • Advisory Panel formed by program/project sponsors • Working Group Chairs and Science Advisor(s) assigned as needed

3. Standards Developed • Working Group draft standards • Draft standards receive input from all interests until consensus is achieved

• ASF Emerging Challenge/Technology • Individual/Sole Source

In Food & Agriculture, We Set the Standard


Integrating AOAC Consensus Products • Performance Requirements • Guidelines • Sampling Standards • Methods of Analysis • Best Practices • Operational Documents • Reference Materials • Training

Basic Principles

• Transparency • Openness • Balance of Interests • Due Process • Consensus • Appeals

In Food & Agriculture, We Set the Standard


Stakeholder Panel Composition

 All stakeholders are entitled to due process.  All stakeholders can share their perspectives  Anyone with a material interest may participate  Standards are established by a group that lacks dominance by any one interest - BALANCE

Perspective 1

Perspective 2

Perspective 3

 Draft standard must contain input of stakeholders of varying perspectives.

Perspective 6

Perspective 4

Perspective 5

 AOAC will confirm consensus regardless of mechanism used to demonstrates consensus of the stakeholders.

In Food & Agriculture, We Set the Standard


Industry - Food



A robust standard must contain input of stakeholders of varying perspectives.








Industry – Technology Provider

Industry – Dietary Supplement

In Food & Agriculture, We Set the Standard

In Food & Agriculture, We Set the Standard


Community Consensus

Advisory Panel Meeting

WG Meetings

Works with AOAC to define scope of work to be launched, including development of new programs and/or new working groups (WGs)

WG begin work and draft consensus documents via web conference and reach general consensus on draft standard between AOAC meetings

WG chair(s) present final draft standard along with reconciled comments for deliberation and consensus.

At AOAC program 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)

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

Launching New WG Activities

In Food & Agriculture, We Set the Standard


• Priorities & Scope • Financial Support

Working Group (WG) • Launch and develop draft standard • Draft standard posted for broader input

In Food & Agriculture, We Set the Standard


Working Group (WG) •Reconcile comments •Present new draft standard Stakeholders •Provide input •Demonstrate consensus

In Food & Agriculture, We Set the Standard


Process Audit • Ensures voting/ballot results Publications •Published in OMA

In Food & Agriculture, We Set the Standard


AOAC carefully documents the actions of Integrated Science Programs

AOAC will prepare summaries of the meetings

Communicate summaries to participants/stakeholders, etc…. Publish summaries in the Referee section of AOAC’s Inside Laboratory Management

AOAC publishes its consensus standards and Official Methods

Official Methods of Analysis of AOAC INTERNATIONAL Journal of AOAC INTERNATIONAL

AOAC publishes the status of standards and methods in the Referee section of AOAC’s Inside Laboratory Management

In Food & Agriculture, We Set the Standard


These organization form the AOAC AIMS Advisory Panel which advises on program direction and priorities.

If you wish to expand on this program’s work, please join the Advisory Panel by supporting the program .

In Food & Agriculture, We Set the Standard

In Food & Agriculture, We Set the Standard

OPTIMIZATION OF VIABILITY TREATMENT ESSENTIAL FOR ACCURATE DROPLET DIGITAL PCR ENUMERATION OF PROBIOTICS. Presented by: Anthony Kiefer Reference: Kiefer, A., Tang, P., Arndt, S., Fallico, V., and Wong, C. (2020). Optimization of Viability Treatment Essential for Accurate Droplet Digital PCR Enumeration of Probiotics. Front. Microbiol. 11. doi:10.3389/fmicb.2020.01811.


• ddPCR represents the latest development in DNA quantification technology.

• ddPCR partitions the DNA sample into oil droplets and uses a Poisson distribution to extrapolate absolute counts from the number of partitions showing amplification of single-copy gene targets (Hindson et al., 2011).

(Codony et al., 2015)

• Viability ddPCR (v-ddPCR) using a combination of PMA and EMA was shown to enumerate several commercial probiotic strains with high correlation to plate counts.

• V-ddPCR offers improvements to agar plate counting methods including generating faster (4–8 vs. 24–72 h) and more accurate (1–3% vs. 12–20% CV) results (Hansen et al., 2020).

• V-ddPCR offers improvements over quantitative PCR such as less sensitivity to inhibitions (Huggett et al., 2013), lower limit of detection (Qian et al., 2016) and does not require a standard curve (Hindson et al., 2011).

