Molecular characterization of the waterborne pathogens Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, Cyclospora cayetanensis and Eimeria spp. in wastewater and sewage in Guangzhou, China.

BACKGROUND: The waterborne pathogens Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi and Cyclospora cayetanensis can cause intestinal diseases in humans. An understanding of their occurrence and transport in the environment is essential for accurate quantitative microbial risk assessment. METHODS: A total of 238 influent samples were collected from four wastewater treatment plants (WWTPs) and 88 samples from eight sewer locations in Guangzhou, China. PCR-based tools were used to detect and genetically characterize Cryptosporidium spp., G. duodenalis and E. bieneusi. Eimeria spp. and Cyclospora spp. were also analyzed to assess the sources of Cryptosporidium spp., G. duodenalis and E. bieneusi in wastewater. RESULTS: The overall occurrence rates in the WWTP and sewer samples were 14.3% (34/238) and 13.6% (12/88) for Cryptosporidium spp., 55.5% (132/238) and 33.0% (29/88) for G. duodenalis, 56.3% (134/238) and 26.1% (23/88) for E. bieneusi and 45.4% (108/238) and 47.7% (42/88) for Eimeria spp., respectively. Altogether, 11 Cryptosporidium species and genotypes, six G. duodenalis genotypes, 11 E. bieneusi genotypes and four C. cayetanensis were found, together with the presence of nine Eimeria species. The common occurrence of Cryptosporidium rat genotype IV, C. muris and Eimeria papillata and E. nieschulzi suggested that rodents were significant sources of the enteric pathogens detected in the wastewater samples. CONCLUSIONS: While the dominant Cryptosporidium spp. detected in the raw wastewater sampled in this study are not pathogenic to humans, the widely detected G. duodenalis assemblage A and E. bieneusi genotypes D and Type IV are well-known zoonotic pathogens. Further studies are needed to monitor the occurrence of these waterborne pathogens in WWTPs to better understand their transmission and environmental transport in China.

Human cyclosporiasis.

Cyclospora species are socioeconomically important protistan pathogens. Cyclospora cayetanensis is usually transmitted via food or water to a human host via the faecal-oral route and can cause the gastrointestinal disease cyclosporiasis, which can be complicated by extra-intestinal disorders, particularly in immune-compromised people. Although more than 2 million children die each year from diarrhoeal diseases worldwide, it is not known to what extent cyclosporiasis is involved. Few epidemiological data are available on Cyclospora as a water-borne and food-borne pathogen in both underprivileged communities and developed countries. To gain an improved understanding of human cyclosporiasis, this Review describes the background of Cyclospora, summarises salient aspects of the pathogenesis, epidemiology, diagnosis, treatment, and control of cyclosporiasis, and explores what is known about its prevalence and geographical distribution. The findings show that the effect on human health of cyclosporiasis is likely underestimated, and recommendations are made about areas of future research and the prevention and control of this disease within an international collaborative context.

Evaluation of two DNA extraction methods for the PCR-based detection of eukaryotic enteric pathogens in fecal samples.

OBJECTIVE: Efficient and easy-to-use DNA extraction and purification methods are critical in implementing PCR-based diagnosis of pathogens. In order to optimize the routine clinical laboratory diagnosis of eukaryotic enteric pathogens, we compare, via quantitative PCR cycle threshold (Ct) values, the efficiency of two DNA extraction kits: the semi-automated EZ1® (Qiagen) and the manual QIAamp® DNA Stool Mini Kit (Qiagen), on six protozoa: Blastocystis spp., Cryptosporidium parvum/hominis, Cyclospora cayetanensis, Dientamoeba fragilis, Giardia intestinalis and Cystoisospora belli and one microsporidia: Enterocytozoon bieneusi. RESULTS: Whereas EZ1® (Qiagen) and QIAamp® DNA Stool Mini Kit (Qiagen) yielded similar performances for the detection of Cryptosporidium spp. and D. fragilis, significant lower Ct values (p < 0.002) pointed out a better performance of EZ1® on the five remaining pathogens. DNA extraction using the semi-automated EZ1® procedure was faster and as efficient as the manual procedure in the seven eukaryotic enteric pathogens tested. This procedure is suitable for DNA extraction from stools in both clinical laboratory diagnosis and epidemiological study settings.

