ArticleLiterature Review

# Evolution of monitoring for Giardia and Cryptosporidium in water

Authors:
• University of Nicosia Medical School
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## Abstract

This review describes the evolution of monitoring methodology for Cryptosporidium and Giardia in water since the 1970’s. Methods in current use for Giardia and Cryptosporidium in water are highlighted, though attention is given to all available published methods by country and continent. The review is intended to stimulate research leading to future improvements and further developments in monitoring methodology for Giardia, Cryptosporidium and other waterborne protozoan parasites in water.

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... Hundreds of efforts have been described in addressing selected components of the problems encountered in the monitoring of Cryptosporidium in water with the objectives to define the quantity, viability, and human infectivity of oocysts [16]. This provides a quantitative understanding of the public health risks concerning water supply and water treatment strategies, which can provide foundations for water reservoir management, treatment designs, operation, supply, monitoring, and regulation [17]. ...
... The provision of low-cost, user-friendly, and sustainable recovery methods for Cryptosporidium in high (50-100 L) and standard-volume (5-10 L) water samples like flotation and flocculation have been previously described [16,[18][19][20][21][22][23]. However, Cryptosporidium identification in water samples still proves to be challenging and requires expertise in identification through a plethora of technical methodologies [24]. ...
... The higher oocyst count in the current study can be attributed to several factors. Methodologically, the direct filtration, elution, and pelleting of lowvolume (50 mL) aquatic matrices supposedly minimized the potential loss of oocysts if processed using multiple sequential steps [16,34], while the standardized smear area of 1 cm diameter (25 µL), systematic, and exhaustive microscopy (200 fields per 1 cm-diameter smear area) aided in increased detection (Additional file 3: Table S3), where the results provided a clear picture of the distribution of Cryptosporidium spp. oocysts in environmental freshwater systems all over the country. ...
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Background Cryptosporidium is a waterborne global pathogen causing diarrhea primarily in infants and immunocompromised individuals. The Philippines is a tropical country susceptible to the influences of climate change and water crises. To date, the country has no existing epidemiologic data, regulation, or strategy for monitoring Cryptosporidium in freshwater systems. We, therefore, endeavored to provide evidence on the multi-spatial contamination of Cryptosporidium in environmental aquatic matrices using low-cost, user-friendly, and sustainable strategies and submit implications on the presence of Cryptosporidium in freshwater systems in a climate, health, and regulatory framework. Results Here, we present the microscopic detection of Cryptosporidium oocysts in low-volume (50 mL) environmental samples of surface water (SW), sediments (BW), and substrate-associated biofilm (SAB) and in 1 L bulk SW investigated by PCR. The multi-spatial distribution of Cryptosporidium oocysts in the low-volume (50 ml) aquatic matrices based on microscopy was highest at 69% (20/29) in SW and lowest at 50% (13/26) in BW. Immunofluorescence technique provided the highest microscopic positivity rate with 59% (17/29), 38% (10/26), and 50% (10/20) detection in SW, BW, and SAB, respectively. The detection and identification of Cryptosporidium in 1 L bulk SW by PCR and sequence analysis was recorded in total at 21% (6/29) in sampling sites where the differential identification of C. parvum, C. hominis, and Cryptosporidium spp. was 7% (2/29), 10% (3/29), and 3% (1/29), respectively. Conclusions We report the microscopical and first molecular epidemiologic data of Cryptosporidium from the most significant environmental freshwater systems in the Philippines. The presence of the two main human and animal pathogenic species C. parvum and C. hominis from the largest lakes and major water reservoirs in the country calls for sustainable solutions in safeguarding the quality of freshwater resources in a climate, health, and regulatory approach.
... Este fato também alerta para a questão de que populações que consomem água tratada apenas pelo processo de desinfecção (cloração), ou que consomem água de estações de tratamento -ETAs que não realizam um controle rigoroso da eficiência do processo de filtração ou que apresentam deficiências operacionais, podem estar sob maior risco de adquirir infecções por esses agentes patogênicos (ADLER et al., 2017;EFSTRATIOU et al. 2017;OLIVEIRA, 2017;OGURA, 2018). ...
