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Cyclospora cayetanensis is an emerging pathogen that is endemic in developing countries and responsible for many large foodborne cyclosporiasis outbreaks in North America since 1990s. Because of the lack of typing targets, the genetic diversity and population genetics of C. cayetanensis have not been investigated. In this study, we undertook a population genetic analysis of multilocus sequence typing data we recently collected from 64 C. cayetanensis specimens. Despite the extensive genetic heterogeneity in the overall C. cayetanensis population, there were significant intra- and inter-genic linkage disequilibria (LD). A disappearance of LD was observed when only multilocus genotypes were included in the population genetic analysis, indicative of an epidemic nature of C. cayetanensis. Geographical segregation-associated sub-structuring was observed between specimens from China and those from Peru and the United States. The two subpopulations had reduced LD, indicating the likely occurrence of genetic exchange among isolates in endemic areas. Further analyses of specimens from other geographical regions are necessary to fully understand the population genetics of C. cayetanensis.

Background Cyclospora cayetanensis is an important enteric pathogen, causing diarrhea and food-borne cyclosporiasis outbreaks. For effective outbreak identification and investigation, it is essential to rapidly assess the genetic heterogeneity of C. cayetanensis specimens from cluster cases and identify the likely occurrence of outbreaks. Methods In this study, we developed a quantitative PCR (qPCR) targeting the polymorphic link region between copies of the mitochondrial genome of C. cayetanensis, and evaluated the genetic heterogeneity among 36 specimens from six countries using melt curve, gel electrophoresis, and sequence analyses of the qPCR products. Results All specimens were amplified successfully in the qPCR and produced melt peaks with different Tm values in the melt curve analysis. In gel electrophoresis of the qPCR products, the specimens yielded bands of variable sizes. Nine genotypes were identified by DNA sequencing of the qPCR products. Geographical segregation of genotypes was observed among specimens analyzed, which could be useful in geographical source-tracking. Conclusions The length and nucleotide sequence variations in the mitochondrial genome marker allow rapid assessment of the genetic heterogeneity among C. cayetanensis specimens by melt curve, gel electrophoresis, or DNA sequence analysis of qPCR products. The sequence data generated could be helpful in the initial source-tracking of the pathogen.

Faecal samples were collected from cats kept as pets (n = 120) and stray cats (n = 135) in Central Europe (Czech Republic, Poland and Slovakia) and screened for the presence of Cryptosporidium spp., Giardia intestinalis (Kunstler, 1882), Encephalitozoon spp. and Enterocytozoon bieneusi Desportes, Le Charpentier, Galian, Bernard, Cochand-Priollet, Lavergne, Ravisse et Modigliani, 1985 by PCR analysis of the small-subunit of rRNA (Cryptosporidium spp. and G. intestinalis) and ITS (microsporidia) genes. Sequence analysis of targeted genes revealed the presence of C. felis Iseki, 1979, G. intestinalis assemblage F, E. cuniculi Levaditi, Nicolau et Schoen, 1923 genotype II, and E. bieneusi genotype D. There was no correlation between the occurrence of detected parasites and sex, presence of diarrhoea or drug treatment (drug containing pyrantel and praziquantel). Compared to pet cats (7%), stray cats (30%) were statistically more frequently infected with protist parasites and overall may present a greater risk to human health.

Cyclospora cayetanensis, a coccidian parasite, can cause gastrointestinal illness in humans and is characterized by watery and persistent diarrhea and abdominal pain. Cyclosporiasis has been associated with traveler's diarrhea. The infection is acquired through food and waterborne transmission, particularly by consumption of contaminated fresh fruits and vegetables. In the present study, stool samples from 8,877 children were examined for ova and parasites at the Pediatric Hospital of Morelia in Michoacán, Mexico, during 2000–2009. Sixty children (0.67%) had Cyclospora in their stools. Diarrhea (45.8%), abdominal pain (39.6%), and vomiting (18.8%) were the most frequent symptoms of cases with cyclosporiasis. Most of the cases (93.3%) were observed during June–August, the rainy season. In 45 children, Cyclospora was the only parasitic pathogen detected (75%); 15 children were co-infected with commensal, pathogenic, or both groups of parasites. Our findings suggest that C. cayetanensis is endemic to Michoacán and shows characteristically temporal patterns.

