Synopses

Cyclospora: An Enigma Worth Unraveling

Charles R. Sterling and Ynés R. Ortega University of Arizona, Tucson, Arizona, USA

In part, cayetanensis owes its recognition as an emerging pathogen to the increased use of staining methods for detecting enteric parasites such as . First reported in patients in New Guinea in 1977 but thought to be a coccidian parasite of the genus , C. cayetanensis received little attention until it was again described in 1985 in New York and Peru. In the early 1990s, human infection associated with waterborne transmission of C. cayetanensis was suspected; foodborne transmission was likewise suggested in early studies. The parasite was associated with several disease outbreaks in the United States during 1996 and 1997. This article reviews current knowledge about C. cayetanensis (including its association with waterborne and foodborne transmission), unresolved issues, and research needs.

Cyclospora Overview an ocular micrometer, oocysts of Cyclospora is a protozoan might be easily confused with those of parasite (subphylum , subclass Cryptosporidium or other fecal artifacts that Coccidiasina, order Eucoccidiorida, family Eimeri- stain acid-fast positive, as was the case in a idae). The organism’s link to the pseudo-outbreak of cyclosporiasis reported in extends to the genus level through use of Florida (6). Cyclospora oocysts are easily molecular phylogenetic analysis techniques (1). observed by phase contrast microscopy, and the Collected data link infection to a single host— algal-like morula appearance is evident in fresh humans. In 1993, asexual meronts were stool specimens. A useful and distinguishing described from jejunal enterocytes of humans feature is oocyst autofluorescence, which (2). In 1997, two types of meronts and sexual appears blue by Epi-illumination and a 365-nm stages were observed in jejunal enterocytes of dichroic exciter filter and green by a 450-nm to biopsy specimens from infected patients excret- 490-nm dichroic filter. ing oocysts, confirming that the entire life cycle Susceptible humans are infected by ingest- could be completed within a single host (3); ing sporulated oocysts. While unknown, the infected persons excrete unsporulated oocysts. infectious dose is presumed to be low. Symptoms In the laboratory, oocysts are induced to of infection may include watery diarrhea, mild to sporulate in potassium dichromate in a petri dish severe nausea, anorexia, abdominal cramping, at ambient temperatures (25°C to 30°C). After 1 fatigue, and weight loss. Diarrhea can be week and up to 2 weeks, approximately 40% of intermittent and protracted (3,7,8). Persons with oocysts contain two sporocysts with two no previous immunity as well as very young sporozoites in each (4). Excystation of sporulated children in developing countries are likely to oocysts occurs in vitro when oocysts are exhibit symptoms. Limited data suggest that in subjected to bile salts and sodium taurocholate disease-endemic countries, frequent exposure and mechanical pressure from a glass tube mortar may predispose to asymptomatic infection in and pestle (5). These findings suggest that direct children and absence of infection in adults (9). person-to-person transmission is unlikely. Symptomatic infections can be treated with Oocysts measure 8 µm to 10 µm in diameter trimethoprim-sulfamethoxazole (Bactrim) (9-11). and stain variably acid-fast. Without the use of Cyclospora infections have been confirmed in North, Central, and South America, the Caribbean, England, eastern Europe, Africa, the Address for correspondence: Charles R. Sterling, Department Indian subcontinent, Southeast Asia, and of Veterinary Science and Microbiology, University of Arizona, Tucson, AZ 85721, USA; fax: 520-621-2799; e-mail: Australia (12). In the United States, England, [email protected]. and Australia, most cases were first observed in