(Hansen et al., 2020)

(Bio-Rad, 2022)


From Previous Literature: • The use of a single concentration of PMA or EMA has been shown to accurately measure loss in viability when comparing different treatment groups such as untreated and heat/isopropanol killed cells (Nkuipou-Kenfack et al., 2013; Nogva et al., 2003). • In contrast, several studies report the need to adjust PMA or EMA concentration based several factors including sample turbidity, pH, salt concentration, excess dead cells, and in accordance with the genus, species and even strain of interest ((Fittipaldi et al., 2012; Gobert et al., 2018). This Study: • This study investigated the efficiency of a mixture of PMA and EMA (PE51), at specified concentrations, to exclude DNA from freeze dried probiotic products. • PE51 efficiency was evaluated across the two most common probiotic genera, Bifidobacterium (Bi-07) and Lactobacillus (La-14) when a common DNA target was utilized, and when utilizing two different assay targets within the same strain. • Lastly, we investigated the potential of v-ddPCR assays, when paired with PE51, to accurately enumerate freeze dried probiotic products.

Pre- Treatment Efficiency

PE51 Efficiency by Genus

PE51 Efficiency by Assay Target

Overall PE51 Efficiency

v-ddPCR vs. Plate Count


• Four Assays were utilized for this study, two existing strain specific assays and two developed for this study on the GroEL gene.

• Strain specific primer and probe assays are designed around unique or highly distinctive genetic regions capable of distinguishing at or near the strain level and were first reported by Hansen et al. (2020). • GroEL assays designed for this study are in the same location on the GroEL gene for Bi-07 and La-14, they were designed to have similar amplicon length, GC content, and Tm to strain specific counterparts.

• No statistical difference between GroEL and strain specific assays as measured by t-ddPCR in La-14 (p = 0.1102) or Bi-07 (p = 0.4186) across six random lots.


• PE51 efficiency was analyzed over a 2-log concentration gradient. • Significant exclusion capacity was observed on all assays, even at the lowest concentrations tested (42 nM), much lower than the concentrations suggested by the manufacturer or as reported in previous literature (50 µM/100 µM) (Biotium, 2019; Nocker and Camper, 2006). • Significant differences continue to be observed at higher concentrations with results dropping well below viable counts obtained through plate count enumeration. • Agrees with previous literature on PMA and EMA (Kobayashi et al., 2009; Kramer et al., 2009; Nogva et al., 2003; Oketič et al., 2015; Yáñez et al., 2011) and calls into question protocols and products which recommend the use of a single concentration of dye for distinguishing viable cells and instead supports the concept of dye optimization.

PE51 EFFICIENCY BY GENUS • Despite similar pre-viability treatment efficiency as measured by t-ddPCR and

averaging approximately 200% higher viability when measured by plate count enumeration (La-14 = 2.81E + 11, Bi-07 = 8.37E + 11), Bi-07 exhibited significantly lower v-ddPCR results when compared to La-14 across the range of PE51 concentrations analyzed. • Likely associated with physiological differences such as membrane permeability and efflux efficiency (Nocker et al., 2006). • Similar findings have been reported even at the species level when utilizing EMA with identical primer sets (Kobayashi et al., 2009).


• PE51 efficiency was next assessed using two different assays per strain (GroEL vs. Strain Specific).

• Treatment and method variability were controlled by performing the enumeration with both assays on the same treated samples.

• v-ddPCR results indicated a significant difference for both La-14 and Bi-07 when PE51 treated samples were enumerated utilizing GroEL or strain specific targets.

• Previous research has suggested sequence specificity and secondary nucleic acid structures

may affect the binding efficiency of DNA intercalating dyes (Hardwick et al., 1984; Parshionikar et al., 2010).


• Optimized strain specific v-ddPCR assays for both La-14 and Bi-07 were demonstrated to agree with plate count enumeration results. GroEL assays, with equivalent PE51 concentrations, were significantly different from plate count in La-14 (p = 0.0039), but not in Bi-07 (p = 0.0725). • Previous research demonstrates that viability dyes used in too high or too low a concentration can over or underestimate viable counts (Løvdal et al., 2011; Nkuipou- Kenfack et al., 2013). • Plate count enumeration has a long history of use in probiotic formulation of products, quality measures, clinical dosages, and research and development efforts (Fenster et al., 2019). • For these reasons, we find that the use of alternative methods that reproduce plate count results are of greater value to the probiotic industry.