Population genetic studies have been used to understand the transmission of pathogens in humans and animals, especially the role of zoonotic infections and evolution and dispersal of virulent subtypes. In this study, we analysed the genetic diversity and population structure of Cryptosporidium meleagridis, the only known Cryptosporidium species that infects both avian and mammalian hosts and is responsible for approximately 10% of human cryptosporidiosis in some areas. A total of 62 C. meleagridis specimens from children, AIDS patients, and birds in Lima, Peru were characterised by sequence analysis of the ssrRNA gene and five minisatellite, microsatellite and polymorphic markers in chromosome 6, including the 60 kDa glycoprotein (gp60), 47 kDa glycoprotein (CP47), a serine repeat antigen (MSC6-5), retinitis pigmentosa GTPase regulator (RPGR) and thrombospondin protein 8 (TSP8). The multilocus sequence analysis identified concurrent infections with Cryptosporidium hominis in four AIDS patients and three children. Unique subtypes of C. meleagridis ranged from eight at the gp60 locus (gene diversity -Hd=0.651), three at the RPGR (Hd=0.556), three at the MSC6-5 locus (Hd=0.242), two at TSP8 (Hd=0.198), to one at CP47 (monomorphic), much lower than that of C. hominis in the same area. Intragenic linkage disequilibrium was strong and complete at all gene loci. Intergenic linkage disequilibrium was highly significant (P

Globalisation is a manmade phenomenon encompassing the spread and movement of everything, animate and inanimate, material and intangible, around the planet. The intentions of globalisation may be worthy – but may also have unintended consequences. Pathogens may also be spread, enabling their establishment in new niches and exposing new human and animal populations to infection. The plethora of foodborne parasites that could be distributed by globalisation has only recently been acknowledged and will provide challenges for clinicians, veterinarians, diagnosticians, and everyone concerned with food safety. Globalisation may also provide the resources to overcome some of these challenges. It will facilitate sharing of methods and approaches, and establishment of systems and databases that enable control of parasites entering the global food chain.

Background The transitional period between the oncosphere and the cysticercus of Taenia solium is the postoncospheral (PO) form, which has not yet been completely characterized. The aim of this work was to standardize a method to obtain T. solium PO forms by in vitro cultivation. We studied the morphology of the PO form and compared the expression of antigenic proteins among the PO form, oncosphere, and cysticerci stages. Methodology/Principal Findings T. solium activated oncospheres were co-cultured with ten cell lines to obtain PO forms, which we studied at three stages of development–days 15, 30, and 60. A high percentage (32%) of PO forms was obtained using HCT-8 cells in comparison to the other cell lines. The morphology was observed by bright field, scanning, and transmission electron microscopy. Morphology of the PO form changed over time, with the six hooks commonly seen in the oncosphere stage disappearing in the PO forms, and vesicles and microtriches observed in the tegument. The PO forms grew as they aged, reaching a diameter of 2.5 mm at 60 days of culture. 15–30 day PO forms developed into mature cysticerci when inoculated into rats. Antigenic proteins expressed in the PO forms are also expressed by the oncosphere and cysticerci stages, with more cysticerci antigenic proteins expressed as the PO forms ages. Conclusions/Significance This is the first report of an in vitro production method of T. solium PO forms. The changes observed in protein expression may be useful in identifying new targets for vaccine development. In vitro culture of PO form will aid in understanding the host-parasite relationship, since the structural changes of the developing PO forms may reflect the parasite’s immunoprotective mechanisms. A wider application of this method could significantly reduce the use of animals, and thus the costs and time required for further experimental investigations., Author Summary Neurocysticercosis is caused by T. solium, which is a neglected disease. The postoncospheral (PO) form is an intermediate form between the oncosphere, which is the larva, and the fully developed cysticercus, which is a cyst with a scolex. The morphology, development, and protein and antigen expression of the PO form have not previously been characterized. Here, we report the novel in vitro cultivation of T. solium PO forms and characterize the morphology, development, and expression of antigenic proteins. This new method will allow for better study of this transitional form, which is very difficult to study in the intermediate host. With the increased availability of secreted proteins and antigens, in vitro cultivation will help improve diagnostic assays and provide new targets for vaccine development to block transmission.

