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PubMed1998

Identification of Cyclospora cayetanensis in stool using different stains.

Cyclospora cayetanensis, a newly emerging coccidian protozoa is world-wide in distribution. In the present study, different concentrations and staining techniques were used for identification of Cyclospora. Formol-ether sedimentation and Sheather's sugar flotation were used as concentration techniques and the different stains used were: the modified Ziehl-Neelsen, Giemsa, safranin-methylene blue, modifications of trichrome stain, calcoflour white and finally phenol-auramine. The safranin stain was the best, as it stained all the oocysts of Cyclospora uniformly, besides being rapid and easily applicable in the laboratories. Phenol-auramine stained the oocysts well, where both the wall and internal contents fluoresced brightly. With the calcoflour white stain, only the wall of oocysts took that fluorescent stain. The modified Ziehl-Neelsen stained some of the oocysts well, yet great variability in the staining pattern was noticed. Cyclospora oocysts were not efficiently stained with either trichrome modifications or Giemsa stains.

Animals↗
PubMed2001

Sequence variability in the first internal transcribed spacer region within and among Cyclospora species is consistent with polyparasitism.

Cyclospora cayetanensis is a coccidian parasite which causes severe gastroenteritis in humans. Molecular information on this newly emerging pathogen is scarce. Our objectives were to assess genetic variation within and between human-associated C. cayetanensis and baboon-associated Cyclospora papionis by examining the internal transcribed spacer (ITS) region of the ribosomal RNA operon, and to develop an efficient polymerase chain reaction- (PCR)-based method to distinguish C. cayetanensis from other closely related organisms. For these purposes, we studied C. cayetanensis ITS-1 nucleotide variability in 24 human faecal samples from five geographic locations and C. papionis ITS-1 variability in four baboon faecal samples from Tanzania. In addition, a continuous sequence encompassing ITS-1, 5.8S rDNA and ITS-2 was determined from two C. cayetanensis samples. The results indicate that C. cayetanensis and C. papionis have distinct ITS-1 sequences, but identical 5.8S rDNA sequences. ITS-1 is highly variable within and between samples, but variability does not correlate with geographic origin of the samples. Despite this variability, conserved species-specific ITS-1 sequences were identified and a single-round, C. cayetanensis-specific PCR-based assay with a sensitivity of one to ten oocysts was developed. This consistent and remarkable diversity among Cyclospora spp. ITS-1 sequences argues for polyparasitism and simultaneous transmission of multiple strains.

Animals↗
PubMed2002

Cyclospora spp.

PURPOSE OF REVIEW: Infection with Cyclospora cayetanensis continues to pose many questions, both in endemic populations and in travelers and food-borne outbreaks. The present review discusses existing knowledge but focuses more on what is yet to be learned about this infection. RECENT FINDINGS: Information on the parasite in endemic settings continues to be gathered, and similarities to and differences from other intestinal coccidia, especially Cryptosporidium spp., are becoming clearer. Food-borne outbreaks in North America continue despite efforts to identify and limit importation of particular items, such as berries, at certain times of the year. Study of Cyclospora spp. found in east African primates has shed some light on human infection but raises many new questions regarding the biology of the organism. SUMMARY: Despite new information being gathered regarding Cyclospora spp., including infection rates in various age and population groups, significant gaps remain in our knowledge of such basic issues as the factors that influence infectivity, seasonality, mode of food contamination, and geographic distribution. These gaps highlight the need for continued study on a variety of fronts, including surveillance, and clinical and basic biology.

Animals↗
PubMed2003

Targeting single-nucleotide polymorphisms in the 18S rRNA gene to differentiate Cyclospora species from Eimeria species by multiplex PCR.

Cyclospora cayetanensis is a coccidian parasite that causes protracted diarrheal illness in humans. C. cayetanensis is the only species of this genus thus far associated with human illness, although Cyclospora species from other primates have been named. The current method to detect the parasite uses a nested PCR assay to amplify a 294-bp region of the small subunit rRNA gene, followed by restriction fragment length polymorphism (RFLP) or DNA sequence analysis. Since the amplicons generated from C. cayetanensis and Eimeria species are the same size, the latter step is required to distinguish between these different species. The current PCR-RFLP protocol, however, cannot distinguish between C. cayetanensis and these new isolates. The differential identification of such pathogenic and nonpathogenic parasites is essential in assessing the risks to human health from microorganisms that may be potential contaminants in food and water sources. Therefore, to expand the utility of PCR to detect and identify these parasites in a multiplex assay, a series of genus- and species-specific forward primers were designed that are able to distinguish sites of limited sequence heterogeneity in the target gene. The most effective of these unique primers were those that identified single-nucleotide polymorphisms (SNPs) at the 3' end of the primer. Under more stringent annealing and elongation conditions, these SNP primers were able to differentiate between C. cayetanensis, nonhuman primate species of Cyclospora, and Eimeria species. As a diagnostic tool, the SNP PCR protocol described here presents a more rapid and sensitive alternative to the currently available PCR-RFLP detection method. In addition, the specificity of these diagnostic primers removes the uncertainty that can be associated with analyses of foods or environmental sources suspected of harboring potential human parasitic pathogens.

