Attempts to establish experimental Cyclospora cayetanensis infection in laboratory animals.

Attempts were made to develop an animal model for Cyclospora cayetanensis to identify a practical laboratory host for studying human cyclosporiasis. Oocysts collected from stool of infected humans in the United States, Haiti, Guatemala, Peru, and Nepal were held in potassium dichromate solution to allow development of sporozoites. The following animal types were inoculated: 9 strains of mice, including adult and neonatal immunocompetent and immune-deficient inbred and outbred strains, rats, sandrats, chickens, ducks, rabbits, jirds, hamsters, ferrets, pigs, dogs, owl monkeys, rhesus monkeys, and cynomolgus monkeys. Most animals were inoculated by gavage, although some of the primates were fed oocysts on food items. The animals were examined for signs of infection, particularly diarrhea, and stool samples were examined for 4-6 wk after inoculation. None of the animals developed patent infections or signs of infection. We conclude that none of the animals tested is susceptible to infection with C. cayetanensis.

Intervening transcribed spacer region 1 variability in Cyclospora cayetanensis.

Cyclospora cayetanensis is an apicomplexan protozoan parasite which has emerged as an important cause of epidemic and endemic diarrhea. Water-borne as well as food-borne outbreaks have occurred, including a large number of U.S. cases associated with raspberries imported from Guatemala. Molecular markers exist for tracing the epidemiology of many of the bacterial pathogens associated with water-borne or food-borne diarrhea, such as serotyping and pulsed-field electrophoresis. However, there are currently no molecular markers available for C. cayetanensis. The intervening transcribed spacer (ITS) regions between the small- and large-subunit rRNA genes demonstrate much greater sequence variability than the small-subunit rRNA sequence itself and have been useful for the molecular typing of other organisms. Thus, ITS1 variability might allow the identification of different genotypes of C. cayetanensis. In order to determine the degree of ITS1 variability among C. cayetanensis isolates, the ITS1 sequences of C. cayetanensis isolates from a variety of sources, including raspberry-associated cases, cases from Guatemala, and pooled and individual isolates from Peru, were obtained. The ITS1 sequences of all five raspberry-associated isolates were identical, consistent with their origin from a single source. In contrast, one of the two Guatemala isolates and two Peruvian isolates contained multiple ITS1 sequences. These multiple sequences could represent multiple clones from a single clinical source or, more likely, variability of the ITS1 region within the genome of a single clone.

Enteropathogens and other factors associated with severe disease in children with acute watery diarrhea in Lima, Peru.

To evaluate enteropathogens and other factors associated with severe disease in children with diarrhea, 381 children <5 years of age with diarrhea and moderate to severe dehydration (in-patients) and 381 age-, sex-, and date-of-visit-matched children with mild diarrhea (out-patients) presenting to a hospital in Peru, were studied. Rotavirus was detected in 52% of the in-patients and 35% of the out-patients (odds ratio [OR]=2.3, 95% confidence interval [95% CI]= 1.6-3.2); 95% of the rotaviruses among in-patients were of serotypes G1-G4. The risk of severe diarrhea was particularly great in children who were not exclusively breast-fed in early infancy and who also lacked piped water in their homes (for children with both characteristics OR=6.8, 95% CI=3.6-12.8). The high prevalence of rotavirus and its association with severe diarrhea underscores the need for rotavirus vaccines. Interventions to educate mothers and improve access to safe water should augment the impact of rotavirus vaccines in preventing severe diarrhea.

Cryptosporidium, enterocytozoon, and cyclospora infections in pediatric and adult patients with diarrhea in Tanzania.

Cryptosporidiosis, microsporidiosis, and cyclosporiasis were studied in four groups of Tanzanian inpatients: adults with AIDS-associated diarrhea, children with chronic diarrhea (of whom 23 of 59 were positive [+] for human immunodeficiency virus [HIV]), children with acute diarrhea (of whom 15 of 55 were HIV+), and HIV control children without diarrhea. Cryptosporidium was identified in specimens from 6/86 adults, 5/59 children with chronic diarrhea (3/5, HIV+), 7/55 children with acute diarrhea (0/7, HIV+), and 0/20 control children. Among children with acute diarrhea, 7/7 with cryptosporidiosis were malnourished, compared with 10/48 without cryptosporidiosis (P < .01). Enterocytozoon was identified in specimens from 3/86 adults, 2/59 children with chronic diarrhea (1 HIV+), 0/55 children with acute diarrhea, and 4/20 control children. All four controls were underweight (P < .01). Cyclospora was identified in specimens from one adult and one child with acute diarrhea (HIV-). Thus, Cryptosporidium was the most frequent and Cyclospora the least frequent pathogen identified. Cryptosporidium and Enterocytozoon were associated with malnutrition. Asymptomatic fecal shedding of Enterocytozoon in otherwise healthy, HIV children has not been described previously.