Detection of Cryptosporidium and Cyclospora Oocysts from Environmental Water for Drinking and Recreational Activities in Sarawak, Malaysia.

Cryptosporidiosis and cyclosporiasis are caused by waterborne coccidian protozoan parasites of the genera Cryptosporidium and Cyclospora, respectively. This study was conducted to detect Cryptosporidium and Cyclospora oocysts from environmental water abstracted by drinking water treatment plants and recreational activities in Sarawak, Malaysia. Water samples (12 each) were collected from Sungai Sarawak Kanan in Bau and Sungai Sarawak Kiri in Batu Kitang, respectively. In addition, 6 water samples each were collected from Ranchan Recreational Park and UNIMAS Lake at Universiti Malaysia Sarawak, Kota Samarahan, respectively. Water physicochemical parameters were also recorded. All samples were concentrated by the iron sulfate flocculation method followed by the sucrose floatation technique. Cryptosporidium and Cyclospora were detected by modified Ziehl-Neelsen technique. Correlation of the parasites distribution with water physicochemical parameters was analysed using bivariate Pearson correlation. Based on the 24 total samples of environmental water abstracted by drinking water treatment plants, all the samples (24/24; 100%) were positive with Cryptosporidium, and only 2 samples (2/24; 8.33%) were positive with Cyclospora. Based on the 12 total samples of water for recreational activities, 4 samples (4/12; 33%) were positive with Cryptosporidium, while 2 samples (2/12; 17%) were positive with Cyclospora. Cryptosporidium oocysts were negatively correlated with dissolved oxygen (DO).

Detection of Cryptosporidium parvum and Cyclospora cayetanensis infections among people living in a slum area in Kathmandu valley, Nepal.

OBJECTIVE: The aim of this study is to determine the prevalence of Cyclospora cayetanensis and Cryptosporidium parvum infections among people living a slum in Kathmandu valley, Nepal. RESULTS: Ten different parasites were detected in the stool samples; the prevalence of any parasite was in 27.1% (71/262). The prevalence of C. cayetanensis and C. parvum were 14.1% (10/71) and 5.6% (4/71), respectively. This study showed high prevalence of intestinal parasitic infections along with the coccidian parasites in the slum area of Kathmandu Valley.

Comparative genomics reveals Cyclospora cayetanensis possesses coccidia-like metabolism and invasion components but unique surface antigens.

BACKGROUND: Cyclospora cayetanensis is an apicomplexan that causes diarrhea in humans. The investigation of foodborne outbreaks of cyclosporiasis has been hampered by a lack of genetic data and poor understanding of pathogen biology. In this study we sequenced the genome of C. cayetanensis and inferred its metabolism and invasion components based on comparative genomic analysis. RESULTS: The genome organization, metabolic capabilities and potential invasion mechanism of C. cayetanensis are very similar to those of Eimeria tenella. Propanoyl-CoA degradation, GPI anchor biosynthesis, and N-glycosylation are some apparent metabolic differences between C. cayetanensis and E. tenella. Unlike Eimeria spp., there are no active LTR-retrotransposons identified in C. cayetanensis. The similar repertoire of host cell invasion-related proteins possessed by all coccidia suggests that C. cayetanensis has an invasion process similar to the one in T. gondii and E. tenella. However, the significant reduction in the number of identifiable rhoptry protein kinases, phosphatases and serine protease inhibitors indicates that monoxenous coccidia, especially C. cayetanensis, have limited capabilities or use a different system to regulate host cell nuclear activities. C. cayetanensis does not possess any cluster of genes encoding the TA4-type SAG surface antigens seen in E. tenella, and may use a different family of surface antigens in initial host cell interactions. CONCLUSIONS: Our findings indicate that C. cayetanensis possesses coccidia-like metabolism and invasion components but unique surface antigens. Amino acid metabolism and post-translation modifications of proteins are some major differences between C. cayetanensis and other apicomplexans. The whole genome sequence data of C. cayetanensis improve our understanding of the biology and evolution of this major foodborne pathogen and facilitate the development of intervention measures and advanced diagnostic tools.

