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Most cases of cyclosporiasis in the United States have been associated with consumption of contaminated imported foods. Based on the reported cases, Cyclospora does not appear to be endemic to the United States. Raspberries were also implicated in a multistate outbreak in the United States in and in the Georgia and Pennsylvania outbreaks in In the Pennsylvania outbreak, a wedding cake was significantly associated with the illness RR, 5.

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In this outbreak, the origin of raspberries was not identified. One farm in Guatemala, one Mexican farm, and U. One peculiarity of this outbreak was that the raspberries used to make the cake filling were exposed to freezing conditions, as was the cake before it was served at the event, suggesting that either Cyclospora is more resistant to freezing than other parasites or the raspberry topology played a protective role for this parasite.

In , a chicken pasta salad containing basil RR, 4. In this outbreak, Cyclospora was identified in the chicken pasta salad and basil by PCR and microscopy. The fresh produce linked to this outbreak was provided by farms in Mexico and the United States. Snow peas imported from Guatemala were also associated with a Cyclospora outbreak in the United States In , an outbreak was documented in British Columbia, Canada, with 17 cases of cyclosporiasis.

Interestingly, the Thai basil was imported from the United States Characteristics of polymorphic areas of the genomes of microorganisms have been studied extensively to better understand their role in epidemiology. Tracing the source of infection can be facilitated by genomic comparison between isolates. Moreover, Cyclospora may be identified clearly and differentiated from other protozoan parasites and from other microorganisms involved in coinfections that result in food-borne or waterborne outbreaks.

In addition, sequence analysis of the 18S ribosomal DNA rDNA has shown that Cyclospora strains from humans and baboons belong to a distinct monophyletic group with 1. In a different study, C. However, it should be noted that the 18S rRNA gene is highly conserved and that multiple copies could explain low sequence variability; therefore, this region may not be useful to differentiate between isolates of C. In studies conducted using the highly variable ITS1 region, C. Considerable variability was determined for C. Internal transcribed spacer ITS sequence variation was greater for U.

The high sequence variability observed in samples obtained from persons in the United States without a recent travel history overseas suggests the possibility of an endemic focus for C. The ITS1 sequences in five isolates obtained from the outbreak in the United States Floridian isolates were identical, while different ITS1 sequences were present in Guatemalan and Peruvian isolates.

Seven clones of Guatemalan isolates were identical to Floridian isolates 1. In all cases, coinfection or the presence of multiple copies may explain variability in the ITS1 region. Although current PCR protocols can be effective in detecting Cyclospora in different samples, these methods cannot distinguish between sporulated and unsporulated oocysts 82 , limiting their use in biological or epidemiological studies.

Epidemiological studies would also be facilitated if there were serological screening tests for Cyclospora. These assays would be very useful in outbreak investigations, where asymptomatic or convalescent-phase cases are not usually reported Samples suspected to contain Cyclospora can be examined using microscopy, molecular detection techniques, or sporulation studies. Samples can be stored in 2. Cyclospora oocysts can be observed in wet preparations by light and epifluorescence microscopy. Fecal smears can be stained and observed using a light microscope or by molecular analysis, using frozen samples or samples stored in potassium dichromate.

Proficiency in identification of Cyclospora has been a challenge since the early s. As a result, a pseudo-outbreak of Cyclospora was reported in the United States. Information that patients have obtained from the Internet and media reports has enhanced the reporting of food-borne outbreaks in the United States Cyclospora can be identified by phase-contrast microscopy or bright-field microscopy during examination of specimens for the ova and parasites.

Oocysts, when excreted, are spherical refractile bodies with a central morula Fig. Detection of Cyclospora oocysts. The images show unsporulated A and sporulated B oocysts and oocysts detected by autofluorescence C or modified acid-fast stain D. Like other coccidia, e. The intensity of oocyst fluorescence may be influenced by the time and storage conditions of the fecal sample. This characteristic has been used to confirm the diagnosis of Cyclospora and also to purify the parasite from human fecal samples by flow cytometry.

Detection of fluorescence has also been suggested as a useful alternative for screening large numbers of samples in the event of an outbreak Cyclospora oocysts stain variably with MZN acid-fast stain. Some oocysts stain dark red, whereas others stain pale pink or do not take the stain at all. Other stains used in parasite detection, such as Giemsa, trichrome, and Gram-chromothrope stains, do not stain Cyclospora oocysts The amplified product of about bases cannot differentiate Cyclospora from Eimeria species.

Digestion of the amplified products using the restriction enzyme MnlI PCR-restriction fragment length polymorphism [PCR-RFLP] followed by visualization by gel electrophoresis results in patterns that can discriminate these two genera of parasites. Compared to conventional microscopy assays, PCR is a more sensitive diagnostic tool Hussein 92 examined fecal samples of children with diarrhea; The amplified product of the PCR assay was a bp segment.

Serological assays to determine human exposure to Cyclospora are not yet available. However, attempts to determine a serological immune response were done using immunofluorescent-antibody IFA microscopy Preliminary studies were conducted in China to determine cellular and humoral immune function in patients with cyclosporiasis. The assays used oocysts produced in guinea pigs. This animal model and serological testing need to be reproduced and validated by other scientists A Western blot assay has been developed and can identify acute- from convalescent-phase cyclosporiasis Y.

Ortega, unpublished data. A serious limitation of this assay is the large number of oocysts needed and the lack of an animal model in which Cyclospora can be propagated. Cyclospora oocysts can be concentrated prior to microscopic observation or as a primary step in parasite purification, and ethyl acetate-formalin sedimentation has been used frequently. If the goal is to concentrate viable oocysts, a modification of this procedure includes replacing the formalin with saline solution.

