1

Response to questions posed by the Food and Drug Administration 

(FDA): Cyclospora cayetanensis in Produce

National Advisory Committee on Microbiological Criteria for Foods1

TABLE OF CONTENTS

Executive Summary................................................................................................................................. 2

Charge from FDA to NACMCF .............................................................................................................. 3

Sources and Routes................................................................................................................................ 9

Q4: Foods associated with outbreaks............................................................................................ 9

Q11: Transfer and attachment........................................................................................................ 13

Q14: The role of vectors................................................................................................................... 14

Q15: The role of farm workers........................................................................................................ 16

Prevalence/Persistence and Indicators............................................................................................ 18

Q1: Prevalence, incidence, and burden ....................................................................................... 18

Q2: Seasonality, incidence, and prevalence............................................................................... 20

Q10: Persistence/survival in food and the environment ......................................................... 23

Q13: Indicator organisms ................................................................................................................ 24

Analytical Methods ................................................................................................................................ 25

Q3: Sampling data ............................................................................................................................. 26

Q6: Approaches for characterizing ............................................................................................... 28

Q7: Current available test methods for detecting and/or isolating ......................................30

Q8: Viability of oocysts .................................................................................................................... 39

Control Strategies and Surrogates.................................................................................................... 40

Q5: Current monitoring and management strategies ............................................................. 40

Q9: Preventive measures................................................................................................................. 42

Q12: Possible surrogates ................................................................................................................ 46

Q16: Maintenance and conveyance of wastewater................................................................... 49

Q17: Prevention in food ................................................................................................................... 51

1 Participating agencies include the U. S. Department of Agriculture, Food Safety and Inspection Service; U.S. 

Department of Health and Human Services, Food and Drug Administration, and Centers for Disease Control and 

Prevention; U.S. Department of Commerce, National Marine Fisheries Service; and U.S. Department of Defense, 

Veterinary Service Activity. Disclaimer: Mention of trade names or commercial products in this publication is solely 

for the purpose of providing specific information and does not imply recommendation or endorsement by the U. S. 

Department of Agriculture and other participating agencies.
2

Q15b: Strategies use to mitigate the contamination from farm workers............................54

Relevant Factors and Data Gaps ....................................................................................................... 55

Q18: Relevant factors, available data, and data gaps for quantitative risk assessment 55

References............................................................................................................................................... 58

Appendix A: Glossary.......................................................................................................................... 76

Appendix B: Additional Tables........................................................................................................... 78

Executive Summary of Findings

Cyclospora cayetanensis (C. cayetanensis) is a coccidian protozoan parasite, belonging 

to the phylum Apicomplexa, order Eucoccidiorida, family Eimeriidae, described between 1993 to 

1994 as a newly identified human gastrointestinal pathogen. Within the genus Cyclospora, 

only C. cayetanensis is known to infect humans. However, recent advances in genomics 

separated C. cayetanensis into 3 proposed species, with the two new proposed species also 

considered parasitic to humans (Cyclospora ashfordi sp. nov. and Cyclospora henanensis sp. 

nov.). For the purpose of this document and to reflect the proposed status of the new 

nomenclature “C. cayetanensis” refers to all three species of Cyclospora parasitic in humans. 

The parasite produces oocysts that are resistant to harsh environmental conditions and many 

chemical treatments commonly used to reduce the presence of bacterial pathogens in the 

specialty crop production environment and in agricultural inputs (e.g., agricultural water). C. 

cayetanensis is the etiologic agent of cyclosporiasis, its host range is limited to humans. 

Detected in association with human illness in many parts of the world, C. cayetanensis 

previously was considered to be a pathogen acquired during childhood in developing nations. In 

the United States, cyclosporiasis was previously associated with international travel or 

consumption of contaminated imported foods. In recent years, the U.S. has seen an increase in 

cases and positive samples associated with domestically grown produce, both as raw 

agricultural commodities and fresh cut. Laborers with the history of recent travel to countries 

where C. cayetanensis is endemic have not been ruled out as the sources of the pathogen in 

these outbreaks. Since 2016, the number of cyclosporiasis cases has increased approximately 

3-fold, often linked to the consumption of leafy herbs and ready-to-eat salads. Fecal 

contamination from symptomatic or asymptomatic carriers is, ultimately, the only known source 

of C. cayetanensis. The hypothesis that C. cayetanensis has become endemic in the production 

regions of the U.S. remains to be robustly supported. The hypothesis that farm workers with a 

history of recent travel to areas where the parasite is common are the likeliest source of the 

pathogen has not been ruled out. C. cayetanensis likely spreads via the fecal-environment-oral 

route when sanitation controls break down. Efforts have been made to develop molecular 

detection methods for C. cayetanensis in both food and environmental samples. However, due 
3

to the high degree of genome-level conservation between C. cayetanensis and its close 

relatives that are not pathogenic in humans, results of some environmental surveys that relied 

solely on the PCR-based detection of ribosomal RNA genes likely overestimated the prevalence 

of C. cayetanensis. There remain significant knowledge and data gaps that hamper the 

implementation of effective measures to prevent the contamination of produce with the oocysts 

of this parasite. Awareness of the factors that can contribute to C. cayetanensis contamination 

of domestically grown and imported produce is key to developing an effective prevention and 

management strategy.

RECOMMENDATIONS: 

1. To facilitate future research (e.g., validation of surrogates, studies on environmental 

persistence and attachment) and identification and validation of control strategies, the 

Committee urges development of a practical method to propagate C. cayetanensis

oocysts under laboratory settings. 

2. Because of the limited availability of C. cayetanensis oocysts, research with surrogates –

and specifically with the close relative Eimeria – can be informative for identifying control 

strategies and learning about persistence in the production environment. 

3. Method development for the detection of C. cayetanensis in food and environmental 

samples should include the evaluation of multiple genetic targets representing different 

regions of the genome. Modifications to current molecular methods for the detection of 

C. cayetanensis should be thoroughly validated for impacts on specificity before using 

modified methods on food or environmental samples. Conversely, detection methods 

should be designed to be robust, reproducible, and tolerant of minor modifications in the 

methodologies (e.g., brand of equipment or reagents, minor deviations in PCR 

conditions, etc.) without sacrificing specificity or sensitivity. 

4. Given that the hypothesized likeliest source of the parasite in the food production 

environment (individuals with a history of recent travel to areas where infections with C. 

cayetanensis are common or other exposures to the parasite), preventative measures 

should center around clear sanitation guidelines, ensuring on-site capacity for 

implementing sanitation protocols (i.e., readily accessible hand washing stations with 

soap, etc.) and periodic training of the employees. 

Charge from FDA to NACMCF

Background

Cyclospora spp. are protozoan parasites in the phylum Apicomplexan that can parasitize 

different species of mammals with remarkable host-specificity. Cyclospora has a complex life 

cycle and can only multiply within the infected hosts. Among the Cyclospora species, 
4

only Cyclospora cayetanensis is known to infect humans; all other species are associated with 

infections in other animals. This parasite is characterized by environmentally hardy oocysts that 

are shed in stools of infected persons. These oocysts are shed unsporulated and are not 

infectious. Once released into the environment, unsporulated oocysts require approximately 7 to 

14 days under certain environmental conditions to sporulate and become infectious. The 

oocysts are thought to be transferred to the surface of foods through environmental routes (e.g., 

through human fecal pollution carried by agricultural water) and subsequently infect the host 

after produce is consumed. Once consumed, the sporulated oocysts replicate in the human 

gastrointestinal tract and continue the infection cycle as unsporulated oocysts are shed in 

stool. The cycle continues as human fecal pollution again contaminates the environment. A 

limitation to widespread C. cayetanensis research is the inability to directly culture or propagate

the organism. Researchers rely solely on acquired oocysts to conduct research. Some work 

has been done to use surrogate organisms to mimic the life cycle of C. cayetanensis, however 

with limited positive results. 

A positive C. cayetanensis finding is indicative of the presence of human fecal contamination, as 

humans are the only known reservoir. Cyclosporiasis is characterized by symptoms such as 

explosive diarrhea, vomiting, fatigue, and weight loss. C. cayetanensis has become a major 

public health and food safety concern during the last few years. Outbreaks of cyclosporiasis 

affect thousands of individuals in the U.S. annually, with a steady increase in reported cases 

over recent years. In 2020, CDC reported 1,241 laboratory-confirmed cases of cyclosporiasis in 

people who had no history of international travel and experienced illness onset during May 1-

August 31, the typical period during which cyclosporiasis illnesses increase in the US. In 2019 

and 2018, there were 2,408 and 2,299 cases reported each year, respectively. Comparatively, 

between 2000–2017, the total number of cases reported for cyclosporiasis in the US was 1,730. 

Additionally, cyclosporiasis typically results in symptomatic illness in the general population 

regardless of age in the US, whereas in endemic areas, young children and 

immunocompromised individuals are most at risk for severe illness. Outbreaks and cases of 

cyclosporiasis generally occur during the warmer months of May – September for the northern 

hemisphere, and November – March for the southern hemisphere. Historically, among 

outbreaks where a source could be identified, they have been linked to ingestion of 

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

Several efforts have been implemented to develop molecular detection methods for C. 

cayetanensis in both food and environmental samples. These methods have been used to 

assist epidemiological investigations and surveys to estimate the prevalence of C. 

cayetanensis in commodities and growing regions. Despite these scientific efforts, there are 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.

Charge Questions:
5

FDA is seeking information on the factors that can contribute to C. cayetanensis 

contamination of domestically grown and imported produce, and recommendations for 

developing an effective prevention and management strategy.

1. What is known about the prevalence, incidence, and burden of disease of cyclosporiasis 

in the U.S. and internationally? 

a) 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.?

b) What is the diversity of Cyclospora cayetanensis genotypes in the US and 

internationally?

c) What factors (e.g., food safety practices, location of the farms) may contribute to 

contamination with Cyclospora cayetanensis? 

d) 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? 

a) Extrinsic factors that may influence sporulation and survival (e.g., extrinsic factors 

influencing sporulation and survival); 

b) Environmental factors influencing movement (e.g., rainfall);

c) Other?

3. What sampling data exists for Cyclospora cayetanensis in food products and 

environmental samples, domestically and internationally?

a) What trends have been observed?

b) What methods of detection were used?

4. What types of foods have been attributed to outbreaks of cyclosporiasis domestically 

and internationally and what (if any) contributing factors, sources or routes of 

contamination that have been identified?

5. Is monitoring for Cyclospora cayetanensis by testing food products, agricultural 

environment and agricultural inputs being applied as a management strategy currently 

(e.g., by industry, government)? 

a) Are there best practices for monitoring for the presence of Cyclospora cayetanensis 

in agricultural production (including matrices [e.g., water, product], frequency, timing 

of sample collection (pre- vs. post-harvest), and sample numbers)? 

b) Has monitoring led to development and implementation of effective preventive 

measures? If so, how effective have they been? 

6. What are available approaches for characterizing the relatedness of different strains of 

Cyclospora cayetanensis (e.g., subtyping)?
6

7. What are currently available test methods (and comparative sensitivity/specificity) for 

detecting and/or isolating Cyclospora cayetanensis in different matrices (e.g., food, 

water, environmental samples)? What type of validation has the method(s) undergone? 

What are the matrices for which the methods have been validated?

8. What information exists on assessing viability of oocysts?

9. What preventive measures exist for the control of Cyclospora cayetanensis (e.g., using 

filtration)?

a) How effective have they been?

b) What are the impediments to development of effective preventive measures for 

Cyclospora cayetanensis and how can they be overcome?

10. What is known about Cyclospora cayetanensis persistence/survival in food, such as 

produce, and the environment (e.g., soil, water, food contact surfaces)? 

11. What is known about transfer and attachment of Cyclospora cayetanensis from 

environmental samples (water and soil) to produce?

12. What other coccidian parasites could serve as a surrogate research model for 

Cyclospora cayetanensis behavior (e.g., for evaluation of control measures)? 

13. Are there indicator organisms that can be used to determine the likely presence or 

absence of Cyclospora cayetanensis in various matrices?

14. What is known about the role of vectors (such as non-human organisms), if any, in the 

transmission of Cyclospora cayetanensis? 

15. 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? 

a) 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)? 

b) What are strategies that have been utilized to mitigate the contamination from farm 

workers? Have efforts to mitigate contamination from farm workers been successful?

16. 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)?

a) 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?
7

b) Does municipal water treatment adequately reduce, control or eliminate Cyclospora 

cayetanensis? 

c) Can effective municipal water treatments systems be scaled to treat agricultural 

water used in produce production?

d) How do practices compare for domestic growers versus international growers who 

export to the U.S.?

17. 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?

a) 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.

b) What is a recommended protocol for evaluating the effectiveness of control 

measures against Cyclospora cayetanensis?

18. 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?
8

COMMITTEE RESPONSES

Approach by the committee:

A number of comprehensive reviews of peer-reviewed literature on Cyclospora have been 

published recently and consulted by this committee. However, in this rapidly evolving field, a 

reliance on only peer-reviewed publications was deemed limiting by this Committee. Therefore, 

in addition to the peer-reviewed studies accessible via PubMed, the committee consulted 

scientific reports (such as those found in the databases of completed or ongoing research 

projects found in the United States Department of Agriculture Current Research Information

System (USDA CRIS) database and in the database maintained by the Center for Produce 

Safety), the Committee accessed documents released by federal agencies into the public 

domain and heard semi-structured testimonies from academic, federal and industry researchers 

working on C. cayetanensis and other parasites. Results of these findings are presented in this 

report. 

The Committee notes an on-going conversation about the nomenclature of Cyclospora and a 

proposal to separate C. cayetanensis into 3 species (with the addition of Cyclospora ashfordi

sp. nov. and Cyclospora henanensis sp. nov.). All three of these species are parasitic to 

humans (Barratt et al. 2023). However, because all prior research functionally defined C. 

cayetanensis as the only member of the genus responsible for the human cyclosporiasis, and to 

reflect that the new nomenclature is not widely accepted, and all prior publications referred to 

this parasite as “Cyclospora cayetanensis”, or “C. cayetanensis” the rest of this report will 

continue to refer to these organisms as “Cyclospora cayetanensis” or “C. cayetanensis”.

Finally, the Committee notes recent peer-reviewed and non-peer reviewed studies from 

academic and federal laboratories that demonstrated limitations of the detection of C. 

cayetanensis relying solely on the PCR primers designed to amplify 18S regions of the 

organisms rRNA genes and/or internal transcribed spacer (ITS) When PCR products from 

environmental samples amplified with primers targeting regions of the 18S rRNA genes were 

sequenced, the majority of them (>90%) were identified as loci of the Eimeria spp. parasitic in 

various animals, but not humans (Ortega 2022, Mattioli 2022) or failed to result in a sequenced 

PCR product matching a sequence from C. cayetanensis at least under some conditions (Kniel 

2022, Lalonde 2022). Sequencing of the loci amplified using primers targeting the ITS region 

resulted in 3/16 confirmations by sequencing (Temesgen et al. 2022). Therefore, throughout 

this report, when discussing environmental and food samples, the detection of amplicons in a 

PCR reaction (unless a secondary positive identification step was performed), does not confirm 

the presence of C. cayetanensis nor a presumptive presence of the parasite, regardless of the 

conclusions drawn by the authors of the original publications at the time of the original 

publication. 

The Committee organized the charge questions into 5 groups: (1) Sources and Routes (Q4, 

Q11, Q14 and Q15); (2) Prevalence/Persistence and indicators (Q1, Q2, Q10 and Q13); (3) 

Analytical Methods (Q3, Q6, Q7 and Q8); (4) Control Strategies and surrogates (Q5, Q9, Q12, 

Q15b, Q16 and Q17); and (5) Relevant Factors & Data Gaps (Q18).
9

Sources and Routes

Q4: Foods associated with outbreaks

What types of foods have been attributed to outbreaks of cyclosporiasis domestically 

and internationally and what (if any) contributing factors, sources or routes of 

contamination that have been identified? 

Cyclosporiasis outbreaks have been linked to fresh fruits and vegetables, and foods containing 

them. From a total of 70 outbreaks reported globally, 55 were caused by fresh produce. The 

fruits and vegetables most frequently related to Cyclospora infections were: raspberries (34%), 

basil (31%), cilantro (10%) and salad mixes (10%). Sugar snap peas, lettuce, blueberries, 

blackberries, carrots, mangos, mint, scallions, mixed vegetable trays, and fruit salads were also 

associated with cyclosporiasis outbreaks (in some investigations, a single food vehicle was not 

identified). During the summer months cyclosporiasis increases in both endemic and nonendemic regions. Research is needed to identify factors that influence the transmission of the 

parasite to fresh fruits and vegetables. 

In the United States, the first cyclosporiasis outbreak happened in hospital workers in Illinois in 

1990, which the authors attributed to the tap water in a storage tank that may have experienced 

a pump failure (Huang et al. 1995). While that report is often cited as the first case of 

domestically acquired cyclosporiasis, it is important to note that the diagnosis was based solely 

on a microscopic observation of spherical bodies 8-11 um in diameter, and neither the 

methodology nor key epidemiological data linking the outbreak to the water tank were reported. 

Most studies indicate that the fecal--oral route via transmission through contaminated water 

and/or food is most likely for C. cayetanensis (Fig. 1). Direct fecal-oral transmission is less 

likely given the observation that fecally shed oocysts (which are themselves not known to be 

infectious) need to sporulate into infectious spores in response to a yet unknown environmental 

or chemical cue. Therefore, the route of transmission is more accurately described as “fecalenvironment-oral”. In the absence of known vertebrate or invertebrate vector (see discussion 

on vectors below), the only reasonable routes of transfer involve fecally-contaminated 

agricultural water or fecally-contaminated deposits on or in direct vicinity of the harvested 

product. 
10

Fig. 1. Lifecycle of C. cayetanensis. Reproduced with 

permission from Center for Disease Control and Prevention, originally published in 

https://www.cdc.gov/dpdx/cyclosporiasis/index.html 

Most recorded Cyclospora foodborne outbreaks where a source was identified had been linked 

to the consumption of fresh fruits and vegetables (Almeria, Cinar and Dubey 2019, Hadjilouka 

and Tsaltas 2020). The first documented case of transmission via a food product was reported 

in 1995 when raspberries imported from Guatemala were linked to 38 cases in the U.S. 

(Herwaldt 2000). From the investigations of the outbreaks linked to raspberries in the 1990’s, it 

was not determined if the contamination came from direct human contact (e.g., worker hands), 

animals, or indirect human contact through contaminated water from poorly constructed or 

maintained wells, or from run off during the rainy season. Insecticides or fungicides mixed with 

contaminated water were also suspected. Investigation of a large raspberry outbreak in 1996 

(1465 cases) linked to Guatemalan farms, no positive results for C. cayetanensis were obtained 

from any of the environmental samples (Herwaldt and Ackers 1997). Prior to the first raspberries 

outbreak, water had been the only known vehicle for transmission of the parasite and to this 

date no food category other than fresh or fresh-cut produce has been associated with this 

parasite (Almeria et al. 2019). 

 

More than 70 outbreaks in different parts of the world have been reported since 1989, and from 

those in 1990 to 2021, 55 have been suspected or confirmed to be linked to fresh produce 

(Almeria et al. 2019 and Table 1). In the last decade, five outbreaks were caused by fresh 

produce imported from Mexico, including three events due to contaminated cilantro (CDC 

2018c; CDC 2020; CDC 2021). From those outbreaks in which the vehicle was identified 

between 1995 to 2019, basil consumption was reported in 34% of outbreaks and raspberries 

were the vehicle in 31% of events (Hadjilouka and Tsaltas 2020). Cilantro was the third 

individual fresh produce commodity most frequently associated with cyclosporiasis outbreaks, 

and different salad mixes caused eight outbreaks (Almeria et al. 2019) . Other fruits and 

vegetables reported to be linked to C. cayetanensis transmission include snow snap peas, 

blackberries, blueberries, salad mixes, fruit mixes, scallions, carrots, and mangos.
11

Table 1. Most common food linked to recorded C. cayetanensis outbreaks. Table was based on 

data from (Almeria et al. 2019a; CDC, 2020; CDC, 2021).

