Detection of Cyclospora cayetanensis on bagged pre-cut salad mixes within their shelf-life and after sell by date by the U.S. food and drug administration validated method.

Recently, outbreaks of Cyclospora cayetanensis in the U.S. were linked to the consumption of a variety of salads containing romaine and/or iceberg lettuce, carrots and/or red cabbage. The Bacteriological Analytical Manual (BAM) Chapter 19b method was validated for the detection of C. cayetanensis in carrots, cabbage and romaine lettuce, but has not been previously evaluated in ready-to-eat (RTE) salad mixes. In addition, the only samples available for traceback investigations are sometimes leftovers in bad conditions. This study evaluated the validated BAM method for detection of C. cayetanensis in two different RTE mixed salads (mix 1: romaine and iceberg lettuces, carrots, and red cabbage and mix 2: romaine and iceberg lettuces, carrots, red cabbage, radish, and pea pods) in good condition and after their sell by date. Individual samples (25 g) were seeded with five and 200 C. cayetanensis oocysts. Unseeded produce was used as negative control. The method included washing of the produce, concentration and extraction of C. cayetanensis DNA and molecular detection of C. cayetanensis 18 S rRNA gene. As few as five oocysts were detected in both fresh and after sell by date mix salads. All unseeded samples were negative, and all samples of both salad types seeded with 200 oocysts were positive. In samples seeded with 200 oocysts, average 18 S rRNA C. cayetanensis CT values were significantly higher in fresh salad mix 1 compared to fresh salad mix 2; CT values were significantly higher in the after sell by date salads compared to their respective fresh mixes (p < 0.05). In conclusion, the BAM method was able to detect as few as five oocysts even in after sell by date RTE mix salads. However, the differences in detection observed, highlight the importance of evaluating the performance of the validated C. cayetanensis detection method in different food matrices and conditions, in advance for future outbreak investigations.

Contamination of fresh produce sold on the Italian market with Cyclospora cayetanensis and Echinococcus multilocularis.

To investigate the presence of Cyclospora cayetanensis, Toxoplasma gondii and Echinococcus spp. in fresh produce sold in Italy, 324 locally produced 'ready-to-eat' (RTE) mixed-salad packages belonging to three brands and 324 berries packages (blueberries and blackberries imported from Peru and Mexico, respectively, and raspberries grown in Italy) were purchased at retail. Nine individual packages from each of the six types of fresh produce were collected monthly for one year, and with the same produce pooled, this resulted in a total of 72 pools for the whole year. Using microscopy (FLOTAC), a Cyclospora-like oocyst was detected in a blueberry sample and a taeniid egg was detected in a RTE-salad sample. Molecular tools confirmed these to be C. cayetanensis and Echinococcus multilocularis, respectively. Toxoplasma gondii was not detected in any of the samples. This study shows for the first time in Europe that imported berries on the Italian market may be contaminated with C. cayetanensis and RTE salads grown in Italy with E. multilocularis. The results indicate a new epidemiological scenario and highlight that current management of fresh produce, locally produced or imported, does not ensure products are free from parasite contamination.

Modifications of the U.S. food and drug administration validated method for detection of Cyclospora cayetanensis oocysts in prepared dishes: Mexican-style salsas and guacamole.

Although multiple outbreak clusters of Cyclospora cayetanensis have been traced back to consumption of dishes in Mexican-style restaurants, the FDA Bacteriological Analytical Manual (BAM) does not currently provide methods to detect C. cayetanensis in dishes that contain multiple produce ingredients, such as salsas and guacamole. These complex food matrices also may contain high levels of fats, which can interfere with the detection. Several modifications to the BAM Chapter 19b method (washing produce, DNA extraction, and a TaqMan real-time PCR assay targeting the 18S rRNA gene of C. cayetanensis) were assessed with the goal to detect as few as 5 oocysts of C. cayetanensis in 25 g samples of commercial salsa/pico de gallo, guacamole, and salsa verde. Both freshly prepared and frozen versions of these foods were seeded with 5, 10 and 200 oocysts. For salsa samples, using a gentler washing step than recommended by BAM, we achieved detection of 5 oocysts in the samples (81.8%, n = 11). Increasing the amount of Alconox® in the wash solution to 1%, rather than the 0.1% used in BAM, and adjusting the DNA extraction protocol to process large wash pellets, enabled detection of 5 oocysts in guacamole. To reach the desired level of detection in salsa verde, two types of modifications were necessary: gentler washing and DNA extraction modifications. The use of these same method modifications on previously frozen food samples, provided levels of detection similar to those achieved with fresh dishes. Our modifications enabled robust and reproducible detection of C. cayetanensis in multi-ingredient Mexican dishes, detecting as few as 5 oocysts in 25 g samples. Validating and deploying effective methods to detect C. cayetanensis in high risk fresh produce and prepared dishes are critically important for prevalence studies and outbreak investigations of this parasite.