Other procedures have been used in which ethyl acetate is replaced by the FeKal CON-trate system After this initial concentration step, oocysts can also be purified using discontinuous sucrose gradients. Whenever highly purified oocysts are needed, usually for research purposes, an additional purification step using cesium chloride can be used Ortega, personal observation. A modified aqueous detachment solution containing 0. A discontinuous Percoll gradient was reported to recover more oocysts than ethyl acetate and discontinuous sucrose gradients The Percoll solution was composed of Percoll and 1.

The fecal suspension was then placed on top of the bilayer. The sample yielded more positive samples than did Sheather's sucrose and Formol-ether. This study also described the application of the method for recovering other parasites, such as Cryptosporidium , Giardia , and Entamoeba , and other protozoan oocysts and cysts It is important to know if parasites or oocysts in foods or water can cause infection if ingested. It is also important to determine parasite viability when testing inactivation strategies that should neutralize the infectious potential of the parasite.

Currently, there are no animal models or in vitro cultivation methods to determine the infectious potential of Cyclospora. Two alternatives have been proposed to determine the viability of Cyclospora oocysts.

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The first method is to determine if unsporulated oocysts sporulate followed by excystation. The second alternative is electrorotation, which has been reported to correlate well with vital dyes and morphological indicators for Giardia oocysts 47 , Two parameters were considered, namely, direction of rotation and rotational velocity. This technique has been proposed to determine oocyst viability and compares favorably to oocyst sporulation detection. The reader should keep in mind that oocyst sporulation does not necessarily correlate with oocyst infectivity. The ideal method would correlate sporulation with infectivity by use of animal models.

As noted above, in spite of major efforts, attempts to infect animals with Cyclospora oocysts have been unsuccessful. In a later study, 63 children enrolled in a double-blind, placebo-controlled trial received the same 3-day course of therapy. The mean time with detectable Cyclospora oocysts was 4.

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In these patients, the infection resolved after extending treatment for an additional week. The eradication of Cyclospora in stools was directly correlated with clinical improvement Other antibiotics, such as azithromycin 40 , norfloxacin, tinidazole, nalidixic acid, diloxanide fluorate, and quinacrine , have also been tested for Cyclospora treatment, all without success. In a randomized control trial, ciprofloxacin mg BID for 7 days was tested in a randomized control trial of HIV-positive patients with cyclosporiasis Diarrhea ceased in all patients receiving TMP-SMX at mg BID median time to cessation of diarrhea was 3 days and in 10 of 11 patients receiving ciprofloxacin median time to cessation, 4 days.

After 7 days, all patients receiving TMP-SMX were negative for Cyclospora upon stool examination, whereas four of the patients receiving ciprofloxacin were still positive.

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No side effects were observed in any of the patients in the study. Ciprofloxacin can be used as an alternative therapy, especially in patients who are allergic to sulfa products ; however, anecdotal evidence of treatment failure with ciprofloxacin has also been reported A 7-day regimen with nitazoxanide has been proposed as an effective alternative for treatment of cyclosporiasis in a patient with sulfa allergy 54 , There are reports that suggest waterborne transmission of Cyclospora.

An elderly man with Cyclospora infection was in contact with a sewage backup in the basement of a house, and another person in the same house later developed persistent diarrhea that was suspected to have been caused by Cyclospora In another suspected waterborne case, a patient without travel history reported having cleaned a salt water aquarium by oral siphoning 4 days prior to developing symptoms In a hospital-based outbreak in Chicago, drinking water was associated with Cyclospora infections Water used either for irrigation or for processing vegetables has been found to contain Cyclospora oocysts.

A study by Tram et al.

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Oocysts were isolated from all varieties of farm-grown herbs examined basil, coriander, lettuce, marjoram, and Vietnamese mint. Cyclospora contamination was observed before the rainy season November to April but not during the rainy season May to October. Interestingly, no human cases were detected in a hospital-based surveillance study performed in the same period. Cyclospora has also been detected in wastewater , Eight of 11 water samples from a primary oxidation lagoon in Peru contained Cyclospora oocysts These samples were concentrated using Envirocheck capsules and Hannifin filters.

Environmental water collected from rivers and lakes in Vietman, Guatemala, and Egypt was also positive for Cyclospora 14 , 65 , These contradictions suggest that the nested PCR method gives false-positive results, as amplification of other coccidia present in river water or environmental samples occurs.

One of the possible vehicles of Cyclospora contamination of produce could result from use of contaminated water for application of pesticides. Extended incubation 1 week in Benomyl, however, reduces sporulation.

Cyclospora can be isolated from irrigation and drinking water by filtration, using Hannifin polypropylene cartridge filters or Envirocheck capsules After centrifugation, pellets are stored in 2. These temperatures can be reached in shorter periods than those with conventional heating. Fresh produce samples in Phnom Penh, Cambodia, were examined for the presence of Cyclospora.

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High concentrations of thermotolerant coliforms ThC , intestinal helminth eggs, and protozoa were found in a wastewater-fed lake where water spinach was grown. Calvo et al. The highest prevalence of fecal coliforms was identified during the rainy season. Cyclospora was identified only in lettuce during the dry season. In Peru, Cyclospora was identified in vegetables surveyed on three occasions Of vegetables analyzed, 2 1.

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Lettuce, mint, and black mint were contaminated with Cyclospora oocysts. Sherchand et al. Since Cyclospora has been associated with outbreaks of food-borne infections, methods for food sample preparation and detection of the parasite have been a research priority. Robertson et al. Although recovery efficiencies were not improved, this method resulted in cleaner samples that were of smaller volume. Other studies used water, saline solution, glycine buffer, pH 5.