Animals↗
PubMedDate not supplied

Cyclospora cayetanensis in a patient with travelers' diarrhea: case report and review.

In recent years, several studies have shown that Cyclospora cayetanensis is a worldwide intestinal pathogen, and it has been implicated in a number of sporadic cases and epidemic outbreaks of diarrheal illness in several endemic areas. The parasite is associated with prolonged and relapsing watery diarrhea in immunocompetent persons, as well as in AIDS patients. Most reports of Cyclospora infection concern travelers visiting endemic countries. In European countries, a few cases of Cyclospora cayetanensis infection have been reported, and almost all of them were observed in persons after foreign travel.

Adult↗
PubMed2006

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.

Animals↗
PubMedDate not supplied

[Emergence of a new pathogen: Cyclospora cayetanensis in patients infected with human immunodeficiency virus].

The emergence of a new pathogen Cyclospora cayetanensis as a cause of clinical disease in immunosuppressed hosts is related with prolonged, severe and highly recurrent diarrheas. This paper reports two Cuban cases of cyclosporiasis associated with infection from human immunodeficiency virus in which non sporulated oocysts of Cyclospora cayetanensis were detected in feces by modified Zielhl Neelsen's technique. The most significant clinical symptoms were chronic diarrheas and loss of body weight, with CD4 levels below 200 cells per mm3. The occurrence of severe digestive symptoms in patients with Cyclospora cayetanensi and important immune compromise backed up the concept that this pathogen may act as a new opportunistic pathogen in patients with HIV.

AIDS-Related Opportunistic Infections↗
PubMed1999

Cyclospora cayetanensis--an emerging coccidian parasite.

Over the last decade increasing number of enteritis cases have been attributed to infection with a new coccidian sp that was named Cyclospora cayetanensis in 1993. Diarrhoea caused by this agent is clinically indistinguishable from cryptosporidiasis, isosporiasis and microsporidiasis but cyclospora infection are often very prolonged (upto 15 weeks) and may cause severe weight loss. Diagnosis is important because unlike diarrhoea caused by cryptosporidium and microsporidium, treatment with co-trimoxazole is effective. We report here a case of cyclosporiasis, to increase awareness of possibility of cyclospora infection in patients with prolonged diarrhoea. It should be considered in assessment of patients with unexplained prolonged diarrhoeal illness.

Animals↗
PubMedDate not supplied

Cyclospora cayetanensis associated with diarrhea in a patient with idiopathic compensated hepatic cirrhosis.

A 52 year-old male patient with idiopathic hepatic cirrhosis complaining of diarrhea and weakness was accepted to the gastroenterology clinic. In order to find out the causative etiologic agent of diarrhea, stool samples were examined by different methods and stained using modified Kinyoun's acid-fast stain. Following examination, approximately 9 microns diameter, acid-fast variable wrinkled spheres were seen and diagnosed as Cyclospora cayetanensis. Confirmation of the diagnosis was established by fluorescent microscope (380 to 420 nm excitation filter), which showed bright green to intense blue autofluorescent oocysts. It has been shown that, Cyclospora cayetanensis is a coccidian parasite mainly found in immunocompromised patients and that it may be the agent of prolonged diarrhea. Only three cyclosporiosis cases have been previously reported in our country; all three cases were AIDS patients. We report here a further case of Cyclospora cayetanensis infection in a patient with hepatic cirrhosis and we consider that this is the first case, which was reported in hepatic cirrhosis.

Cyclospora↗
PubMed2003

Prevalence and pathogenic role of Cyclospora cayetanensis in a Venezuelan community.

The prevalence and pathogenic role of Cyclospora cayetanensis among 212 subjects (age range = two months to 70 years) in an impoverished community in Venezuela were assessed retrospectively. For identification of the coccidium, modified Ziehl-Neelsen carbolfuchsin staining of formalin-ether concentrates was used. For other pathogenic parasites, iron-hematoxylin-stained smears and formalin-ether concentrates were examined. Cyclospora infections were identified in 13 (6.1%) subjects with a high percentage of asymptomatic carriers (11 of 13, 84.6%). Only two (15.4%) infants had diarrhea and the coccidium as the single detectable pathogenic parasite. The findings suggest that Cyclospora infections are relatively common and often asymptomatic in this region.

Adolescent↗
PubMed2004

Detection of Cyclospora cayetanensis in animal fecal isolates from Nepal using an FTA filter-base polymerase chain reaction method.