Enteric protozoa in the developed world: a public health perspective.

Several enteric protozoa cause severe morbidity and mortality in both humans and animals worldwide. In developed settings, enteric protozoa are often ignored as a cause of diarrheal illness due to better hygiene conditions, and as such, very little effort is used toward laboratory diagnosis. Although these protozoa contribute to the high burden of infectious diseases, estimates of their true prevalence are sometimes affected by the lack of sensitive diagnostic techniques to detect them in clinical and environmental specimens. Despite recent advances in the epidemiology, molecular biology, and treatment of protozoan illnesses, gaps in knowledge still exist, requiring further research. There is evidence that climate-related changes will contribute to their burden due to displacement of ecosystems and human and animal populations, increases in atmospheric temperature, flooding and other environmental conditions suitable for transmission, and the need for the reuse of alternative water sources to meet growing population needs. This review discusses the common enteric protozoa from a public health perspective, highlighting their epidemiology, modes of transmission, prevention, and control. It also discusses the potential impact of climate changes on their epidemiology and the issues surrounding waterborne transmission and suggests a multidisciplinary approach to their prevention and control.

[Cyclospora cayetanensis: biology, environmental distribution and transfer]. / Cyclospora cayetanensis: biología, distribución ambiental y transferencia.

Cyclospora cayetanensis is an apicomplexan protozoan that has emerged as an important pathogen causing endemic or epidemic diarrheal disease worldwide. In industrialized countries, the parasite has been recognized as the causative agent of several outbreaks of diarrheal illness mostly associated with produce imported from endemic areas. In developing countries, human cyclosporosis is widely distributed. Infection rates from 0% to 41.6% have been described in the general population. However, the epidemiology, biology, and ecology of C. cayetanensis are not fully understood. The life cycle is not completely characterized, although it appears to require a single human host to be accomplished. The role of animals as natural reservoirs of the parasite remains to be determined. Little information is available concerning the environmental distribution and vehicles of transmission of C. cayetanensis. Contaminated water, foods or soil can be vehicles of spread of the parasite. The significant uncertainties that remain in the knowledge of C. cayetanensis highlight the need for continuing research in several areas, including its basic biology and environmental distribution.

Travelers' diarrhea in Nepal: an update on the pathogens and antibiotic resistance.

BACKGROUND: Diarrhea is the most common illness among travelers and expatriates in Nepal. Published data on the etiology of travelers' diarrhea (TD) in Nepal are over 13 years old and no prior data exist on antibiotic susceptibility for currently used drugs. We investigated the etiology of diarrhea and antimicrobial susceptibility pattern of bacterial pathogens and compared the results to previous work from the same clinical setting. METHODS: A total of 381 cases and 176 controls were enrolled between March 2001 and 2003 in a case-control study. Enrollees were over age 18 years from high socioeconomic countries visiting or living in Nepal. Stool samples were assessed by microbiologic, molecular identification, and enzyme immunoassay (EIA) methods, and antimicrobial susceptibility was determined by disk diffusion. Risk factors were assessed by questionnaires. RESULTS: At least one enteropathogen was identified in 263 of 381 (69%) cases and 47 of 176 (27%) controls (p ≤ 0.001). Pathogens significantly detected among cases were Campylobacter (17%), enterotoxigenic Escherichia coli (ETEC) (15%), Shigella (13%), and Giardia (11%). Cyclospora was detected only in cases (8%) mainly during monsoon season. Although 71% of Campylobacter isolates were resistant to ciprofloxacin, 80% of bacterial isolates overall were sensitive to either ciprofloxacin or azithromycin while 20% were intermediately sensitive or resistant. No bacterial isolates were resistant to both drugs. CONCLUSIONS: The most common pathogens causing TD in Nepal were Campylobacter, ETEC, and Shigella. Because resistance to fluoroquinolone or azithromycin was similar, one of these drugs could be used as empiric therapy for TD with the other reserved for treatment failures.