The microbial diversity of wastewater used for irrigation and fertilization was assessed using specific polymerase chain reaction (PCR) assays to detect and genotype several pathogenic protists including Cryptosporidium spp., Giardia duodenalis, Cyclospora spp., Eimeria spp. and Enterocytozoon bieneusi. A total of 220 wastewater samples (110 raw, 110 treated) and 12 sludge samples were collected from 2005 to 2008 from 18 treatment plants located throughout Tunisia. Except for Cyclospora, which was detected only once, E. bieneusi (61%), G. duodenalis (28%), Cryptosporidium spp. (27%) and Eimeria spp. (45%) were frequently observed in wastewater and sludge. Sequencing of PCR products showed that C. hominis, C. andersoni, G. duodenalis sub-assemblage A-II and E. bieneusi genotypes D and IV were the most prevalent. An analysis of the distribution of 209 internal transcribed spacer sequences of E. bieneusi originating from wastewater at the 18 treatment plants showed a similar genetic diversity, regardless of the geographical location. The identification of these parasite species and genotypes and of host-specific Eimeria species indicates that the microbial quality of wastewater was impacted by humans, livestock and rodents. Given the public health risks that some of these parasites represent, guidelines on wastewater usage are needed to minimize human exposure to these pathogens.

One-sentence summary for table of contents: Different genotypes and subtypes are linked to different clinical manifestations., To determine whether clinical manifestations are associated with genotypes or subtypes of Cryptosporidium spp., we studied a 4-year longitudinal birth cohort of 533 children in Peru. A total of 156 infection episodes were found in 109 children. Data from first infections showed that C. hominis was associated with diarrhea, nausea, vomiting, general malaise, and increased oocyst shedding intensity and duration. In contrast, C. parvum, C. meleagridis, C. canis, and C. felis were associated with diarrhea only. C. hominis subtype families were identified (Ia, Ib, Id, and Ie); all were associated with diarrhea. Ib was also associated with nausea, vomiting, and general malaise. All C. parvum specimens belonged to subtype family IIc. Analysis of risk factors did not show associations with specific Cryptosporidium spp. genotypes or subtypes. These findings strongly suggest that Cryptosporidium spp. and subtypes are linked to different clinical manifestations in children.

Protozoal diseases are increasingly recognized as the cause of diarrhoeal outbreaks in both developed and developing countries. Cyclospora cayetanensis has been responsible for several epidemics in the last decade. In March 2005, an outbreak of diarrhoea was identified in recruits at the Ancon Naval Base in Lima, Peru. A case-control study was carried out. The overall diarrhoea attack rate was 53% (45/85). Complete data from 52 recruits were available for the analysis; 37 met the criteria for case and 15 for control. The epidemic curve indicated a point source transmission, with cases occurring over 9 days with a peak on the fifth day. Cyclospora cayetanensis was found in 7/37(18.9%) cases and 1/15 (6.7%) controls via standard microscopic techniques. PCR for C. cayetanensis detected 20/35 (57.1%) cases and 3/15 (20%) controls, demonstrating the improved diagnostic yield of this technique. This is the second report to characterize an outbreak of diarrhoea due to C. cayetanensis in Peru among a local population. The epidemiology and clinical course were similar to other reported outbreaks in developed regions. PCR greatly increased the number of C. cayetanensis cases detected during this outbreak, allowing the correct identification of its aetiology.

We performed a cross-sectional study to determine the epidemiology of Cryptosporidium in human immunodeficiency virus (HIV)-infected persons at 3 diagnostic levels: microscopy, genotypes of Cryptosporidium, and subtype families of C. hominis and C. parvum. The study enrolled 2,490 HIV-infected persons in Lima, Peru, and 230 were microscopy positive for Cryptosporidium infection. Specimens from 193 participants were available for genotyping. They had C. hominis (141 persons), C. parvum (22 persons), C. meleagridis (17 persons), C. canis (6 persons), C. felis (6 persons), and C. suis (1 person) infection. Although microscopy results showed that Cryptosporidium infections were associated with diarrhea, only infections with C. canis, C. felis, and subtype family Id of C. hominis were associated with diarrhea, and infection with C. parvum was associated with chronic diarrhea and vomiting. These results demonstrate that different Cryptosporidium genotypes and subtype families are linked to different clinical manifestations.