Food vehicle

No. of outbreaks

(confirmed and 

suspected)

Years

Countries where 

the cases were 

reported

Size of 

outbreaks

(No. 

cases)

Raspberries 12 1995, 1996, 1997, 

1998, 2001, 2009

Canada, USA, 

Spain,

13 -1,465

Basil 11 1997, 1999, 2001, 

2004, 2005, 2006, 

2007, 2010, 2018, 

2019

Canada, USA, 28 - 582

Salad mixes 

(including 

vegetable 

trays and 

coleslaw)

8 1997, 2000, 2001, 

2013, 2016, 2018, 

2020, 2021

Canada, 

Germany, 

Mexico, USA,

25 - 711

Cilantro 6 2003, 2004, 2013, 

2014, 2015, 2018

Canada, USA 8 - 546

Berry/fruit 

mixes

6 1997, 1998, 1999, 

2009, 2019

Canada, USA 8 – 104

Lettuce 2 2010, 2014, Australia, USA, 227, 266

Snap peas 2 2008, 2009 Sweden, USA 4, 18

The first year when Cyclospora infections were suspected to have a domestic origin in the U.S. 

was in 1997, when 185 cases were reported after attending an event and consumed 

contaminated basil (CDC 1997). In 2001, 17 cases of cyclosporiasis were reported in British 

Columbia, Canada. The investigation found that 11 of 12 (92%) cases had consumed Thai 

basil, which had been imported from the U.S. (Ortega and Sanchez 2010). In 2017, from more 

than 1,060 cases of laboratory-confirmed cyclosporiasis (CDC 2017), 597 of those patients 

reported no international travel. In 2018, several outbreaks were recorded (CDC 2018a) (CDC 

2018b) including an event associated with pre-packaged mixed vegetable (broccoli, cauliflower, 

carrots, and dill dip) trays. While the specific vehicle of transmission was not identified, these 

produce items appeared to have been grown domestically. The second major outbreak in 2018 

with domestically produced vegetables involved 511 laboratory-confirmed cases in 15 states, 

caused by romaine lettuce and carrot salad mix served at a fast-food chain and produced by a 

fresh cut processor company (CDC 2018b). 

In addition to those two outbreaks, there were clusters of cases linked to cilantro and basil 

reportedly grown in the U.S. In 2019, from 2,409 cyclosporiasis cases distributed among 
12

multiple restaurant and event clusters, only 10% of the patients were linked to consumption of 

fresh basil imported from Mexico (CDC 2019). In 2020, another multi-state outbreak that 

involved 701 cases was caused by salad mixes containing iceberg lettuce, carrots, and red 

cabbage, distributed by the same fresh produce company from 2018 (CDC 2020). In 2021, 

1,020 confirmed cases were reported with no history of international travel, including two 

outbreaks of 40 and 130 illnesses, respectively, in which the patients reported consuming 

different leafy greens (CDC 2021). 

Pasteurized foods or foods thoroughly heated before consumption have not been associated 

with cyclosporiasis in the U.S. From the 154 outbreaks listed in the National Outbreak Reporting 

System (NORS) from 1971 to 2021, none of them lists food that was subjected to processing 

other than cutting and bagging (CDC 2023). Shellfish have been proposed to concentrate 

oocysts from contaminated waters. Controlled laboratory studies with fresh-water clams 

(Corbicula fluminea) showed that 48 to 100% of the clams retained Cyclospora oocysts for up to 

13 days (Graczyk et al. 1998). In surveys of natural exposure of invertebrates to C. 

cayetanensis, filter feeder shellfish such as mussels and clams were found to be positive for 

oocysts of C. cayetanensis (Aksoy et al. 2014, Ghozzi et al. 2017). Although, the review 

authors concluded that shellfish were unlikely to be significant to the epidemiology of 

cyclosporiasis because the mollusks did not travel large distances, it was noted that sampling 

shellfish for C. cayetanensis oocysts may be more efficient than sampling large volumes of 

water (Totton et al. 2021).

For this report, the Committee distinguishes associations with environmental conditions 

between countries/regions where C. cayetanesis is endemic (and transmission is via fecalenvironmental-oral route) versus those where cases of cyclosporiasis are linked to exotic 

introductions (via travel, or interactions with imported product). 

Seasonality has been identified as one of the factors affecting the incidence of Cyclospora 

infections in the areas where C. cayetanensis is endemic (Li et al. 2020). In most countries, 

especially in the Northern hemisphere, during the summer months the cases increase markedly, 

but other climate factors such as rainfall seem to differ in some regions of the world (Almeria et

al. 2019). The seasonality of traditionally non-endemic countries, such as the U.S., has 

resembled seasonal patterns of endemic countries from which produce is exported or those of 

popular travel destination, such as Mexico. Increased incidence between May to September has 

continued in the U.S. in the last four years in domestically acquired outbreak cases (CDC 

2018a, CDC 2021). This coincidence in the seasonality of the presumptive domestically 

acquired cyclosporiasis cases is curious, but it is unclear whether it coincides or correlates with 

increased summer travel, migration of seasonal labor force, import of certain commodities to 

supplement domestic production during “shoulder seasons” or some other factor that has not 

yet been considered. Intriguingly, a study by Barratt et al. (2022) suggests that distinct 

genotypes (or species) of Cyclospora are responsible for partially overlapping seasonally 

occurring outbreaks of cyclosporiasis.
13

In countries where cyclosporiasis is endemic, consumption of contaminated water has been 

consistently identified as the most important risk factor for infections (Almeria et al. 2019a). 

Studies in Venezuela and Nepal have also reported a relationship between exposure to soil 

contaminated with human feces, exposure to livestock, and consumption of fruits and 

vegetables (Bhandari et al. 2015, Chacín-Bonilla 2008). Bhandari et al. (2015) is the only report 

that found a group of patients in which the odds ratio (OR) was significant for exposure to 

livestock. This observation seemingly contradicts the prevailing hypothesis of the host range for 

C. cayetanensis and may mask an underlying livestock management practice where exposure 

to C. cayetanensis is likely. In the U.S., the majority of cases used to be linked to ingestion of 

imported fresh produce or to international travel, but in recent years the proportion of cases that 

do not have an identified connection with international origin is increasing (CDC 2018a, CDC 

2019, CDC 2021).

Since 2018, as the implicated crops have been predominantly grown in the U.S., leafy greens 

have emerged as one of the most common vehicles, as compared to earlier years when 

imported produce was more frequently associated with outbreaks (CDC 2018c). Despite the 

periodic seasonal occurrence of outbreaks every year, the routes by which fresh produce 

becomes contaminated have not been elucidated. The possibility that infected field laborers 

were the source of food contamination has not been ruled out.

Q11: Transfer and attachment

What is known about transfer and attachment of C. cayetanensis from environmental 

samples (water and soil) to produce?

A literature survey was completed on the detection, epidemiology, and control of C. 

cayetanensis on produce, water, and soil. The review indicated that out of 38 studies, 13 were 

conducted on produce, 24 were conducted on water and only one study was conducted on a 

soil sample (Totton et al. 2021). The CDC has conducted multiple epidemiology studies during 

or after an outbreak period and have yet to conclusively determine if transfer is primarily through 

direct contact of contaminated surfaces, worker's hands, or indirect contact from food contact 

surfaces or water sources such as irrigation water, protective sprays, or wash water. The lack of 

conclusive results about source transfer represents a major knowledge gap and more studies 

are needed to better understand whether/how C. cayetanensis oocysts are transferred from 

water and/or soil to produce. The attachment of C. cayetanensis to plant surfaces is not fully 

understood but it may be enhanced by the physical structure of the plants and surface adhesive 

structures produced by the parasite (Tefera et al. 2018). The physical attachment of better 

studied parasites to non-host surfaces may offer models that shed light on attachment and 

transfer of Cyclospora to/from plants. 

Cyclospora oocysts are considered comparatively more “sticky” than Cryptosporidium oocysts, 

due to specific adhesins (Tefera et al. 2018). Cyclospora, Toxoplasma, Eimeria and other 

parasites in the Apicomplexa phylum use adhesins to promote recognition, attachment, and 

invasion of the host cells. The parasite could produce modified versions of the naturally 

occurring surface glycans in plants to increase affinity and specificity. It is suggested that the 
14

affinity is partially determined by the surface molecules on the parasite and host that act in a 

concerted receptor-ligand manner (Boulanger et al. 2010). 

In Chandra et al. (2014), oocysts were dislodged with water from basil leaves, but were more 

efficiently recovered using acidified water or surfactant. This result may suggest that some 

parasite surface structures were involved in the covalent or physical attachment to plant 

surfaces. Given that little is known about Cyclospora attachment, there is a potential opportunity 

to conduct a comparative analysis with Eimeria and what is known about attachment of Eimeria 

to animal cells. Eimeria attachment mechanisms have been extensively studied, but attachment 

to animal cells almost certainly involves different mechanisms than attachment to plant cells 

(Fuller and McDougald 2002). This is an opportunity for further research to determine whether 

this is an example of a mechanism used to attach to both plant and animal hosts, recognizing 

that different life stages of the organism interact with the animal and plant hosts. However, given 

the currently limited availability of C. cayetanensis oocysts, these studies may need to be deprioritized.

Q14: The role of vectors

What is known about the role of vectors (such as non-human organisms), if any, in the 

transmission of C. cayetanensis?

C. cayetanensis is known to infect only humans, and humans are the only known naturally 

occurring host for C. cayetanensis. However, the involvement of animals should not be 

discounted in the epidemiology of cyclosporiasis associated with fresh produce. Exposure to 

domestic animals/livestock has been implicated as a risk factor for cyclosporiasis. Living closely 

with birds, guinea pigs, rabbits (Bern et al. 2002), poultry (el-Karamany, Zaher and elBahnasawy 2005), and cattle (Bhandari et al. 2015) were found to be possible hygienic factors

associated with the elevated incidence of cyclosporiasis. The design of these correlative 

studies leaves uncertainty regarding whether the elevated risk stems directly from interactions 

with livestock or if possible concealed hygiene practices within the observation structure might 

be influencing the outcomes. While Cyclospora and Eimeria species appear to be host-limited, 

the role of animals as potential vectors cannot be ruled out due to scarce and inconclusive 

evidence presented below. Therefore, the Committee’s recommendations focus on other routes.

When C. cayetanensis was first recognized as an infective agent in human outbreaks, surveys 

were conducted in an attempt to determine whether there was a zoonotic source of the parasite. 

Garcia-Lopez et al, (García-López, Rodríguez-Tovar and Medina-De la Garza 1996) found what 

were assumed to be C. cayetanensis oocysts in fecal samples pooled from 600 4–6-week-old 

chickens and a second pooled fecal sample of 50 6-8-week-old chickens. The identification was 

based on oocyst morphology, positive acid-fast staining, positive autofluorescence under UV 

light and sporulation after 10 days of incubation. The authors hypothesized that it could have 

been a related organism, and – in retrospect – this was the likeliest conclusion, with the 

researchers having almost certainly observed a closely related Eimeria spp., a common poultry 

parasite.
15

Yai et al. (Yai et al. 1997) reported on two cases of dogs with unexplained diarrhea that yielded 

characteristic Cyclospora oocysts using light microscopy. The authors suggested that this 

contact with dogs may be important in human cyclosporiasis. Zhao et al. (2021) provided a 

literature review of animal surveys for Cyclospora-like organisms in a variety of animals 

including dogs, birds, cattle, insects, poultry, non-human-primates, rodents, sheep/goats, and 

shellfish. A variety of methods were used in these studies to identify Cyclospora-like organisms 

ranging from light microscopy with staining to distinct types of PCR. Although Cyclospora-like 

oocysts were observed microscopically or samples were tested by PCR and amplicons were 

detected in the reactions in these studies, infection of any animal by C. cayetanensis was not 

confirmed. 

A wide range of primates, reptiles, rodents, and insects may serve as primary hosts to 19 

different species of Cyclospora (Onstad et al. 2019, Giangaspero and Gasser 2019). Incidents 

have been reported of Cyclospora found outside of the primary host organism such as in 

shellfish and non-host primates in the wild and captivity, although infections of the non-host 

organisms were not demonstrated (Graczyk, Ortega and Conn 1998, Li et al. 2015, Marangi et 

al. 2015, Chu et al. 2004). Eberhard et al. (1999) identified three different Cyclospora species 

from oocysts in baboon and monkey stool samples. However, sporulated oocysts could not be 

identified due to the preservation process (Eberhard et al. 1999). Infection of these animals by 

C. cayetanensis was not confirmed by biopsy of the small intestine or it was not performed in 

any of these studies (Totton et al. 2021). Several experimental studies attempted to infect other

animal species with C. cayetanensis. The results of those experiments suggested that after 4-6 

weeks, there were no signs of infection, a result that suggests that none of the animals tested 

were susceptible to infection with C. cayetanensis (Eberhard et al. 2000). In reviewing surveys 

of natural exposure of vertebrates to C. cayetanensis, no publications were found that examined 

fish, reptiles or amphibians' exposure to C. cayetanensis oocysts (Totton et al. 2021). 

The hypothesis that C. cayetanensis is transmitted by coprophagous animals (as paratenic or 

transient hosts) was tested using a soil nematode model. Huamanchay et al. (Huamanchay et 

al. 2004) reported that while the soil nematode Caenorhabditis elegans was able to ingest 

Cryptosporidium parvum oocysts, oocysts of C. cayetanensis were not ingested by the 

nematode. The authors hypothesized that the observed difference was due to the much larger 

size of the Cyclospora oocysts. Despite this outcome, the authors noted that there was 

possibility that other nematodes may be able to ingest C. cayetanensis oocysts and that the role 

of other free-living nematodes in the mechanical transport of C. cayetanensis oocysts from the 

soil to fresh produce needs to be investigated. Since insects such as houseflies are attracted to 

human feces, insects could be an area for future research (Totton et al. 2021).The fact that 

coprophagous animals present in crop production environment (dogs, coyotes and some birds) 

are also a host to their own host-adapted close relatives of C. cayetanensis complicates 

interpretation of the surveys given difficulties in interpreting PCR and microscopy data without a 

confirmatory sequencing step.

Totton et al. (2021) reviewed the role of animal vectors in the epidemiology of cyclosporiasis. In 

the review of natural or experimental studies of infection of animals by C. cayetanensis, the 
16

authors included only studies that were specific to C. cayetanensis and used PCR in nonlaboratory studies to identify DNA consistent with C. cayetanensis. They used this method of 

selecting studies to be included in the review because a variety of Cyclospora species infect 

animals and identification by microscopy is not sufficient to accurately identify C. cayetanensis. 

The authors also recommended that future studies use PCR coupled with DNA sequencing to 

confirm C. cayetanensis because PCR primers may cross-react with other protozoa leading to 

misidentification. Solarczyk et al. (2021) also reviewed the zoonotic implications of Cyclospora

and recommended using morphometric analysis along with sporulation analysis as a primary 

method in zoonotic surveys. These reports clearly stress the importance of primer design and 

specificity for C. cayetanensis to minimize false positives. 

Q15: The role of farm workers 

What role do farm workers play in the transfer of C. cayetanensis contamination during 

pre-harvest, harvest, and post-harvest handling? Are there particular approaches that 

would result in selective identification of the genotypes of public health concern? 

a) 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)? 

b) What are strategies that have been utilized to mitigate the contamination from 

farm workers? Have efforts to mitigate contamination from farm workers been 

successful?

Produce consumed in the US may originate from the regions where C. cayetanensis is endemic 

and non-endemic. We make this distinction because in the areas where this parasite is 

endemic, sources of the parasite (laborers along the entire supply chain, irrigation water, 

organic fertilizers, and other inputs) and the inoculum load are almost certainly different from 

those in the areas where C. cayetanensis is not considered to be endemic. Because C. 

cayetanensis is a host-limited parasite, human fecal contamination is the only ultimate source of 

oocysts in the production and processing environment. In the areas where C. cayetanensis is 

endemic, oocysts are likely common in agricultural water. As discussed in this report, there is 

not sufficient evidence to ascertain that C. cayetanensis has become endemically established in 

the U.S. (See responses to Q1 and Q4, and elsewhere in this report). In most crop production 

environments of the United States, feces of individuals who are symptomatic or asymptomatic 

carriers of C. cayetanensis are potential sources of the oocysts in the production or processing 

environment.

Hygienic practices of farm workers are a key focus area for prevention of the transmission of 

cyclosporiasis. Farm workers may be temporary seasonal workers hired for weeding, irrigation, 

harvesting and packing of fresh produce items in many agricultural regions of the United States. 

These farm workers may have been asymptomatic during the harvest period. Therefore, it is 

critical that farmworkers are well trained in appropriate hygienic practices, that necessary 

equipment is available including well-managed toilet facilities, gloves, and aprons, and that 

there is an awareness of nearby sources of potential human fecal contamination into farm water 

sources.
17

Because C. cayetanensis is a host-limited parasite, human fecal contamination is the only 

ultimate source of oocysts in the production and processing environment. In the areas where C. 

cayetanensis is endemic, oocysts are likely common in agricultural water. In most crop 

production environments of the United States, feces of laborers who are symptomatic or 

asymptomatic carriers of C. cayetanensis are potential sources of the oocysts in the production 

or processing environment.

In 2012-2015 between April 1 and August 31, the CDC and State Health Departments identified 

multiple outbreaks traced to cilantro harvested from farms in Puebla, Mexico. Since none of the 

outbreaks were confined to a single farm, pack date, ship date and/or lot code, the FDA 

concluded that the contamination was from a larger source (Abanyie et al. 2015). Suggested 

sources of the parasite included fecal contamination of growing areas, irrigation of fields with 

water contaminated with sewage, cleaning, or cooling produce with contaminated water, poor 

hygienic practices of workers that harvest and process the produce, and lack of adequate 

cleaning and sanitizing of equipment that encounters the product. Inspections of 11 farms and 

pack houses found human feces and toilet paper in the growing fields and around facilities; 

inadequately maintained and supplied toilet and hand washing facilities (no soap, no toilet 

paper, no running water, no paper towels) or a complete lack of toilet and hand washing 

facilities; food-contact surfaces (such as plastic crates used to transport cilantro or tables where 

cilantro was cut and bundled) were visibly dirty and not washed; and water used for purposes 

such as washing cilantro was vulnerable to contamination from sewage/septic systems 

(Graczyk et al. 1998). In these cases, the transfer was either from direct contact with human 

feces in the field, on supplies, workers hands and/or contaminated wash water. An increase in 

the chance for cross-contamination over a larger volume was observed after the cilantro was cut 

or chopped (Abanyie et al. 2015).

Fresh produce growers, harvesters, processors, and shippers need to be aware of potential 

mechanisms for fresh produce to be contaminated with C. cayetanensis and the best practices 

to manage the potential risk. Farm workers (symptomatic or asymptomatic) may be carriers of 

Cyclospora, although conducting surveys of laborers in the United States will require 

satisfactorily addressing ethical and legal concerns. Food safety programs at growing 

operations that are intended for Cyclospora should include training for workers handling fresh 

produce on general hygiene, “sick worker” policies, personal protective equipment (gloves, 

boots, aprons, etc.) as well as management of sanitary facilities (permanent or temporary), 

assessment of agricultural water for potential human waste contamination, and appropriate 

handling of tools and equipment. There are resources that are and will continue to become 

available from Extension services, private consultants, industry- and consumer-facing 

organizations to fresh produce operations and the respective supply chain that provide training 

materials, best practices, and assessment tools for mitigation of food safety risks associated 

with Cyclospora. 

Cyclospora oocysts shed in the feces of an infected person require maturation (sporulation) 

outside of the host (in the environment) to become infective. Once contaminated feces are in 
18

the production environment, they can contaminate water and soil which could serve as potential 

routes of contamination. Fresh produce growers, harvesters, processors, and handlers must be 

aware that human waste can enter water systems, especially open water sources, overhead or 

furrow irrigation, ditches in which water can accumulate, and sewage system infiltration. Other 

potential sources of human waste contamination to be considered include recreational vehicles 

and portable toilets near a growing field (https://www.afdo.org/wpcontent/uploads/2021/05/Investigating-Fresh-Produce-Cyclospora-Outbreaks.pdf). It remains to 

be determined how effective chemicals typically used in portable toilets, chlorine or other 

sanitizers used to treat agricultural water are against C. cayetanensis. Therefore, it’s critical that 

a growing operation complete an assessment of surrounding land uses, the management of 

nearby permanent or portable toilets as well as other possible points of contamination from 

human feces, such as boots or clothing to build a comprehensive prevention plan.

Prevalence/Persistence and Indicators

Q1: Prevalence, incidence, and burden

What is known about the prevalence, incidence, and burden of disease of cyclosporiasis 

in the U.S. and internationally? 

a) 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.?

b) What is the diversity of C. cayetanensis genotypes in the US and internationally?

c) What factors (e.g., food safety practices, location of the farms) may contribute to 

contamination with C. cayetanensis? 

d) 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?