Cyclospora cayetanensis is an emerging protozoan parasite capable of causing a protracted diarrheal illness in both immunocompromised and immunocompetent individuals. Ingestion of fresh produce and water sources contaminated with mature sporulated oocysts results in acquisition of cyclosporiasis. Currently, no animal model exists for the study of this pathogenic parasite and the only confirmed reservoir host for C. cayetanensis in nature is humans. Previously, Cyclospora-like oocysts had been detected by microscopy in several animals including non-human primates. However, their phylogenetic relationship to C. cayetanensis remained uncertain due to the limited availability of molecular techniques to differentiate and speciate these isolates. In the present study, we examined a series of fecal isolates obtained from dogs, chickens, and monkeys collected between May and September 2002 from several geographic regions of Nepal. All samples were examined by microscopy and a polymerase chain reaction (PCR) for the presence of C. cayetanensis. Both microscopic and conventional PCR/restriction fragment length polymorphism (RFLP) analysis demonstrated the presence of Cyclospora sp. in the fecal samples of two dogs, one chicken, and one monkey. Application of a species-specific multiplex PCR assay confirmed the presence of both Eimeria sp. and C. cayetanensis in the positive chicken sample and only C. cayetanensis in the dog and monkey samples. However, in the absence of tissue analysis, the assignment of these animals as a natural reservoir host for C. cayetanensis remains to be determined.

Animals↗
PubMed2004

[Cyclospora cayetanensis in patients with AIDS and chronic diarrhea].

Cyclospora spp. is a protozoan parasite responsible for significant gastrointestinal disease in patients infected with the human immunodeficiency virus. We report the clinical features of two patients with chronic diarrhea and intestinal cyclosporosis caused by Cyclospora cayetanensis. The average value for CD4 count in these patients was lower than or equal to 100 cells/mm3. The oocysts were detected in smears from stool samples stained with modified acid-fast or safranin technique. Light microscopy revealed parasites in the enterocytes and these parasites were associated with villous atrophy. Cyclospora cayetanensis infection might be an important cause of diarrhea in patients with AIDS in Argentina.

Acquired Immunodeficiency Syndrome↗
PubMed2006

Co-infection with Cryptosporidium parvum and Cyclospora cayetanensis in immunocompromised patients.

Forty nine stool specimens collected from severe diarrheic patients. Eight were suffering from Hodgkin's lymphoma, and the rest were suffering from acute lymph plastic leukaemia. All were examined microscopically for protozoan parasites mainly, Cryptosporidium parvum and Cyclospora cayetanensis. Of the patients, 34 (69.4%) were positive and 15 (30.6%) were negative by both microscopy and nested PCR. An additional 12 (24.5%) who were negative by microscopy were positive by nested PCR. Stool examination revealed 16 cases with C. parvum, and 6 with C. cayetanensis, and 3 cases showed mixed infection. The results were compared with the established nested PCR assay to detect DNA directly from stool specimens. The patients <3 years old more affected by Cryptosporidium infection, unlike Cyclospora sp. Infection was in older age groups, which reflected the modes of parasite' transmission.. Diarrheal illness was stronger for Cyclospora than for Cryptosporidium. After the extraction of DNA from stool, a 402-bp fragment of C. parvum, and 602 bp fragment of C. cayetanensis was amplified. The amplified products, 194-bp DNA fragment for C. parvum, and 306 bp DNA fragment of C. cayetanensis were used for the second run. This study indicated that primers were specific for DNA of C. parvum and C. cayetanensis. PCR detected a total of 22 (44.9%) positives for C. parvum infection (6 negative by AF stool examination), and 12 (24.5%) positives for C. cavetanensis. Infection (6 negative by AF stool examination), 7 (14.3%) showed mixed infection (4 negative by AF stool examination), all microscopic negative specimens were positive by successive stool examination. Microscopy exhibited sensitivity of 72.7% for C. parvum, 50% for C. cayetanensis and 100% specificity for both parasites compared to 100% sensitivity and specificity with PCR. So, PCR is more sensitive and easier to interpret but required more hands-on time to perform and is more expensive. However, PCR batch analysis reduces the cost considerably.

Adolescent↗
PubMed1996

Identification of intracellular stages of Cyclospora species by light microscopy of thick sections using hematoxylin.

Cyclospora sp is a recently identified coccidia responsible for enteric infection in humans. Most reports have failed to detect this parasite in intestinal biopsies by light microscopy, although the different stages have been ultrastructurally described in jejunum enterocytes. Very recently, some investigators have reported the detection by light microscopy of parasitophorous vacuoles in intestinal biopsies; however, only transmission electron microscopy (TEM) could clearly identify the parasitic stages. To improve the histological diagnosis without using TEM, we have tested different staining methods in biopsies obtained from a patient infected by the human immunodeficiency virus who was shedding Cyclospora oocysts. Hematoxylin stain alone for 15 minutes on 5 micrometer-thick sections of duodenal biopsies was found to be the most efficient method for observing different stages of the parasite. In particular, the banana-shaped merozoites were visualized and appeared very similar to the human coccidia Isopora belli. This simple technique may be useful in diagnosing Cyclospora infection.