Assay Linearity




Actual Copies/g






Expected Copies/g

La-14 Strain Specific - R 2 = .99 Bi-07 Strain Specific - R 2 = .97

p -value of likelihood ratio test on Assay a

Post-hoc Pairwise Comparison (Tukey HSD)


Standard Error



95% C.I.


(-2.86e11, - 1.48e11) (-2.65e11, - 1.28e11) (-4.78e10, 8.953e10)

GroEL vs SS b






0.0045 **

GroEL vs Plate





SS b vs Plate GroEL vs SS b GroEL vs Plate SS b vs Plate




-- c -- c -- c

-- c -- c -- c

-- c -- c -- c

-- c -- c -- c


0.0725 **


• Introduced the use of PE51 with ddPCR for the detection of viable probiotic cells.

• Confirmed differences in dye efficiency between genera, with probiotics.

• Added assay target to the level of specificity required when optimizing viability treatment.

• Demonstrated the caution that must be taken when broadly applying a single concentration of viability dye to ddPCR assays.

• Demonstrated the ability of optimized v-ddPCR to agree with traditional plate count enumeration results.

• Based on agreement with current methods, improvements offered by ddPCR, and a need to establish a history of testing, we suggest the implementation of v-ddPCR methods into current probiotic product testing.


• Biotium (2019). Product Information: PMA (Propidium Monoazide). Avaliable at: (accessed December 13, 2019). • Bio-Rad (2022). Droplet Digital PCR and Technology. Available at: _knsh_id=_kenshoo_clickid_&WT_mc_id=170125000809&WT_srch=1&gclid=EAIaIQobChMI6t-rpfTF9gIVN21vBB2uVgI4EAAYAyAAEgICUvD_BwE. (accessed March 14, 2022). • Codony, F., Agustí, G., and Allué-Guardia, A. (2015). Cell membrane integrity and distinguishing between metabolically active and inactive cells as a means of improving viability PCR. Molecular and Cellular Probes 29, 190–192. doi:10.1016/j.mcp.2015.03.003. • Fenster, K., Freeburg, B., Hollard, C., Wong, C., Rønhave Laursen, R., and Ouwehand, A. C. (2019). The Production and Delivery of Probiotics: A Review of a Practical Approach. Microorganisms 7, 83. doi:10.3390/microorganisms7030083. • Fittipaldi, M., Nocker, A., and Codony, F. (2012). Progress in understanding preferential detection of live cells using viability dyes in combination with DNA amplification. Journal of Microbiological Methods 91, 276–289. doi:10.1016/j.mimet.2012.08.007. • Gobert, G., Cotillard, A., Fourmestraux, C., Pruvost, L., Miguet, J., and Boyer, M. (2018). Droplet digital PCR improves absolute quantification of viable lactic acid bacteria in faecal samples. Journal of Microbiological Methods 148, 64–73. doi:10.1016/j.mimet.2018.03.004. • Hansen, S. J. Z., Tang, P., Kiefer, A., Galles, K., Wong, C., and Morovic, W. (2020). Droplet Digital PCR Is an Improved Alternative Method for High-Quality Enumeration of Viable Probiotic Strains. Front. Microbiol. 10. doi:10.3389/fmicb.2019.03025. • Hardwick, J. M., Sprecken, R. S. von, Yielding, K. L., and Yielding, L. W. (1984). Ethidium binding sites on plasmid DNA determined by photoaffinity labeling. J. Biol. Chem. 259, 11090–11097. • Hindson, B. J., Ness, K. D., Masquelier, D. A., Belgrader, P., Heredia, N. J., Makarewicz, A. J., et al. (2011). High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number. Anal. Chem. 83, 8604–8610. doi:10.1021/ac202028g.Huggett, J. F., Foy, C. A., Benes, V., Emslie, K., Garson, J. A., Haynes, R., et al. (2013). • The Digital MIQE Guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments. Clinical Chemistry 59, 892–902. doi:10.1373/clinchem.2013.206375.Kiefer, A., Tang, P., Arndt, S., Fallico, V., and Wong, C. (2020).


• Optimization of Viability Treatment Essential for Accurate Droplet Digital PCR Enumeration of Probiotics. Front. Microbiol. 11. doi:10.3389/fmicb.2020.01811.

