Highly sensitive and specific PCR assay for reliable detection of Cyclospora cayetanensis oocysts

Multiple outbreaks of food-borne gastroenteritis caused by the coccidian parasite Cyclospora cayetanensis have been reported annually in North America since 1995. Detection of C. cayetanensis contamination typically relies on laborious and subjective microscopic examination of produce washes. Molecular detection methods based on nested PCR, restriction fragment length polymorphism, or multiplex PCR have been developed for C. cayetanensis; however, they have not been adequately validated for use on food products. Further challenges include reliably extracting DNA from coccidian oocysts since their tough outer wall is resistant to lysis and overcoming PCR inhibitors in sample matrices. We describe preliminary validation of a reliable DNA extraction method for C. cayetanensis oocysts and a sensitive and specific novel PCR assay. The sensitivity and repeatability of the developed methods were evaluated by multiple DNA extractions and PCR amplifications using 1,000-, 100-, 10-, or 1-ooycst aliquots of C. cayetanensis oocysts in water or basil wash sediment. Successful PCR amplification was achieved on 15 and 5 replicates extracted from aliquots containing 1,000 oocysts in water and basil wash, respectively. All 45 replicates of the 100-oocyst aliquots in water and 5 in basil wash were amplified successfully, as were 43/45 and 41/45 of the 10- and 1-oocyst aliquots in water and 9/15 and 2/15 in basil wash, respectively. The developed primers showed no cross-reactivity when tested against bacteria, nematodes, and protozoans, including Eimeria, Giardia, and Cryptosporidium. Our results indicate that these methods are specific, can reliably detect a single oocyst, and overcome many of the limitations of microscopic diagnosis.     

Concentrating Toxoplasma gondii and Cyclospora cayetanensis from surface water and drinking water by continuous separation channel centrifugation

Aims:  To evaluate the effectiveness of continuous separation channel centrifugation for concentrating Toxoplasma gondii and Cyclospora cayetanensis from drinking water and environmental waters.
Methods and Results:  Ready-to-seed vials with known quantities of T. gondii and C. cayetanensis oocysts were prepared by flow cytometry. Oocysts were seeded at densities ranging from 1 to 1000 oocysts l⁻¹ into 10 to 100 l test volumes of finished drinking water, water with manipulated turbidity, and the source waters from nine drinking water utilities. Oocysts were recovered using continuous separation channel centrifugation and counted on membrane filters using epifluorescent microscopy. Recovery efficiencies of both parasites were ≥84% in 10 l volumes of drinking water. In source waters, recoveries ranged from 64% to 100%, with the lowest recoveries in the most turbid waters. Method precision was between 10% and 20% coefficient of variation.
Conclusion:  Toxoplasma gondii and C. cayetanensis are effectively concentrated from various water matrices by continuous separation channel centrifugation.
Significance and Impact of the Study:  Waterborne transmission of T. gondii and C. cayetanensis presents another challenge in producing clean drinking water and protecting public health. Detection of these parasites relies on effectively concentrating oocysts from ambient water, otherwise false negatives may result. Validation data specific to T. gondii and C. cayetanensis concentration methods are limited. Continuous separation channel centrifugation recovers oocysts with high efficiency and precision, the method attributes required to accurately assess the risk of waterborne transmission.     

Comparison of three microscopic techniques for diagnosis of Cyclospora cayetanensis

Cyclospora cayetanensis oocysts in the feces of humans from Kathmandu, Nepal were identified on the basis of their size and other morphological characteristics. We compared the detection of C. cayetanensis oocysts in the feces using three microscopic techniques such as formalin-ether sedimentation, sucrose centrifugal floatation, and direct smear. Standard procedures were used for the formalin-ether sedimentation and the sucrose centrifugal floatation techniques using 0.5 g of feces, however, the direct smear technique was performed using 10 microl of fecal suspension (0.005 g of feces) and observed under the fluorescent microscope. Of the 403 samples examined, 21 samples were positive for oocysts by all three techniques. Therefore, in these 21 samples, the number of oocysts recovered by the three techniques were compared. The highest number of oocyst was obtained by the sucrose centrifugal floatation technique. In contrast, the formalin-ether sedimentation technique was found to be the least reliable concentration technique for the detection of Cyclospora in human feces. Surprisingly, the direct smear technique was found to be an effective and rapid technique for diagnosis of C. cayetanensis making it a technique of choice for routine epidemiological investigation of the prevalence of this infection in human populations.     