... Erros de resultados nos testes ou testes não apropriados e negligência procedentes de análises microbiológicas podem induzir os equívocos na condução de problemas na área ambiental e sérios erros de planejamento e direcionamento de programas de controle das enfermidades de veiculação hídrica . Segundo Efstratiou et al. (2017), um critério ou padrão que pode ser rastreado é, se apenas um protocolo (teste) único seria recomendado para aplicação independentemente das condições da qualidade de todos os corpos d'água, ou seja, implica se unicamente os protocolos, Método 1623.1 /US EPA 2005, poderiam ser articulados para atender a todas as aplicações igualmente de forma efetiva. Os autores, com base também em suas experiências anteriores, acreditam que este é um conceito incorreto, imperfeito e incompleto, o que é reforçado pela recente revisão da literatura realizada pelos mesmos cientistas. ...
Chapter
... Este fato também alerta para a questão de que populações que consomem água tratada apenas pelo processo de desinfecção (cloração), ou que consomem água de estações de tratamento -ETAs que não realizam um controle rigoroso da eficiência do processo de filtração ou que apresentam deficiências operacionais, podem estar sob maior risco de adquirir infecções por esses agentes patogênicos (ADLER et al., 2017;EFSTRATIOU et al. 2017;OLIVEIRA, 2017;OGURA, 2018). ...
... Erros de resultados nos testes ou testes não apropriados e negligência procedentes de análises microbiológicas podem induzir os equívocos na condução de problemas na área ambiental e sérios erros de planejamento e direcionamento de programas de controle das enfermidades de veiculação hídrica . Segundo Efstratiou et al. (2017), um critério ou padrão que pode ser rastreado é, se apenas um protocolo (teste) único seria recomendado para aplicação independentemente das condições da qualidade de todos os corpos d'água, ou seja, implica se unicamente os protocolos, Método 1623.1 /US EPA 2005, poderiam ser articulados para atender a todas as aplicações igualmente de forma efetiva. Os autores, com base também em suas experiências anteriores, acreditam que este é um conceito incorreto, imperfeito e incompleto, o que é reforçado pela recente revisão da literatura realizada pelos mesmos cientistas. ...
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... As little as 10 Giardia cysts can infect hosts who are vulnerable to infection. Therefore, contaminated water and nutrients with cysts can cause epidemics [2,[9][10][11][12]. ...
... Furthermore, studies reported that assemblage E may also be present in humans. Ruminants are seen as the most important source in giardiasis epidemics because their feces production is very high [12][13][14][15][16]. Buffaloes are important ruminant species and are endangered. ...
Article
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Giardia duodenalis (G. duodenalis) is an important zoonotic protozoan agent that causes foodborne and waterborne diarrhea in humans and other mammals. Molecular-based tests are critical in diagnosing giardiasis in humans and animals, identifying species, understanding the zoonotic potential and transmission routes, and evaluating taxonomy. Therefore, this study aimed to investigate the molecular characterization of G. duodenalis in buffaloes in the Van region in Türkiye. Buffaloes are a species that has been poorly studied in this regard. For this purpose, 100 fecal samples were collected from buffaloes in the Van region. The DNA extraction was performed using the GeneMATRIX STOOL DNA Purification Kit from stool samples. The nested PCR test was performed with the appropriate primers from the obtained DNA samples. The obtained bands suitable for sequencing were sent for sequence analysis, and the sequence results were aligned bidirectionally and compared with the database of GenBank by BLAST. As a result of the study, an 11% positivity rate for G. duodenalis was found in buffaloes, and assemblage E and assemblage B were isolated. To our knowledge, assemblage B in buffaloes was reported for the first time in this study. As a result, it was concluded that buffaloes are an important reservoir for waterborne and foodborne giardiasis.
... Cryptosporidiosis can be transmitted from person to person in a variety of ways ( Figure 1). The contaminated food and drink, touching the mouth with contaminated hands, and oral-anal sexual contact can all spread the disease from person to person (1,12). The most prevalent causes of outbreaks include drinking water from a lake or river and swimming in a contaminated pool (12). ...
... The contaminated food and drink, touching the mouth with contaminated hands, and oral-anal sexual contact can all spread the disease from person to person (1,12). The most prevalent causes of outbreaks include drinking water from a lake or river and swimming in a contaminated pool (12). In addition, inhaling oocysts from the contaminated air can infect the respiratory tract and produce respiratory symptoms in both animals and humans (13). ...