Evaluation of the U.S. Food and Drug Administration validated method for detection of Cyclospora cayetanensis in high-risk fresh produce matrices and a method modification for a prepared dish.

The performance of the U.S. Food and Drug Administration (FDA) validated method for regulatory detection of Cyclospora cayetanensis in leafy greens and berries was evaluated in additional high-risk fresh produce items and in a dish prepared with these produce commodities. The method was robust and reproducible in basil, parsley, shredded carrots, shredded cabbage and carrot mix, and could detect as few as 5 oocysts in 25 g samples. Some differences in C. cayetanensis detection were found among the fresh produce analyzed. Significantly lower target gene copy numbers per reaction were obtained with shredded carrots, and shredded cabbage and carrot mix compared to leafy greens, which highlights the importance of evaluating the performance characteristics of validated methods in different food matrices. In the prepared dish, coleslaw with dressing, the method was optimized to detect 5 oocysts in a 25 g sample by using 1.0% Alconox® in the washing solution instead of 0.1% as originally described. These data are important to assess the prevalence of C. cayetanensis in different produce items and to support outbreak investigations.

Detection and prevalence of protozoan parasites in ready-to-eat packaged salads on sale in Italy.

To investigate the prevalence of protozoan contamination by Giardia duodenalis, Cryptosporidium spp., Toxoplasma gondii and Cyclospora cayetanensis, in 'ready to eat' (RTE) salads on sale in Italy, 648 packages were purchased from industrial and local brands. Nine individual packages from each brand were collected per month, pooled and subjected to microscopy and molecular analyses. Microscopic examination of 864 slides detected Cryptosporidium spp. but also Blastocystis hominis and Dientamoeba fragilis. Molecular tools identified G. duodenalis assemblage A, Cryptosporidium parvum and Cryptosporidium ubiquitum, T. gondii Type I and C. cayetanensis. B. hominis and D. fragilis were also molecularly confirmed. The overall prevalence of each protozoan species was 0.6% for G. duodenalis, 0.8% for T. gondii, 0.9% for Cryptosporidium spp., and 1.3% for C. cayetanensis, while prevalence for B. hominis was 0.5% and for D. fragilis 0.2%. Microscopy and/or molecular tools revealed that 4.2% of the samples were contaminated by at least one protozoan species, and 0.6% of samples presented contamination by two protozoan species, with a number of oocysts ranging from 62 to 554 per g of vegetable matter for T. gondii, and 46 to 1.580 for C. cayetanensis. This is Europe's first large-scale study on the presence of protozoans in packaged salads, and shows that RTE sanitation processes do not guarantee a product free from protozoans of fecal origin.

Population-based active surveillance for Cyclospora infection--United States, Foodborne Diseases Active Surveillance Network (FoodNet), 1997-2009.

BACKGROUND: Cyclosporiasis is an enteric disease caused by the parasite Cyclospora cayetanensis. Since the mid-1990 s, the Centers for Disease Control and Prevention has been notified of cases through various reporting and surveillance mechanisms. METHODS: We summarized data regarding laboratory-confirmed cases of Cyclospora infection reported during 1997-2009 via the Foodborne Diseases Active Surveillance Network (FoodNet), which gradually expanded to include 10 sites (Connecticut, Georgia, Maryland, Minnesota, New Mexico, Oregon, Tennessee, and selected counties in California, Colorado, and New York) that represent approximately 15% of the US population. Since 2004, the number of sites has remained constant and data on the international travel history and outbreak status of cases have been collected. RESULTS: A total of 370 cases were reported, 70.3% (260) of which were in residents of Connecticut (134 [36.2%]) and Georgia (126 [34.1%]), which on average during this 13-year period accounted for 29.0% of the total FoodNet population under surveillance. Positive stool specimens were collected in all months of the year, with a peak in June and July (208 cases [56.2%]). Approximately half (48.6%) of the 185 cases reported during 2004-2009 were associated with international travel, known outbreaks, or both. CONCLUSIONS: The reported cases were concentrated in time (spring and summer) and place (2 of 10 sites). The extent to which the geographic concentration reflects higher rates of testing, more sensitive testing methods, or higher exposure/infection rates is unknown. Clinicians should include Cyclospora infection in the differential diagnosis of prolonged or relapsing diarrheal illness and explicitly request stool examinations for this parasite.