Intragenomic sequence variation of the ITS-1 region within a single flow-cytometry-counted Cyclospora cayetanensis oocysts

Cyclospora cayetanensis, a protozoan of emerging concern, causes self-limiting gastroenteritis in immune-competent hosts. It has been established that sequence variability exists in the first internal transcribed spacer region (ITS-1) of the ribosomal DNA operon from collections of oocysts obtained from individual or pooled fecal samples. To determine if single oocysts also exhibited ITS-1 sequence variability, DNA was extracted from individually flow-cytometry-counted oocysts. We determined that ITS-1 sequence variability exists at an individual-genome level for C. cayetanensis and approached or exceeded the variability exhibited among oocyst collections. ITS-1 variability, at the genome level, reduces this region's utility for inferring relationships between strains.     

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.     

Examination of attachment and survival of Toxoplasma gondii oocysts on raspberries and blueberries

The consumption of Toxoplasma gondii oocysts on fresh produce may be a means of its transmission to humans. Cats shed T. gondii oocysts, which contaminate produce directly or contaminate water sources for agricultural irrigation and pesticide and fertilizer applications. Cyclospora cayetanensis is a related coccidial parasite, and outbreaks of diarrhea caused by C. cayetanensis have been associated with the ingestion of contaminated raspberries. The oocysts of these coccidians are similar in size and shape, indicating that they may attach to and be retained on produce in a similar manner. In the present study the attachment and survival of T. gondii oocysts on 2 structurally different types of berries were examined. Raspberries and blueberries were inoculated individually with 1.0 x 10(1) to 2.0 x 10(4) oocysts of sporulated T. gondii. Berries inoculated with 2.0 x 10(4) oocysts were stored at 4 C for up to 8 wk. Oocyst viability and recovery were analyzed by feeding processed material to mice. Mice fed T. gondii-inoculated berries stored at 4 C for 8 wk developed acute infections. In other experiments mice fed raspberries inoculated with > or = 1.0 x 10(1) oocysts became infected, whereas only mice fed blueberries inoculated with > or = 1.0 x 10(3) oocysts became infected. This study demonstrates that T. gondii oocysts can adhere to berries and can be recovered by bioassays in mice and that raspberries retain more inoculated oocysts than do blueberries. The results suggest that T. gondii may serve as a model for C. cayetanensis in food safety studies.     

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.     

Detection and differentiation of coccidian oocysts by real-time PCR and melting curve analysis

Rapid and reliable detection and identification of coccidian oocysts are essential for animal health and foodborne disease outbreak investigations. Traditional microscopy and morphological techniques can identify large and unique oocysts, but they are often subjective and require parasitological expertise. The objective of this study was to develop a real-time quantitative PCR (qPCR) assay using melting curve analysis (MCA) to detect, differentiate, and identify DNA from coccidian species of animal health, zoonotic, and food safety concern. A universal coccidia primer cocktail was designed and employed to amplify DNA from Cryptosporidium parvum, Toxoplasma gondii, Cyclospora cayetanensis, and several species of Eimeria, Sarcocystis, and Isospora using qPCR with SYBR Green detection. MCA was performed following amplification, and melting temperatures (T(m)) were determined for each species based on multiple replicates. A standard curve was constructed from DNA of serial dilutions of T. gondii oocysts to estimate assay sensitivity. The qPCR assay consistently detected DNA from as few as 10 T. gondii oocysts. T(m) data analysis showed that C. cayetanensis, C. parvum, Cryptosporidium muris, T. gondii, Eimeria bovis, Eimeria acervulina, Isospora suis, and Sarcocystis cruzi could each be identified by unique melting curves and could be differentiated based on T(m). DNA of coccidian oocysts in fecal, food, or clinical diagnostic samples could be sensitively detected, reliably differentiated, and identified using qPCR with MCA. This assay may also be used to detect other life-cycle stages of coccidia in tissues, fluids, and other matrices. MCA studies on multiple isolates of each species will further validate the assay and support its application as a routine parasitology screening tool.     

Survey for Cyclospora cayetanensis in domestic animals in an endemic area in Haiti.

From January 1997 through July 1998, we examined stool samples from 327 domestic animals, including pigs, cattle, horses, goats, dogs, cats, guinea pigs, chicken, ducks, turkeys, and pigeons in Leogane, Haiti, for the presence of Cyclospora cayetanensis infection. No coccidian oocysts morphologically compatible with C. cayetanensis were detected in any of the animal samples, despite their living in, or near, households with infected individuals. These results suggest that domestic animals are not reservoir hosts for C. cayetanensis and that in this endemic area, humans are the only natural host for this parasite.     

Sensitivity of PCR detection of Cyclospora cayetanensis in raspberries,basil, and mesclun lettuce

The sensitivity of a polymerase chain reaction (PCR) method for detection of Cyclospora cayetanensis in raspberries, basil, and mesclun lettuce was evaluated. The assay could detect 40 or fewer oocysts per 100 g of raspberries or basil, but had a detection limit of around 1000 per 100 g in mesclun lettuce.     