The response below highlights the distribution of C. cayetanensis infections and illness 

outbreaks both in the United States and internationally. Many cases of cyclosporiasis illness in 

the United States are associated with people who have traveled to other countries. Domestically 

acquired cyclosporiasis illnesses have not been associated with specific geographical areas in 

the Unites States. The question (1b) of diversity of C. cayetanensis genotypes is discussed in 

the Question 6 response. The factors (e.g., food safety practices, location of the farms) that may 

contribute to contamination with C. cayetanensis (Question 1c) are discussed with the 

responses to Questions 4, 14 and 15. Additional question responses discuss factors that can 

prevent contamination. Factors that may be more significant for increasing the incidence of 

cyclosporiasis or detection of Cyclospora (Question 1d) are discussed in the Question 2 

response and elsewhere in this report. 

Distribution of C. cayetanensis infections and illness outbreaks internationally. C. 

cayetanensis infections have been reported globally across 54 countries including 13 recorded 

cases of outbreaks. Even though Cyclospora may have a worldwide distribution, detailed 

epidemiological information is not available in several countries. Most of the available 
19

epidemiological information regarding Cyclospora is from travelers to endemic regions and 

residents of countries, such as Haiti, Guatemala, Peru, and Nepal. Ortega and Sanchez (2010) 

summarized data and information from 198 publications in a review article. Asymptomatic 

infections are more frequent in endemic areas and younger children report more severe 

symptoms. The infections and severity of the disease tends to be milder as the children grow 

older. For example, the prevalence of Cyclospora in children in Peru with ages from 1 to 2.5 

years was 18%, whereas the prevalence was 6% in children with ages from 1 month to 1.5 

years. The authors hypothesize that the age at which children are exposed to the parasite 

influences the prevalence rate of Cyclospora (Ortega et al. 1993). Five percent of the Nepalese 

children aged six to 60 months and who had diarrhea were infected with Cyclospora, while only 

two percent of asymptomatic children had cyclosporiasis (Hoge et al. 1995). Giangaspero and 

Gasser (2019) provided an assessment of the prevalence of C. cayetanensis infection in 

humans. Coprological or molecular tests were used for the detection of the parasite. They report 

higher prevalence rates of cyclosporiasis in endemic countries with 5.6 % in China, 9.2% in 

Nepal, 17.4% in Turkey and up to 22% in India. Similarly, prevalence rates of 7.9% in Haiti, 

10.8% in Brazil, 24.2% in Venezuela and up to 41.6% in Peru were reported for Latin America. 

Among African countries, prevalence rates of 10% in Egypt and 7.2% in South Africa were 

reported. Lower prevalence rates were reported in non-endemic countries, 0.1 % in Czech 

Republic, 1.9% in Canada and 2.2% in Germany; although there was much higher prevalence 

rate of 27.5% in Italy (Giangaspero and Gasser 2019).

Cyclospora infections are commonly reported in endemic regions with lower socioeconomic 

conditions, although developed countries also have documented cases of large outbreaks. The 

highest prevalence of Cyclospora among susceptible populations was documented in 

immunocompetent individuals with diarrhea (Li et al. 2020). Cyclosporiasis is generally selflimiting in most immunocompetent persons, however, it can lead to severe or chronic diarrhea in 

some patients. The parasite may colonize other organs in patients with immune compromised 

conditions (Mansfield and Gajadhar, 2004). However, one group reported a very low incidence 

rate of Cyclospora in malnourished children and people with HIV/AIDS, a finding which seems 

to contradict the results of other published reports (Pratdesaba et al. 2001). Ramezanzadeh et 

al. (2022) concluded that the prevalence of C. cayetanensis infections among people with HIV 

and/or AIDS is higher, and this sub-population is more prone to gastrointestinal disease and 

diarrhea due to infection.

Epidemiological studies conducted in Guatemala at three raspberry farms, two of which were 

involved in the 1996 cyclosporiasis outbreak in the U.S., showed that children were five times 

more likely to be infected with Cyclospora than adults, while adults with AIDS reported higher 

infection rates (Ortega and Sanchez 2010). Infections were higher during spring raspberry 

harvest season which is typically during the warmest months. The overall prevalence of

Cyclospora was 2.3% with higher detection between May and August, with the highest detection 

rate of 6.7% in June. Estimates of 15,000 or more Cyclospora-like oocysts per ten liters of river 

water were reported from May to July. High levels of fecal contamination were also noted in the 

rivers during these months (Bern et al. 1999). 
20

Distribution of C. cayetanensis infections and illness outbreaks in the United States. Hall 

et al. (2012) summarized the data from laboratory-confirmed cases of Cyclospora infections 

recorded in the Foodborne Diseases Active Surveillance Network (FoodNet) in the United 

States from 1997-2009. The FoodNet sites included Connecticut, Georgia, Maryland, 

Minnesota, New Mexico, Oregon, Tennessee and selected counties in California, Colorado and 

New York and represented about 15% of the U.S. population. 70.3% (260 out of 370 cases) of 

the cases were from residents of Connecticut (134 cases) and Georgia (126 cases), which 

accounted for 29% of the FoodNet population (Hall et al. 2012)

About a third of the 1,110 laboratory-confirmed sporadic cases of cyclosporiasis in the United

States, recorded by the CDC from 1997 to 2008, were linked to people who traveled outside the 

United States and 69.8% of the domestically acquired cases were from April to August. Among 

the domestically acquired cases, 31.2% lived in Florida (124 persons), 16.1% lived in New York 

City or other places in New York state (15 persons) (Hall et al. 2011). It is unknown if the higher 

incidence rates in some of the geographic areas are due to more sensitive test methods or 

higher exposure and infection rates. The over-representation of cases in certain areas of the 

country is concerning and causes and public health consequences of it need to be investigated. 

The authors report that due to lack of robust testing methods and molecular tools, some of 

these outbreaks associated cases could not be linked to other cases. The median age of the 

patients was 44 years (range of 3 months to 96 years), with 50.5% female, 47.2% male and 

2.3% of unknown gender. Gender does not have a significant effect on Cyclospora infection rate 

(Hall et al. 2011). Cyclospora seems to be particularly affecting children in areas where water 

and food sanitation are poor or inadequate (Bern 2002). 

Documented international travel two weeks before the onset of symptoms was reported in 

33.5% of case patients (372), while 35.9% had no reported travel outside the U.S and 30.6% 

reported unknown travel history. Of note, cyclosporiasis is currently a reportable disease in 46

states, the District of Columbia, and New York City (CDC 2022). The available epidemiological 

information was not conclusive to associate if these sporadic domestically acquired cases were 

due to identified or unidentified outbreaks.

The five-year surveillance data from the United States for the period 2011- 2015 shows 

increased cases of cylcosporiasis during spring and summer months. Casillas et al. (2019) 

reported that five of the ten outbreaks of cyclosporiasis investigated during this period were 

linked to foods of international origin. Barratt et al. (2022) suggest that distinct genotypes (or 

species) of Cyclospora may be responsible for the outbreaks occurring earlier and later in the 

summer, this report is discussed in more detail below.

Q2: Seasonality, incidence, and prevalence

How does the seasonality, incidence and prevalence of cyclosporiasis compare 

throughout the United States and internationally and what factors may contribute? 

a) Extrinsic factors that may influence sporulation and survival (e.g., extrinsic 

factors influencing sporulation and survival); 

b) Environmental factors influencing movement (e.g., rainfall);
21

c) Other factors?

Cyclosporiasis exhibits a seasonal pattern globally. In the United States, the peak season 

occurs from May to August. The seasonality of infections varies geographically, and infections 

can be more prevalent in dry seasons or in rainy seasons. Detection frequencies of Cyclospora

oocysts throughout the year can vary and may not correlate to patterns of seasonal infections. 

The factors contributing to the seasonality of cyclosporiasis are not fully known, and the 

variations across regions cannot be attributed to a single common factor, although recent 

evidence suggests that distinct genotypes (or species) of Cyclospora may be responsible for the 

outbreaks occurring in different seasons (Barratt et al. 2023). The sporulation and survival of 

Cyclospora cayetanensis can be influenced by various external factors. While the application of 

some cold and hot temperatures affects sporulation and survival, exposure to some commonly 

used pesticides and antimicrobial chemicals has been shown to have a limited effect. 

Environmental factors influencing seasonality of incidence or prevalence of 

cyclosporiasis. Cyclospora cayetanensis infection follows a distinct seasonal pattern globally 

as evidenced by several studies (Hall et al. 2012, Bern et al. 1999,). The prevalence of infection 

by C. cayetanensis rises during periods of elevated rainfall and warm weather in Guatemala, 

Honduras, Mexico, Jordan, Nepal, and China. (Almeria et al 2019) Nevertheless, this seasonal 

variation differs among various regions, probably impacted by human activities, environmental 

contamination, and the unique conditions that promote sporulation in each specific area. It is 

unknown why there are extended periods without humans presenting with cyclosporiasis even 

while C. cayetanensis remains detectable in the environment. How the organism survives in the 

environment over these extended periods is also unknown. The seasonality of this disease is 

thought to be attributed to environmental factors such as temperature, rainfall amounts, 

humidity, and photoperiod. Notably, this pattern cannot be solely attributed to rainfall, given the 

evident seasonal fluctuations even in arid environments (Almeria et al. 2019). Nonetheless, in 

Peru and Turkey, the occurrence of infection is more widespread when there is a lack of rainfall, 

typically during the drier and hotter months., Infections in Haiti tend to arise during the driest and 

coolest periods of the year, or alternatively during the cooler wet season in Indonesia. In India, 

clinical cases showed a higher frequency during the summer prior to the rainy period (Almeria et 

al. 2019). As a result, pinpointing a unified factor to account for the observed seasonal 

variations becomes challenging.

In a study undertaken in Colombia by Frickmann et al. (2021), a lower number of individuals (2 

out of 16, or 12.5%) presented with gastrointestinal symptoms during the rainy season, in 

contrast to the dry season (6 out of 15, or 40%), even though parasite loads were higher in the 

rainy season. The prevalence of C. cayetanensis among Colombian indigenous people remains 

significant during the dry season. The coexistence of minimal gastrointestinal symptoms 

alongside heightened parasite loads suggests the likelihood of colonization rather than infection. 

The correlation between parasite detection in the environment and its clinical manifestation in 

the population remains unexplained.

Cyclosporiasis cases are reported throughout the year in the U.S., but there is an increase in 

domestically acquired cases from May to August. Between 1992 and 1995, during periods 

without outbreaks, the prevalence of Cyclospora infection in the general population of North 

America and the United Kingdom was below 0.5% (Herwaldt 2000). However, there were 

variations in the prevalence of infection across different regions within the U.S. From 1997 to 
22

2009, out of the 370 cases of Cyclospora infection that were confirmed through laboratory 

analysis and reported via the Foodborne Diseases Active Surveillance Network, the majority 

(70.3%) were clustered in Georgia and Connecticut (Hall et al. 2012). Between 2004 and 2009, 

approximately 37.8% (70 out of 185) of the cases were categorized as infections acquired within 

the country (Hall et al. 2012, Almeria et al. 2019). It's worth highlighting that although 

cyclosporiasis isn't regarded as endemic in the U.S., there exists a potential for specific 

localized regions to exhibit low-level endemicity (Casillas et al. 2019). Additional research into 

domestic prevalence, environmental contamination, and endemicity could be considered. 

The factors contributing to the seasonality of cyclosporiasis are not fully known, and the 

variations across regions cannot be attributed to a single common factor. There remain 

unidentified factors associated with the apparent absence of symptomatic human infection over 

extended durations, despite specific biological conditions required for the parasites to survive 

during these prolonged periods and the presence of the parasite in the environment (Almeria et 

al. 2019). In non-endemic industrialized countries, isolated cases and outbreaks are primarily 

associated with international travel and the consumption of contaminated imported produce 

originating from regions where the infection is endemic (Almeria et al. 2019), although an 

increase in cases associated with the consumption of domestically-grown produce has been on 

the rise. Barratt et al (2023) suggested that, at least in part, the seasonality of domestic 

illnesses can be explained by the distinct genotypes (or species) of Cyclospora, with the 

Lineage A being responsible for the illnesses appearing earlier in the season and peaking 

around June, and Lineage B most prevalent among illnesses later in the season peaking around 

July. While this is an intriguing hypothesis, it is important to note that each of these American 

lineages included at least one isolate common to areas of Mexico and/or Central America where 

the majority of the US seasonal labor force originates. These genomic data should be analyzed 

in the systems context which includes seasonal crop production patterns.

Extrinsic factors that may influence sporulation and survival of oocysts. Cyclospora 

oocysts develop within enterocytes, are excreted in feces without undergoing sporulation, and 

need to undergo sporulation to become capable of infecting a host. Transmission usually occurs 

through the ingestion of oocysts found in fecally-contaminated water or produce. Transmission 

directly from person to person is improbable since the excreted oocysts are non-infectious, 

necessitating sporulation outside the host to become capable of causing infection. The median 

incubation period spans about one week, within which the organism infiltrates the enterocytes of 

the small intestine. (Almeria et al. 2019). It's worth mentioning that oocysts from certain 

individuals experiencing severe diarrhea might not go through the process of sporulation.

(Almeria et al. 2019)

The sporulation and survival of Cyclospora cayetanensis can be influenced by various external 

factors. For example, under laboratory conditions at temperatures of 22°C and 30°C, 

sporulation of Cyclospora oocysts stored in deionized water or potassium dichromate typically 

takes place within 7–14 days outside the host (Tucker et al. 2022). However, exposure of 

oocysts to temperatures of 37°C for 4 days or 50°C for 1 hour has been observed to induce 

sporulation. Conversely, storage at 4°C or 37°C for 14 days delays sporulation, with only 12% of 

Cyclospora spp. sourced from humans and baboons sporulating under such conditions. 
23

Interestingly, oocysts that were stored at 4°C for one to two months sporulated when 

subsequently stored for six to seven days at 30°C (Tucker et al. 2022). 

The effects of temperature, including freezing and heating conditions, on the sporulation of C. 

cayetanensis were investigated in dairy products and basil (Sathyanarayanan and Ortega, 

2006). Sporulation was observed in these matrices at 23°C, but extreme temperatures led to the 

inactivation of oocysts. No sporulation occurred at temperatures of -70°C, 70°C, and 100°C for 

both water and basil samples. Similarly, dairy products did not exhibit sporulation when cooked 

at 70°C, frozen at -70°C for 1 hour, or exposed to -15°C for 24 hours. Basil kept at -20°C for two 

days and water stored for four days also did not support oocyst sporulation. Additionally, the use 

of recommended concentration levels of pesticides, including fungicides and insecticides, or 

combinations of these products did not affect the sporulation of C. cayetanensis. Due to the 

limited understanding of the mechanisms triggering sporulation in C. cayetanensis oocysts and 

the factors influencing their survival, it would be prudent to investigate factors that affect 

sporulation and survival in similar parasites. Particularly, the examination of surrogate 

organisms in future challenge studies could provide valuable insights into the study of C. 

cayetanensis (Sathyanarayanan and Ortega, 2006).

Q10: Persistence/survival in food and the environment 

What is known about Cyclospora cayetanensis persistence/survival in food, such as 

produce, and the environment (e.g., soil, water, food contact surfaces)?

C. cayetanensis oocysts have been detected in several types of water. These include 

chlorinated and unchlorinated drinking water, food/agricultural process water, wastewater, 

recreational waters, and well water. Furthermore, this organism has been detected in areas 

where soil can contact human feces and in areas where there is a lack of personal hygiene. 

Further research is needed to elucidate survival times and sporulation rates in water, soil, and 

food or agriculture process environments.

Water and soil contaminated with fecal matter could transmit C. cayetanensis infection. In 

endemic areas, drinking water is a known risk factor for Cyclospora infection. The detection of 

C. cayetanensis oocysts in several types of water—including chlorinated water, and wastewater 

in both endemic and in non-endemic areas suggests that Cyclospora could spread thorough 

drinking and recreational waters (Almeria et al. 2019) (Rabold et al. 1994) (Kwakye-Nuako et al. 

2007). Chlorine and other water disinfectants are not effective against Cyclospora oocysts and 

the oocysts can pass through physical barriers (Mansfield and Gajadhar, 2004). Studies 

conducted in Guatemala concluded that drinking untreated water and soil contact were 

significant risk factors for cyclosporiasis, among children <2 years of age. These studies also 

found that among 182 people in the cohort, four farm workers had asymptomatic cyclosporiasis 

(Bern et al. 1999). 

Exposure to recreational water contaminated with Cyclospora may also be a source of infection 

(Bilung et al. 2017). Nine percent of water samples (20 out of 233) collected along a river in 

Spain over a one-year period tested positive for Cyclospora spp. with 17/20 positive in a qPCR 

with primers amplifying 116-bp fragments in the internal transcribed spacer 2 (ITS-2) gene 
24

(Lalonde and Gajadhar, 2008) (Galvan et al. 2012). Nine of 48 samples of influent and effluent 

water from wastewater treatment plants in Arizona showed the presence of C. cayetanensis. 

The authors reported that they did not determine the efficacy of the removal of Cyclospora in the 

treatment process (Kitajima et al. 2014). These studies show that water contaminated with feces 

could be a potential source of Cyclospora.

Soil is a potential and important mode of transmission and source of infection for C. 

cayetanensis. Contaminated soil is a risk factor in both developing and developed countries 

(Mansfield and Gajadhar, 2004). In Venezuela, for example, most cases of C. cayetanensis 

were recorded in areas with poor hygiene and extreme poverty. Contact with soil contaminated 

with human feces was strongly associated with Cyclospora infection with higher prevalence 

rates when hand washing water was lacking in agricultural operations. In Italy, soil was found to 

be positive for oocysts (11.8% positive samples, 6/51) (Giangaspero et al. 2015a). Higher rates 

of infection have been reported in areas where inadequate sanitary facilities, poor personal 

hygiene and soil contaminated with human feces were noted as risk factors.

Q13: Indicator organisms

Are there indicator organisms that can be used to determine the likely presence or 

absence of C. cayetanensis in various matrices?

An indicator organism is a microorganism or group of microorganisms that may indicate a 

possible presence of a pathogen of concern, that are typically present in much lower numbers 

than indicators or that conditions under which an indicator increases in numbers may favor 

pathogen growth (Busta et al. 2003). Indicator organisms for parasites such as C. cayetanensis

are difficult to identify. Since C. cayetanensis can only originate from human feces, an indicator 

of human fecal pollution is likely to provide a practical solution. The committee acknowledges 

the multitude of studies on advantages and limitations of indicators of human fecal 

contamination, as well as at least a dozen existing indicators and at least a dozen proposed 

indicators of human fecal contamination. 

A study by Mattioli et al. (2022) attempted to correlate the presence of a fecal indicator bacteria, 

Escherichia coli, and human-specific fecal molecular markers, Bacteroides HF183 and 

crAssphage with the presence of C. cayetanensis in the crop production environment. 

However, while this study detected the presence of some of these markers of human fecal 

pollution, C. cayetanensis was not detected in any of the samples. This outcome should not be 

considered discouraging as indicators often overestimate the potential for the presence of 

fecally-shed human pathogens. Given that essentially nothing is known about persistence of C. 

cayetanensis in the environment and given that oocysts of the parasites are currently in a 

limited supply to conduct correlational or comparative studies with well-characterized indicator 

organisms, it is unclear how productive efforts to identify a “perfect indicator” for the presence of 

C. cayetanensis would be. Collectively, these results indicate that future efforts should continue 

programs on risk-based management, not on efforts to manage hazards, whether potential or 

perceived as potential. 
25

Analytical Methods

Isolation, Concentration, Detection and Confirmation

As discussed throughout this report, C. cayetanensis is a parasite with a host range that is 

limited to humans, while many other animals host very closely related organisms that are 

nonpathogenic in humans. This, therefore, highlights the primary challenge with the isolation, 

concentration, and detection of C. cayetanensis: any Cyclospora isolated from a human fecal 

sample is almost certainly C. cayetanensis (because humans act as “biological concentrators” 

of the parasite), however, environmental isolates of Cyclospora could have originated from a 

nearly infinite number of potential hosts of non-human parasites. Because a Cyclospora from a 

human sample is almost certainly C. cayetanensis, a fairly generic target (such as the 18S 

ribosomal RNA gene) for the typing at the genus level is practically sufficient (to distinguish from 

other eukaryotic or procaryotic causes of gastrointestinal symptoms). However, using 18S 

ribosomal RNA genes as targets for environmental samples has led researchers to erroneous 

conclusions about prevalence of C. cayetanensis in environmental samples collected in regions 

where the parasite is not endemic. These limitations were highlighted by recent studies of 

Mattioli et al. (2022), Kniel (2022) and a retrospective re-analysis of samples previously thought 

to be C. cayetanensis by Ortega (2022). In two of these studies (Mattioli et al. 2022 and Ortega 

2022), a nearly 90% false-positive rate for PCR-based assays using a common method 

targeting 18S ribosomal RNA genes was observed, and Kniel (2022) carefully documented 

factors and conditions under which PCR products obtained using 18S primers failed to yield 

conclusive sequencing results. As an alternative target, a PCR-based method targeting internal 

transcribed spacer 1 (ITS-1) region was reported to have 102% efficiency, linearity and 

reproducibility using purified templates and samples. When this method was used on berries 

from commercial trade in Norway, only ~19% of amplicons from the PCR reactions with ITS-1 

primers were confirmed by sequencing as those belonging to C. cayetanensis. Collectively, 

these results highlight the need for a more robust and reproducible method for the detection of 

the parasite to the species level in environmental samples. 