Article
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Cryptosporidiosis is an infectious emerging zoonotic protozoan disease occurring in both human and animal populations all over the world. Cryptosporidiosis is caused by Cryptosporidium , a tiny parasite that affects humans as well many species of animals. Although C ryptosporidium parvum and Cryptosporidium hominis (formerly known as C. parvum anthroponotic genotype or genotype 1) are the most common species causing disease in human beings, Cryptosporidium felis , Cryptosporidium meleagridis , Cryptosporidium canis , and Cryptosporidium muris infections have also been reported. Waterborne transmission via drinking water or a swimming pool is common, and outbreaks have been reported in several countries. The most common symptom of cryptosporidiosis in humans is watery diarrhea. People with weakened immune systems are more susceptible to contract serious, long-term, and even fatal infections. The laboratory endeavor is required to make a precise diagnosis of the disease. The demonstration of oocysts of Cryptosporidium in the fecal samples is still used as a simple, easy, and cost-effective technique for the diagnosis of disease. As a preventative and control measure, appropriate hygienic practices must be practiced everywhere. Furthermore, the veterinarians play a crucial role in the treatment of disease in domestic animals.
... Surveillance data shows a lack of coverage in the analysis of the presence of protozoa that should be strengthened to improve microbial risk analysis, and the frequency of diagnoses would provide more accurate data. There are aspects of the USEPA method 1623 that must be considered in surveillance and microbial risk analysis: The technique does not determine the viability of the (oo)cysts; they may be intact in their morphology, so they may not be infectious (Efstratiou et al., 2017). The technique has limitations to identify the species, which is a necessary information considering that there are some species of Cryptosporidium spp. ...
... For example, a comparative study by Morgan et al. (1998) between microscopy and PCR techniques to detect Cryptosporidium spp concluded that PCR yields higher sensitivity, specificity, and genotyping capacity. However, the costeffectiveness, processing times, and technical and operational capacity of the available laboratories must be evaluated for its implementation, mainly in developing countries (Efstratiou et al., 2017). ...
Article
Cryptosporidium spp. and Giardia spp. cause zoonotic diseases as well diseases by contamination of human fecal material, being various reported outbreaks associated with water. The infecting (oo) cyst forms of these parasites are highly resistant to water treatments such as chlorine disinfection and fast filtration. The objective of this study was to assess the microbial risk of infection and symptomatic illness by the ingestion of Giardia spp. and Cryptosporidium spp. in water for human consumption in Colombia, based on the results of water quality surveillance. The detection method was according to the USEPA method 1623. Concentration data of the different points of distribution were grouped according to the pathogen and type of treatment (no treatment; chlorine treatment; chlorine treatment + coagulant). Annual microbial risks of infection and symptomatic diseases were estimated using the quantitative microbial risk assessment approach that included parasite concentrations, the dose‐response model, the ingestion rates of water by children and adults and the morbidity rate of the diseases. The mean annual microbial risk of infection for Giardia spp. was 29.8% for treated water and 50.4% for untreated water, while being 6.0% and 17.7% respectively for Cryptosporidium spp. Microbial risk of symptomatic illness for Giardia spp, was 8.2% for treated water and 13.9% for untreated water, while being 3.6% and 10.6% respectively for Cryptosporidium spp. The estimated annual microbial risks of infection exceeded the acceptable value of 10‐4 (0.01%) recommended by USEPA. Results obtained in this study suggest the need to reduce the microbial risk of infection to protozoan parasites by improving the water treatment, by adopting better handling practices for livestock manure and treatment processes of human feces.
... These intracellular apicomplexan parasites are also infectious to humans and a very wide range of animals, with felids serving as the only definitive hosts (Djurković-Djaković et al., 2019). Direct microscopic examination, staining, immunofluorescence microscopy and PCR are all commonly used to detect protozoan parasites on fresh produce and in water samples (Berrouch et al., 2020;Efstratiou et al., 2017b;Li et al., 2020). There are, however, limitations to the use of both microscopical methods and PCR. ...
... Newer molecular methods for the detection of food and waterborne parasites include quantitative PCR (qPCR), loop-mediated isothermal amplification (LAMP), fluorescence in situ hybridization (FISH) and aptasensors (Ahmed and Karanis, 2018;Berrouch et al., 2020;Chalmers et al., 2020;Efstratiou et al., 2017b;Hassan et al., 2020;Lalonde et al., 2021). While these methods are rapid and highly sensitive in the detection of parasites, some require access to specialized instruments that are not necessarily available to all researchers, especially in remote or developing regions. ...