Cyclospora: an enigma worth unraveling.

In part, Cyclospora cayetanensis owes its recognition as an emerging pathogen to the increased use of staining methods for detecting enteric parasites such as Cryptosporidium. First reported in patients in New Guinea in 1977 but thought to be a coccidian parasite of the genus Isospora, 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.

Detection of Cyclospora cayetanensis in wastewater.

Cyclospora cayetanensis causes diarrheal disease worldwide without a confirmed mode of transmission. Wastewater was examined for the presence of this organism. Oocysts were detected microscopically, and their identity was confirmed by molecular techniques. These findings verify that current techniques can isolate Cyclospora oocysts and suggest that fecally contaminated water may act as a vehicle of transmission.

Cyclospora cayetanensis.

Cyclospora cayetanensis is a coccidian pathogen in humans. Cyclosporiasis is characterized by mild to severe nausea, anorexia, abdominal cramping, and watery diarrhea. Cyclospora has now been described from patients with protracted diarrheal illness in North, Central and South America, the Caribbean, Africa, Bangladesh, south-east Asia, Australia, England, and eastern Europe, and is characterized by marked seasonality. Routes of transmission are still unknown, although the fecal-oral route, either directly or via water, is probably the major one. A recent outbreak in the USA suggested transmission of Cyclospora by ingestion of contaminated berries. Cyclospora oocysts can be detected by phase contrast microscopy, modified acid-fast staining, autofluorescence, and amplification by the polymerase chain reaction. Oocysts are not sporulated when excreted in the feces, and sporulated oocysts are needed for infection. Each sporulated oocyst contains two sporocysts and each sporocyst contains two sporozoites. Humans seem to be the only host for this parasite. Histopathological examination of jejunal biopsies from infected individuals showed mild to moderate acute inflammation of the lamina propria and surface epithelial disarray. Parasitophorous vacuoles containing sexual and asexual forms of Cycl. cayetanensis were located in the cytoplasm of epithelial cells. Cyclospora infections can be treated successfully with trimethoprim-sulfamethoxazole.

Recovery of waterborne oocysts of Cyclospora cayetanensis by Asian freshwater clams (Corbicula fluminea).

Asian freshwater clams (Corbicula fluminea) were exposed for 24 hr in 38 liters of water contaminated with 1.0 x 10(5) Cyclospora cayetanensis oocysts (2.6 x 10(3) oocysts/L). The hemolyph and gill smears of 30 clams were examined by acid-fast stain on days 1, 3, 5, 7, 10, 13, and 18 postexposure (PE). Since no oocysts were detected in the water 24 hr after contamination by the membrane filter-dissolution method, the oocyst retention rate was 4.6 X 10(2) oocysts/clam. The prevalence of oocyst-positive clams significantly decreased (P < 0.01) from 93% to 47% during 13 days PE. None of the clams contained oocysts on day 18 PE; no oocysts were detected in the clam feces. The numbers of oocysts recovered from six clam size classes varied and significantly decreased with smaller clam size (P < 0.01). The lowest prevalence values of oocyst-positive clams, 45% and 34%, were observed in the two lowest size classes: 12.1-14.0 mm and 14.1-16.0 mm, respectively. The prevalence values in the remaining four classes ranged from 84% to 100%. The sampling program demonstrated that the population of 180 clams examined during the study up to 13 day PE could be assessed for C. cayetanensis positivity by random testing of a minimum of 75 clams (42%). When the two lowest clam size classes are eliminated, the population of 114 clams could be assessed by sampling a minimum of 32 clams (28%). The results demonstrate that Corbicula fluminea can recover waterborne oocysts of C. cayetanensis, and could be used as biological indicators of contamination of water with C. cayetanensis oocysts.

Pathologic and clinical findings in patients with cyclosporiasis and a description of intracellular parasite life-cycle stages.

Cyclospora cayetanensis has been observed in the feces of persons with prolonged diarrhea. A description of the symptoms and histopathologic findings for patients with cyclosporiasis is presented. The intracellular life-cycle stages of these parasites in the enterocytes of patients will also be described. Seventeen Peruvian patients positive for Cyclospora organisms were surveyed and underwent endoscopy, and their symptoms were recorded. Patients presented with gastrointestinal symptoms, including diarrhea, flatulence, weight loss, abdominal discomfort, and nausea. Jejunal biopsies showed an altered mucosal architecture with shortening and widening of the intestinal villi due to diffuse edema and infiltration by a mixed inflammatory cell infiltrate. There was reactive hyperemia with vascular dilatation and congestion of villous capillaries. Parasitophorous vacuoles contained sexual and asexual forms. Type I and II meronts, with 8-12 and 4 fully differentiated merozoites, respectively, were found at the luminal end of epithelial cells. These findings demonstrate the complete developmental cycle associated with host changes due to Cyclospora organisms.