In the absence of a robust, specific, and reproducible single-step method for the detection of C. 

cayetanensis, there remains the need for confirmatory molecular methods of the PCR-positive 

samples from implicated foods and potential contamination sources. A genotyping system for 

Cyclospora based on eight genetic markers has been applied to human clinical samples 

(Nascimento et al 2020). This approach, although helps to discriminate between clinical cases, 

still requires development for food sampling and improved cluster detection.

Whole-genome sequencing is impractical for routine molecular surveillance of C. cayetanensis 

outbreaks because of the inability to culture the organism (which makes it difficult to obtain 

sufficient DNA mass from samples) and due to its large genome (44 megabases). To address 

these issues, researchers have focused on the development of new methods based on potential 

genomically-derived markers for strain-level identification (Nascimento et al. 2020, Gopinath et 

al. 2018). One approach has been to apply bioinformatic analyses to public mitochondrial 

genome assemblies to create a reference genome which can then be used in the application of 

subtyping C. cayetanensis strains during foodborne outbreak investigations (Nascimento et al. 
26

2020). In addition, it is worth exploring other options for the detection and differentiation of C. 

cayetanensis such as infrared-functionalized microbalance sensor (Maloney et al. 2023).

Q3: Sampling data

What sampling data exists for Cyclospora cayetanensis in food products and 

environmental samples, domestically and internationally?

a) What trends have been observed?

b) What methods of detection were used?

Summary of Question 3 Response

Currently, there are no international standards for testing for C. cayetanensis in the environment 

and food products. Since the FDA BAM Chapter 19c detection method was validated, most 

studies have used either this method or a modified methodology, however, this method targets 

18S ribosomal RNA genes, and the limitations of this approach have been discussed in this 

report. However, the prevailing consensus is that more methods need to be developed that are 

able to isolate the small numbers of oocysts from environmental samples, in addition to the 

various food matrices. Even when C. cayetanensis is detected in environmental samples, 

additional confirmatory testing is required due to the significantly high number of false positives 

from cross-reactions with related parasites that are not pathogenic in humans. 

Q3a). What trends have been observed?

As discussed earlier in this report, data collected from regions and countries where C. 

cayetanensis is endemic should not be co-interpreted with the data from the regions where the 

parasite has not been established endemically. Seasonal trends and epidemiological trends for 

the areas where C. cayetanensis is endemic have been discussed elsewhere in this report. 

Epidemiological trends in the US have been discussed in response to other questions. 

Prior to the reports of Mattioli et al. (2022), Kniel (2022) and Ortega (2022), detection of 18S 

ribosomal RNA gene amplicons in environmental samples has been interpreted to indicate the 

presence of C. cayetanensis in an environmental (primarily, water) sample. Given a ~90% false 

positive rate of a common PCR-based method for the detection reliant on primers for 18S 

ribosomal RNA genes, discussing trends based on the results of studies in which a definitive 

confirmatory step (such as amplicon sequencing) was not performed is premature. 

Q3b). What methods of detection were used?

Currently, there are no recognized International Organization for Standardization methods for 

detecting C. cayetanensis in foods and the environment. Most studies have been conducted 

using BAM 19b, BAM 19c, or a modification (Lalonde et al 2022). Recent molecular studies 

have provided data that suggests current 18S-based methodologies may not be sufficiently 

specific to distinguish between C. cayetanensis from Eimeria or Isospora species (Mattioli et al. 

2022, Ortega 2022, Kniel 2022).

The Center for Produce Safety (CPS) sponsored a study, from January 2020 through April 

2022, to examine the sources and prevalence of C. cayetanensis in irrigation water, harvested 
27

produce (using spent packinghouse water in dump tanks as a proxy for the produce), and 

municipal wastewater in the Southeastern Coastal Plains region in Georgia (Mattioli 2022 and 

Table 2). 

At the start of the project in 2020, the researchers collected samples from the surface-fed 

holding ponds once a month during the fallow and growing periods and twice a month during 

harvesting. In 2021, the sampling frequency was increased to twice a month during the fallow 

and growing seasons. The researchers collected weekly samples from the spent packinghouse 

water in the dump tanks, the spent water being used as a proxy for harvested produce. The 

samples were either filtered onsite or, if the turbidity was too high, the samples were sent to the 

CDC. Municipal wastewater sludge samples were taken from the thickener sludge and from the 

return activated sludge from the aeration basin. Dead-end Ultrafiltration was used to 

concentrate holding pond water samples and continuous flow centrifugation was used to 

concentrate dump water from the packinghouses. Sludge and portable toilet samples were 

concentrated via centrifugation.

All samples underwent DNA extraction followed by quantitative PCR (qPCR). BAM Chapter 

19C defines a positive as any sample that has at least one of the three qPCR replicates below a 

Cq of 40. The researchers deviated from the cutoff Cq value in BAM 19C. Instead, they used a 

Cq of ≤ 37, to reduce the number of false positives. Samples with at least one replicate with a Cq 

≤ 37 were submitted to the CDC Parasitic Diseases Branch for attempted genotyping. This was 

useful to eliminate false-positive results. Of the 217 samples from eight surface-fed holding 

ponds, 18S rRNA amplicons were detected in 59 (27%). 18S rRNA amplicons were detected in 

only one of 46 (2%) dump tank water samples. No 18S rRNA amplicons were detected in the 37 

samples from the on-farm portable toilets. Of the total of 76 sludge samples, the 18S rRNA 

locus was amplified in nine (20%) sludge from the thickener and nine (30%) return activated 

sludge. However, of the samples submitted for amplification, only one sample matched C. 

cayetanensis haplotypes from clinical specimens, which indicated low level community 

shedding. Despite positive 18S rRNA amplicon detections, their sequencing failed to confirm 

amplicons as those belonging to C. cayetanensis. Giangaspero et al (2015b) conducted the first 

comprehensive molecular survey, over a two-year period from 2012 to 2014, looking for C. 

cayetanensis in southern Italy. They examined water (treated water from the municipal 

treatment plants, drinking water, and well water used for irrigation), eight types of vegetables 

and fruits (cucumber, lettuce, fennel, celery, tomato, melon, endive, and chicory), farm soil from 

the bases of the selected vegetables and fruits, and human fecal samples, that had been 

submitted to the main area hospital. The water samples were filtered through a 1 μm yarnwound cartridge filter which was backflushed three times then concentrated using centrifugation. 

Likewise, soil and produce samples were placed in suspension, centrifuged, and filtered through 

double gauze and the filtrate again centrifuged. All sedimented pellets were subjected to 

Percoll-sucrose flotation and DNA extraction. The samples were tested using qPCR-coupled 

single strand conformation polymorphism (SSCP) analysis and DNA sequencing. Giangaspero 

et al (2015a) detected Cyclospora DNA in 21.3% of treated water samples and 6.2% of well 

water samples but did not detect Cyclospora DNA in drinking water samples. Detection rates in 

soil and produce samples were 11.8% and 12.2% respectively (Giangaspero et al. 2015c and 
28

Table 3). The survey did not use controls, therefore, as seen with other studies, it is difficult to 

determine if the positive samples cross-reactions and, therefore, false positives.

Table 2. Results of surveys for Cyclospora Mattioli et al (2022)

Sample source n 18S rRNA 

detected

% 

Detection

Confirmed C. 

cayetanensis

Irrigation ponds 217 59 27 0

Packinghouse dump 

tanks 46 1 2 0

Municipal wastewater 

sludge (RAS/REC) 46/30 9/9 20%/30% 7

Portable toilets 37 0 0 0

Table 3. Results of Cyclospora surveys in Southern Italy, Survey Results, Giangaspero et al 

(2015)

Sample source n % Detected 

Cyclospora DNA

Well water used for irrigation 16 6.2

Treated water from municipal 

treatment plants

94 21.3

Drinking water 3 0

Farm soil surrounding 

produce

51 11.8

Produce (vegetable and fruit 

crops)

49 12.2

Human feces (submitted to 

main hospital)

40 27.5

Q6: Approaches for characterizing

What are available approaches for characterizing the relatedness of different strains of C. 

cayetanensis (e.g., subtyping)?

Clearly understanding the relatedness of strains and species of C. cayetanensis has at least two 

practical implications. First, there is a need to define genomic targets for the specific detection 

and differentiation of strains of Cyclospora capable of causing human illness, as currently 

available tools based on the amplification of 18S rRNA genes fail to do so reproducibly and 

robustly. Second, the question of endemicity of C. cayetanensis in the U.S. remains open (with 

environmental sampling data being called into question and the uncertainty with the 

interpretation of sewage data as the main argument for endemicity). Robust and conclusive 

molecular evidence will be required to address links between clinical and environmental C. 

cayetanensis strains. For example, does clustering of C. cayetanensis from domestic outbreaks 

with seemingly random isolates from a number of countries where the pathogen is endemic and 
29

most agriculture labor force originate argue for the “exotic introduction” hypothesis for the origin 

of the parasite in each outbreak? If C. cayetanensis has established endemically in some areas 

of the United States, how soon should we expect genome-level separation of the “US isolates”, 

given that the parasite has a diploid genome and undergoes sexual reproduction? The presence 

of distinct regional clusters is supported by current studies (Barratt et al. 2022), however, it 

remains to be elucidated whether it took years or decades for these regionally distinct 

genotypes to evolve. Given that C. cayetanensis can only reproduce inside the human host and 

given a relatively low prevalence of human cyclosporiasis in the United States, the temporal 

scale of evolution of the geographically distinct strains may be longer than what is expected for 

areas where the pathogen is endemic and cycles rapidly through human populations. 

Recently, whole genome assemblies, complete mitochondrial and apicoplast genomes of C. 

cayetanensis have become available (Cinar et al. 2016). Cluster analysis of specific C. 

cayetanensis apicoplast genomes revealed tight grouping of C. cayetanensis with Eimeria and 

Toxoplasma, separated from distant species such as Plasmodium and Babesia. Single 

nucleotide polymorphisms (SNPs) and identified DNA sequence repeats may be useful as 

genetic markers for identification and differentiation of C. cayetanensis isolates found and could 

facilitate outbreak investigations (Cinar et al. 2016). The mitochondrial genome and apicoplast 

genomes of C. cayetanensis have a high similarity to Eimeria spp (Tucker et al, 2022 and 

references therein, Cinar et al. 2016), which has complicated PCR-based detection of the 

parasite in environmental samples. The nuclear genome of C. cayetanensis has important 

differences that help to differentiate this organism from other apicomplexans. Human C. 

cayetanensis isolates from around the world have noticeable geographic clusters. C. 

cayetanensis genotyping methods, using targeted amplicon sequencing, are useful for 

epidemiological trace-back investigations (Cinar et al. 2020). Molecular typing of C. 

cayetanensis in produce and clinical samples can distinguish between case clusters and may 

be helpful for cyclosporiasis outbreak investigations (Zhang et al. 2021).

To supplement the epidemiological data with genetic information, Yanta et al. (2022) genotyped 

isolates from stool samples in 169 Canadian cyclosporiasis cases which occurred between 

2010 to 2021. An eight-marker targeted amplicon deep (TADS) scheme specific to C. 

cayetanensis as previously described by the U.S. Centers for Disease Control and Prevention 

(CDC) was used. Their study focused on evaluating the genotyping performance and genetic 

clustering of the Canadian C. cayetanensis isolates and reports that genotype information was 

successfully collected with at least part of one of the markers in the TADS assay for 97.9% of 

specimens, and 81.1% of cyclosporiasis cases met the minimum requirements to genetically 

cluster into 20 groups. The authors conclude that examining cyclosporiasis cases genetically 

will be a valuable tool for supplementing epidemiological outbreak investigations and further 

research is required to expand the number of discriminatory markers to improve genetic 

clustering. Barratt et al. (2023) reported genotyping thousands of US isolates and one from 

China (strain CHN_HEN01) and revealed two lineages. From March 2018 to October 2020, a 

total of 3459 C. cayetanensis genotypes were sequenced from fecal specimens collected from 

patients who received a diagnosis of cyclosporiasis in the U.S. or Canada, and from 4 
30

specimens collected before 2018 ((Nascimento et al. 2020); (Barratt et al. 2021), (Barratt et al. 

2022)). 

Their retrospective examination of epidemiologic data revealed associations between lineage 

and the geographical distribution of U.S. infections plus strong temporal associations. With the 

multiple lines of evidence for speciation within human infecting Cyclospora, the authors provide 

an updated taxonomic description of C. cayetanensis and describe two novel species as 

etiological agents of human cyclosporiasis: Cyclospora ashfordi sp. nov. and Cyclospora 

henanensis sp. nov. (Apicomplexa: Eimeriidae). The Barratt et al. (2023) study may be the first 

study suggesting the existence of two “American” (but not limited to US) species/genotypes of 

Cyclospora, likely reflective of the trade routes for fresh produce and the origin of the domestic 

agricultural labor force. 

A clustering of the US isolates with those from Mexico and Guatemala (where a significant 

number of the non-permanent US agriculture laborers originate) was reported by Leonard et al 

(2023). In contrast, in both studies (Barratt et al. 2023 and Leonard et al. 2023), isolates from 

Asian neighboring countries (Nepal, China and Indonesia) cluster tightly and separately. 

With the recent advances in sequencing technologies such as next generation sequencing

(NGS) and availability of efficient genome assembly programs, whole genome assemblies, 

complete mitochondrial and apicoplast genomes of C. cayetanensis have become available 

(Cinar et al. 2016). Whole-genome sequence data from C. cayetanensis protozoa enabled the 

development of a multilocus sequence typing (MLST) tool for characterizing isolates in outbreak 

investigations. The high resolution of the typing tool and the apparent presence of geographic 

clusters might facilitate the identification of outbreaks and infection sources. (Guo et al. 2016). 

One method based on MLST has been recently developed by CDC researchers. This method 

relies on the amplification of 8 genetic markers followed by deep sequencing and bioinformatic 

analysis of the data. This method has been used to characterize haplotypes of C. cayetanensis 

for molecular epidemiology purposes (Nascimento et al. 2020, Barratt et al. 2021, Barratt et al. 

2022). This method has been implemented at FDA to be used on characterization of C. 

cayetanensis haplotypes of DNA extracted from produce and water that is found to be positive

for the presence of C. cayetanensis. In 2021 this MLST approach was applied to environmental 

samples collected from a canal in Florida. Characterization of C. cayetanensis haplotypes using 

this approach was possible in 6 of the 8 samples analyzed (FDA/CDC report from August 13th,

2021- unpublished data). This was a follow-up of the work done as part of an investigation to 

identify the root cause of the 2020 bagged salad outbreak. In 2022, FDA began to apply the 

CDC genotyping method, with modifications, to C. cayetanensis collected from produce and 

water samples, which will enable the linkage of human illness to suspect food items (Viazios et 

al. 2022). 

Q7: Current available test methods for detecting and/or isolating

What are currently available test methods (and comparative sensitivity/specificity) for 

detecting and/or isolating C. cayetanensis in different matrices (e.g., food, water, 
31

environmental samples)? What type of validation has the method(s) undergone? What 

are the matrices for which the methods have been validated?

Detection methods

C. cayetanensis is an unculturable parasite, therefore all analytical methods used for the 

detection and characterization of C. cayetanensis in different types of samples rely on 

microscopy techniques, detection of the parasite’s DNA (e.g., PCR methods), and/or DNA 

sequencing analysis of suitable genetic markers.

In clinical settings, Cyclospora infection is diagnosed by examining stool specimens using 

various microscopy techniques and/or by PCR assays designed to detect the parasite in stool 

(CDC 2023). Symptomatic patients are known to at times shed low numbers of Cyclospora

oocysts, therefore sample preparation techniques to concentrate the oocysts, such as the 

formalin-ethyl acetate sedimentation technique, are typically used to increase the chances of 

detection (CDC 2023). Smears of the resulting sediment can be stained and examined 

microscopically using modified acid-fast or modified (“hot”) safranin techniques, although 

Cyclospora oocysts may not uniformly stain and appear either stained or unstained in 

microscopic fields when using the modified acid-fast technique. Alternatively, an ultraviolet (UV) 

fluorescence microscope (set at 330-365 nm or 450-490 nm) can be used to view Cyclospora

oocysts since they autofluorescence (CDC 2023). Although these microscopy techniques are 

effective for clinical diagnosis, they cannot distinguish oocysts of C. cayetanensis from 

morphologically identical oocysts of other Cyclospora species which may be present in food, 

environmental, or other zoonotic samples (Eberhard, Pieniazek and Arrowood 1997, Eberhard 

et al. 1999).

C. cayetanensis oocysts are expected to be present in exceedingly low numbers in food and 

environmental samples, when present at all. Unlike clinical samples, food and environmental 

samples are expected to contain significant background populations of other parasites, including 

non-pathogenic species of Cyclospora and closely genetically related Apicomplexan parasites, 

such as species of the genus Eimeria. Due to the limitations of microscopy techniques to detect 

C. cayetanensis oocysts in food and environmental samples, molecular methods, such as PCR, 

represent the most feasible approach for detection in these matrices (Lalonde and Gajadhar 

2008, Murphy et al. 2017, Durigan, Murphy and da Silva 2020, Kahler et al. 2021, Barlaam et al. 

2021, Lalonde, Oakley and Fries 2022). Sample preparation techniques, such as flocculation, 

floatation, filtration, and centrifugation, have been used alone or in combination to concentrate 

oocysts and improve the chances of detecting C. cayetanensis in food and environmental 

samples. Challenges to developing molecular detection methods for C. cayetanensis in food 

and environmental samples include matrix complexity (including the potential presence of PCR 

inhibitors), the inability to culture the organism in vitro, and the lack of genomic sequences for C. 

cayetanensis and other closely related organisms (Balan et al. 2023). 
32

PCR methods targeting the 18S rRNA genes

Currently, FDA has validated two methods for the detection of C. cayetanensis, one that is 

specific for detection in fresh produce and another that is specific for detection in agricultural 

water. These methods employ various sample preparation techniques followed by a quantitative 

real-time PCR (qPCR) targeting the 18S rRNA gene with a species-specific probe and an 

internal amplification control. 

The current FDA method for the detection of C. cayetanensis oocysts on fresh produce 

(Chapter 19b of the FDA BAM) (FDA 2023a) was validated in a collaborative study to detect as 

few as five oocysts inoculated onto 25 g samples of cilantro or 50 g samples of raspberries 

(Murphy et al. 2017). This method uses a procedure to recover inoculated oocysts from produce 

previously demonstrated to significantly improve the recovery of C. cayetanensis oocysts from 

basil and lettuce and a commercial DNA extraction kit (Shields, Lee and Murphy 2012, Murphy 

et al. 2017). The collaborative study included a comparison of the nested PCR from FDA’s 

previous method with the qPCR in the current method. Although the nested PCR detected C. 

cayetanensis at the 5-oocyst inoculation level in a few more inoculated samples than the qPCR 

method, analysis of uninoculated samples using the nested PCR resulted in a false-positive rate 

of 2.6% for cilantro samples and 5.0% for raspberry samples, whereas there were no falsepositives observed for the qPCR (Murphy et al. 2017). The performance of the current FDA 

method for the detection of C. cayetanensis oocysts on fresh produce has since been verified 

for other produce matrices, such as shredded carrots, basil, parsley, cilantro, blackberries, 

strawberries, blueberries, shredded cabbage, romaine lettuce, spring mix, coleslaw, and green 

onions (Almeria et al. 2018). 