Mixed Cryptosporidium infections were detected in 7 of 21 patients with a diagnosis of rare Cryptosporidium canis or C. felis infections; 6 patients were infected with 2 Cryptosporidium spp. and 1 patient with 3 species. Mixed infections may occur more frequently than previously believed and should be considered when assessing cryptosporidiosis.

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.

Poor hygiene and improper food preparation practices in consumers' homes have previously been demonstrated as contributing to foodborne diseases. To address potential cross-contamination by kitchen utensils in the home, a series of studies was conducted to determine the extent to which the use of a knife or grater on fresh produce would lead to the utensil's contamination with Escherichia coli O157:H7 or Salmonella enterica. When shredding inoculated carrots (ca. 5.3 log CFU/carrot), all graters became contaminated and the number of E. coli O157:H7 present on the utensil was significantly greater than Salmonella (p 0.05). Contamination of knives after slicing inoculated produce (4.9-5.4 log CFU/produce item) could only be detected by enrichment culture. After slicing tomatoes, honeydew melons, strawberries, cucumbers, and cantaloupes, the average prevalence of knife contamination by the two pathogens was 43%, 17%, 15%, 7%, and 3%, respectively. No significant increase in the incidence or level of contamination occurred on the utensils when residues were present (p 0.05); however, subsequent contamination of 7 produce items processed with the contaminated utensils did occur. These results highlight the necessity of proper sanitization of these utensils when used in preparation of raw produce.

We compared the epidemiologic characteristics of cyclosporiasis and cryptosporidiosis in data from a cohort study of diarrhea in a periurban community near Lima, Peru. Children had an average of 0.20 episodes of cyclosporiasis/year and 0.22 episodes of cryptosporidiosis/year of follow-up. The incidence of cryptosporidiosis peaked at 0.42 for 1-year-old children and declined to 0.06 episodes/child-year for 5- to 9-year-old children. In contrast, the incidence of cyclosporiasis was fairly constant among 1- to 9-year-old children (0.21 to 0.28 episodes/child-year). Likelihood of diarrhea decreased significantly with each episode of cyclosporiasis; for cryptosporidiosis, this trend was not statistically significant. Both infections were more frequent during the warm season (December to May) than the cooler season (June to November). Cryptosporidiosis was more frequent in children from houses without a latrine or toilet. Cyclosporiasis was associated with ownership of domestic animals, especially birds, guinea pigs, and rabbits.

During the past century, the microbiological safety of the US food supply has improved; however, many foodborne illnesses and outbreaks occur annually. Hence, opportunities for the food industry to improve the safety of both domestic and imported food exist through the adoption of risk-based preventive measures. Challenging food safety issues that are on the horizon include demographic changes to a population whose immune system is more susceptible to foodborne and opportunistic pathogens, climate changes that will shift where food is produced, and consumers' preferences for raw and minimally processed foods. Increased environmental and product testing and anonymous data sharing by the food industry with the public health community would aid in identifying system weaknesses and enabling more targeted corrective and preventive actions. Clinicians will continue to play a major role in reducing foodborne illnesses by diagnosing and reporting cases and in helping to educate the consumer about food safety practices.

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.

Cyclospora cayetanensis infection continues to be a major cause of diarrhea particularly in immunosuppressed patients. The emergence of new related parasite pathogens, is an issue of public health concern. We report here a case where a Cyclosporalike organism was the cause of diarrhea in an immunocompetent patient from Venezuela.

An unusual Enterocytozoon bieneusi genotype was found in seven guinea pigs and a 2-year-old child in the same household. The genetic uniqueness of the parasite, its wide occurrence in other guinea pigs in the community, and its absence in other children in the community suggest the possibility of zoonotic transmission of the infection to the study child.

In a longitudinal cohort diarrhea study, a girl living in Lima, Peru, and her brother and dog were diagnosed with Cryptosporidium canis infections during the same period. Both children had transient diarrhea, but the dog was asymptomatic. This is the first report of possible transmission of cryptosporidiosis between humans and dogs.