Isolation of Cryptosporidium parvum and Cyclospora cayetanensis from vegetables collected in markets of an endemic region in Peru.

Cryptosporidium parvum and Cyclospora cayetanensis are protozoan pathogens that cause prolonged diarrhea in both immunocompetent and immunocompromised hosts. Cryptosporidium parvum can be transmitted via the fecal-oral route, while the exact mechanisms of transmission of Cyclospora cayetanensis have not been fully determined. Humans appear to be the sole host for the latter and a distinct seasonality has been observed in endemic areas around the world. Samples of vegetables were collected at several small markets in a periurban slum in Peru during the seasons of high and low incidence. The vegetables were washed, the supernatants were collected and centrifuged, and the pellets were resuspended in a solution of 2.5% potassium dichromate. Pellets were examined using direct microscopic observation, acid-fast staining, and immunofluorescent assays for C. parvum and Cyclospora cayetanensis oocysts. Samples were collected during three time periods: the season of low incidence, the beginning of the season of high incidence, and end of the season of high incidence. Of the total vegetables examined, 14.5% contained C. parvum oocysts and 1.8% had Cyclospora oocysts. Thus, market vegetables may provide a route by which Cryptosporidium and Cyclospora can be transmitted. Our study also suggests that washing vegetables does not completely remove Cryptosporidium and Cyclospora oocysts.

A new coccidian parasite (Apicomplexa: Eimeriidae) from humans.

A new coccidian parasite has been found in stool specimens of humans with and without diarrhea. The oocyst of this parasite measures 8.6 microns in diameter (7.7-9.9 microns), with ovoid sporocysts 4.0 x 6.3 (3.3-4.4 x 5.5-7.1) microns. Each oocyst has 2 sporocysts and each sporocyst contains 2 sporozoites. Based on these characteristics and the structures observed by electron microscopy, this parasite has been classified in the genus Cyclospora. We propose the name Cyclospora cayetanensis n. sp. for this new human parasite.

Cyclospora species--a new protozoan pathogen of humans.

BACKGROUND: Organisms referred to as "cyanobacterium-like bodies" have now been identified worldwide in the feces of both immunocompetent and immunocompromised patients with diarrhea. Organisms with a similar appearance have been isolated from Peruvian patients since 1985. From 1988 to 1991 we studied prospectively two cohorts of infants and young children infected with this organism. We now attempt to identify it. METHODS: Fecal samples were collected weekly from the children and examined with the use of acid-fast staining and staining with a monoclonal antibody specific for cryptosporidium. Stools positive for cyanobacterium-like bodies were preserved in potassium dichromate and exposed to conditions allowing coccidian sporulation and excystation. Both unsporulated and sporulated oocysts were fixed by freeze-substitution techniques and then examined by electron microscopy. RESULTS: Organisms isolated from the feces of Peruvian patients and two patients from the United States were identified as belonging to the coccidian genus cyclospora, after sporulation and excystation of the oocysts according to standard techniques. Complete sporulation occurred within 5 to 13 days in oocysts maintained in potassium dichromate at 25 or 32 degrees C. Complete excystation resulted in the liberation of two sporozoites from the two sporocysts within each oocyst (cryptosporidia have four naked sporozoites within each oocyst). The presence of organelles characteristic of coccidian organisms was confirmed by electron microscopy. CONCLUSIONS: We have identified organisms of the genus cyclospora that are remarkably similar to cryptosporidia in their morphologic features and the diarrheal disease that they produce in humans. The complete life cycle and epidemiology of this new protozoan parasite remain to be described.

Restriction fragment length polymorphism analysis of Cryptosporidium parvum isolates of bovine and human origin.

Cryptosporidium parvum oocysts isolated from different hosts and geographical areas were compared by restriction endonuclease analysis of repetitive DNA: Iowa (bovine), Florida (bovine), New York (bovine), Peru (human), Brazil (human), and Mexico (human). Southern blot hybridization analysis was performed using the restriction endonuclease enzyme Eco RI and the DNA probe pV47-2. The probe hybridized with 18 bands present in all the isolates. The Brazilian, Mexican, and Peruvian human isolates had an additional common band of 4.3 kbp that was absent in the bovine isolates. Two extra bands of 14 and 12 kbp were present in the Brazilian isolate whereas the Mexican isolate had an extra band of 14 kbp. When the Iowa and Peru C. parvum isolates were passed twice through calves, oocysts recovered from both passages showed identical banding patterns, suggesting that recombination of the repetitive sequences was not altered during sexual reproduction. The DNA digested with other restriction endonucleases were tested confirming differences between isolates. A genomic DNA library is currently being produced to better define isolate variation in C. parvum.