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BACKGROUND: Cyclospora cayetanensis is a protozoan parasite that causes intestinal illness in humans worldwide. Despite its global distribution, most genomic data for C. cayetanensis has been obtained from isolates collected in the United States, leaving genetic variability among globally distributed isolates underexplored. RESULTS: In the present study, the genome of an isolate of C. cayetanensis obtained from a child with diarrhea living in Mexico was sequenced and assembled. Evaluation of the assembly using a lineage typing system recently developed by the Centers for Disease Control and Prevention revealed that this isolate is lineage A. CONCLUSIONS: Given that the only other whole genome assembly available from Mexico was classified as lineage B, the data presented here represent an important step in expanding our knowledge of the diversity of C. cayetanensis isolates from Mexico at the genomic level.

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Cyclospora cayetanensis infection has emerged as a significant public health concern worldwide. Developed countries are generally considered non-endemic for infection. However, sporadic cases and non-travel-related outbreaks of C. cayetanensis infections associated with domestically grown produce are becoming more common in developed countries. Cyclospora cayetanensis has been detected in fresh produce, surface water, wastewater, irrigation water, and soil in these countries, suggesting that the parasite may be more common in areas with advanced sanitation than previously thought and illustrating the potential risk for exposure and indigenous/autochthonous infections. The evidence suggests the possibility of foci of endemicity in developed countries, particularly in communities where sanitary conditions are compromised, and raises transmission issues that require further research to better define the risks for infection, how widespread C. cayetanensis may be in these areas, and to guide interventions against this infection. The main purpose of the present opinion was to evaluate the presence of cyclosporiasis in developed countries, which is a very important and ongoing issue in food safety.

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BACKGROUND: Transmission dynamics of Cyclospora cayetanensis in endemic areas and the factors associated with soil contamination remain unclear. The effects of environmental factors on Cyclospora have been insufficiently studied, particularly in South America, thus a Venezuelan community was studied to profile risk factors for infection. METHODS: A cross-sectional stool survey of 732 individuals was conducted. For Cyclospora screening, an acid-fast-stained smear of formalin-ethyl acetate concentrate and ultraviolet (UV) epifluorescence examination of a wet mount were used. Water (n=14), soil (n=50) and produce (n=77) samples were collected, processed and examined by UV epifluorescence. Data were analysed using multivariate logistic regression. RESULTS: Cyclospora infections were identified in 73 (9.9%) subjects. Variables associated with the infection were age ≤10 y (odds ratio [OR] 14), hut living (OR 5), well water use (OR 18.5), drinking untreated water (OR 7.6), toilet absence (OR 8), having contact with faeces-contaminated soil (OR 4) and poultry exposure (OR 3). Infections (63%) were clustered in 25 huts. Oocysts were identified in 28.6%, 18% and 3.9% of the water, soil and produce samples, respectively. CONCLUSIONS: There was an explicit association of Cyclospora infection with extreme poverty and soil transmission reflecting the household socio-economic correlate of cyclosporiasis in this community.

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BACKGROUND: Cyclosporiasis has a marked seasonality. Few community-based studies have addressed this issue and there are no reports from Venezuela. A study was conducted to determine the seasonal variation of infection in a community from Falcon State, Venezuela. METHODS: A sample of 732 individuals was collected for 1 y. Stools were examined with modified Ziehl-Neelsen carbolfuchsin staining of ethyl acetate-formalin concentrates and ultraviolet epiflorescence of wet mounts. RESULTS: Cyclospora prevalence was 9.9% (73/732) with monthly variation from 0% to 35.3%. A trend of increased infections coinciding with the rainy time was observed (p<0.001). CONCLUSIONS: Cyclosporiasis is common in this area with high endemicity during the rainy periods.

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Fecal-orally transmitted cyclosporiasis is frequent in remote resource-limited settings in Central and South America with poor hygiene conditions. In this study, we aimed at assessing seasonal effects on the epidemiology of colonization or infection with C. cayetanensis in Colombian indigenous people living under very restricted conditions. In the rainy season between July and November and in the dry season between January and April, stool samples from indigenous people with and without gastrointestinal symptoms were collected and screened for C. cayetanensis applying in-house real-time polymerase chain reaction (PCR). In the rainy season and in the dry season, positive PCR results were observed for 11.8% (16/136) and 5.1% (15/292), respectively, with cycle threshold (Ct) values of 30.6 (±3.4) and 34.4 (±1.6), respectively. Despite higher parasite loads in the rainy season, fewer individuals (2/16, 12.5%) reported gastrointestinal symptoms compared to the dry season (6/15, 40%). In conclusion, considerable prevalence of C. cayetanensis in Colombian indigenous people persists in the dry season. Low proportions of gastrointestinal symptoms along with higher parasite loads make colonization likely rather than infection.