Endogenous Developmental Cycle of the Human Coccidian Cyclospora cayetanensis.

Cyclospora cayetanensis is a coccidian parasite of humans of known and growing importance. However, we are surprisingly naïve as to our understanding of how to diagnose it and how it develops inside the human body. Here we provide details of the developmental stages of C. cayetanensis in the gallbladder of a 33-yr-old male with human immunodeficiency virus. The gallbladder was removed surgically in 2001 because of severe abdominal pain. For the present study, the archived paraffin block of gallbladder was processed for light microscopy and transmission electron microscopy (TEM). Histological sections were examined after staining with hematoxylin and eosin (HE) or using the periodic acid Schiff (PAS) reaction. Immature and mature asexual stages, gamonts, and oocysts were seen in epithelial cells, both in the superficial epithelium and in glands. The merozoites were present singly, in pairs, and 3 or more in a single parasitophorous vacuole in the host cytoplasm. Up to 6 nuclei were seen in immature schizonts without evidence of merozoite formation. Mature schizonts were 7.6 × 5.1 µm and contained up to 10, 3-4 µm long merozoites. Merozoites were 0.6 to 2.0 µm wide, and their shape varied from pear-shaped to slender. Merozoites were generally PAS-positive; however, some were intensely positive, some had only minute granules, while others were PAS-negative. The microgamonts (male) were 6.6 × 5.2 µm and contained fewer than 20 microgametes around a residual body. The microgametes were up to 2 µm long and were flagellated. Macrogamonts (female) contained distinctive eosinophilic wall-forming bodies that varied in size and were less than 1 µm in HE-stained sections. Macrogamonts were 5.8-6.5 × 5.3-6.5 µm. Oocysts in sections were unsporulated and had a diameter of 5.7-7.5 µm. The TEM examination confirmed the histologic findings. The DNA extracted from paraffin sections was confirmed as C. cayetanensis with real-time PCR. The detailed description of the life cycle stages of C. cayetanensis reported here in an immunosuppressed patient could facilitate histopathologic diagnosis of this parasite. We have shown that the parasite's development more closely resembles that of Cystoisospora than Eimeria and that the parasite has multiple nuclei per immature meront indicating schizogony, and we have undermined evidence for a Type II meront.

Efficacy of gaseous chlorine dioxide as a sanitizer against Cryptosporidium parvum, Cyclospora cayetanensis, and Encephalitozoon intestinalis on produce.

The efficacy of gaseous chlorine dioxide to reduce parasite and bacterial burden in produce was studied. Basil and lettuce leaves were inoculated with Cryptosporidium parvum and Cyclospora cayetanensis oocysts, Encephalitozoon intestinalis spores, and a cocktail of two isolates of nalidixic acid-resistant Escherichia coli O157:H7. The inoculated samples were then treated for 20 min with gaseous chlorine dioxide at 4.1 mg/liter. Cryptosporidium had a 2.6 and 3.31 most-probable-number log reduction in basil and lettuce, respectively. Reduction of Encephalitozoon in basil and lettuce was 3.58 and 4.58 CFU/g respectively. E. coli loads were significantly reduced (2.45 to 3.97 log), whereas Cyclospora sporulation was not affected by this treatment.

Presence of Parasites in Environmental Waters in Samsun and Its Districts.