Article
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Protozoan parasites in food or water samples are generally detected using microscopy or PCR followed by Sanger sequencing. However, microscopy is subjective, requires a high degree of expertise and has limited sensitivity, while DNA sequencing requires expensive and specialized equipment and facilities. This study describes a cloth-based hybridization array system (CHAS) that is an alternative to Sanger sequencing to confirm PCR-positive samples. CHAS is an inexpensive, rapid and reliable method for the simultaneous detection of multiple protozoan parasite species based on the colorimetric detection of PCR amplicons on a polyester cloth. PCR primers and CHAS hybridization probes were developed to detect the protozoan parasites Giardia duodenalis, Cryptosporidium spp. and Toxoplasma gondii. In addition, CHAS probes were designed for the differentiation of G. duodenalis Assemblages A and B. In artificially contaminated fresh produce (lettuce, parsley) and water samples (river water, wastewater), this CHAS assay allowed for the successful detection of G. duodenalis, Cryptosporidium spp., and T. gondii. The present study demonstrates that the CHAS detection method is a simple and inexpensive alternative to DNA sequencing for the confirmation of PCR-positive results in laboratories testing for parasites in food or water samples. This assay may also be beneficial in developing countries, where DNA sequencing facilities may not be readily available.
... In 1999, USEPA developed the 1622 method for detecting Cryptosporidium and in 2002 the 1623 method for detecting simultaneous Giardia and Cryptosporidium using immunomagnetic separation, updated in 2012 to version 1623.1 which became the most accepted procedure for monitoring these two pathogens in water. Through this method, recoveries of 30-50% for Cryptosporidium and 40-60% for Giardia are found, which allows limits of quantification and detection of 0.2-0.5 oo(cysts)/L in 10 L of sample (Efstratiou et al., 2017b). These methods do not discriminate among genotypes and subtypes, neither point to infectivity. ...
... The protozoa tend to be in very low concentrations in water, and the isolation and quantification techniques have limitations in terms of specificity and, mainly, of sensitivity. There is also a low recovery that is around 30-40% (Efstratiou et al., 2017b). For this reason, monitoring is carried out in raw water, and based on this, in the estimate of removal through treatment, the cyst and oocyst concentration in drinking water is estimated. ...
Article
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The objectives of this research are to evaluate Giardia and Cryptosporidium contamination in surface water supply in Rio Grande do Sul (RS) State in South Brazil in the years 2016 to 2020, assess seasonality, and to infer the population that may have been exposed to these protozoa through drinking water based on drinking water treatment efficiency. Data were obtained through the drinking water surveillance national information system. From 204 DWT plants in the state, 66 have been analyzed for protozoa. A total of 2304 analyses of protozoa in raw water were evaluated, of which 223 had both Giardia spp. cysts and/or Cryptosporidium spp. oocysts in concentrations that varied from 0.1 to 21.5/L. A total of 2,712,125 people from 48 cities were at risk of having the presence of pathogenic protozoa in their drinking water. The probability of finding these protozoa was higher in winter. Giardia cysts were more likely to be found in a period without rain, suggesting that sewage was the main source of contamination. It is concluded that the springs of Rio Grande do Sul are impacted and the circulation of pathogenic protozoa through the territory is endemic with a probable source of contamination to sewage and livestock activity.
... Cryptosporidium is reported secondary cause of diarrhoea only behind rotavirus. The leading parasitic protozoa causing waterborne enteric disease outbreaks at global level are Cryptosporidium and Giardia lamblia (Efstratiou et al., 2017). Originating from faeces of infected hosts they can be transmitted to new hosts via oral-faecal route upon contact with contaminated soil, water, feed and food. ...
... Lack of awareness, personal hygiene and sanitation facilities, poverty, indiscriminate eating habits are favourable infestation conditions for Cryptosporidium and Giardia lamblia infections. The prevalence for both Cryptosporidium and Giardia lamblia, is higher in developing countries as compared to developed countries (Mahmoudi et al., 2017;Efstratiou at al., 2017). Interesting is the future study of statistics regarding the measures taken to prevent the disease in children and to treat the sick. ...