Most recently, however, when independently validating FDA BAM 19b Method, Centre for FoodBorne and Animal Parasitology of the Canadian Food Inspection Agency (CFIA) was able to 

reliably detect only 200 C. cayetansesis oocysts spiked into 25g samples of berries, leafy 

greens and various salads; when 10 oocysts were spiked into 25g samples, median sensitivity 

of detection was 30%, and 8 out of 9 tests failed when only 5 oocysts were spiked into 25g fresh 

produce samples. The CFIA authors comment that these differences could be due to the age of 

oocysts used in spiking and/or “to the fact that The FDA procedure for preparing the spike stock 

did not involve verification of the oocyst numbers after serial dilution to the required spiking 

concentration”. In Lalonde et al (2022), the number of oocysts in each stock was confirmed by 

microscopy (Lalonde et al. 2022). It should be noted that similar limits for reliable detection (200 

oocysts per sample) were reported to the subcommittee by a representative from one of the 

commercial testing labs using FDA BAM 19b Method. 

The current FDA method for the detection of C. cayetanensis oocysts in agricultural water 

(Chapter 19c of the FDA BAM) (FDA 2023a) was validated in a multi-laboratory study to detect 

as few as six oocysts in ten liters of irrigation water (Murphy et al. 2017, Durigan et al. 2020). 

The previous FDA BAM method (Chapter 19a) was found to be ineffective at handling 

agricultural waters with high turbidity during a C. cayetanensis outbreak in 2013 (Durigan et al

2023). The current method uses hollow fibers in a dead-end ultrafiltration (DEUF) configuration 

to recover inoculated oocysts from large volumes of agricultural water. A DNA purification step 

was added after DNA extraction to overcome PCR inhibitors commonly found in agricultural 
33

waters and optimize performance (Durigan et al. 2020). In addition, the qPCR was evaluated 

using a panel of DNA samples from selected foodborne bacterial and parasitic pathogens: 

Cryptosporidium parvum, Cryptosporidium hominis, Eimeria acervulina, Eimeria tenella, Eimeria 

maxima, Entamoeba histolytica, Giardia duodenalis, Blastocystis hominis, Plasmodium 

falciparum, Toxoplasma gondii, Salmonella spp., Escherichia coli, and Trypanosoma cruzi

(Durigan et al. 2020). The purification method was used on 6 samples from open water sources 

in Maryland, and PCR-based detection targeting 18S rDNA using a modified FDA's BAM, 

chapter 19B method. In 3/6 samples, amplicons were detected (Ct values ranging between 33 

and 36, vs Ct values from 27 to 31 for stool samples from patients that were used as positive 

controls). However, products resulting from the amplification of the 18S rRNA genes 

environmental samples were not sequenced, with questions about specificity of detection (in 

light of recent studies highlighted in this report) remaining unresolved. It is of note that in a 

different study (Durigan et al. 2022) which focused on amplifying another target gene (mit3), 

sequencing of PCR products resulted in sequences with ~ 99% identity with C. cayetanensis

sequences in GenBank.

Alternative to the BAM qPCRs, a nested PCR assay targeting the 18S rRNA gene has recently 

been described for the detection of C. cayetanensis oocysts in fresh berries and soil from berry 

farms in Mexico (Resendiz-Nava et al. 2020). The primer sets used for the nested PCR reaction 

were the same as used in the BAM qPCR method for fresh produce. Sensitivity of the nested 

PCR (established only with blueberries) was as few as 50 oocysts per 50 g berry sample, and 

Sanger sequencing and phylogenetic analysis was used on the amplicon from the second PCR 

to confirm the presence of C. cayetanensis. To further promote sensitivity, bovine serum 

albumin was added to the PCR reactions to offset potential inhibitory substances commonly 

present in environmental samples (Resendiz-Nava et al. 2020). When evaluated by the nested 

PCR assay, 16.6% (1/6), 36.4% (4/11) and 20.0% (1/5) of blueberry, blackberry, and farm soil 

samples, respectively, tested positive for C. cayetanensis and Sanger sequencing of the nested 

amplicons matched database sequences of C. cayetanensis (Resendiz-Nava et al. 2020). 

Although nested PCRs may allow for sensitive detection, the transfer of amplicon from the first 

PCR reaction to the second PCR introduces additional opportunity for cross contamination 

within the laboratory. 

Targeting the 18S rRNA gene for qPCR detection of C. cayetanensis has several advantages 

and challenges. The 18S rRNA gene is conserved among the coccidian group and C. 

cayetanensis has been previously estimated to contain 18 copies per genome (Murphy et al. 

2018). Considering that a sporulated C. cayetanensis oocyst in the environment would contain 

four genomes, targeting the numerous copies of the 18S rRNA gene allows more sensitive 

detection than targeting a single-copy gene. However, given the conserved nature of the 18S 

rRNA gene among coccidia/Apicomplexa, even slight modifications to the qPCR described in 

the FDA BAM methods for fresh produce and agricultural waters may negatively affect the 

assay’s specificity. For example, one study demonstrated that some modifications to the qPCR 

master mix from that described in the FDA BAM method for the detection of C. cayetanensis on 

fresh produce resulted in cross-reactivity with several Eimeria spp. and Isospora suis, whereas 

strict adherence to the method verified specificity on various types of produce (Lalonde et al. 

2022). Specifically, when Lalonde et al (2022) carried out experiments to validate the FDA BAM 
34

19b method using fresh produce samples spiked with oocysts, the composition of the PCR mix 

impacted outcomes of the reaction: off-target amplification was observed when two BioRad 

reaction mixes were used, but the use of selected PCR mixes from Qiagen and Applied 

Biosystems (including TaqMan Fast Advanced Master Mix, recommended by the FDA BAM 

19b) resulted in amplification of C. cayetanensis as determined by an exclusivity panel of 

closely related organisms. It should also be noted that sequencing of the ~100-bp amplicons 

obtained from the reactions containing DNA extracted from samples in which 200 C. 

cayetanensis oocysts were spiked onto fresh produce did not result in sequences that 

correspond uniquely to C. cayetanensis. The authors cited technical difficulties with sequencing 

of the ~100-bp fragments, although no details on the sequencing method (beyond using kits for 

PCR purification and a reference software for fragment assembly) were provided. In another 

study (Kniel 2022), when two qPCR reagent mixes (a discontinued product from Qiagen listed in 

the FDA BAM 19B at the time of the report’s publication and a manufacturer suggested 

replacement not recommended by FDA BAM19B at the same time), no significant differences in 

the outcomes were detected when parallel environmental samples were used in reactions. 

This highlights the importance of conducting robust validation of any modifications to the FDA 

BAM methods for the detection of C. cayetanensis before attempting to use the modified 

method for evaluating food or environmental samples. 

Several recent, non-peer reviewed studies have attempted to combine the use of PCR methods 

targeting the 18S rRNA gene with amplicon sequencing or MLST typing methods for confirming 

the specific detection of C. cayetenansis (Kniel 2022, Mattioli et al. 2022; Ortega 2022; 

FDA/CDC report from August 13, 2021- unpublished data). These studies have produced 

different results with respect to confirmed detection of C. cayetanensis, which further indicates 

the sensitivity of PCR methods targeting the 18S rRNA gene to method modifications and the 

need for robust validation of any modifications before use on food and environmental samples. 

In one of these non-peer reviewed studies referenced above, samples of lettuce, cilantro, 

parsley, and basil (total of 767 samples) were obtained from seven markets in Florida and 

analyzed using qPCR (via a modified BAM 19B protocol) and nested PCR (Li et al. 2007) 

(Ortega 2022). Amplicons were detected in 21 samples analyzed by nested PCR and in 2 

samples analyzed by the modified BAM qPCR. Amplicons from the nested PCR were 

sequenced, and none were confirmed as C. cayetanensis. The report does not discuss attempts 

to sequence amplicons from the qPCR from the modified BAM protocol. Additionally, surface 

waters collected in California (426 samples) and Florida (370 samples) during 2020-2021 were 

analyzed using qPCR (via a modified BAM 19B protocol) and nested PCR (Li et al 2007) 

(Ortega, 2022). Presumptive Cyclospora was detected in 27 and 56 water samples from Florida 

and California, respectively, when the samples were analyzed using the modified BAM qPCR, 

and in 44 and 27 samples, respectively, when analyzed using nested PCR (Ortega, 2022). 

Amplicons from the nested PCR were sequenced and C. cayetanensis was not confirmed in any 

of the samples. It should be noted that the nested PCR referenced in this study was reported to 

generate an approximately 500-bp amplicon from both Cyclospora spp. and Eimeria spp. (Li et 

al. 2007), and it is unclear what validation of the modifications to the BAM qPCR were 

conducted and if those modifications affected the specificity of the method. Further, no 

performance characteristics of the methods used were provided, for example results from 
35

verification of reproducibility, limits of detection, and specificity/sensitivity with respect to the 

matrices analyzed.

In another non-peer reviewed study, samples of irrigation water, packinghouse spent dump tank 

water, on-farm portable toilets, and municipal wastewater sludge in the Southeastern Coastal 

Plain of Georgia were analyzed for C. cayetanensis using a modified BAM qPCR (Mattioli et al. 

2022). The 18S rRNA target was detected by the modified BAM qPCR in 59 of 217 samples of 

irrigation water, 1 of 46 samples of packinghouse spent dump tank water, and 0 out of 37 

samples from on-farm portable toilets. Of these qPCR-positive samples, none were confirmed 

as C. cayetanensis by MLST (Mattioli et al. 2022). Additionally, the 18S rRNA target amplicon 

was detected in 9 of 46 samples of return activated sludge (RAS) and 9 of 30 samples of sludge 

collected from the thickener (REC). The report identifies 29 RAS/REC samples that were 

analyzed by MLST (the 18 qPCR-positives, plus 8 additional RAS and 3 additional REC 

samples for which only one replicate demonstrated amplification with a Cq value ≤ 37), however 

these 29 samples are not fully reconciled in the results. The report indicates that no 

amplification of any typing markers was observed in 18 samples, whereas amplification of at 

least one typing marker was observed for 9 samples (although the associated table in the report 

included data for only 8 samples). Of the 8 samples with MLST results, 1 sample was identified 

as non-C. cayetanensis (suspected cross-reaction) and 7 samples were confirmed as C. 

cayetanensis (with 1 of these samples clustered with clinical specimens from 2018 to 2021) 

(Mattioli et al. 2022). The investigators noted limitations with the use of MLST on environmental 

samples and as with the previous study, it is unclear what validation of the modifications to the 

BAM qPCR were conducted and if those modifications affected the specificity of the method. In 

addition, it is not clear if the sensitivity of the MLST was evaluated on well-characterized C. 

cayetanensis positive environmental samples given that the validated BAM qPCR has been 

validated in a multi-laboratory study to detect as few as six oocysts in ten liters of irrigation 

water (Murphy et al. 2017, Durigan et al. 2020). 

In contrast, the FDA and CDC conducted a follow-up investigation to a 2020 outbreak of C. 

cayetanensis associated with bagged salad (FDA/CDC report from August 13, 2021-

unpublished data). As part of that investigation, environmental samples (surface water) 

collected from various locations in Florida and California were analyzed for C. cayetanensis

using the BAM qPCR without modifications. To evaluate the potential utility of the MLST for 

confirming environmental samples, eight (8) BAM qPCR-positive samples with lower Ct values, 

more than one triplicate positive, and positivity at a 1:10 dilution of the DNA extraction 

(representing 2 locations in California and 3 locations in Florida) were submitted to the CDC 

Parasitic Disease Branch for MLST analysis. C. cayetanensis was confirmed in all 8 samples by 

the MLST method, with sufficient information generated in 6 of the samples to identify 

haplotypes. Two (2) of these samples (surface water samples collected from 2 different canals 

in Florida) clustered with haplotypes associated with the bagged salad mix outbreak from the 

2020 season, whereas the other 4 samples did not cluster with any sequences from the 2018 to 

2021 outbreak seasons (FDA/CDC report from August 13, 2021 – unpublished data). The qPCR 

used in this study was conducted as prescribed in the BAM without modifications, and the 

application of the MLST to the selected qPCR-positive samples confirmed both specific 

amplification in the qPCR and the presence of C. cayetanensis in the samples. 
36

The performance characteristics of the MLST method on environmental samples needs to be 

determined through proper validation studies before this approach should be used to confirm 

the presence of C. cayetanensis in PCR-positive samples. Further, the MLST method (or any 

other sequencing/typing method) should only be used following a fully validated PCR method for 

primary detection. Mattiolli et al. (2022) notes in their study that “it remains unknown whether 

the samples with suspected cross-reactions had qPCR amplification due to closely related 

Eimeriidae species within the samples outcompeting any C. cayetanensis that may have also 

been present in the sample, or whether C. cayetanensis was detected by qPCR but was present 

at levels too low or in a matrix too complex to be detectable by the subsequent typing assays”, 

however, this possibility is only valid if the primers’ affinity for the target and off-target template 

were very similar The MLST method discussed in this section was previously used to genotype 

specimens from laboratory-confirmed cyclosporiasis cases that occurred between May and 

August of 2020 (Barratt et al. 2022). A total of 1019 specimens from laboratory-confirmed cases 

were analyzed by MLST, but sequence data of sufficient quality for genotyping was obtained in 

only 816 specimens (Barratt et al. 2022). These limitations of the MLST method when applied to 

laboratory-confirmed clinical specimens would be expected to present greater challenges when 

applying the method to environmental samples where the expected concentration of C. 

cayetanensis oocysts would be very low, the populations of closely related parasites (e.g., 

Eimeria spp. and Isospora spp.) would be high, and the matrix would be more complex. 

These discrepancies make it difficult to interpret results among studies where food and 

environmental samples were evaluated for the presence of C. cayetanensis using PCR methods 

that target the 18S rRNA gene. 

Modifications to PCR methods, including those targeting the 18S rRNA gene of C. 

cayetanensis, may impact positively or negatively the performance of the method. It is likely that 

some literature studies that used such modified methods without determining the impact of 

those modifications through proper validation studies have overestimated the prevalence of C. 

cayetanensis in food and environmental samples. 

PCR methods targeting the mitochondrial genome

Recently described methods for detecting C. cayetanensis in fresh produce and agricultural 

water have included PCR assays targeting the C. cayetanensis oxidase gene (Cox3; a multicopy gene) located within the mitochondrial genome. Two such methods are available on the 

FDA’s website as “Other Analytical Methods of Interest to the Foods Program” (FDA 2023b). 

These two methods are virtually identical to the FDA BAM methods for the detection of C. 

cayetanensis in fresh produce and agricultural water except the qPCR assay targeting the 18S 

rRNA gene in the BAM methods has been replaced with the Mit1C qPCR assay (which also 

includes an IAC). As such, the Mit1C qPCR is positioned as a stand-alone detection assay to be 

used as an alternative to the current BAM qPCR. The target for the Mit1C assay (a 205 bp 

region) was identified in silico using BLAST searches against available sequences of C. 

cayetanensis and other genera/species (e.g., Eimeria spp. and Isospora spp.) in the 

Apicomplexa phylum (Shipley, Arida and Almeria 2022). The Mit1C qPCR assay was validated 

in a single-laboratory study to detect as few as 5 C. cayetanensis oocysts in 25 g samples of 

cilantro or romaine lettuce, and 50 g samples of raspberries (Balan et al. 2023). In the study, the 
37

Mit1C qPCR demonstrated specific amplification when used to evaluate cilantro and romaine 

lettuce samples spiked with oocysts of two Eimeria spp. (E. acervulina and E. tenella) alone or 

at a 2:1 ratio with C. cayetanensis oocysts (Balan et al. 2023). However, the genetic diversity of 

food and environmental samples extends beyond the mitochondrial genome sequence data 

currently available and the scope of this study, therefore further evaluation of Mit1C qPCR 

specificity is needed. 

A flotation concentration method using saturated sucrose solution followed by the FDA BAM 

qPCR assay (targeting the 18S rRNA gene) was recently investigated for isolation and detection 

of silt loam soil samples inoculated with C. cayetanensis oocysts (Shipley et al. 2022). 

Additionally, this study compared the flotation method to three commercial DNA isolation kits 

and compared detection using the Mit1C qPCR with the BAM qPCR. The flotation method 

resulted in greater sensitivity of detection than the three commercial DNA isolation kits, and the 

method was reported capable of detecting 10 oocysts in 10 g of soil (Shipley et al. 2022). The 

Mit1C qPCR was only evaluated at the 100 oocysts per 10 g soil inoculation level, however 

when compared to the BAM qPCR the Mit1C qPCR achieved detection at lower Ct values 

indicating better detection (Shipley et al. 2022). In the study, all inoculated samples evaluated 

by the Mit1C assay tested positive and all negative control (uninoculated) samples tested 

negative. 

Detecting parasites from various types of soil samples has historically been difficult, therefore 

further studies using the flotation concentration method followed by either the BAM qPCR or the 

Mit1C qPCR for the detection of C. cayetanensis oocysts in samples of silt loam soil, sandy clay 

loam soil, and a commercial potting mix were performed (Arida, Shipley and Almeria 2023). 

Similar to the previous study, both qPCRs provided specific detection of as few as 10 oocysts 

per 10 g sample (both silt loam and sandy clay loam soils) with the Mit1C qPCR achieving 

detection in a higher (but not statistically significant) number of samples but at a significantly 

lower Ct value (reported as Cq) (Arida et al. 2023). Modification to the flotation method was 

required to optimize recovery and detection of C. cayetanensis oocysts from the potting mix with 

both the BAM qPCR and Mit1C qPCR detecting as few as 20 oocysts in 5 g samples. It should 

be noted that the unseeded (negative control) sample of potting mix returned an “undetermined” 

result by the Mit1C qPCR assay. Amplicons from the Mit1C qPCR on soil samples inoculated 

with 100 oocysts were successfully sequenced to confirm alignment with C. cayetanensis, 

however such attempts at sequencing amplicons from the samples inoculated at 20 and 10 

oocysts were unsuccessful (Arida et al. 2023).

Additionally, a conventional PCR (Mit3PCR) has also been recently described for the detection 

of C. cayetanensis in food and water samples that targets a 182-bp fragment of the Cox3 gene 

(the same mitochondrial target as the Mit1C qPCR assay) (Durigan et al. 2022). Mit3PCR was 

proposed to be used complementary to the FDA BAM methods to confirm BAM qPCR-positive 

samples, with any amplicon bands generated by the Mit3PCR subsequently analyzed by DNA 

sequencing (Durigan et al. 2022). The sensitivity of the mit3PCR method was confirmed to be 

equivalent to that of both FDA BAM qPCR methods (unpublished data from a single laboratory 

validation performed by FDA in 2018). The specificity of mit3PCR was evaluated using a panel 

of DNA samples from selected foodborne bacterial and parasitic pathogens: Cryptosporidium 

parvum, Cryptosporidium hominis, Cyclospora papionis, Eimeria acervulina, Eimeria 
38

tenella, Eimeria maxima, Entamoeba histolytica, Giardia duodenalis, Blastocystis 

hominis, Plasmodium falciparum, Neospora caninum, Toxoplasma gondii, Salmonella 

spp., Escherichia coli, and Trypanosoma cruzi. No cross reactivity with this DNA panel was 

observed. In addition, the specificity of the 182-bp region amplified by the mit3PCR was also 

confirmed by sequence comparison with other Eimeriidae species. Therefore, the sequences of 

any amplicons generated due to cross-reaction with background taxa in mixed samples could 

be used to resolve the specificity by comparison with a database of mitochondria genomes 

(Durigan et al. 2022). While the genetic diversity of environmental samples extends beyond this 

limited DNA panel as well as the mitochondrial genome sequence data currently available this 

additional molecular marker would add to the target repertoire of qPCR-based screening 

strategies for food and water samples.

Future method development and evaluation for the detection of C. cayetanensis in food and 

environmental samples

The close genetic relatedness of C. cayetanensis with other coccidia/Apicomplexa, such as 

other Cyclospora spp. and Eimeria spp., and the limited genomic sequences of coccidia 

currently available have clearly complicated the development of DNA/RNA-based detection 

methods with the desired degree of specificity and robustness for widespread laboratory use. 