OBJECTIVE: The aim of this study was to detect the presence of parasites in environmental waters in Samsun and its districts. METHODS: At the center of Samsun, 13 stations were determined. The research was performed between March 2012 and February 2013, and every month, water samples were collected on the dates stated. The samples were stained with Kinyoun acid-fast, modified trichrome, and trichrome dyes after examining with the direct bond. The preparations were evaluated in terms of parasitologic under a light microscope. RESULTS: Totally, 180 of 228 water samples analyzed were from streams; of these, 48 were drinking water samples. The following were found: 142 Giardia spp., 132 Cryptosporidium spp., 56 Cyclospora spp., 38 microsporidia, 47 Blastocystis spp., 38 Entamoeba coli cysts, 18 Dientamoeba, 9 Chilomastix, 9 Strongyloides spp., and 6 hookworms. CONCLUSION: The widespread use of animal husbandry and agriculture in the region and the use of stream surroundings as a grazing area increase the presence of some determined protozoa during a certain period. Parasitological studies in humans and animals in the region should be conducted, and control programs should be applied.

Co-infection of Giardia intestinalis and Cyclospora cayetanensis in an immunocompetent patient with prolonged diarrhea: case report.

Cyclospora cayetanensis is an agent of emerging infectious disease, and a recognized cause of diarrhea in some patients. Also, the flagellated protozoan, Giardia intestinalis, induces a diarrheal illness of the small intestine. Cases of cyclosporiasis are frequently missed, primarily due to the fact that the parasite can be quite difficult to detect in human fecal samples, despite an increasing amount of data regarding this parasite. On the other hand, G. intestinalis can be readily recognized via the microscopic visualization of its trophozoite or cyst forms in stained preparations or unstained wet mounts. In this report, we describe an uncommon case of co-infection with G. intestinalis and C. cayetanensis in an immunocompetent patient with prolonged diarrhea, living in a non-tropical region of Turkey.

Microwave inactivation of Cyclospora cayetanensis sporulation and viability of Cryptosporidium parvum oocysts.

The efficacy of microwave heating on the viability of Cryptosporidium parvum oocysts and on the sporulation of Cyclospora cayetanensis oocysts for various periods of cooking times (0, 10, 15, 20, 30, and 45 s) at 100% power was determined. Cyclospora oocysts were stored in 2.5% dichromate at 23 degrees C for 2 weeks, and sporulation rates were then determined. The 4',6-diamidino-2-phenylindole and propidium iodide vital stain and the neonate animal infectivity assay determined Cryptosporidium oocyst viability. Cryptosporidium oocysts could be completely inactivated with as little as 20 s of cooking time, whereas Cyclospora sporulation was observed up to 45 s. Two of the examined microwave ovens were more effective at reducing sporulation and viability than the third one. Because of the variability of temperature achieved by the various ovens, cooking time was not an accurate parameter for parasite inactivation. Cryptosporidium oocysts could be inactivated only when temperatures of 80 degrees C or higher were reached in the microwave ovens.

Inactivation of protozoan parasites in food, water, and environmental systems.

Protozoan parasites can survive under ambient and refrigerated storage conditions when associated with a range of substrates. Consequently, various treatments have been used to inactivate protozoan parasites (Giardia, Cryptosporidium, and Cyclospora) in food, water, and environmental systems. Physical treatments that affect survival or removal of protozoan parasites include freezing, heating, filtration, sedimentation, UV light, irradiation, high pressure, and ultrasound. Ozone is a more effective chemical disinfectant than chlorine or chlorine dioxide for inactivation of protozoan parasites in water systems. However, sequential inactivation treatments can optimize existing treatments through synergistic effects. Careful selection of methods to evaluate inactivation treatments is needed because many studies that have employed vital dye stains and in vitro excystation have produced underestimations of the effectiveness of these treatments.

Detection of Cyclospora cayetanensis using a quantitative real-time PCR assay.

Cyclospora cayetanensis, a coccidian parasite, with a fecal-oral life cycle, has become recognized worldwide as an emerging human pathogen. Clinical manifestations include prolonged gastroenteritis. While most cases of infection with C. cayetanensis in the United States have been associated with foodborne transmission, waterborne transmission has also been implicated. We report on the development and application of a real-time, quantitative polymerase chain reaction assay for the detection of C. cayetanensis oocysts, which is the first reported use of this technique for this organism. Both a species-specific primer set and dual fluorescent-labeled C. cayetanensis hybridization probe were designed using the inherent genetic uniqueness of the 18S ribosomal gene sequence of C. cayetanensis. The real-time polymerase chain reaction assay has been optimized to specifically detect the DNA from as few as 1 oocyst of C. cayetanensis per 5 microl reaction volume.