Little is known about the emergence and spread of virulent subtypes of Cryptosporidium hominis, the predominant species responsible for human cryptosporidiosis. We conducted sequence analyses of 32 genetic loci of 53 C. hominis specimens isolated from a longitudinally followed cohort of children living in a small community. We identified by linkage disequilibrium and recombination analyses only limited genetic recombination, which occurred exclusively within the 60-kDa glycoprotein gene subtype IbA10G2, a predominant subtype for outbreaks in industrialized nations and a virulent subtype in the study community. Intensive transmission of virulent subtype IbA10G2 in the study area might have resulted in genetic recombination with other subtypes. Moreover, we identified selection for IbA10G2 at a 129-kb region around the 60-kDa glycoprotein gene in chromosome 6. These findings improve our understanding of the origin and evolution of C. hominis subtypes and the spread of virulent subtypes.

Cyclospora cayetanensis is an apicocomplexan parasite that infects the gastrointestinal tract and causes acute diarrheal disease in humans. In recent years, this human-pathogenic parasite has led to several foodborne outbreaks in the United States and Canada, mostly associated with imported produce. Understanding the biology and epidemiology of C. cayetanensis is difficult because little is known about its origin, possible zoonotic reservoirs, and genetic relationships with other coccidian parasites. Recently, we developed a 70kDa heat shock protein (HSP70) gene based nested PCR protocol for detection of C. cayetanensis parasite and sequenced the PCR products of 16 human isolates from Nepal, Mexico, and Peru. In this study, we have characterized the regions of 18S ribosomal RNA (rRNA) gene of 17 human C. cayetanensis isolates for molecular detection, and also to ascertain the genetic diversity of this parasite. The 18S rRNA primer sets were further tested by PCR amplification followed by nucleotide sequencing of the PCR amplified products of previously characterized C. cayetanensis isolates from three endemic regions at HSP70 locus. Although no genetic polymorphism was observed at the regions of HSP70 locus characterized in our previous study, the data analysis of this study revealed a minor genetic diversity at the 18S rRNA locus among the C. cayetanensis isolates. The 18S rRNA gene-based nested PCR protocol provides a useful genetic marker for the detection of C. cayetanensis parasite and confirms it as a genetically distinct species in genus Cyclospora. The results also supported lack of geographic segregation and existence of genetically homogeneous population for the C. cayetanensis parasites both at the HSP70 as well as at the18S rRNA loci.

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.

Human noroviruses and hepatitis A virus are responsible for numerous outbreaks associated with handling fresh produce. In this study, physical removal of hepatitis A virus and murine norovirus, a human norovirus surrogate, from contaminated produce items (honeydew melons, cantaloupes, carrots, and celery) by scrubbing under running water with a nylon brush or scouring pad and by peeling (carrots and celery) with a peeler was investigated. The degree and extent of utensil contamination with viruses during these operations in the presence and absence of food residue also was investigated. Scrubbing or peeling produce initially inoculated with ∼5.5 log PFU of each virus resulted in significant levels of virus removal, ranging from 0.93 to 2.85 log PFU. However, utensil cross-contamination occurred, with2 log PFU of virus transferred from a single produce item. After preparation of a contaminated produce item, utensil cross-contamination resulted in virus detection on seven successively prepared produce items. Produce residue accumulation on utensils variably impacted virus transfer to utensil surfaces. Results indicate that scrubbing and peeling produce can reduce levels of viruses on contaminated produce, but the importance of utensil sanitation to prevent cross-contamination is highlighted. Findings also provide important information for modeling virus cross-contamination during food preparation.

We have described the development of a 2-step nested PCR protocol based on the characterization of the 70-kDa heat shock protein (HSP70) gene for rapid detection of the human-pathogenic Cyclospora cayetanensis parasite. We tested and validated these newly designed primer sets by PCR amplification followed by nucleotide sequencing of PCR-amplified HSP70 fragments belonging to 16 human C. cayetanensis isolates from 3 different endemic regions that include Nepal, Mexico, and Peru. No genetic polymorphism was observed among the isolates at the characterized regions of the HSP70 locus. This newly developed HSP70 gene-based nested PCR protocol provides another useful genetic marker for the rapid detection of C. cayetanensis in the future.