For these reasons, the specificity of genetic targets for detecting C. cayetanensis that are 

identified in silico should be evaluated using a robust exclusivity panel of DNA from closely 

related coccidia as well as a wide range of food and environmental samples. As discussed in 

sections above, the current BAM qPCR methods require strict adherence to the defined PCR 

conditions/procedures for specific detection of C. cayetanensis. Any modifications to PCR 

conditions/procedures in established official methods should be thoroughly evaluated to 

determine if the modification(s) negatively affected specificity before the method is further 

developed by the addition of steps to confirm and/or genetically characterize PCR-positive 

samples. Continued research to identify additional genetic targets for specific detection of C. 

cayetanensis in food and environmental samples should be continued as well as the 

development of detection methodology that is more tolerant to minor modifications without 

sacrificing specificity. 

Future method development for the detection of C. cayetanensis in food and environmental 

samples should include the evaluation of multiple genetic targets representing different regions 

of the genome. However, secondary genetic targets to confirm initial PCR-positive samples 

should also be specific for C. cayetanensis to avoid amplification bias when testing complex 

samples expected to contain a diverse population of coccidia/apicomplexan protozoa, such as 

food and environmental samples. DNA sequencing analysis (e.g., NGS or Sanger sequencing) 

of one or more amplicons should be considered for further confirmation of the presence of C. 

cayetanensis and/or characterization at the species or haplotype/genotype levels (Durigan et al. 

2020, Kahler et al. 2021, Lalonde et al. 2022). However, amplicon sequencing is unlikely to be 

a useful method when a required management decision needs to be done quickly (i.e., whether 

or not to harvest a field within the next 3-5 days). 
39

At the time of this Committee report, the International Standards Organization (ISO) has 

approved a project proposal within TC34/SC9 to develop an international standard for detection 

of C. cayetanensis in fresh leafy green vegetables and berry fruits, with possible application to 

other fresh produce (https://www.iso.org/standard/83574.html). Although the approved ISO 

proposal does not include the determination of C. cayetanensis to the genotype or haplotype 

level, the development of this international standard should consider the recommendations of 

this report for future method development, specifically the inclusion of multiple genetic targets 

representing different regions of the genome and the use of DNA sequencing of secondary 

target amplicons to further confirm the presence of C. cayetanensis. 

Q8: Viability of oocysts

What information exists on assessing viability of oocysts? 

The viability of recovered oocysts is needed to assess the public health risks of foodborne 

transmission. Currently, the viability of Cyclospora oocysts can only be assessed by analysis of 

the sporulation rates of the oocysts (Almeria et al. 2019). The sporulation viability of oocysts 

refers to the ability of the oocysts to undergo sporulation, which is the process of forming 

sporozoites, the infective stage of the parasite. Sporulation was often used as an indicator of 

viability of C. cayetanensis oocysts. Assessing the sporulation viability provides information on 

the ability of the oocysts to develop into the infectious form. 

Compared with the development of detection techniques for Cyclospora, the assessment of 

viability of Cyclospora oocysts was much slower. One of the barriers for the assessment of 

viability of oocyst was that the sporulation of Cyclospora takes much longer than other 

protozoan parasites, with an incubation of 7 to 12 days at 25 to 30°C (Sathyanarayanan and 

Ortega, 2006). 

Microscopy-based technique was used to assess the sporulation viability of oocysts of 

Cyclospora. Some other methods, like vital dye assays (e.g., DAPI) (Almeria et al. 2019) and 

Electrorotation method (Dalton et al. 2001), which was either not commercially available or still 

at the research stage (Almeria et al. 2019). 

Some molecular methods such as PCR (polymerase chain reaction) or qPCR (quantitative 

PCR) were useful to detect oocysts and to investigate the source of contamination. In the past, 

those methods cannot be used to verify viability of oocysts. A recent study by Tucker and 

colleagues (Tucker et al. 2021) reported that genes differentially expressed in E. acervulina

during sporulation, in mature and immature oocysts were identified and their homologs were 

detected in C. cayetanensis. It is reasonable to hypothesize that these could be useful targets 

for mRNA-based assays for viability of the propagules.

Another promising development of assessment of viability of Cyclospora oocysts was to use 

artificial intelligence and machine learning to speed detection of Cyclospora cayetanensis’ 

infectious life stage. Researchers are using a library of images of viable and nonviable oocysts 
40

to “teach” a machine to make the differentiation by robotic microscopy and image analysis 

(Center for Produce Safety, 2023).

Control Strategies and Surrogates

Q5: Current monitoring and management strategies

Is monitoring for Cyclospora cayetanensis by testing food products, agricultural 

environment and agricultural inputs being applied as a management strategy currently 

(e.g., by industry, government)? 

a) Are there best practices for monitoring the presence of Cyclospora cayetanensis 

in agricultural production (including matrices [e.g., water, product], frequency, 

timing of sample collection (pre- vs. post-harvest), and sample numbers)? 

b) Has monitoring led to development and implementation of effective preventive 

measures? If so, how effective have they been?

Summary response. Currently, widespread environmental monitoring of agricultural 

environments and agricultural inputs, even in endemic areas, is not routinely conducted. The 

challenge, with environmental monitoring, lies in the low prevalence of the parasite in the 

environment, low recovery rate for oocysts, and the use of testing methodologies that have not 

been fully validated. The limiting factor for environmental monitoring is the lack of commercially 

available rapid test kits, that are low cost and can detect very low oocysts concentrations. In the 

interim, emphasis should be on, and enforcing, improved worker hygiene and sanitary practices, 

to include toileting habits, and routine testing of irrigation water supplies for human fecal 

contamination at the farm and packing facilities.

Q5 a) In regions where C. cayetanensis is endemic, a more comprehensive set of best practices 

could be put in place to monitor for C. cayetanensis in the production environment (WHO/FAO 

2017). We recognize that even in the areas where C. cayetanensis is endemic, in the small 

number of studies that have been done so far, amplicons resulting from PCR reactions aimed at 

detecting 18S rDNA from C. cayetanensis were detected in only 1-4% of locally grown produce

(Barlaam et al. 2021, Caradonna et al. 2017, Giangaspero et al. 2015, Ortega et al. 1997, Sim 

et al. 2017). 18s rDNA amplicons were detected by Resendiz-Nava et al (2020) in 20% of soil 

samples from Mexican farms; and by Giangaspero in 12% soil samples and 6 to 21% of well 

and municipal treated water (Resendiz-Nava et al 2020, Giangaspero et al 2015). ResendizNava et al confirmed the presence of C. cayetanenis using Sanger sequencing and phylogenic 

analysis, comparing the amplicons with 18S rRNA gene sequences from the GenBank 

database. Giangaspero et al. (2015) used single-strand conformation polymorphism and the 

BLAST tool to compare amplicons against known reference sequences. 

Given the low level of prevalence in food samples even in the areas where C. cayetanensis is 

endemic, risk-based sampling would be advisable. The Code of Hygienic Practice for Fresh 

Fruits and Vegetables, Codex Alimentarius CXC 53-2003, provides guidance in accordance with 

Good Agricultural Practices and Good Hygienic Practices to control hazards, beginning with 

primary production at the farm. C. cayetanensis is listed among the microbiological pathogens 
41

of concern. Risk-based and risk-appropriate testing of agricultural water may be advisable;

however, this Committee is not convinced that testing specifically for C. cayetanensis using 

existing methodologies and abundance of closely related organisms that are not pathogenic to 

humans is more practical than the risk-based tests for the presence of human fecal pollution. 

Growers should consider testing irrigation water for microbial and chemical contamination for 

identified risks, at a frequency determined by water source, with the consideration risk of 

environmental contamination, such as flooding, the type of irrigation or application method, and 

the use of manure, biosolids, and natural fertilizers. 

Growing operations should consider evaluating hazards posed by the agricultural workers who 

may be symptomatic or asymptomatic carriers and consider medical examinations as 

appropriate. Agricultural workers should be encouraged to report diarrheal diseases and 

incentivized to seek treatment. Growing operations should emphasize and reinforce training in 

health, hygiene, and sanitation. An adequate number of functional sanitary toileting facilities and 

handwashing stations close to work areas in the growing fields and packinghouses should be 

available. (Codex Alimentarius, CXC 53-2003) Codex Alimentarius Guidelines CAC/GL 88-2016 

provides guidelines to control food-borne parasites, although C. cayetanensis is not specifically 

mentioned by name. (Reference is Guidelines on the Application of General Principles of Food 

Hygiene to the Control of Foodborne Parasites, CAC/GL 88-2016, Codex Alimentarius 

International Food Standards, Food and Agricultural Organization of the United Nations and 

World Health Organization, Adopted 2016).

However, in the areas where C. cayetanensis is not endemic (such as production areas in the 

continental U.S.), proposed sampling and preventative measures must recognize extremely low 

detection rates in environmental and food samples. Therefore, environmental testing programs 

must take into consideration that humans are the only documented host for C. cayetanensis, 

and contamination with human waste or sewage is the likeliest source of the parasite in the 

production or processing environment. If testing of the final product is considered, a testing 

method needs to be developed to address the following criteria:

• Reliable and cost-effective detection of C. cayetanensis in samples in the presence of 

closely related organisms that are not pathogenic to humans

• Quick laboratory turn-around time to recognize the fact that commodities previously 

linked to the outbreaks of illness are highly perishable, and final product testing is 

typically done on the already harvested commodities

• Ideally, tests should be sufficiently sensitive to allow for a single-step detection, with only 

an occasional need for a second step validation of rare positives.

• Given the low prevalence of C. cayetanensis in domestic environmental samples and the 

final product, a method for detecting small numbers of oocysts from large volumes of 

wash water, for example, needs to be developed. The committee acknowledges that 

median oocysts recovery efficiency using dead-end ultrafiltration (DEUF) and filter-based 

US EPA Method 1623.1 is 17% and 16-22% respectively when 15,000 oocysts were 

seeded into 50L of water (Kahler et al. 2021).
42

• When sampling and testing are done for root cause analysis, an appropriate number of 

samples from a diversity of sites, such as water source, irrigation water, farm soil, and 

field portable toilet facilities should be considered. 

• Given the low prevalence of C. cayetanensis in areas where it is not endemic, a negative 

result from a routine test for C. cayetanensis does not conclusively establish absence as 

the number of oocysts may be below current detection levels. Therefore, testing should 

include indicators of human fecal pollution.

b. Has monitoring led to development and implementation of effective preventive 

measures? If so, how effective have they been?

There is little published information regarding monitoring programs. In 2017, the Canadian Food 

Inspection Agency implemented a national routine surveillance for C. cayetanensis, using the 

BAM Chapter 19b method, in imported and domestic fresh leafy greens, herbs, and berries 

(Chacin-Bonilla and Santin 2023). The Canadian Centre for Foodborne and Animal Parasitology 

detected C. cayetanensis in 0.28% of the survey samples. Chacin-Bonilla and Santin, reiterated 

the recommendations in Codex Alimentarius CXC 53-2003, where the focus should be on 

agricultural worker hygiene and sanitary practices. Resendiz-Nava et al (2020) recommend 

monitoring at the farm and packing facilities. The issue with monitoring at the farm and packing 

facilities remains the low prevalence of C. cayetanensis in the environment, low recovery rates 

for oocysts, and the use of testing methodologies that haven’t been fully validated. 

Q9: Preventive measures 

What preventive measures exist for the control of Cyclospora cayetanensis (e.g., using 

filtration)?

a. How effective have they been?

b. What are the impediments to development of effective preventive measures for C. 

cayetanensis and how can they be overcome?

Measures to control or eliminate C. cayetanensis in food products have generally not been 

identified (Erickson and Ortega 2006, Kniel et al. 2007, Erickson and Ortega 2006, Ortega and 

Sanchez 2010). The resilient nature of the oocyst bilayer cell wall structure and its larger size 

(7.5-10 μm) could make filtration a practical and promising approach to eliminating the risk from 

water, assuming that filtration rates and useful life of filters meet the throughput requirements. 

To date, no water filtration systems have been constructed to eliminate C. cayetanensis while 

also providing the speed and filtering capacity needed to eliminate the oocyst in high-volume 

systems, such as those used for irrigation of field crops (Erickson and Ortega 2006, Kniel 2020). 

Given the challenges of filtration, other treatments for food systems, water, and irrigation 

systems have been studied (Erickson and Ortega 2006, Kniel et al. 2007). 

Other than high heat, most common chemical and physical treatments for fresh produce water 

treatments or used in processing of fresh produce are not sufficient or practical to reduce 

oocyst’s ability to sporulate. The oocysts’ sporulation ability has been evaluated by treating 

them with chemicals common in food processing such as chlorine, peracetic acid, and chlorine 

dioxide; the resilient oocyst cell walls have proven mostly resistant to such treatments (Ortega 
43

et al. 2008, Ortega and Sanchez 2010, Malka and Park 2021, Praeger, Herppich and 

Hassenberg 2018). Post-harvest treatments and processing conditions of temperature 

(high/low), UV, ozone, high-pressure processing (HPP) have all been evaluated to eliminate the 

risk for consumers; most treatments have been unsuccessful, impractical, or only evaluated on 

surrogate organisms with unknown understanding of how C. cayetanensis oocysts may react 

(Kniel et al. 2007,Ortega and Sanchez 2010, Guo, Huang and Chen 2019, Kniel et al. 2007, 

Erickson and Ortega 2006, Kniel et al. 2007). Extreme temperatures (60°C for 1 h or 70°C for 

15 min, 100°C or -70°C temperature treatment) were found to be successful to inhibit 

sporulation; however, these temperatures are not practical for the foods often associated with 

risk; fresh produce, berries, herbs, etc. (Erickson and Ortega 2006, Kniel et al. 2007, Almeria et 

al. 2019, Ortega and Sanchez 2010). On-going research on C. cayetanensis oocysts with more 

novel treatment options is needed to identify effective and functional treatment options for the 

food industry (Kniel 2020, Erickson and Ortega 2006, Malka and Park 2022). 

Preventative Measures. Potential means to prevent future illness from C. cayetanensis are to 

physically remove oocysts that may be found in food, water, and agricultural production 

environments, and/or to render Cyclospora oocysts that may be present in a food or production 

environment non-infectious (Erickson and Ortega 2006). The spherical Cyclospora oocyst 

bilayer cell wall serves as a strong protective barrier minimizing susceptibility to challenging 

environmental conditions and common antimicrobial treatments (Kniel et al. 2007). The physical 

structure of the Cyclospora oocyst is highly resistant to degradation in the environment from 

heat, sunlight, cold, and other environmental pressures (Erickson and Ortega 2006). The 

characteristics that allow for environmental survival also render them challenging to eliminate 

with mitigations in the food industry (Kniel et al. 2007, Erickson and Ortega 2006). Previous 

research has relied upon using oocyst sporulation as an indication of whether oocysts remain 

infectious post treatment or surrogate experiments with related parasites since no effective in 

vitro or in vivo methods have been discovered to be able to test infectivity directly (Ortega and 

Sanchez 2010). Below are brief summaries of preventative measures and treatments that have 

been explored for efficacy against consumer exposure from C. cayetanensis within the food 

system. 

Fresh produce represents a high percentage of foods associated with past Cyclospora 

outbreaks; numerous events were attributed to pre-packaged salads, berries and herbs 

(Temesgen et al. 2021). Products consumed fresh represent a challenge for food safety due to 

the limited number of interventions available to control microbial risk while maintaining the 

attributes demanded by the consumer (e.g., freshness, texture, color) (Kniel et al. 2007). In 

addition to the lack of many mitigation methods for fresh produce, managing parasite risk is 

further complicated in that many foodborne parasites such as Giardia cysts, Cryptosporidium 

and Cyclospora oocysts have been observed to harbor physical structures that facilitate 

adherence to surfaces; consequently, physical removal from food surfaces is difficult 

(Temesgen et al. 2021).

Filtration. Filtration relies upon the physical removal of oocysts from a sample or environment 

as opposed to rending the oocyst noninfectious; it is an approach reliant on elimination either 

from water wash systems that may be in a production environment, or from water distribution 
44

and irrigation systems used for crop production. Cyclospora oocysts’ size (7.5-10 μm) makes 

them a more favorable candidate for physical separation methods than bacteria and viruses 

which are smaller and subsequently more difficult to physically remove from an environment or 

process (Erickson and Ortega 2006). A 2020 study using Cryptosporidium parvum (4.5 μm 

oocysts) and Eimeria tenella (19-22 μm) as surrogates for C. cayetanensis found success in 

removing oocysts from contaminated pond water using sand and reported the use zero-valent 

iron (ZVI) filtration for combined physical removal and oocyst inactivation (Kim et al. 2020). 

Sand filtration physically captures oocysts, while the addition of ZVI is intended to impact 

oocyst viability. Cryptosporidium parvum in that study represented a parasite closer in size to C. 

cayetanensis (7.5-10 μm), and the researchers observed a 4.3 log reduction using a ZVI sand 

column versus sand only (Kniel 2020). Treatment of Eimeria tenella, a much larger oocyst, 

resulted in a 6-log reduction for the combined ZVI sand column, and only a 2.3 log reduction by 

sand alone (Kim et al. 2020). Filtration remains a promising approach to control Cyclospora 

cayetanensis within water systems; however more research is needed to identify systems to 

effectively remove oocysts while also accommodating the volume of water needed in industrial 

wash and irrigation networks. 

Washing. While industry and produce consumers have long relied on washing to clean produce 

items prior to consumption, these treatments have historically been utilized to remove soil, 

insects, and other substances prior to consumption, but were not designed for the removal of 

pathogens. Research on produce wash systems has shown that microorganism removal 

efficacy can vary immensely based on matrix, target organism(s), and whether a combination of 

washing (physical application) with other treatments such as chlorination, chemical treatment, 

ozone, etc. are applied (Temesgen et al. 2021). In 2021, researchers studied the potential of 

three consumer wash systems (running water, 1.75% acetic acid solution, washing followed by 

a salad spinner) applied to fresh blueberries and raspberries inoculated with C. parvum oocysts, 

C. cayetanensis oocysts, G. duodenalis cysts to better understand if washing just prior to 

consumption would be sufficient to remove foodborne parasite risk (Temesgen et al. 2021). 

Results indicated that 80% of the C. parvum and G. duodenalis oocysts were removed by each 

of the washing methods on either berry matrix; raspberries being more difficult to remove 

oocysts than blueberries. C. cayetanensis across all treatments was found to remain on the 

berries to a much greater percentage than the other parasites studied (Temesgen et al. 2021). 

Cyclospora’s greater adherence led the researchers to hypothesize that the adherence may be 

related to the specific adhesins found within this organism’s oocyst cell structure that are unique 

compared to the other parasites studied (Temesgen et al. 2021). Industrial wash systems have 

not yet been studied in respect to C. cayetanensis, and washing is an area requiring further 

research. 

Chemical Sanitizers (chlorine/peracetic). Fresh produce industry relies on chemical 

sanitizers to prevent cross-contamination of the product during post-harvest processing. 

Chlorine is one of the most common and universally effective sanitizers used in the food and 

agriculture industries (Erickson and Ortega 2006, Malka and Park 2022). Unlike bacteria and 

viruses which are susceptible to destruction following chlorine treatment, Cyclospora oocysts 
45

have not been shown to be susceptible to common chlorine treatments used for disinfection and 

sanitization within the food industry (Erickson and Ortega 2006, Malka and Park 2022).

Chlorine Dioxide. Chlorine dioxide (ClO2) in gaseous and aqueous forms has been found to be 

a useful tool in the food and fresh produce industry due to its bactericidal effects on bacterial 

foodborne pathogens, its efficacy over a wider range of pH values (pH 3-8) than other 

disinfectant systems, and unlike other chlorine-based sanitizers it does not create dangerous 

halogenated byproducts ((Ortega et al. 2008, Malka and Park 2021, Praeger, Herppich and 

Hassenberg 2018), FDA 2008). However, oocysts of C. cayetanensis artificially inoculated onto 

basil and lettuce were able to withstand the exposure to gaseous chlorine dioxide at 4.1 mg/liter 

for 20 min without losing the ability to sporulate (Ortega et al. 2008, Ortega and Sanchez 2010).

Temperature. Cyclospora oocysts treated in the laboratory with high temperatures, 60°C for 1 h 

or 70°C for 15 min, 100°C were found to no longer be capable of sporulation (Erickson and 

Ortega 2006, Almeria et al. 2019). In one study, researchers observed that a slight reduction in 

wash water temperature to 50°C for 1 hour maintained C. cayetanensis sporulation abilities 

(Erickson and Ortega 2006). Application of high wash water temperature could be a means of 

inactivation for oocysts; however, such treatments would not be practical for fresh produce 

treatments as item quality would degrade and no longer meet market expectations (Kniel et al. 