• Kobayashi, H., Oethinger, M., Tuohy, M. J., Hall, G. S., and Bauer, T. W. (2009). Unsuitable distinction between viable and dead Staphylococcus aureus and Staphylococcus epidermidis by ethidium bromide monoazide. Letters in Applied Microbiology 48, 633–638. doi:10.1111/j.1472-765X.2009.02585.x. • Kramer, M., Obermajer, N., Bogovič Matijašić , B., Rogelj, I., and Kmetec, V. (2009). Quantification of live and dead probiotic bacteria in lyophilised product by real-time PCR and by flow cytometry. Appl Microbiol Biotechnol 84, 1137–1147. doi:10.1007/s00253-009-2068-7. • Løvdal, T., Hovda, M. B., Björkblom, B., and Møller, S. G. (2011). Propidium monoazide combined with real-time quantitative PCR underestimates heat- killed Listeria innocua. Journal of Microbiological Methods 85, 164–169. doi:10.1016/j.mimet.2011.01.027. • Nkuipou-Kenfack, E., Engel, H., Fakih, S., and Nocker, A. (2013). Improving efficiency of viability-PCR for selective detection of live cells. Journal of Microbiological Methods 93, 20–24. doi:10.1016/j.mimet.2013.01.018. • Nocker, A., and Camper, A. K. (2006). Selective Removal of DNA from Dead Cells of Mixed Bacterial Communities by Use of Ethidium Monoazide. Appl. Environ. Microbiol. 72, 1997–2004. doi:10.1128/AEM.72.3.1997-2004.2006. • Nogva, H. K., Drømtorp, S. M., Nissen, H., and Rudi, K. (2003). Ethidium Monoazide for DNA- Based Differentiation of Viable and Dead Bacteria by 5′ - Nuclease PCR. BioTechniques 34, 804–813. doi:10.2144/03344rr02. • Oketič , K., Matijašić , B. B., Obermajer, T., Radulović , Z., Lević , S., Mirković , N., et al. (2015). Evaluation of propidium monoazide real-time PCR for enumeration of probiotic lactobacilli microencapsulated in calcium alginate beads. Beneficial Microbes 6, 573–581. doi:10.3920/BM2014.0095. • Parshionikar, S., Laseke, I., and Fout, G. S. (2010). Use of Propidium Monoazide in Reverse Transcriptase PCR To Distinguish between Infectious and Noninfectious Enteric Viruses in Water Samples. Appl. Environ. Microbiol. 76, 4318–4326. doi:10.1128/AEM.02800-09. • Qian, L., Song, H., and Cai, W. (2016). Determination of Bifidobacterium and Lactobacillus in breast milk of healthy women by digital PCR. Beneficial Microbes 7, 559–569. doi:10.3920/BM2015.0195. • Yáñez, M. A., Nocker, A., Soria-Soria, E., Múrtula, R., Martínez, L., and Catalán, V. (2011). Quantification of viable Legionella pneumophila cells using propidium monoazide combined with quantitative PCR. Journal of Microbiological Methods 85, 124–130. doi:10.1016/j.mimet.2011.02.004.


IFF Probiotic Development Team

Peipei Tang

Samuel Arndt

Connie Wong

Vincenzo Fallico

Anthony Kiefer

AIMS - Analytical International Methods & Standards Program

Erin Crowley, Q Laboratories Morgan Wallace, Rheonix

March 16, 2022


• ISPAM History- How did we get here?

• First of many- Non-culturables, Viable but Not Culturable – New and Emerging contaminants – First will be Cyclospora

• Industry and Regulatory Engagement – CDC Typing Method – FDA BAM Chapter 19B – NACMCF • Technology Applications- DDPCR, V-DDPCR • Objectives of WG – Validation Criteria – Suitable Reference Material


ISPAM History

• Formed in 2011

– 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 – Charter expanded to address a variety of other high-profile needs through the formation of working groups • Sunset in 2019 after many accomplishments


ISPAM History


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


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 – Viruses – 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


Cyclospora Background

• Cyclospora cayetanensis - coccidian protozoan parasite • Newly recognized - described between 1993 to 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; all other species are associated with infections in other animals. • Cyclospora has a complex life cycle and can only multiply within the infected hosts.



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 – Contaminated berries, fresh cilantro, basil and, more recently, ready- to-eat bagged salads.


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.