Foodborne parasites are characterized as being highly resistant to sanitizers used by the food industry. In 2009, a study reported the effectiveness of levulinic acid in combination with sodium dodecyl sulfate (SDS) in killing foodborne bacteria. Because of their innocuous properties, we studied the effects of levulinic acid and SDS at various concentrations appropriate for use in foods, on the viability of Cryptosporidium parvum and Encephalitozoon intestinalis. The viability of Cryptosporidium and E. intestinalis was determined by in vitro cultivation using the HCT-8 and RK-13 cell lines, respectively. Two Escherichia coli O157:H7 isolates were also used in the present study: strain 932 (a human isolate from a 1992 Oregon meat outbreak) and strain E 0018 (isolated from calf feces). Different concentrations and combinations of levulinic acid and SDS were tested for their ability to reduce infectivity of C. parvum oocysts (10(5)), E. intestinalis spores (10(6)), and E. coli O157:H7 (10(7)/ml) when in suspension. Microsporidian spores were treated for 30 and 60 min at 20 ± 2°C. None of the combinations of levulinic acid and SDS were effective at inactivating the spores or oocysts. When Cryptosporidium oocysts were treated with higher concentrations (3% levulinic acid-2% SDS and 2% levulinic acid-1% SDS) for 30, 60, and 120 min, viability was unaffected. E. coli O157:H7, used as a control, was highly sensitive to the various concentrations and exposure times tested. SDS and levulinic acid alone had very limited effect on E. coli O157:H7 viability, but in combination they were highly effective at 30 and 60 min of incubation. In conclusion, Cryptosporidium and microsporidia are not inactivated when treated for various periods of time with 2% levulinic acid-1% SDS or 3% levulinic acid-2% SDS at 20°C, suggesting that this novel sanitizer cannot be used to eliminate parasitic contaminants in foods.

The association of wealth and infections with Giardia, Cryptosporidium, Cyclospora, and microsporidia were examined in a longitudinal cohort conducted in Peru from 2001 to 2006. Data from 492 participants were daily clinical manifestations, weekly copro-parasitological diagnosis, and housing characteristics and assets owned (48 variables), and these data were used to construct a global wealth index using principal component analysis. Data were analyzed using continuous and categorical (wealth tertiles) models. Participant's mean age was 3.43 years (range = 0–12 years), with average follow-up of 993 days. Univariate and multivariate analyses identified significant associations between wealth and infections with Giardia and microsporidia. Participants with greater wealth indexes were associated with protection against Giardia (P < 0.001) and persistent Giardia infections (> 14 days). For microsporidia, greater wealth was protective (P = 0.066 continuous and P = 0.042 by tertiles). Contrarily, infections with Cryptosporidium and Cyclospora were independent of wealth. Thus, subtle differences in wealth may affect the frequency of specific parasitic infections within low-income communities.

We were delighted to read that Drs Galan-Puchades and Fuentes took the opportunity of our recent globalisation and foodborne parasites article [1] to draw attention to the importance, and probable spread, of the Asian tapeworm, Taenia asiatica [2].

Background. Giardia lamblia is ubiquitous in multiple communities of nonindustrialized nations. Genotypes A1, A2, and B (Nash groups 1, 2, and 3, respectively) are found in humans, whereas genotypes C and D are typically found in dogs. However, genotypes A and B have occasionally been identified in dogs. Methods. Fecal Giardia isolates from 22 families and their dogs, living in Pampas de San Juan, were collected over 7 weeks in 2002 and 6 weeks in 2003. Samples were genotyped, followed by sequencing and haplotyping of many of these isolates by using loci on chromosomes 3 and 5. Results. Human infections were all caused by isolates of genotypes A2 and B. Human coinfections with genotypes A2 and B were common, and the reassortment pattern of different subtypes of A2 isolates supports prior observations that suggested recombination among genotype A2 isolates. All dogs had genotypes C and/or D, with one exception of a dog with a mixed B/D genotype infection. Conclusions. In a region of high endemicity where infected dogs and humans constantly commingle, different genotypes of Giardia are almost always found in dogs and humans, suggesting that zoonotic transmission is very uncommon.