Cyclospora cayetanensis: a review, focusing on some of the remaining questions about cyclosporiasis.

Cyclosporiasis is a protracted, relapsing gastroenteritis and has a short recorded history. Cyclospora cayetanensis is an enigmatic parasite since its discovery highlights the need for isolation of cases of infection that could be part of widespread outbreaks. It is associated with diarrhoea among children in developing countries in the Americas, where C. cayetanensis is endemic; traveller's diarrhoea and/or food and waterborne outbreaks sometimes occur in the developed countries. In SubSaharan Africa and Egypt, cyclosporiasis has been reported to occur in both immunocompromised and immunocompetent patients. Zoonotic species of Cyclospora have also been identified worldwide in primates, indicating likely endemicity of this underreported disease. This can be attributed to the lack of awareness in the public and medical profession concerning the disease, which is, therefore, not routinely tested at the health centres. The correlation between the density of water contamination and the prevalence of cyclosporiasis among the individuals of each area is significant. No doubt, water is the main vehicle of transmission in the present community. Soil contact and poultry are significant risk factors. All literature on C. cayetanensis, cyclosporiasis worldwide, and endemic cyclosporiasis was searched from libraries, colleagues and internet. Although cyclosporiasis is considered an enigma worth unravelling, with many aspects of this disease and its transmission having been uncovered only recently, the situation has been rapidly changing since the disease first came to medical attention in the 1970s.

Diarrea del viajero por Cyclospora cayetanensis / Cyclospora cayetanensis in a patient with traveller´s diarrhoea

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Update on Cyclospora cayetanensis, a food-borne and waterborne parasite.

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.

Brote de Cyclospora cayetanensis en viajeros a Cuba / Cyclospora cayetanensis outbreak in travelers to Cuba

Descritos inicialmente en viajeros, los brotes epidémicos de Cyclospora cayetanensis fueron posteriormente asociados a productos alimentarios importados. En este artículo se describe un brote de ciclos poriasis en viajeros españoles. Se rellenó un cuestionario específico, tras la identificación de Cyclospora en heces. Los síntomas de pirosis aparecieron en el 57% de los casos (4/7). La presencia de sintomatología péptica durante la diarrea del viajero puede hacer sospechar la presencia deciclosporiasis (AU)

Initially described in travelers, outbreaks of cyclosporiasis were soon linked to imported food products. An outbreak of cyclosporiasis in Spanish travelers is described. After identification of Cyclospora cayetanensis in stool analyses, a specific questionnaire was completed. Pyrosis was described in 57% of cases (4/7). Peptic symptoms can bea useful clue to indicate the diagnosis of cyclosporiasis in patients with travelers’ diarrhea (AU)

Cyclospora cayetanensis: un protozoo intestinal emergente / Cyclospora cayetanensis: an emerging intestinal protozoan

Resumen Cyclospora cayetanensis es un protozoo patógeno emergente causante de diarrea el cual se ha estado notificando cada vez con mayor frecuencia a escala mundial en personas de todas las edades, independientemente de su estado inmunológico. Esta coccidia ha sido descrita como causa de diarrea del viajero y de brotes de diarrea debido a la ingestión de frutas y vegetales, particularmente de áreas tropicales. La enfermedad que causa, cyclosporosis, está caracterizada principalmente como un síndrome de diarrea aguda autolimitada que puede ocasionar deshidratación y llegar hasta provocar diarrea prolongada o crónica. Otras manifestaciones como perdida de peso, anorexia, fatiga, astenia, borborismos, flatulencia y distensión abdominal pueden estar presentes, lo que hace esta afección indistinguible de otras diarreas infecciosas causadas por parásitos intestinales. El diagnóstico depende básicamente de la identificación del microorganismo en muestras fecales. Su tratamiento consiste en mantener el equilibrio hidromineral y electrolítico, al tiempo que se prescribe tratamiento antibiótico entre ellos se usa fundamentalmente el trimetoprín- sulfametoxazol, aunque se ha reportado la utilidad de otras drogas entre las que se cuentan la ciprofloxacina y la nitazoxanida. Esta revisión presenta diferentes aspectos actuales del manejo de la infección por Cyclospora cayetanensis y pone en relieve interrogantes sobre esta parasitosis donde factores relacionados con el protozoo, el ambiente y el susceptible quedan por esclarecer.