2007). 

Past cyclosporiasis outbreaks have been associated with consumption of foods that 

have been stored refrigerated and frozen, providing an indication that freezing and cold 

temperatures alone may not be sufficient to inactivate Cyclospora oocysts (Ortega and Sanchez 

2010). Experimentally, Cyclospora oocysts remain capable of sporulation following -15°C 

treatment for 24 hours in dairy matrices, and -20°C for 48 hours on basil and for 4 days in -20°C 

water samples (Ortega and Sanchez 2010, Almeria et al. 2019). In one study, -70°C 

temperature treatment of basil and water samples inoculated with Cyclospora oocysts was also 

found to be successful in prohibiting oocyst sporulation (Ortega and Sanchez 2010). These 

extreme temperatures would not be practical with current industry practices, nor product 

expectations for consumers. While no studies were found that specifically researched 

refrigeration temperatures consistent with cold-chain and consumer storage, one reference 

noted that Cyclospora oocysts stored at 4°C for one or two months were capable of sporulating 

following a 30°C exposure for 6-7 days (Almeria et al. 2019). 

UV. Ultraviolet (UV) light consists of short wavelengths of light (250-270 nm) that has been used 

for its antimicrobial properties on bacteria, viruses, yeasts, molds and parasites in a variety of 

food, water and produce matrices (Guo, Huang and Chen 2019, Kniel et al. 2007). While direct 

exploration of Cyclospora has not been completed, UV treatment of a closely related bird 

parasite Eimeria acervulina yielded variable outcomes that were dependent on the UV exposure 

and the inoculum level of oocysts (Kniel et al. 2007).

Ozone. No published research to date has been completed to C. cayetanensis response 

following ozone exposure and further research is needed to determine if ozone would be 
46

effective on preventing sporulation, however in prior laboratory studies ozone treatments were 

effective against Giardia lamblia and Cryptosporidium parvum (Erickson and Ortega 2006, 

Khalifa et al. 2001).

High pressure processing (HPP). HPP treatment with 550 MPa at 40°C for 2 minutes was 

found effective to inactivate oocysts on fresh basil and raspberries inoculated with Eimeria 

acervulina, a potential Cyclospora surrogate (Kniel et al. 2007). Findings on surrogates are 

suggestive that HPP may be effective at rendering Cyclospora oocysts nonviable on food 

matrices where HPP is a potential application. 

Future needs. As evident from the studies and treatments referenced in the preceding 

sections, direct research for C. cayetanensis is limited, and many conclusions and hypotheses

on efficacy of treatments for Cyclospora have been drawn from studies completed on related 

parasites and surrogates. Major impediments for identifying or developing preventative 

measures against Cyclospora include the limited availability of Cyclospora oocysts for 

researchers, the inability to culture oocysts in the laboratory, and lack of consensus on the most 

appropriate surrogates for this organism. When considering the collection of studies and 

approaches that have been completed regarding preventative measures against Cyclospora, 

few studies have yet to identify commercially viable measures against this parasite for the food 

and produce industry. Of the studies that identified promising measures (e.g., 70-100°C, HPP, 

filtration) most would not be practical within the produce industry’s supply chain. Given the 

percentage of cyclosporiasis outbreaks associated with fresh produce items, the lack of 

identified treatments amenable for fresh produce is problematic and warrants further 

exploration. Future research is needed to better understand the organism and ultimately help 

identify measures to reduce consumer risk from this organism. For this research to be 

successful, solutions must first be found regarding C. cayetanensis oocysts availability and/or 

identifying the most appropriate surrogates for this organism.

Q12: Possible surrogates

What other coccidian parasites could serve as a surrogate research model 

for Cyclospora cayetanensis behavior (e.g., for evaluation of control measures)?

Three other Apicomplexan parasites have been proposed as surrogates for C. cayetanensis: 

Eimeria, Toxoplasma and Cryptosporidium. Eimeria is probably the best surrogate because of 

its taxonomic closeness, the existence of an animal model and the extensive molecular tools 

available. T. gondii has also been widely characterized, but safety issues for lab workers, the 

public concern for use of cats for research, taxonomically farther and different life cycles limit 

their equivalence. C. parvum has less similarities to be considered a representative surrogate of 

C. cayetanensis. 

 

Several publications have provided a set of criteria to guide researchers in the selection of 

surrogate organisms. Busta et al (2003) distinguished between indicator and surrogate 

organisms, defining the latter as a unique tool that is specifically utilized to evaluate the effects 

and responses of a target organism to selected processing treatments. A list of twelve ideal 
47

traits for potential surrogate microorganisms was outlined in the same publication. Those 

included:1) Nonpathogenic Inactivation characteristics and kinetics useful to predict those of the 

target organism; 2) similar responses to pH, temperature and oxygen as the to the target 

microorganisms when exposed to raw fruit and vegetable; 3) growth characteristics that are 

consistent and stable; 4) cultivated easily to obtain relatively high-count populations and 

inoculum population changes very little from preparation to utilization; 5) easily quantified using 

rapid, sensitive, inexpensive detection systems; 6) easily differentiated from other 

microorganisms; 7) similar attachment characteristics to those of the target microorganisms; 8) 

genetically stable so results can be reproducible, by multiple laboratories; 9) does not have 

persistence characteristics to become a spoilage organism in the environment or products 

where it is applied; and 10) susceptibility to injury similar to that of target pathogen.

Harris et al. (Harris et al. 2012) and Harris et al. (Harris et al. 2013) provided similar 

recommendations to the use of surrogates for agricultural water and un-treated soil 

amendments of animal origin to be used in fresh produce fields. These included: “(i) similar 

characteristics to those of the pathogen of concern such as growth, inactivation kinetics, 

attachment capacity, susceptibility to sublethal stress injury, and resuscitation; (ii) inducible 

stress tolerance resistance traits (pH, heat, desiccation, osmotic pressure, etc.); (iii) ease of 

detection; and (iv) differential or unique phenotypic and/or genotypic traits from background 

isolates” (Harris et al. 2012). The latter reference also included a compilation of surrogate 

microorganisms reported in the literature, but none of these included parasite surrogates. It 

should be noted that among bacteria several surrogate strains may meet most of them, for 

parasite surrogates it is challenging to identify organisms according to those desirable traits.

The concept of surrogate microorganism was also defined by Sinclair et al (2012) in a more 

general scope as: “an organism, particle, or substance used to study the fate of a pathogen in a 

specific environment” (Sinclair et al. 2012). This paper outlined a detailed conceptual decision 

framework for selecting a surrogate and listed four possible types of surrogate benchmarking 

and validation experiments. Based on this set of proposed experiments, validation of surrogates 

can only occur if both the potential surrogate and the target microorganisms can be compared 

under the same experimental conditions. The other benchmarking options described depend on 

whether the target organism can be reliably grown and detected and if the surrogate organism is 

known to have the greatest resistance of its category.

Cyclospora cayetanensis is a coccidian protozoa classified under the family Eimeriidae (Ortega 

2019). Eimeria species are the coccidian parasites more closely related to Cyclospora (Dubey, 

Khan and Rosenthal 2022) and both genera have a fecal-oral cycle, and they infect 

predominantly one host. Eimeria species are economically relevant parasites because they 

infect poultry, cattle and other livestock causing significant losses to agriculture (Thompson and 

Rosenthal 2020). Eimeria species have been extensively characterized and there have been as 

many as 1,800 species identified that infect multiple vertebrate species (Burrell et al. 2020). 

Interestingly, not a single Eimeria species has been reported to infect humans, but because C. 

cayetanensis is so genetically similar, it was considered as the “human Eimeria.” E. tenella and 

E. acervuline are the two most common poultry coccidia which life cycle, pathogenesis, and 
48

invasion mechanism have been extensively described (Venkatas and Adeleke 2019). Because 

of those similarities and the lack of limited laboratory tools to study Cyclospora, Eimeria is 

considered the best surrogate for C. cayetanensis.

In the most recent review paper that addressed the potential of Eimeria as a Cyclospora

surrogate, Tucker and coworkers conducted a systematic assessment that supported its 

utilization (Tucker et al. 2022 and Table 4). That analysis recognized that the two major 

limitations to make significant research progress to address the public health threat that C. 

cayetanensis represents are the scarcity of oocysts and the lack of a viable animal model. 

According to the same study, Eimeria acervulina and other poultry parasites meet several of the 

desirable criteria for a surrogate organism described above. Another recently published study, 

the genomic and genetic closeness of E. acervulina with C. cayetanensis, was also confirmed 

using gene expression in maturing oocysts (Tucker et al. 2021). Because of the evidence 

presented these two papers, there should be little doubt that E. acervulina is the most viable 

surrogate. 

In addition to Eimeria species, Toxoplasma gondii, another coccidian parasite in the family 

Sarcocystidae has also been investigated as Cyclospora surrogate (Lee and Lee 2001, Dubey 

et al. 1998). Lee and Lee reported that a gamma irradiation dose greater than 1 kGy was 

necessary for the complete inactivation of 650 E. acervulina oocysts inoculated on fresh 

raspberries (Lee and Lee 2001). Those results were similar to previous studies conducted with 

T. gondii and E. tenella (Dubey et al. 1998, Gilbert et al. 1998). 

Toxoplasma gondii was used as a surrogate for C. cayetanensis on raspberries (Assurian et al. 

2020). T. gondii as a Cyclospora surrogate poses several advantages that include its extensive 

characterization, well defined models of gene annotation, availability of in vitro and animal study 

models, and a large network of scientist (Anderson, B., personal communication). An additional 

positive aspect for using this parasite as surrogate is the high resistance of its oocysts to 

inactivation, which can serve as a safety factor in developing interventions against Cyclospora. 

In contrast, adoption of T. gondii as surrogate presents serious challenges. Public concern 

about the use of cats for research has led to a reduced availability of oocysts for research. 

Because it is a human parasite, its utilization in research may be a risk for laboratory workers. 

The lack of evolutionary relatedness and different life cycles are probably the strongest 

argument against the use of T. gondii as Cyclospora surrogate.

The availability of a well-tested animal model for Eimeria presents one of the factors that favors 

adoption of this parasite as Cyclospora surrogate (Tucker et al. 2022). In 1995, a detailed 

protocol to grow and recover Eimeria oocysts from less than 5-day-old baby chicks was 

published (Shirley 1995). In addition to requiring the use of very young chicks, this method is 

based on the use of coccidia-free animals. Because the different Eimeria species impact 

different sites in the chicken GI tract, the recovery of merozoites was well described. This 

method continues to be used to this date by researchers developing in-vitro cell culture methods 

(Marugan-Hernandez et al. 2020). Chicken epithelial and kidney cell lines have been tested with 
49

promising results and in the near future, a chicken-free method for harvesting oocysts may be 

available for researchers (Bussière et al. 2018).

Infections by C. cayetanensis can also lead to coinfections of opportunistic pathogens (OP) 

such as Cryptosporidium, therefore, if suspected, screening for C. cayetanensis when there is 

presence of OP should be considered. Cryptosporidium presents some favorable traits to be 

considered a surrogate such as extensive characterization, availability of oocysts, developed 

tools and similar tolerance levels. However, its life cycle differences, large taxonomic 

separation, and multiple hosts limit the extrapolation of C. parvum to C. cayetanensis. 

Given the current limited availability of C. cayetanensis oocysts, validation of surrogates 

remains challenging. 

Table 4. Comparison of Eimeria, Toxoplasma gondii and Cryptosporidium parvum as possible 

surrogates for Cyclospora cayetanensis. 

Trait Eimeria Toxoplasma gondii Cryptosporidium 

parvum

Taxonomic family Emeriidae Sarcocystidae Cryptosporidiidae

Existing animal 

model Baby chicks Cats, controversial Multiple

Life cycle Very similar Different Different

Lab worker risk Small Significant Moderate

Oocyst tolerance Similar More tolerant More tolerant

Number of hosts Single Multiple Multiple

Q16: Maintenance and conveyance of wastewater

Are there practices for the maintenance and conveyance of wastewater, septage or 

human waste that may increase the incidence of C. cayetanensis contamination? Are 

there practices that may be useful in the management of waste to reduce the potential for 

contamination by C. cayetanensis (e.g., third-party toilet service or municipal wastewater 

treatment)?

a) Which wastewater, septage, and human waste treatments in the U.S. are effective 

against C. cayetanensis? Which treatments may not be effective against C. 

cayetanensis?

b) Does municipal water treatment adequately reduce, control or eliminate C. 

cayetanensis? 

c) Can effective municipal water treatments systems be scaled to treat agricultural 

water used in produce production?

d) How do practices compare for domestic growers versus international growers 

who export to the U.S.?

As discussed throughout this report, C. cayetanensis is routinely detected in wastewater and 

sewage in regions where it is endemic and non-endemic. Oocysts have been detected in 

wastewater and sewage in the U.S., thus representing public health concerns. Available 
50

information suggests that physical removal methods, such as sand-filtration, as well as 

exposure to sunlight (UV) need to be further tested to evaluate measurable but variable 

reductions of parasite load (at least based on studies with surrogates) once appropriate 

treatment conditions are fully evaluated under conditions (volumes, temperatures, other 

environmental parameters). Improvements in detection methods as well as studies with 

surrogate protozoa may assist our understanding in the future. Although agricultural water 

sources may serve as a potential contamination route, there is little information on what 

practices a grower can deploy to reduce risk beyond physical removal. Without reliable data on 

the effectiveness of municipal water treatment for the removal of C. cayetanensis, the 

Committee did not have sufficient information to address Question 16c. The Committee was not 

able to access reliable data on the wastewater treatment from areas outside of the U.S., and 

therefore Q16d is unanswered. 

a) Which wastewater, septage, and human waste treatments in the U.S are effective 

against Cyclospora cayetanensis?

Parasite occurrence and concentration, including C. cayetanensis, in wastewater can vary 

greatly and is highly dependent on many factors, including source, season, human population 

demographics and population prevalence rates. Generally, in the U.S., the prevalence and 

concentration of C. cayetanensis is expected to be low (Zarlenga and Trout 2004). This low 

prevalence makes it difficult to measure the effectiveness of the many and varied wastewater, 

septage and human waste treatment processes in use across the US. Limited data are available 

specific to C. cayetanensis reduction related to chemical, biological, and physical wastewater 

treatments. Chemicals commonly used in water treatment, such as chlorine or chlorine dioxide 

gas, have limited effect on C. cayetanensis (Ortega et al. 2008).

Research on physical methods, such as sand filtration, support effectiveness in reducing 

Cyclospora spp. oocysts. For example, a study in rural areas of Nepal measured the impact of 

diarrheal disease following introduction of sand-filtered drinking water. With respect to C. 

cayetanensis, the researchers noted 88.2% removal rate of oocysts (confirmed by microscopy) 

in water samples and a reduction of 4.9% in diarrheal disease within the community.

While the research on inactivation of C. cayetanensis in wastewater, septage and human 

wastewater could be important, it will be hampered by the lack of available oocysts. The use of 

various surrogates is more practical, but it is important to recognize that no surrogate will 

respond to treatment exactly as the target organism. On-going studies indicate that Eimeria

spp. exhibit reasonable sensitivity to some common treatments (Tucker et al. 2021).

b) Does municipal water treatment adequately reduce, control or eliminate C. 

cayetanensis?

Naganathan et al. (2022) completed a comprehensive review and meta-analysis of C. 

cayetanensis in water. Their search identified 33 articles which met the researchers’ criteria for 

inclusion in their analysis of the prevalence of Cyclospora cayetanensis in different types of 

water. In brief, the authors demonstrated that when all analyses are combined (92 prevalence 

estimates from 33 studies), Cyclospora cayetanensis prevalence was estimated to be 6.9% in 
51

global water samples. The authors noted constraints on their analyses including a bias toward 

datasets from endemic areas, and the use of data from studies in which only a single step 

detection was used (which likely to have overestimated the numbers of C. cayetanensis in 

samples). However, despite constraints they estimated that household or drinking water 

prevalence was 5.12%, and water used for irrigation had the highest prevalence at 17.1%.

A study of two wastewater treatment plants in Arizona found C. cayetanensis in both the influent 

and effluent (Kitajima et al. 2014). Nine of the 48 water samples collected from 2011-2012 were 

positive for C. cayetanensis using a novel qPCR technique; however, the authors did not 

determine the removal efficiency of the wastewater treatment plants. The authors concluded 

that existing regulations for water treatment are insufficient to protect the public from C. 

cayetanensis because there are no regulations for managing C. cayetanensis in drinking water 

or wastewater. Further work is needed to understand the specific wastewater treatment 

practices that would demonstrate sufficient effectiveness to benefit public health in the context 

of the contributing population.

c) Can effective municipal water treatment systems be scaled to treat agricultural 

water used in produce production? 

Water scarcity is a major public health problem which impacts billions of people around the 

globe and is an issue that is only becoming worse due to climate change. Parasite removal can 

vary considerably in wastewater treatment plants (WTP) for reasons previously discussed. A 

five-year study of the efficacy of pathogen removal for a California water reuse facility supported 

a very low prevalence of Cryptosporidium spp. oocysts, Giardia spp. cysts, and Cyclospora spp.

oocysts in the effluent, 2.3, 0.3 and 0.034 cysts/L, respectively. Importantly, these 

concentrations were not considered a health risk. Water reuse considerations are critical to the 

evaluation of the effectiveness of municipal water treatment systems and the downstream 

applications of reclaimed water, including agricultural applications. In the aforementioned study, 

the expectation was that the water intended for agriculture met California Title 22 standards 

consistently. Assuming that this standard is sufficient to manage the potential health risk, then it 

provides a model for other municipal systems to consider.

Finally, our understanding of the effectiveness of municipal water treatment systems and their 

applicability to treat agricultural water is limited by the methodology applied to data gathering. 

There remain important considerations for the specificity and sensitivity of the detection 

methods and until resolved there will be limitations to scientific approaches to evaluations of not 

only the risk presented by agricultural water for specific crops and corresponding irrigation 

systems but also effective risk reduction measures.

Q17: Prevention in food

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?

a) 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.
52

b) What is a recommended protocol for evaluating the effectiveness of control 

measures against Cyclospora cayetanensis? 

Control measures for C. cayetanensis should target both the sporulated and unsporulated 

forms. Control strategies should start with measures at the farm and food production levels with 

the provision of proper handwashing and toileting facilities for workers. The detection methods 

used will need to also be evaluated in the presence of Cyclospora spp. that are not human 

pathogens to ensure that any positive samples represent contamination specifically by C. 

cayetanensis. 

Because the sporulated oocyst is the infectious form of the parasite, reduction or prevention of 

oocyst sporulation may be a way to control C. cayetanensis. However, because the 

unsporulated oocyst can become sporulated, the best control strategy would target both stages 

of the parasite lifecycle to reduce or prevent dissemination of C. cayetanensis. Furthermore, 

there is currently no standardized way to distinguish between the unsporulated and sporulated 

C. cayetanensis oocyst except microscopy. However, a molecular technique that relies on 

detection of differentially expressed genes between mature and immature oocysts in model 

organism E. acervulina was recently published (Tucker et al. 2021). That same study reported 

that C. cayetanensis has genes similar to differentially expressed genes identified in E. 

acervulina during sporulation (Tucker et al. 2021). Nonetheless, the identified genes from the 

Tucker et al. study would need to be validated for the capacity to discriminate between 

unsporulated and sporulated C. cayetanensis before they could be used. 

Because no non-human reservoir for C. cayetanensis has been identified, the most appropriate 

point at which reduction of parasite contamination is likely to succeed is in the environment 

around produce production. Environmental controls that can be implemented include inspecting 

delivery vehicles and packaging materials for cleanliness, inspecting produce for damage and 

filth, removing foreign matter, and maintaining records that allow traceback 

(https://www.fda.gov/food/foodborne-pathogens/cyclospora-prevention-response-and-researchaction-plan). Proper toileting facilities and hand washing procedures for field workers and food 

handlers should also decrease the contamination of the environment and food with C. 

cayetanensis. Field workers that are sick should be able to stay home until they have recovered. 