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travelers returning from the areas listed above lagoons adjacent to an area of endemic disease in (7,13,14). As more indigenous cases are Lima, Peru (20); their presence was confirmed by reported from all areas, however, a cosmopoli- microscopy and PCR. Water from these sewage tan distribution of Cyclospora appears pos- lagoons is used to irrigate pasture land, corn fields, sible. A seasonal distribution of infection, and trees. In other parts of Lima, water from coinciding with wet or warm months of the such lagoons is used to irrigate vegetable crops. year, has also been suggested (15). Association with Foodborne Transmission Association with Waterborne While the Nepal study conducted in 1992 Transmission strongly suggested waterborne transmission of While the organism causing Cyclospora Cyclospora, only 28% of infected patients infection was still being identified, an outbreak reported drinking untreated water or milk occurred in the staff of a Chicago hospital in 1990 possibly contaminated with untreated water (16). Infection was confirmed in 11 of 21 persons (18). Therefore, other modes of transmission exhibiting diarrheal symptoms and lasted up to were likely, although none was identified. 9 weeks with alternating cycles of disease and Foodborne transmission was suspected when remission. Epidemiologically, infections were consumption of raw or undercooked meat and associated with drinking tap water (in a poultry products was reported as part of case resident’s dormitory) possibly contaminated histories before the infectious organism was with stagnant water from a rooftop storage identified as Cyclospora (21,22). Foodborne reservoir. In an isolated incident (also in transmission was first suggested in 1995 when Chicago), an 8-year-old child became ill and the illness of an airline pilot was associated with passed oocysts in the feces 1 week after food prepared in a Haitian kitchen and brought swimming in Lake Michigan (7). In another on board the airplane (23). Cyclospora is endemic isolated incident, a man from Utah became ill in Haiti; this study underscored that this type of with severe watery diarrhea and passed oocysts illness could be acquired from meals brought on after cleaning his basement, which had been board without visiting the country in which flooded by sewage backup following heavy rains infection originated. (8). The man’s house was located near a dairy Foodborne transmission of Cyclospora in the farm and much of the sewage backup was United States, first reported in 1995, was widely attributed to water runoff from this site. In yet reported in 1996 and 1997 (24-28). Some reports another isolated incident in the United States, early in 1996 implicated strawberries, but as consumption of well water was implicated in the more epidemiologic information was gathered, infection of one of three patients in Massachu- attention shifted to raspberries. In 1996, a total setts (17). of 1,465 cases of cyclosporiasis were reported Two outbreaks of Cyclospora infection in from 20 states (predominantly east of the Rocky Nepal have also been linked to waterborne Mountains), the District of Columbia, and two transmission (18,19). In the first outbreak in Canadian provinces (24). Almost half (725 cases) 1992, expatriates, who were more likely to drink were event associated; the remaining (740 cases) untreated water or milk reconstituted with were sporadic (i.e., not epidemiologically linked water, became ill with diarrhea and passed to other cases); 978 (67%) cases were laboratory oocysts. The infections occurred during the confirmed; 55 clusters of cases were associated summer, which coincided with annual epidemics with social events. A total of 3,035 persons among the expatriates. The second waterborne attended these events; 1,339 (44.1%) were disease outbreak occurred in 12 of 14 British interviewed, and of these 735 (54.1%) were soldiers, despite chlorination of the water designated case-patients. Cyclospora infection involved. In this outbreak, Cyclospora oocysts was laboratory confirmed in 238 (32.8%) cases. were demonstrated for the first time in drinking Raspberries were definitely served at 50 events water, which consisted of a mixture of river and and possibly at four more. Even in the municipal water. documented 740 sporadic cases in 1996, many Though not directly connected with water- patients recalled eating some type of berries. Of associated disease outbreaks, Cyclospora oocysts the 54 cluster events at which raspberries were have been isolated from wastewater in sewage or may have been served, well-documented

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traceback data as to the source were uncovered environmental conditions. All these factors for 29; of these, 21 were definitely traceable to affect transmission. raspberries imported from Guatemala, and an Because of technologic limitations, Cyclospora additional eight may have originated there. oocysts have only been recovered in very limited Twenty-five (86%) of the 29 well-documented numbers from water sources and vegetables events were traceable to one (versus more than (19,20,29,30). A heavy reliance has been placed one) exporter per event. Further tracings on techniques used for isolating Cryptosporidium, showed that as few as five Guatemalan farms which are inadequate (31). Very little is known could have accounted for the 25 events traceable about conditions that may favor the survival of to a single exporter per event. In part because of Cyclospora. Preliminary studies have shown previous links with waterborne transmission, it that oocysts subjected to -20°C for 24 hours and was postulated that the berries were contami- exposure to 60°C for 1 hour cannot be induced to nated when sprayed with insecticides or sporulate. Oocyst storage at 4°C or 37°C for 14 fungicides mixed with water containing sporu- days retards sporulation (32). The most lated oocysts. intriguing environmental issue is oocyst sporula- As of August 1997, 1,450 cases of tion time. The report that confirmed the identity cyclosporiasis (550 laboratory confirmed) were of Cyclospora indicates that the organism reported (28). Many cases were cluster- requires 1 to 2 weeks to completely sporulate and associated and involved raspberries linked to become infectious under ambient conditions of Guatemala. In addition, 25 confirmed and 20 25°C to 30°C (5). Oocysts maintained at 4°C can possible clusters of cases of cyclosporiasis were sporulate within 6 months (4). These periods are associated with consumption of food that longer than those reported for most ; contained fresh basil. An additional two clusters therefore, direct person-to-person transmission of cases in Florida were linked with eating is unlikely. Also, (if confirmed under changing mesclun lettuce (28). In each situation, the conditions) a prolonged sporulation time would outbreaks were linked to non-Guatemalan imply that oocysts favor a moist environment, fresh produce. ideally water. Early in the Guatemalan berry Cyclospora oocysts have been isolated from investigations, water used to irrigate plants was vegetables from a disease-endemic area of Lima, thought to play a role in contaminating Peru, and from Nepal (29,30). Although the raspberries with oocysts. This notion, which number of oocysts recovered was small, would likely apply only to berries grown with encountered in only a few samples, and not spray irrigation, however, has largely been associated with any known disease outbreak, the discarded since direct contact exposure to implication was clear: foodborne transmission by excessive moisture promotes rapid fruit deterio- this route could occur. In addition, oocysts ration and most raspberries grown in Guatemala experimentally seeded on vegetables could not rely on drip irrigation. The exact method of easily be removed by washing (30). Washing of contamination is not known, and even though vegetables, even though highly recommended as use of insecticides and fungicides made with a means of reducing risk for infection, may oocyst-contaminated water has been hypoth- therefore not totally eliminate the risk. esized, its role has yet to be confirmed. If this hypothesis is true, how these agents might affect Unresolved Issues oocyst viability is also not known. Another Unresolved issues concerning Cyclospora unresolved issue is how the water might have fall into three broad categories: environmental become contaminated. survival, transmission to humans, and epidemi- ology. The boundaries of these categories Transmission to Humans frequently overlap. The primary issues concerning transmission of Cyclospora to humans are infectious dose and Environmental Survival species specificity. For most coccidia that infect The biggest issues of concern in this category humans and animals (e.g., Cryptosporidium are oocyst distribution in the environment, [33]), the infectious dose is presumed to be low oocyst survival under changing conditions, and (34). What we know about the waterborne oocyst sporulation times under changing transmission of Cryptosporidium and how few of