Article
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The aim is to present a summarized study of the available literature on Cryptosporidium and Giardia lamblia for Middle Eastern countries, in order to identify trends in human cryptosporidiosis and childhood morbidity, and to raise awareness among residents. This is necessary in order to address the gap in preventive measures required to mitigate the overall effect attributed to associated illness and its impact in the already water stressed Middle Eastern countries. To assess seroprevalence of Cryptosporidium and Giardia lamblia in Middle Eastern countries systematic review was carried out based on online articles published from 2010-2018. PubMed, Web of Science, Google Scholar, Science Direct, Scopus, World Bank and WHO report and scientific database were explored. The current study highlights the existing subsequent epidemiology, its seroprevalence distribution, genetic diversity across Middle Eastern countries since 2009. This study therefore will provide the platform for future research work and development in comprehending Cryptosporidium and Giardia lamblia epidemiology in Middle Eastern countries. It was found that lack of awareness, personal hygiene and sanitation facilities, poverty, indiscriminate eating habits are favourable infestation conditions for Cryptosporidium and Giardia lamblia infections. The prevalence for both Cryptosporidium and Giardia lamblia, is higher in developing countries as compared to developed countries. The originality is that it is the only study of its kind in the region, as such studies are still lacking in Middle East countries as compared to other European, Asian, American continents and countries.
... Cryptosporidium and Giardia are genera of parasitic protozoa responsible for worldwide outbreaks of enteric diseases that have been reported throughout decades (Efstratiou et al. 2017;Ligda et al. 2020). Monitoring cysts and oocysts (i.e. ...
... Immunomagnetic separation (IMS) is a technique that has stood out in various water and wastewater matrices (Hsu & Huang 2000;McCuin & Clancy 2005) and has been endorsed by Methods 1623.1 and 1693 (USEPA 2012(USEPA , 2014. Selective concentration by density gradient techniques, listed for further information in a fairly recent review in recovery methods (Efstratiou et al. 2017), could be an alternative for situations in which IMS costs renders its application impractical. ...
Article
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Filter backwash water (FBW) is a prominent residue from water treatment plants (WTPs) that is often disposed into water bodies or recycled within the WTP without due disinfection. FBW usually contains particles within a size range that includes pathogenic protozoa, as the infective forms of Giardia and Cryptosporidium, parasites responsible for waterborne diseases outbreaks. Quantifying (oo)cysts is essential for addressing this matter, as it might assist research on giardiasis and cryptosporidiosis epidemiology, as well as shed light onto disinfection technologies for FBW. However, (oo)cyst recovery from FBW and other complex matrices still lacks a standard protocol and entails specialized professionals and expensive material. Seeking to provide insight in a reduced-cost recovery method, this study analysed the recovery efficiency (RE) obtained by acid flocculation with ferric sulphate, a common coagulant, on bench-scale simulated FBW. Steps included concentration by flocculation, centrifugation, and quantification by immunofluorescence. Although recovery was sufficient for Cryptosporidium parvum (40.59%), Method 1623.1 recommendations were not reached for Giardia muris (1.76%). Coefficients of variation obtained for both organisms were not satisfactory, highlighting the variability to which environmental matrices are subjected and why defining a methodology for (oo)cyst recovery in WTP residues is important. Highlights C. parvum oocyst recovery efficiency (RE) complied with Method 1623.1.; G. muris RE from FBW was insufficient and statistically different from C. parvum's.; Coefficients of variation for both microorganisms were higher than Method 1623.1 limits.;
... Efstratiou et al. [19] surveyed the main concentration methods: filtration methods, which include membranes, ultrafiltration, microfiber, and nanofiltration; flocculation-sedimentation with calcium carbonate (CaCO 3 ), Al 2 (SO 4 ) 3 , ferric sulphate (Fe 2 (SO 4 ) 3, and formaldehyde with ethyl acetate or ether; and direct centrifugation (DC) methods batch or continuous flow. The calcium carbonate flocculation (CCF) method is recommended for high turbidity water because of the lower-cost procedures [13,14,20,21]. ...
... The purification step is performed by density gradient, cytometry or immunomagnetic separation (IMS) [19]. Among these methods, IMS consists of (oo)cyst attachment on magnetic microspheres. ...