Objective. A higher incidence of infectious disease has been documented in U.S. regions bordering Mexico compared with non-border areas. We assessed the prevalence of important gastrointestinal infections in Ciudad Juarez, Mexico, and El Paso, Texas, the largest binational community along the U.S.-Mexico border. Methods. Fecal specimens from a sample of the asymptomatic population representing all ages were tested for Helicobacter pylori (H. pylori), Cryptosporidium spp., Giardia spp., and other intestinal parasitic pathogens using flotation, immunoassays, and/or polymerase chain reaction. We also measured indicators of microbiological contamination of drinking water, hands of food preparers, and kitchen surfaces. Results. Overall, of the 386 participants, H. pylori was present in 38.2%, Taenia spp. in 3.3%, Giardia spp. in 2.7%, Cryptosporidium spp. in 1.9%, Entamoeba dispar in 1.3%, and Ascaris lumbricoides and Necator americanus in 0.3% of the study subjects; Cyclospora spp. and Entamoeba histolytica were not found. H. pylori infection was associated with handwashing (prevalence ratio [PR] = 1.3, 95% confidence interval [CI] 1.0, 1.8). Taenia spp. was found more often on the U.S. side (PR=8.6, 95% CI 2.3, 30.8). We did not find an association between these infections and the occurrence of total coliforms or fecal coliforms on kitchen surfaces. In addition, Escherichia coli was not found in any drinking water sample. Conclusion. The study results indicated that H. pylori and Taenia spp. infections may be highly prevalent along the U.S.-Mexico border. Additional research is necessary to adequately characterize the prevalence, as well as determine whether interventions that reduce these infections are warranted.

SUMMARY The coccidian parasite Cyclospora cayetanensis is recognized as an emerging pathogen that causes protracted diarrhea in humans. The first cases of Cyclospora infection were reported in the late 1970s and were observed among expatriates and travelers in regions where infections are endemic. Since then, Cyclospora has been considered a cause of traveler's diarrhea. Epidemiological investigations were reported and examined in areas of endemicity even before the true identity of Cyclospora was elucidated. Cyclospora was fully characterized in the early 1990s, but it was not until the 1995 Cyclospora outbreak in the United States and Canada that it caught the attention of the public and physicians. The biology, clinical presentation, epidemiology, diagnosis, treatment, and control of cyclosporiasis are reviewed, with a focus on diagnostic assays currently being used for clinical and environmental samples. Challenges and limitations in working with Cyclospora are also discussed.

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.

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.

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.

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.

Twenty-two coprolites of human origin, collected from excavations along the north-central coast of Peru, were examined using fluorescent microscopy for the presence of fecal parasites, with emphasis on Cryptosporidium sp., Giardia sp., and Cyclospora sp. Three samples were positive. One coprolite dated between ca. 2,375 and 1,525 BC contained Giardia sp. cysts. This coprolite corresponded to the Peruvian preceramic period. Another positive coprolite ca. AD 770–830 corresponded to Epoch 3 of the Middle Horizon and contained Cryptosporidium sp. oocysts. The third positive coprolite (corresponding to the Middle Horizon, ca. AD 500–900) contained Giardia sp. cysts. This report demonstrates that Giardia sp. and Cryptosporidium sp. were present in Peruvian coastal populations for at least 4,300 and 1,100 BP.

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.

To determine the prevalence of intestinal parasites and risk factors for infection associated with diarrhea in HIV-infected patients in Harare Zimbabwe a prospective observational study was carried out. Single stool samples were collected from 88 HIV- infected individuals presenting with diarrhea of more than 1 week duration. Stools were examined for intestinal parasites using modified acid fast stain fluorescence-labeled monoclonal antibody for Cryptosporidium parvum as well as a modified trichrome stain and a polymerase chain reaction (PCR)-based protocol for Enterocytozoon bieneusi. C. parvum was detected in 9% (7 out of 82) of samples evaluated but no Cyclospora was detected. E. bieneusi was detected in 18% (10 out of 55) of stools examined by trichrome staining and in 51% (28 out of 55) of stools examined by PCR. Risk factors for E. bieneusi infection were: living in rural areas consumption of non-piped water contact with cow dung and household contact with an individual with diarrhea. E. bieneusi infection was common in HIV-infected patients with diarrhea in Zimbabwe and may be acquired through person-to-person and fecal- oral transmission. (authors modified)

To evaluate enteropathogens and other factors associated with severe disease in children with diarrhea, 381 children

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.

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.

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.

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.

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.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.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.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.