Cryptosporidium parvum and Cyclospora cayetanensis: a review of laboratory methods for detection of these waterborne parasites

Cryptosporidium and Cyclospora are obligate, intracellular, coccidian protozoan parasites that infest the gastrointestinal tract of humans and animals causing severe diarrhea illness. In this paper, we present an overview of the conventional and more novel techniques that are currently available to detect Cryptosporidium and Cyclospora in water. Conventional techniques and new immunological and genetic/molecular methods make it possible to assess the occurrence, prevalence, virulence (to a lesser extent), viability, levels, and sources of waterborne protozoa. Concentration, purification, and detection are the three key steps in all methods that have been approved for routine monitoring of waterborne oocysts. These steps have been optimized to such an extent that low levels of naturally occurring Cryptosporidium oocysts can be efficiently recovered from water. The filtration systems developed in the US and Europe trap oocysts more effectively and are part of the standard methodologies for environmental monitoring of Cryptosporidium oocysts in source and treated water. Purification techniques such as immunomagnetic separation and flow cytometry with fluorescent activated cell sorting impart high capture efficiency and selective separation of oocysts from sample debris. Monoclonal antibodies with higher avidity and specificity to oocysts in water concentrates have significantly improved the detection and enumeration steps.To date, PCR-based detection methods allow us to differentiate the human pathogenic Cryptosporidium parasites from those that do not infect humans, and to track the source of oocyst contamination in the environment. Cell culture techniques are now used to examine oocyst viability. While fewer studies have focused on Cyclospora cayetanensis, the parasite has been successfully detected in drinking water and wastewater using current methods to recover Cryptosporidium oocysts. More research is needed for monitoring of Cyclospora in the environment. Meanwhile, molecular methods (e.g. molecular markers such as intervening transcribed spacer regions), which can identify different genotypes of C. cayetanensis, show good promise for detection of this emerging coccidian parasite in water.     

Effects of high pressure processing on Toxoplasma gondii oocysts on raspberries

Oocysts are the environmentally resistant life stage of Toxoplasma gondii. Humans can become infected by accidentally ingesting the oocysts in water or from contaminated produce. Severe disease can occur in immunocompromised individuals, and nonimmune pregnant women can infect their offspring. Chronic infection is associated with decreased mental functions, vision and hearing problems, and some mental disorders such as schizophrenia. High pressure processing (HPP) is a commercial method used to treat food to eliminate pathogens. Treatment of produce to eliminate viable T. gondii oocysts would provide a means to protect consumers. The present study was done to better define the effects of HPP on oocysts placed on raspberries. Raspberries were chosen because they are a known source of a related human intestinal parasite, Cyclospora cayetanensis. Raspberries were inoculated with 5 x 10(4) oocysts of the VEG strain of T. gondii for 20 hr prior to HPP. Individual raspberries were exposed to 500 MPa, 400 MPa, 340 MPa, 300 MPa, 270 MPa, 250 MPa, 200 MPA, 100 MPa, or no MPa treatment for 60 sec in a commercial HPP unit (1 MPa = 10 atm = 147 psi). Treatment of raspberries with 340 MPa for 60 sec was needed to render oocysts spot inoculated on the raspberries noninfectious for mice. Treatment of raspberries with 200 MPa or less for 60 sec was not effective in rendering oocysts noninfectious for mice.     

Ingestion of Cryptosporidium oocysts by Caenorhabditis elegans

Cryptosporidium parvum has been associated with outbreaks of human illness by consumption of contaminated water, fresh fruits, and vegetables. Free-living nematodes may play a role in pathogen transmission in the environment. Caenorhabditis elegans is a free-living soil nematode that has been extensively studied and serves as a good model to study possible transmission of C. parvum oocysts that may come into contact with produce before harvest. The objective of this study was to determine whether C. elegans could serve as a potential mechanical vector for transport of infectious C. parvum and Cyclospora cayetanensis in agricultural settings and whether C. elegans could ingest, excrete, and protect oocysts from desiccation. Seventy to 85% of worms ingested between 0 and 500 oocysts after 1 and 2 hr incubation with oocysts. Most of the nematodes ingested between 101 and 200 oocysts after 2 hr. Intact oocysts and empty shells were excreted by nematodes. Infectivity was determined by the neonatal assay with different treatments of worms (intact or homogenized) or oocysts or both. Adult C. elegans containing C. parvum kept in water were infective for mice. In conclusion, C. elegans adults can ingest and excrete C. parvum oocysts. Caenorhabditis elegans containing C. parvum oocysts can infect mice but does not seem to protect oocysts from extreme desiccation at 23 C incubation of a day or longer. Cyclospora oocysts were not ingested by C. elegans. The role of free-living nematodes in produce contamination needs to be further examined.     