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


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)


FDA Bacteriological Analytical Manual

Revised in 2016 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


CDC- Advanced Molecular Detection and Prototype Typing


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


Questions from FDA to NACMCF

1. What is known about the prevalence, incidence, and burden of disease of cyclosporiasis in the U.S. and internationally? 1. Are there specific segments of the U.S. population that may be at higher risk for infection? What is the geographic distribution of cases in the U.S.? 2. What is the diversity of Cyclospora cayetanensis genotypes in the US and internationally? 3. What factors (e.g., food safety practices, location of the farms) may contribute to contamination with Cyclospora cayetanensis ? 4. Are certain factors (e.g., type of food, seasonality, where the food is produced, degree of hand contact during growing and harvesting) more significant than others? 2. How does the seasonality, incidence and prevalence of cyclosporiasis compare throughout the United States and internationally and what factors may contribute? 1. Extrinsic factors that may influence sporulation and survival (e.g., extrinsic factors influencing sporulation and survival); 2. Environmental factors influencing movement (e.g., rainfall); 3. Other? 16

Questions from FDA to NACMCF

3. What sampling data exists for Cyclospora cayetanensis in food products and environmental samples, domestically and internationally? 1. What trends have been observed? 2. What methods of detection were used? 4. What preventive measures exist for the control of Cyclospora cayetanensis (e.g., using filtration)? 1. How effective have they been? 2. What are the impediments to development of effective preventive measures for Cyclospora cayetanensis and how can they be overcome?


Questions from FDA to NACMCF

5 . What is known about Cyclospora cayetanensis persistence/survival in food, such as produce, and the environment (e.g., soil, water, food contact surfaces)? 6. What is known about transfer and attachment of Cyclospora cayetanensis from environmental samples (water and soil) to produce? 7. What other coccidian parasites could serve as a surrogate research model for Cyclospora cayetanensis behavior (e.g., for evaluation of control measures)? 8. Are there indicator organisms that can be used to determine the likely presence or absence of Cyclospora cayetanensis in various matrices? 9. What is known about the role of vectors (such as non-human organisms), if any, in the transmission of Cyclospora cayetanensis ?


Questions from FDA to NACMCF

10. What role do farm workers play in the transfer of Cyclospora cayetanensis contamination during pre-harvest, harvest and post-harvest handling? Are there particular approaches that would result in selective identification of the serotypes of public health concern? • How might farm workers serve as both sources and routes of contamination (such as through contamination of agricultural water, or transfer of contaminated soil to food contact surfaces or produce)? • What are strategies that have been utilized to mitigate the contamination from farm workers? Have efforts to mitigate contamination from farm workers been successful? • Are there practices for the maintenance and conveyance of wastewater, septage or human waste that may increase the incidence of Cyclospora cayetanensis contamination? Are there practices that may be useful in the management of waste to reduce the potential for contamination by Cyclospora cayetanensis (e.g., third-party toilet service or municipal wastewater treatment)? • Which wastewater, septage, and human waste treatments in the U.S. are effective against Cyclospora cayetanensis ? Which treatments may not be effective against Cyclospora cayetanensis ? • Does municipal water treatment adequately reduce, control or eliminate Cyclospora cayetanensis ? • Can effective municipal water treatments systems be scaled to treat agricultural water used in produce production? • How do practices compare for domestic growers versus international growers who export to the U.S.? 19

Questions from FDA to NACMCF

11. What elements or points in the parasite's life cycle are potential targets of strategies to disrupt its progression, eliminate or destroy oocysts, stop dissemination into the environment, and prevent food contamination? • What are control measures that should be evaluated for effectiveness against Cyclospora cayetanensis ? Including control measures that can be applied to the environment and/or foods that may be consumed in the raw form. • What is a recommended protocol for evaluating the effectiveness of control measures against Cyclospora cayetanensis ? 12. What are the relevant factors, available data, and data gaps needed to develop an informative quantitative risk assessment model for Cyclospora cayetanensis contamination and risk of illness?


International Fresh Produce Association- Virtual Town Hall


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


Questions for the Working Groups

What sampling data exists for Cyclospora cayetanensis in food products and environmental samples, domestically and internationally?

What types of foods have been attributed to outbreaks? Contributing factors or routes of contamination identified?

What methods of detection were used?

Is monitoring for Cyclospora by testing food products, agricultural environment and ag product inputs being applied as a management strategy currently?

Are there best practices for monitoring? (e.g. water vs product, pre vs post harvest, sample numbers)


Questions for the Working Groups

What are available approaches for characterizing the relatedness of different strains of Cyclospora catayenensis (subtyping)?

What type of validation has the method undergone? What matrices have been validated?

What is the comparative sensitivity/specificity of existing available methods?

What information do we have on assessing viability of oocysts?

Can U.S. methods drive the development of harmonized standards?