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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.

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Resumen Cyclospora cayetanensis es un coccidio intestinal relacionado con brotes epidémicos debido al consumo de alimentos y agua contaminados con ooquistes esporulados. Predomina en regiones tropicales y subtropicales y puede causar síntomas gastrointestinales que son más graves en inmunocomprometidos, en los que puede causar infecciones extraintestinales. El diagnóstico se realiza por la observación microscópica de ooquistes que presentan un tamaño entre 8 y 10 micrones, refringentes y con glóbulos internos. Se confirma con coloración ácido resistente; los ooquistes se tiñen de color fucsia y tienen la capacidad de autofluorescer. En este informe se describe el diagnóstico de Cyclospora cayetanensis en un paciente inmunocomprometido, oriundo de Perú, que reside actualmente en un barrio vulnerable de la Ciudad de Buenos Aires. Este informe representaría el sexto caso diagnosticado en la Argentina.

Abstract Cyclospora cayetanensis is an intestinal coccidium related to epidemic outbreaks due to consumption of food and water contaminated with sporulated oocysts. It predominates in tropical and subtropical regions and may cause gastrointestinal symptoms which are more severe in immunocompromised patients, to whom it may cause extraintestinal infections. Diagnosis is made by the microscopic observation of oocysts between 8 and 10 microns in size, with refractile globules inside of them. This diagnosis is confirmed by acid-fast staining where oocysts are observed red-stained. With ultraviolet epifluorescence, they stand out as bright blue or green staining circles. The aim of this report is to communicate the diagnosis of C. cayetanensis in an immunocompromised patient, born in Peru, but currently living in a vulnerable neighbourhood of Buenos Aires city. To our knowledge, this would be the sixth case of C. cayetanensis diagnosed in Argentina.

Resumo Cyclospora cayetanensis é um coccídio intestinal relacionado com surtos epidêmicos devido ao consumo de alimentos e água contaminados com oocistos esporulados. Predomina nas regiões tropicais e subtropicais e pode provocar sintomas gastrointestinais mais graves em pacientes imunodeprimidos, nos quais pode causar infecções extraintestinais. O diagnóstico é feito através da observação microscópica de oocistos com tamanho entre 8 e 10 mícrons, refringentes e com glóbulos internos. O diagnóstico é confirmado com coloração ácido-resistente; os oocistos são tingidos de cor fúcsia e eles têm a capacidade de auto-fluorescer. Este relatório descreve o diagnóstico de Cyclospora cayetanensis em um paciente imunodeprimido, nativo do Peru, atualmente residindo em um bairro vulnerável da cidade de Buenos Aires. Este relatório representaria o sexto caso diagnosticado na Argentina.

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Foodborne outbreaks caused by parasites have long been a public health issue. Among the available contamination detection methods, qPCR is one of the most sensitive and specific. However, it can be cumbersome and error-prone, if used by unexperienced users. Moreover, qPCR reagents usually require freezer temperatures for transportation and storage. We present a gelified reaction format that allows the reagents to be stored at 2-8 °C for up to 90 days without losing performance. The gelification process eliminates most operator mistakes during reaction setup, and renders the qPCR plates ready-to-use. The new reaction makeup was evaluated using artificially contaminated samples of distinct food matrices for sensitivity, specificity, repeatability, reproducibility, and stability. Samples consisted of cilantro leaves and raspberry fruits spiked with Cyclospora cayetanensis oocysts, as well as açai pulp and sugarcane juice tainted with Trypanosoma cruzi trypomastigotes. No significant difference between the gelified and the non-gelified qPCR was found. Our results suggest that gelifying the assay may help to achieve more reproducible qPCR data across laboratories, thus supporting surveillance actions. (170 words).

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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.

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This study shows a clinical case report of a kidney transplant patient who traveled from Mexico to The Netherlands and ate green vegetables in an international food restaurant. After 5 days, he started having diarrhea, nausea, colic, and a physical feeling of malaise. The patient only received symptomatic treatment after showing the characteristic symptoms of traveler's diarrhea. When he returned to Mexico, the clinical picture worsened, and he was hospitalized. Clinical analyses indicated dehydration and acute kidney injury stage II. Coproparasitoscopic study showed the presence of Cyclospora cayetanensis. Parenteral solutions, gastric mucosal protector, ciprofloxacin, and a soft diet were administrated as treatment. The patient was discharged 72 h later with an improvement of the kidney function.

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