Cyclospora cayetanesis.

The novelty of C cayetanensis has led to some misconceptions about how best to detect its presence in stool examinations. Some reports have implied that the organism can only be seen on stained specimens, which is not true. The unstained organism can easily be identified by its characteristic size and internal structures. However, not doing a concentration procedure can reduce the chances of detecting C cayentanensis by up to 40%. Finally, there have been false positive reports of C cayetanensis when stained artifacts were mistaken for the real organism. The best way to become comfortable with the laboratory diagnosis of C cayentanensis is to obtain some known positive samples and practice identifying the oocysts using a variety of methods. The clinical syndrome associated with C cayentanensis is recognizable. The patient will usually have prominent anorexia, fatigue, nausea, and gas. Diarrhea, after the initial severe bout, is often intermittent, and submitted specimens may be formed despite a persistent feeling of being unwell.

Coccidial contamination of raspberries: mock contamination with Eimeria acervulina as a model for decontamination treatment studies.

Numerous outbreaks have been reported since 1995 in the United States and Canada that were linked to the consumption of imported fresh raspberries contaminated with Cyclospora. Because Cyclospora has no laboratory animal hosts, Eimeria acervulina, a common chicken coccidium similar in characteristics to Cyclospora, was used as a surrogate to test decontamination treatments. Raspberries were mock contaminated with E. acervulina-sporulated oocysts in a water suspension, then exposed to washing, freezing, heat, or irradiation before they were fed to chicks. The presence of oocysts in the contaminated raspberries was confirmed either by duodenal lesions or oocysts in cecal contents 5 days postinoculation (PI) or in fecal contents 6 days PI, after 24 h of fecal collection. Washing of raspberries was generally not adequate in removing coccidial contamination, but freezing and heat treatment appeared effective. Gamma irradiation of E. acervulina-sporulated oocysts at a dose of 0.5 kGy was partially effective, but it was completely effective at 1.0 kGy and higher. We suggest that E. acervulina, for mock contamination of raspberries and subsequent decontamination treatments, is easy to handle, safe, and economical to study.

Cyclospora cayetanensis, a food- and waterborne coccidian parasite.

Food- and waterborne coccidia including Cryptosporidium parvum, Cyclospora cayetanensis, Sarcocystis hominis and Sarcocystis suihominis, and Isospora belli are cyst-forming apicomplexan protozoa that cause intracellular infections, predominantly in the epithelial cells of the intestine. They are transmitted by oocysts from person-to-person by the fecal-oral route or via contaminated water or food. The most common symptom of infection is diarrhea, however, asymptomatic infections occur. Infections are associated with intestinal inflammation, with pathological lesions such as villus blunting, and abnormal function such as malabsorption. Mild-to-moderate, self-limiting diarrhea is common in healthy individuals ingesting infective stages of these organisms. However, patients with immune dysfunction can have severe intestinal injury and prolonged diarrhea. Diagnosis in many cases is made by a microscopic examination of the stool, and the use of appropriate staining techniques, but more recently molecular methods for detection are used increasingly. Effective antimicrobial treatment for prolonged infection in immunocompromised patients is available for most of these infections. These gastrointestinal coccidial pathogens have important similarities in epidemiology, disease pathogenesis, clinical manifestations, diagnosis, and treatment. Although there are many other cyst-forming coccidia of public health, veterinary and/or economic importance, discussion in this chapter will be limited to C. cayetanensis, as an important example of the group. Aspects of the biology, epidemiology, diagnosis, disease, treatment and control are considered. This parasite is considered to be an emerging pathogen. From 1990 to 2000, there were 11 foodborne outbreaks of cyclosporosis in North America that affected at least 3600 people. There are many outstanding questions regarding this parasite and under-reporting is common because general diagnostic methods for intestinal parasites are inadequate for detection of Cyclospora.

Cyclospora cayetanensis: oocyst characteristics and excystation.