Get Involved

Benefits Include:

– Developing strategies and priorities to address the analytical challenges presented by important, emerging food-borne microorganisms

– Providing consensus-driven, internationally aligned validation criteria

– Developing standards and methods to protect producers and consumers alike to maintain the reputation of products and ultimately improve the quality and safety of the food supply, and

– Reviewing New Technologies as Solutions


Welcome to the AOAC INTERNATIONAL 2022 Midyear Meeting! Thank you to our sponsors:

In Food & Agriculture, We Set the Standard

 Save money on publications, events, and education  Free access to the Journal of AOAC INTERNATIONAL and Inside Laboratory Management magazine  18 Regional Sections worldwide  2 Technical Divisions  2,300+ individual members  Lab managers, analytical chemists, microbiologists, food scientists, and more  85+ organizational members  Businesses, gov agencies, academic institutions



In Food & Agriculture, We Set the Standard

Organizational Membership: organizational-membership/ Individual Membership: individual-membership/


AOAC Membership Team: +1 (301) 924-7077 ext. 170

In Food & Agriculture, We Set the Standard

In Food & Agriculture, We Set the Standard

 AOAC’s premier program for developing food testing analytical science methods that are recognized and legally defensible worldwide  Evaluates methods across AOAC’s integrated science programs for adoption as First Action or Final Action Official Methods  Approved methods are published in the Official Methods of Analysis publication


In Food & Agriculture, We Set the Standard

Standards Developed/In Progress

 Biothreat agents  Botanicals & dietary supplements  Cannabis & hemp  Endocrine disruptors  Food authenticity

 Food allergens and glutens  Heat generated toxicants  Infant formula & adult Nutritionals  Foodborne pathogens  Veterinary drug residues


Global Standards Engagement

 Observer status in CODEX Alimentarius  Active member of the Interagency Meeting  Liaison agreement with ISO/TC 34

 Liaison arrangement with International Dairy Federation  ANSI-accredited US TAG to ISO/TC 34 (Food products)  MOU and joint participation with IAFNS  MOU with ARSO

In Food & Agriculture, We Set the Standard

 Helps labs compete in the global marketplace by demonstrating that they meet standards for accuracy, reliability, and compliance  A2LA-accredited program  Offers programs in microbiology, listeria, salmonella in liquid egg, food chemistry, pesticide residues, and more

In Food & Agriculture, We Set the Standard

NEW IN 2022: Cannabis and Hemp Proficiency Testing Program First Round: November 2022 To Enroll or for more information, contact AOAC PT Staff

In Food & Agriculture, We Set the Standard

 New program from the AOAC Research Institute  Certifies performance claims of application notes, user guides, or laboratory developed methods  Based on the same science required by the Performance Tested Methods (PTM) and Official Methods programs ptm/reviewed-and-recognized-program

In Food & Agriculture, We Set the Standard

 AOAC Research Institute program  Provides independent third-party review and certification for proprietary test method performance  Helps companies and their customers safeguard their businesses, mitigate risk, and protect consumers


In Food & Agriculture, We Set the Standard

Meeting attendees save 33% on the 21 st edition of the Official Methods of Analysis of AOAC INTERNATIONAL !  Use promo code 33OFFOMA

In Food & Agriculture, We Set the Standard

Get involved with the Journal of AOAC INTERNATIONAL! Seeking volunteers for:  Editor-in-Chief  Drug Formulations Section Editor  Statistical Analysis and Chemometrics Section Editor

In Food & Agriculture, We Set the Standard

 Food Authenticity Methods  Infant Formula  Marine and Freshwater Toxins  Metals  Microbiology  Mycotoxins  Pesticides  Vet Drug Residues  Water and Wastewater

 Agricultural Materials  Biothreat Agents  Cannabis and Hemp  Color Additives  Dietary Supplements and Botanicals  Environmental, Industrial, and Emerging Contaminants  Food Additive Safety  Food Allergens


In Food & Agriculture, We Set the Standard

 Analytical International Methods and Standards Program (launching during the MYM!)  Working group to develop voluntary consensus standards for the determination of pyrrolizidine alkaloids in teas, herbal infusions, herbs, seed spices and botanical dietary supplement ingredients  Working group to develop voluntary consensus standards for color additives from natural sources

NEW INITIATIVES AT AOAC – WE NEED YOUR SUPPORT! solutions/new-initiatives/

In Food & Agriculture, We Set the Standard


In Food & Agriculture, We Set the Standard

COMING SOON: AOAC’S New Botanical & Dietary Supplement Integrity Program!

Visit to learn about the program’s first working group to develop standards for folic acid in chewable gels, soft gels, and tablets.

In Food & Agriculture, We Set the Standard

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