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its oocysts are usually isolated from water is traceback issues associated with clusters of likely true for Cyclospora (35). However, only cyclosporiasis cases and potential indigenous two foodborne outbreaks of infections within the United States and elsewhere. have been reported (one involved fresh pressed Unraveling the first issue involves tracing cider and the other chicken salad) (36,37). imported fruits or vegetables in a forward Cryptosporidium is immediately infectious upon direction (possible distribution sites) as well as passage from an infected person, and oocysts are tracing them back (to their originating sites). In usually passed in large numbers if the person is the raspberry-associated outbreaks of 1996, good symptomatic. Unlike what has been reported for traceback data were obtainable for 29 of 55 Cyclospora to date, Cryptosporidium oocysts are clusters. All sites (except one) were east of the ubiquitous in the environment and could easily Rocky Mountains. For the 25 events traceable to contaminate foods, especially vegetables. In one one (versus more than one) exporter per event, study, Cryptosporidium oocysts were recovered 33 (85%) of 36 shipments entered through more frequently from vegetables than Cyclospora Miami, Florida (24). If berries were also being oocysts (30). In addition, Cryptosporidium distributed in large quantities to other, largely infectious to humans has many known animal western regions of the country during this hosts (38). period, would we not expect more infections in The issue of potential animal hosts for western regions? This point, along with the Cyclospora has not been resolved. Cyclospora- attendant epidemiologic investigations, helped like organisms have been recovered from ducks, dissociate strawberries from reported Cyclospora chickens, dogs, and primates (39-41). Only in infections. California strawberry growers were primates has there been any concrete evidence as likely or more likely to ship strawberries identifying the agent as a species of the genus within their own region of the United States as Cyclospora, and whether it is the same as they were to ship them elsewhere, yet most C. cayetanensis is not known (41). For the other infections occurred in eastern regions of the animal species mentioned, recovered oocysts, if country. In addition, Guatemalan raspberries they were oocysts of Cyclospora, may have been are imported into the United States in large passing through these hosts. Attempts at finding quantities twice a year, yet no outbreaks animal hosts infected with Cyclospora-like occurred during the winter months when this organisms in human disease-endemic areas have importation occurs, which indicates that the largely failed, as have preliminary attempts at epidemiology of this infection in countries such infecting conventionally used laboratory ani- as Guatemala where the berries are grown needs mals. Some researchers have convincingly further study. shown on the basis of molecular data that The issue of indigenous U.S. infections Cyclospora and are closely related (1): should be investigated. Waterborne and sporadic others have even suggested that Cyclospora cases have occurred in which no association should be considered a mammalian Eimeria could be made to raspberry consumption (7,8,16- species (42). To clarify the taxonomic issue, small 19,24,29). Preliminary data (in one region of the subunit rRNA sequences from Isospora should United States) have linked Cyclospora infection be compared with those of C. cayetanensis and to gardening and working with soil (43). with Cyclospora isolates from nonhuman primates. In addition, conventional and molecu- Dr. Sterling is professor and head of the Depart- ment of Veterinary Science and Microbiology, Univer- lar taxonomists should name the species on the sity of Arizona, Tucson. His laboratory research focuses basis of combined phenotypic and genotypic on cryptosporidiosis in immunologically naive and characteristics. immunocompromised persons, monoclonal and polyclonal antibodies in diagnostic parasitology and im- Epidemiology munotherapy, and Cyclospora and the microsporidia as Even though epidemiologic investigations of emerging pathogens. Cyclospora have been thorough and convincing, Dr. Ortega is assistant research scientist in the De- they raise environmental and transmission partment of Veterinary Science and Microbiology, Uni- issues that require further investigation. The versity of Arizona, Tucson. She was first to identify taxo- nomically Cyclospora, and her research focuses on the two areas we will consider are the relative molecular biology and epidemiology of this organism. geographic restriction of cases and attendant

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