Effects of temperature and different food matrices on Cyclospora cayetanensis oocyst sporulation

Effects of temperature on the sporulation of the parasite Cyclospora cayetanensis were studied in 2 food substrates, dairy and basil. Unsporulated Cyclospora oocysts were subjected to freezing and heating conditions for time periods ranging from 15 min to 1 wk. Oocysts were then removed from the food substrates and placed in 2.5% potassium dichromate for 2 wk to allow viable unsporulated oocysts to differentiate and fully sporulate, and to determine the percentage sporulation as an indicator of viability. Sporulation occurred when oocysts resuspended in dairy substrates were stored within 24 hr at -15 C. When oocysts were placed in water or basil, sporulation occurred after incubation for up to 2 days at -20 C, and up to 4 days at 37 C. Few oocysts sporulated when incubated for 1 hr at 50 C. Sporulation was not observed in basil leaves or water at -70 C, 70 C, and 100 C. Sporulation was not affected when incubated at 4 C and 23 C for up to 1 wk, which was the duration of the experiment in both of the tested substrates.     

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.     

Analysis of parasites by electrorotation

AIMS: The application of the AC electrokinetic technique of electrorotation for studying eukaryotic parasite transmission stages is reviewed. Electrorotation is a noninvasive technique that utilizes electrically energized microelectrode structures within micro-fluidic chambers to probe the physiological structure of micro-organisms. Application of the technique to the transmission life cycle stages of three separate genera of protozoan parasites, Cryptosporidium, Giardia and Cyclospora, and one nematode genus Ascaris, each of significant public health importance, is described. METHODS AND RESULTS: Standard electrorotation apparatus, consisting of micro-fabricated electrodes in a fluidic chip, quadrature sinusoidal signal generator, microscope and image capture system, was used to study each organism. Spectra of cellular rotation rate were recorded as a function of applied electric field frequency and compared with standardized biological tests, where appropriate, to illustrate the effectiveness and versatility of the electrorotation technique. CONCLUSIONS: Electrorotational determination of the viability of individual G. intestinalis cysts, Cryptosporidium parvum and Cyclospora cayetanensis oocysts has been achieved. The sporulation state of Cyclospora cayetanensis oocysts was also readily determined, as was the fertilization state of A. suum ova. SIGNIFICANCE AND IMPACT OF THE STUDY: Electrorotation is a simple, noninvasive and versatile analytical technique suited to a wide range of particle types and capable of incorporation into integrated Lab-on-a-chip devices.     

Viability of Giardia intestinalis cysts and viability and sporulation state of Cyclospora cayetanensis oocysts determined by electrorotation

Electrorotation is a noninvasive technique that is capable of detecting changes in the morphology and physicochemical properties of microorganisms. Electrorotation studies are reported for two intestinal parasites, Giardia intestinalis and Cyclospora cayetanensis. It is concluded that viable and nonviable G. intestinalis cysts can be differentiated by this technique, and support for this conclusion was obtained using a fluorogenic vital dye assay and morphological indicators. The viability of C. cayetanensis oocysts (for which no vital dye assay is currently available) can also be determined by electrorotation, as can their sporulation state. Modeling of the electrorotational response of these organisms was used to determine their dielectric properties and to gain an insight into the changes occurring within them. Electrorotation offers a new, simple, and rapid method for determining the viability of parasites in potable water and food products and as such has important healthcare implications.     

First Italian case of cyclosporiasis in an immunocompetent woman: local acquired infection

Cyclospora cayetanensis is a coccidian agent of chronic diarrhea in humans with a worldwide distribution. We report the first documented case of acquired Cyclosporiasis in Italy. The patient was an immunocompetent woman with no recent history of travel outside the country. Microscopy detected Cyclospora oocysts in a feces sample. PCR detected the pathogen in a second sample, which had tested negative by microscopy. The patient was investigated to detect other microorganisms in feces, such as Salmonella spp., Shigella spp, Campylobacter spp., Yersinia spp, and enteroviruses: all were negative. All symptoms disappeared 72 h after the beginning of therapy.     

PCR-restriction fragment length polymorphism method for detection of Cyclospora cayetanensis in environmental waters without microscopic confirmation

We developed an alternative nested-PCR-restriction fragment length polymorphism (RFLP) protocol for the detection of Cyclospora cayetanensis in environmental samples that obviates the need for microscopic examination. The RFLP method, with the restriction enzyme AluI, differentiates the amplified target sequence from C. cayetanensis from those that may cross-react. This new protocol was used to reexamine a subset (121 of 180) of surface water samples. Samples previously positive when the CYCF3E and CYCR4B primers (33) and RFLP with MnlI (20) were used were also PCR positive with the new primers; however, they were RFLP negative. We verified, by sequencing these amplicons, that while two were most likely other Cyclospora species, they were not C. cayetanensis. We can detect as few as one oocyst seeded into an autoclaved pellet flocculated from 10 liters of surface water. This new protocol should be of great use for environmental microbiologists and public health laboratories.