AIDS-Related Opportunistic Infections↗
PubMed1998

Cyclospora.

Although Cyclospora infection has been documented in humans worldwide since at least 1977, it is only in the past 2 years that this organism has come into prominence as a result of major foodborne outbreaks in the United States and Canada. Cyclospora causes significant gastrointestinal disease in immunocompetent and immunocompromised hosts and can be successfully treated with trimethoprim-sulfamethoxazole. The infection is under-recognized because our methods for diagnosis are rudimentary and insensitive. The mechanisms by which the parasite causes disease, the range of animal hosts, and the natural reservoir are unknown. Cyclospora is a unique coccidian parasite that has just begun to emerge; as yet, we have no clue as to where it comes from or where it hides.

Animals↗
PubMed1999

Molecular characterization of Cyclospora-like organisms from baboons.

Cyclospora organisms are intestinal pathogens of humans that are increasingly recognized in many parts of the world; yet, the reservoirs and host range remain poorly defined. Analysis of 18S ribosomal DNA (rDNA) suggests that the human-associated Cyclospora species (Cyc-hu) is most closely related to the Eimeria species, which are host species-specific. Recently, oocysts identical to those of Cyc-hu were detected in baboon fecal specimens from Tanzania. The 18S rDNA from 3 of these baboon-associated oocyst specimens was amplified and sequenced. Phylogenetic analysis indicated that these baboon-associated Cyclospora-like organisms (Cyc-bab) are nearly identical to each other and are distinct from Cyc-hu (1.6%-1.7% dissimilar); however, these Cyc-bab organisms are the closest known relatives of Cyc-hu. Together, these primate-associated cyclosporans constitute a coherent clade within the diverse group of Eimeria species. These findings raise important questions about the evolutionary relationships of the eimeriids and Cyc-hu host range and should lead to improved polymerase chain reaction-based diagnostics.

Animals↗
PubMed1994

Cyclospora: a newly identified intestinal pathogen of humans.

A number of reports over the last several years have linked a previously unidentified acid-fast organism with prolonged diarrhea in humans. Initially thought to be a cyanobacterium, the organism has been identified as a coccidian protozoan of the genus Cyclospora, and the name Cyclospora cayetanensis has been proposed. Organisms that resemble Cyclospora protozoa have been discovered in human stool samples around the world and have been isolated from children, immunocompetent adults, and human immunodeficiency virus (HIV)-seropositive individuals. The apparently waterborne organisms cause disease predominantly in summer months. In wet mounts of fresh stool specimens, the organisms are wrinkled spheres of 8-9 microns in diameter, with well-defined nonrefractile external walls and internal granular material, and resemble large oocysts of Cryptosporidium species. Organisms fluoresce under ultraviolet illumination. Formalin-preserved oocysts are variably acid-fast, and the results of staining with the modified carbolfuchsin technique (which is used to stain Cryptosporidium species) range from no staining to deep-red staining. The clinical syndrome is characterized by watery diarrhea (approximately 6 stools/day), nausea, anorexia, abdominal cramping, fatigue, and weight loss. Diarrhea appears to be self-limiting in the immunocompetent host but may be prolonged in patients with advanced HIV infection. Symptoms have abated in a handful of people treated with trimethoprim-sulfamethoxazole. Many questions remain to be answered about this newly identified pathogen.

Animals↗
PubMed1995

Cyclospora cayetanensis infection in patients with and without AIDS: biliary disease as another clinical manifestation.

We describe patients with and without AIDS who had Cyclospora cayetanensis infection; these patients were seen at a tertiary care teaching hospital in Mexico City because of diarrheal disease. C. cayetanensis was detected by examination of fresh fecal preparations and acid-fast staining of fecal smears; the presence of other bacteria and parasites was excluded by standard methods. Fecal specimens from 12 patients contained C. cayetanensis. The overall mean duration of illness was 94 days. Seven of the 12 patients had AIDS; these patients presented with more weight loss than did patients without AIDS (P = .04). The patients with AIDS also tended to have a more prolonged illness. Two patients with AIDS had biliary disease that resolved when they received therapy with trimethoprim-sulfamethoxazole for cyclospora infection; the excretion of oocysts also ceased. Our data confirm that C. cayetanensis causes diarrhea in humans and a significant weight loss in patients with AIDS. In addition, Cyclospora could be involved in biliary disease in patients with AIDS.

AIDS-Related Opportunistic Infections↗