In Thailand in 1999-2000, Cyclospora oocysts from two HIV-infected patients and one patient with prolonged diarrhea were detected by formalin-ether concentration technique. Sporulation was performed by mixing stool samples in 2.5% potassium dichromate solution, sporulated oocysts were then treated with various solutions before mechanical rupturing in order to establish excystation, fewer than 10% of the sporulated oocysts could be excysted. Our techniques provided more details of the characteristic appearance of sporocysts and sporozoites within the oocysts (DMSO-modified acid-fast technique with our modification).

Emerging pathogen Cyclospora cayetanensis infection in Nepal.

Cylospora cayetanensis, an emerging parasitic pathogen of human is being increasingly recognized throughout the world, however the means of transmission and the possibility of a reservoir host remain an enigma. A longitudinal study on cyclosporiasis in different parts of Nepal was carried out from April, 1995 until November, 2000. Fecal specimens were collected from symptomatic and asymptomatic patients. The data shows a distinct seasonality with the highest infection rates occurring during the summer and rainy season of the year. Attempts have been made to determine the sources of infection and possible reservoir hosts. Stools were examined from nearly 700 animals such as chickens, pigs, buffalos, cows, dogs, cats, monkeys, rats, mice and pigeons. In addition, vegetable farms around the Kathmandu Valley were examined during the seasonal high and low periods of transmission. C. cayetanensis-like oocysts were found in sewage water and from vegetable washings on five occasions during June, July, August, October, and November. Similarly, C. cayetanensis-like oocysts were recovered from mice, rats, chickens, and dogs. These results suggest that these sources may be important in the transmission of this parasitosis. However, further studies will be required to obtain definitive answers on transmission.

Occurrence of Cryptosporidium, Giardia, and Cyclospora in influent and effluent water at wastewater treatment plants in Arizona.

We investigated the occurrence of Cryptosporidium, Giardia, and Cyclospora at two wastewater treatment plants (WWTPs) in Arizona over a 12-month period, from August 2011 to July 2012. Influent and effluent wastewater samples were collected monthly, and protozoan (oo)cysts were concentrated using an electronegative filter, followed by the detection of protozoa using fluorescent microscopy (Cryptosporidium oocysts and Giardia cysts) and PCR-based methods (Cryptosporidium spp., Giardia intestinalis, and Cyclospora cayetanensis). The concentration of Giardia cysts in the influent was always higher than that of Cryptosporidium oocysts (mean concentration of 4.8-6.4×10(3) versus 7.4×10(1)-1.0×10(2)(oo)cysts/l) with no clear seasonality, and log10 reduction of Giardia cysts was significantly higher than that of Cryptosporidium oocysts for both WWTPs (P<0.05). Log10 reduction of Giardia cysts at the WWTP utilizing activated sludge was significantly higher than the other WWTP using trickling filter (P=0.014), while no statistically significant difference between the two WWTPs was observed for the log10 reduction of Cryptosporidium oocysts (P=0.207). Phylogenetic analysis revealed that G. intestinalis strains identified in wastewater belonged to two assemblages, AII and B, which are potentially infectious to humans. C. cayetanensis was also detected from both influent and effluent using a newly developed quantitative PCR, with the highest influent concentration of 1.2×10(4)copies/l. Our results demonstrated that these protozoan pathogens are prevalent in the study area and that efficacy of the conventional wastewater treatment processes at physically removing (oo)cysts is limited.

[Cyclospora cayetanensis: biology, environmental distribution and transfer]. / Cyclospora cayetanensis: biología, distribución ambiental y transferencia.

Cyclospora cayetanensis is an apicomplexan protozoan that has emerged as an important pathogen causing endemic or epidemic diarrheal disease worldwide. In industrialized countries, the parasite has been recognized as the causative agent of several outbreaks of diarrheal illness mostly associated with produce imported from endemic areas. In developing countries, human cyclosporosis is widely distributed. Infection rates from 0% to 41.6% have been described in the general population. However, the epidemiology, biology, and ecology of C. cayetanensis are not fully understood. The life cycle is not completely characterized, although it appears to require a single human host to be accomplished. The role of animals as natural reservoirs of the parasite remains to be determined. Little information is available concerning the environmental distribution and vehicles of transmission of C. cayetanensis. Contaminated water, foods or soil can be vehicles of spread of the parasite. The significant uncertainties that remain in the knowledge of C. cayetanensis highlight the need for continuing research in several areas, including its basic biology and environmental distribution.