Produce that is to be cut should be washed to reduce microbial contamination from the surface 

onto cut surfaces (Guide for industry, 2008). Because water washes were shown to dislodge 

oocysts from market produce, it is reasonable to hypothesize that washes can reduce the load 

on the product (Duedu et al. 2014; Ortega et al. 1997). Water used for cleaning produce must 

comply with all Federal, State, and local requirements. In addition, if water is reused, the 

cleanest water should be used in the final wash step (https://www.fda.gov/food/foodbornepathogens/cyclospora-prevention-response-and-research-action-plan). The use of temperature 

to prevent oocyst sporulation may require temperatures too extreme to be practical 

(Sathyanarayanan and Ortega 2006). Therefore, a strategy to reduce, destroy or eliminate 

oocysts prior to produce reaching the consumer is important.
53

a) What are control measures that should be evaluated for effectiveness against C. 

cayetanensis? Including control measures that can be applied to the environment 

and/or foods that may be consumed in the raw form.

Control measures that have been shown to be effective against other parasites could be 

evaluated for C. cayetanensis, however we note earlier studies with chlorine, ClO2, or UV had 

limited success (Gaafar 2007; Ortega et al. 2008). In contrast, treatment with magnesium oxide 

(MgO)-coated particles reduced sporulation of unsporulated oocysts, and viability of sporulated 

oocysts by 50% when used at 10 mg/ml for 24 hours (Hussein et al. 2018). Pesticides such as 

captan, benomyl, zineb, malathion, and diazinon did not reduce C. cayetanensis sporulation 

when used as directed (Sathyanarayanan and Ortega 2004). Although, microwave-based 

heating of C. cayetanensis suspended in water resulted in about a 1/4 to 1/3 reduction in 

sporulation when temperature of the water reached approximately 50°C after 10 seconds of 

heating, but temperatures of 95°C reached after 30 seconds of heating did not eliminate 

sporulation (Ortega and Liao 2006). 

Various wash solutions have been tested to determine which would allow recovery and 

detection of C. cayetanensis from artificially inoculated basil by laboratory-based methods 

(Chandra, Torres and Ortega 2014). The solution that was the best at recovering the parasite 

was a 1% HCl-pepsin solution, even better than Alconox detergent. It is unclear from the 

publication what fold-reduction such a treatment would allow since the data were reported as 

the number of samples positive for C. cayetanensis after the wash, and it should be noted that 

neither 1% HCl-pepsin nor Alconox are used for washing fruits and vegetables. However, such 

solutions or their analogues suitable for the purpose could be tested specifically as control 

measures in the future. Some studies on washing methods have already been conducted. For 

example, running water removed roughly 40% of the C. cayetanensis load from raspberries 

whereas washing inside a salad spinner or using a vinegar wash removed more than 80% of the 

parasite (Temesgen et al. 2021). However, for blueberries all three wash methods were greater 

than 95% effective (Temesgen et al. 2021). The parasite was detected by RT-PCR with the 

internal transcribed spacer 1 (ITS-1) region as the target (Temesgen et al. 2021, Temesgen, 

Tysnes and Robertson 2019). These studies suggest that washing by the food preparer should 

result in reduced parasite load.

The lack of in vivo or in vitro methods to test C. cayetanensis viability has prompted researchers 

to use surrogate parasites, such as Eimeria or Toxoplasma species to evaluate other 

treatments. For example, Toxoplasma oocysts irradiated with ≥0.4 kGy sporulated, excysted, 

and infected cells but were not infectious in mice (Dubey et al. 1998). It was recommended, 

therefore, that 0.5 kGy be used to kill coccidian oocysts on fruits and vegetables (Dubey et al. 

1998). However, inactivation of Eimeria acervulina oocysts required 1 kGy (Lee and Lee 2001). 

The use of high-pressure processing (HPP) demonstrated some effectiveness against the 

surrogate E. acervulina and Toxoplasma (Kniel et al. 2007, Lindsay et al. 2008), but has not 

been tested against C. cayetanensis to our knowledge. Additionally, the practicality of using 

HPP for berries is doubtful. 
54

b) What is a recommended protocol for evaluating the effectiveness of control 

measures against C. cayetanensis?

Given the low levels of C. cayetanensis in the final product, establishing reasonable targets for 

reduction are challenging. A further complication is the lack of information on the infectious 

dose (though suspected to be low). However, it is important that a set of reasonable 

preventative or control measures be put in place to minimize or mitigate the risks of this 

pathogen in commodities that have been associated with outbreaks of human illness. Validation 

of control measures is complicated by the lack of C. cayetanensis oocysts readily available for

research. Currently, oocysts are taken from clinical fecal samples. However, more robust 

studies will require consistent access to oocysts. The use of C. cayetanensis oocysts for 

experiments requires the development of approaches for generating oocysts under laboratory 

conditions. Once there is a reliable source of C. cayetanensis, methods for control and 

detection can be tested. For detection of the organism, it would be ideal to ascertain not only 

absence/presence but also whether the organism is viable. To test detection methods, food 

products are spiked with a known quantity of C. cayetanensis, and the RT-PCR methods 

already described are likely adequate in the absence of native background. However, when the 

research moves to food products with unknown levels of C. cayetanensis, it will be important to 

distinguish C. cayetanensis oocysts from any possible Cyclospora contamination of other 

species. Such studies could initially be done with food products spiked with both C. 

cayetanensis and Cyclospora from other animals such as chickens or dogs. 

Q15b: Strategies use to mitigate the contamination from farm workers

What are strategies that have been utilized to mitigate the contamination from farm 

workers? Have efforts to mitigate contamination from farm workers been successful? 

What environmental indicators may be helpful in verification of mitigation practices?

Currently, mitigation for C. cayetanensis includes increased hygiene and protective gear for 

farm workers. It is not clear if those efforts have been successful, as testing is not routinely 

done. Testing for reduction in fecal contamination indicators would be the most practical method 

to verify mitigation practices. 

At present, the primary strategy to mitigate contamination of fresh produce by Cyclospora 

cayetanensis has been to focus on prevention via farm worker training including the topics of 

personal hygiene, clean clothing, and other protective gear, such as gloves and boots, 

equipment management and appropriate sanitary maintenance of toilet facilities. 

Routine water testing for fecal coliforms and/or other markers of human fecal contamination can 

also be used as an indicator of potential risk regarding the presence of other bacterial, viral, or 

parasitic pathogens. Some operations may also use routine health evaluations and clinical 

testing for Cyclospora as a mitigation strategy for the worker populations, in growing regions 

outside of the United States. In a recent paper, L. Chacin-Bonilla and M. Santin (Chacin-Bonilla 

and Santin 2023) proposed that in developed countries, there is a likelihood that endemic 

population foci of Cyclospora infections may exist, most likely in socially and economically 

disadvantaged communities, such as rural farm-worker communities, thus, raising concerns 

regarding transmission issues. The authors believe there would be benefit in exploring the 
55

potential for endemic foci to better define the sources of infection, routes of spreading and 

potentially environmental contamination including produce fields, water sources and animals.

Relevant Factors and Data Gaps – What we know and what we don’t know. 

Q18: Relevant factors, available data, and data gaps for quantitative risk assessment

What are the relevant factors, available data, and data gaps needed to develop an 

informative quantitative risk assessment model for C. cayetanensis contamination and 

risk of illness? 

While fresh or fresh-cut produce has been the only food linked to the cases of domesticallyacquired cyclosporiasis, not all cases of domestically-acquired cyclosporiases have been linked 

to the consumption of fresh or fresh cut produce. It will be critically important to elucidate the 

source of the parasite in these cases to develop appropriate public health measures. 

In developing a framework for controlling this parasite (and other foodborne pathogens), it is 

important to consistently rely on risk-based and not on hazard-based approaches. Risk-based 

and risk-appropriate measures have been the hallmark of the U.S. regulatory process and 

management approaches. A hazard-based approach to regulation should be avoided.

In assessing the risk of cyclosporiasis and establishing an actionable risk assessment 

framework, there are significant data gaps pertaining to sources of C. cayetanensis in the crop 

production environment and its routes of transmission, persistence in the crop production 

environment (especially in the areas where it is not endemic), the utility of indicators, accuracy 

of analytical methods, control strategies and applicability of surrogates to develop control 

measures.

Fresh produce represents a high percentage of foods associated with past Cyclospora

outbreaks; numerous events attributed to processed salads, berries and herbs (Temesgen et al. 

2021). Produce consumed in the US may originate from the regions where C. cayetanensis is 

endemic and non-endemic. As discussed in this report, there is no sufficient evidence to 

ascertain that C. cayetanensis has become endemically established in the U.S. This distinction 

is made to highlight the fact that key considerations in risk assessment such as sources of the 

parasite (laborers along the entire supply chain, irrigation water, organic fertilizers, and other 

inputs) and the inoculum load are almost certainly different from those in the areas where C. 

cayetanensis is not considered to be endemic. 

Products consumed fresh represent a challenge for food safety due to the limited number of 

approaches available to control microbial risk while maintaining the attributes demanded by the 

consumer (e.g., freshness, texture, color) (Kniel et al. 2007). In addition to the lack of many 

mitigation methods for fresh produce, managing parasite risk is further complicated since the 

oocysts of many foodborne parasites, such as Giardia, Cryptosporidium and Cyclospora, have 

been observed to harbor physical structures that facilitate adherence to surfaces; consequently, 

physical removal from food surfaces is even more difficult (Temesgen et al. 2021). Further 

evaluation of the risks to public health for cyclosporiasis illnesses and the detection and control 
56

of Cyclospora cayetanensis in food, water and the environment can be enhanced by addressing 

many of the data or research gaps listed below.

Sources and routes of contamination. 

• C. cayetanensis is a host-limited parasite, and human fecal contamination is the main (if not 

only) source of the oocysts. However, it is not known and critically needed to be understood 

for how long oocysts remain viable and infectious under the diversity of conditions

associated with the fresh produce value chain. 

• Measures of endemicity: A defined criteria and measures are needed to standardize 

designations of endemicity and non-endemicity transmission. This may also aid in the 

identification of pockets of endemicity and assess trends. 

Prevalence and persistence of C. cayetanensis. 

• There appears to be a seasonal pattern in outbreaks where C. cayetanensis is endemic, but 

no specific climate-linked condition has been identified. Even though non-endemic in the 

US, global trade results in products and ingredients imported throughout the year. 

Therefore, year-round vigilance is important, even though no outbreaks, but contaminated 

food, in winter have been reported in the US. 

• Approaches for the mitigation of the risk of transmission of C. cayetanensis will differ in the 

areas where it is endemic vs non-endemic. In areas where C. cayetanensis is not endemic 

(such as many of the production areas in the continental US), it will be important to focus 

mitigation efforts on the likeliest sources of the C. cayetanensis oocysts (i.e., human 

vectors). We note the importance of global trade, and the fact that even domestically 

acquired infections may be ultimately linked to products or ingredients originating from areas 

where it is endemic. 

• The persistence and prevalence of C. cayetanensis oocysts in the post-harvest environment 

is an especially notable data gap.

Indicators for C. cayetanensis.

• As discussed earlier, we acknowledge that no perfect biological or chemical indicator exists 

for C. cayetanensis. However, given the low prevalence of C. cayetanensis, even in the 

areas where it is endemic, there is a need to establish a reasonably reliable indicator. 

Meanwhile, validated indicators of human fecal pollution can serve as convenient indicators.

Analytical methods. 

• The low prevalence of C. cayetanensis in environmental samples (including finished 

product) represents a statistical challenge. Therefore, a method to concentrate oocysts from 

large volumes of water is needed. The committee acknowledges that median oocysts 

recovery efficiency using dead-end ultrafiltration (DEUF) and filter-based US EPA Method 

1623.1 is 17% and 16-22% respectively when 15,000 oocysts were seeded into 50L of 

water (Kahler et al. 2021).

• A lack of availability of oocysts to serve as a positive control can hinder laboratory detection 

method development.
57

• Given the diversity of Cyclospora-like organisms that are not known to be pathogenic to 

humans but share significant homology with target DNA sequences used for the pathogen 

detection, there is a need to develop a tool for C. cayetanensis detection using a simple, 

reproducible, and robust method. Given that the infectious dose of C. cayetanensis is not 

known, a qualitative detection method for non-clinical samples may be sufficient. 

• Methods to determine infectivity or viability of oocysts are lacking, but improved quantitative 

or qualitative methods for detection of oocysts may have a greater public health impact.

Control strategies and mitigation. 

• C. cayetanensis appears to be resistant to common chemical interventions widely used in 

the fresh produce industry. Therefore, additional antimicrobial processes or chemicals 

should be evaluated.

• In the absence of sufficient information on the sources of the pathogen and its routes of 

transmission in the areas where it is not endemic, additional efforts to develop and 

implement Good Agricultural Practices (GAPs) aimed at specific/likely routes of transfer 

should be considered (i.e., Produce can be contaminated due to little or no washing, 

contamination by food handlers, crop irrigation with untreated water, and contaminated soil.)

• In the absence of validated control strategies, a focus on preventative approaches is 

warranted. Post-harvest processing to potentially control C. cayetanensis and others. 

• Further work is needed to understand the specific wastewater treatment practices that would 

demonstrate sufficient effectiveness to benefit public health in the context of the contributing 

population.

• Eimeria spp. (another parasitic protozoa) are the most appropriate surrogate organisms 

known at the time of this report’s writing. These and other organisms should be further 

studied for use as a surrogate for Cyclospora or C. cayetanensis. A lack of reliable access 

to C. cayetanensis oocysts hampers further efforts, and a method for culturing oocysts in the 

laboratory will significantly advance efforts to control it. 

Risk assessment framework. A risk characterization will need to integrate elements of (1) 

hazard identification, 2) exposure assessment, and 3) hazard characterization into an estimation 

of the adverse effects likely to occur in a given population, including attendant uncertainties. An 

infectious dose of C. cayetanensis is not known, and this may be difficult to determine. Excreted 

organisms are not infectious and require maturation for 7 to 14 days in the environment. 

Furthermore, the impact on infectivity is unknown for both the “age” of oocysts and the 

food/water matrix source of contamination. Immunocompromised individuals are at a greater 

risk of infection or illness, and there appears to be immunity in people who have had C. 

cayetanensis as children resulting in asymptomatic infections. 

C. cayetanensis has been detected in chlorinated water, wastewater, irrigation water, and 

produce processing wash water. Foodborne illness outbreaks globally have been linked to the 

consumption of fresh fruits and vegetables. Collaboration should be encouraged between food 

and agricultural industries, academia, states, and local and foreign partners to promote research 

and share data to better understand the prevalence of C. cayetanensis in agricultural water and 

soil. 
58

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Appendix A: Glossary

Term Definition 

18S A component of the eukaryotic ribosomal ribonucleic acid (RNA). 

Detection of the 18S rRNA is used as an indication of the presence 

of a species. The sequence of the 18S rRNA gene is used to 

determine relatedness among organisms. 

Apicomplexa A group that derives its name from the apical complex, a collection 

of anterior structures that allow the parasite to invade host cells and 

establish themselves therein. 

Apicoplast The apicoplast is a secondary plastid organelle unique to most 

species within the phylum Apicomplexa that is essential for survival. 

apicoplast genome Apicoplasts contain their own DNA (35kb circular DNA) that shares 

sequence similarities with plastids (organelles found in the cells of 

photosynthetic organisms like algae and plants). 

Cyclospora 

cayetanensis

A single-celled parasite that is human-specific and transmitted 

through food or water contaminated with human feces. The 

causative agent of cyclosporiasis. 

cyclosporiasis An intestinal disease caused by Cyclospora cayetanensis 

characterized by watery diarrhea. Diarrhea may be persistent in 

some individuals. 

coliform Intestinal bacteria that are indicators of fecal contamination 

Eimeria A genus of parasites that includes some species that cause 

coccidiosis (diarrhea) in animals. 

endemic A geographical location in which an organism is present 

consistently. 

HPP High-pressure processing or high-hydrostatic-pressure processing 

indicator An organism(s) whose presence is used to suggest the presence of 

a pathogen. 

In silico Detected by computer search rather than by experimental 

procedure in the laboratory

mitochondrial genome The mitochondria (organelle that provides energy to the cell) 

contains its own DNA that is separate from the DNA held in the 

nucleus. 

MLST Multilocus sequence typing: a technique in which the DNA 

sequences of parts of several genes are used to divide organisms 

into different groups. 
77

prevalence The fraction or percent of the samples positive for the assessed 

parameter. 

qPCR Quantitative PCR

reference genome A complete assembly of the DNA sequence from a representative 

organism. The sequence is available in a digital database for 

comparison with newly derived sequence data. 

risk-based sampling A method that prioritizes sampling of products considered as having 

a greater likelihood of being positive. This is as opposed to random 

sampling. 

root cause analysis A process used to find the cause of a problem so that solutions may 

be identified. 

sporadic case An illness not associated with an outbreak.

surrogate An organism used to estimate the activity of a pathogen. 
78

Appendix B: Additional Tables

Table 1

Method Advantages Disadvantages

PCR

High sensitivity compared to 

culture and staining (Liu et 

al. 2019)

Ability to test for antimicrobial resistance (Liu et 

al. 2019)

Quickly performed in 3-7 

hours (Giangaspero et al.

2015b)

Increased ability to detect 

fewer common organisms 

such as viruses (Kahler et 

al. 2021)

Shown to be more costeffective with selective use 

than culture and staining 

(Giangaspero et al. 2015b)

Potentially lower specificity 

compared to culture and 

staining (Liu et al. 2019)

Need for a narrow list of 

causative agents to use 

specific primers (Liu et al. 

2019)

Supply costs, machinery 

fees, training expenses 

(Lalonde et al. 2022)

Becomes less cost-effective 

when performed with a 

multi-organism PCR 

approach (Craighead et al. 

2021)

Flow Cytometry Can handle large quantities 

of specimens (QuinteroBetancourt et al. 2002)

Automated (QuinteroBetancourt et al. 2002)

Relative sensitive (Duhain 

et al. 2012)

Very slow (QuinteroBetancourt et al. 2002)

Often not necessary, since 

there are other alternatives

(Quintero-Betancourt et al. 

2002)

Nucleic acid dyes might not 

be as reliable as infectivity 

studies in predicting the 

inactivation of oocysts 

following treatment (Duhain 

et al. 2012)
79

Microscopy: 

was it 

successfully 

used to diff 

viable/infectious 

from noninfectious?

Relatively simple technique 

(Masangkay 2019)

Possible to count the 

number of parasites 

(Masangkay 2019)

More useful than rapid 

diagnostic tests 

(Sathyanarayanan and

Ortega 2006) 

Requires a level of skill 

(Masangkay 2019)

They often lead to falsepositive or false-negative 

results (Sathyanarayanan 

and Ortega 2006) 

 

Table 2 

PCR Flow Cytometer Microscopy

PretreatmentFresh products were purchased 

a maximum of 24 hours prior to 

use, those fresh products were 

sampled and weight in bags with 

a microperforated filter. The 

bags before sealing need to dry 

at room temperature for about 3 

to 4 hours and are finally stored 

overnight at 4 °C prior to 

processing.

 

Water was stored at -80 °C, then 

the contaminated water was 

filtered using a cheesecloth and 

centrifuged at 2125 x g for 30 

minutes, all the water that was in 

the top was discarded. All the 

bottom particles were mixed 

using a pipette.

 

Fresh products are 

washed with ethanol, 

the inoculum was 

placed on the 

surface of the green 

peppers' pieces, 

those pieces were 

placed inside sterile 

tubes, to be dried at 

4 °C for 1 hour each 

tube before 

treatment.

 

Water, there is no 

filter to pretreat 

water, just the use of 

a centrifuge

 

Fresh products were 

washed for 

approximately 6 to 7 

minutes, and each 

vegetative sample 

was eluted y 

vigorous agitation 

followed by 

sonication for 30 

minutes. The 

supernatant was 

discarded, and the 

pellets were washed 

by centrifugation. 

 

Samples of water 

was collected using a 

sterile polyethylene 

cup attached. The 

collected water 
80

 

samples were placed 

inside an ice chest. 

To be transported 

processing within 24 

hours. 

 

 

Quantities Three studies were conducted 

involving different ranges in 

analysis. The first one did not 

have confirmation of the result, 

the second involved different 

studies in which PCR had the 

highest result. Finally, the third 

study had similar results to the 

second one

We could not find 

ranges or numbers 

of analysis 

There were several 

studies in which it 

was determined that 

the range and 

number of analyses

was made by 

comparison between 

two or more 

variables 

Sensitivity High sensitivity including for 

fresh and frozen fruits

Can detect cells 

between 1 and 15 

microns in diameter, 

although it is 

possible to detect 

particles outside of 

this range (0.2 -150 

microns) using 

specialized systems 

 

Low sensitivity in a 

range of 40% and 

50% (Omoruyi, 

Nwodo, Udem, and

Okonkwo 2014)