Cyclosporiasis outbreak, Indonesia.

We describe an outbreak of Cyclospora cayetanensis infection among Dutch participants at a scientific meeting in September 2001 in Bogor, Indonesia. Fifty percent of the investigated participants were positive for C. cayetanensis. To our knowledge, this outbreak is the first caused by C. cayetanensis among susceptible persons in a disease-endemic area.

Foodborne protozoan parasites.

This report addresses Cryptosporidium, Giardia, Cyclospora, and more briefly, Toxoplasma as the main parasitic protozoa of concern to food production worldwide. Other parasitic protozoa may be spread in food or water but are not considered as great a risk to food manufacture. The protozoan parasites Cryptosporidium, Giardia, and Cyclospora have proven potential to cause waterborne and foodborne disease. Toxoplasma gondii has been considered a risk in specific cases, but humans are not its primary host. Cryptosporidium and Giardia are widespread in the environment, particularly the aquatic environment, and major outbreaks of cryptosporidiosis and giardiasis have occurred as a result of contaminated drinking water. Large outbreaks of waterborne cyclosporiasis have not been identified. Cryptosporidium, Giardia, and Cyclospora have potential significance in the preparation and consumption of fresh produce and in catering practice, in which ready-to-eat foods may be served that have not received heat treatment. None of the three organisms Cryptosporidium, Giardia, and Cyclospora has been shown to be a problem for heat processed food or tap water that has undergone appropriate treatment at a water treatment works. All three are sensitive to standard pasteurisation techniques. Although humans are not a primary host for T. gondii, the potential exists for both waterborne and foodborne toxoplasmosis. Parasitic protozoa do not multiply in foods, but they may survive in or on moist foods for months in cool, damp environments. Their ecology makes control of these parasites difficult. For general control of parasitic protozoa in the food chain, the following steps are necessary: - Follow good hygienic practice in food service and catering industries.- Minimise dissemination of cysts and oocysts in the farming environment and via human waste management.- Include these microorganisms in Hazard Analysis Critical Control Point (HACCP) plans of water suppliers, industries or sectors that use fresh produce, and operations in which contaminated process or ingredient water could end up in the product (e.g., where water supplies may become contaminated).

Cyclospora cayetanensis and other intestinal parasites associated with diarrhea in a rural area of Jordan.

Cryptosporidium spp. and Cyclospora cayetanensis have emerged as important causes of epidemic and endemic diarrhea in immunocompetent and immunocompromised hosts. The exact modes of transmission in certain rural areas are still unclear. Reports of water-borne and food-borne outbreaks suggest that fecally contaminated water or food acts as a vehicle of transmission. Two hundred stool samples of patients with gastroenteritis from four health centers in a rural area of Jordan were examined using formalin-ethyl acetate concentration, wet preparation, and modified acid-fast staining methods. Oocysts of C. cayetanensis and Cryptosporidium spp. were found in 6% and 8% of the samples respectively, mainly those of children. Parasites such as Entamoeba histolytica, Giardia lamblia, and other enteropathogens were also observed. The results reflect the seasonality of natural cyclosporiasis and cryptosporidiosis, being higher in the spring. The risk factors that were found by the Fisher test to be significant and might be associated with illness are the source of drinking water, contact with animals, and eating unwashed vegetables ( p<0.028, p<0.0005, p<0.00005 respectively).