Article
Removing and inactivating Cryptosporidium spp. oocysts and Giardia spp. cysts are a challenge for drinking-water treatments, mainly because of their small sizes and resistance to chlorination. Moreover, protozoan detection methods are expensive and subject to high variability and low reproducibility, especially in high turbidity water. This research aimed at detecting and inactivating cysts and oocysts in drinking-water treatment sludge. In jar test treatability assays, 110 NTU study water was treated with polyaluminium chloride to obtain the sludge. The chosen method for detecting cysts and oocysts was direct centrifugation with ICN 7X solution followed by the immunomagnetic separation (IMS) with two acid dissociations. The inactivation of cysts and oocysts was identified by the inclusion of propidium iodide as a vital dye when the cell walls were damaged. This method's analytical quality control, which was performed with the EasySeed® suspension, obtained recovery of 3.3 ± 2.0 % and 24.8 ± 8.0 % for oocysts and cysts, respectively. Besides, the Giardia spp. recovery met the standards recommended for water by the USEPA Method 1623.1. Magnetic microspheres were found attached to the protozoan in the microscope slides after IMS, indicating some limitations of this purification method. The alkaline treatment tested a dose of 27 mg CaO/100 mL for 3 and 5 days, obtaining inactivation of 1.85 and 3.0 log for oocysts and 2.05 and 2.14 log for cysts, respectively. In this context, the alkaline treatment may be feasible to reduce the microbiological risk in water treatment sludge, considering an economic assessment.
... Cryptosporidium and Giardia resist most environment stressors and have low infectious doses, making them significant agents of gastrointestinal illnesses (Sidhu & Toze, 2009). Almost two-thirds of waterborne outbreaks caused by protozoans are attributed to Cryptosporidium (Efstratiou et al., 2017). As a result, Cryptosporidium is prevalent in sewage (Baldursson & Karanis, 2011). ...
... As a result, Cryptosporidium is prevalent in sewage (Baldursson & Karanis, 2011). All Cryptosporidium outbreaks throughout the world have been attributed to C. hominis and C. parvum (Chalmers et al., 2009;Ryan & Power, 2012), except for one in the UK caused by C. cuniculus (Efstratiou et al., 2017). Cryptosporidiosis can be transmitted from cattle to humans (Ng et al., 2012). ...
Article
Compliance with current guidelines for composting based on time-temperature requirements does not always translate into safe compost products. This review’s objective is to assess what pathogens are inactivated during composting and how inactivation could be improved to address that issue. While most pathogens are inactivated by the heat generated during composting, spore-forming bacteria and Group II viruses are likely to survive the process. Other bacteria may also survive the composting process and regrow. Reports of protozoa and helminths inactivation are contradictory due to the lack of cheap and reliable technologies to ascertain their viability. Other factors contribute to pathogen inactivation by promoting the activity of beneficial microorganisms that produce antimicrobial compounds, compete for nutrients or prey on pathogens. Volatile acids are effective against bacteria and viruses by targeting their envelope (if any) and their genome. Ammonia affects most pathogens including Ascaris eggs and exhibits a strong effect against ssRNA viruses. Future research should focus on better understanding the role of the batching recipe and mesophilic and maturation phases in inactivating pathogens. Understanding how pathogens present in a feedstock are inactivated is also crucial to predict what factors are important and optimize the composting process. More appropriate indicators should be used for high-risk wastes containing human or animal wastes. Enterococci have been suggested as an additional indicator but the relation between the presence of enterococci or other indicator and the survival of other types of pathogens should be further investigated.
... Cryptosporidium oocysts transmission can occur following direct or indirect contact with an infected host usually via the faecal-oral route. Person-to-person contact, zoonosis, and the consumption of contaminated food or water are well known mechanisms for faecal-oral transmission [2,3], with a significant risk of infection from the ingestion of a single oocyst [4]. When the oocysts enter the gastrointestinal tract, the invasive Cryptosporidium causes damage to the small intestinal epithelium. ...
... The usage of chlorine as a water disinfectant is known to be effective against many microorganisms; however, Cryptosporidium oocysts are resistant to the effects of chlorine [124] and various environmental stresses, such as extreme temperature variations [40]. The oocysts are small (5 µm) and have a low infectious dose (1-10 oocysts), and reportedly has the ability to maintain viability in water longer than 6-12 months or longer with the capability to cause epidemics, even after the consumption of purified drinking water [2,125,126]. ...
Article
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The present review discusses the burden of cryptosporidiosis in the Gulf Cooperation Council (GCC), which is underreported and underestimated. It emphasizes that the Cryptosporidium parasite is infecting inhabitants and expatriates in the Gulf countries. Children under 5 years are a vulnerable group that is particularly affected by this parasitic disease and can act as carriers, who contribute to the epidemiology of the disease most probably via recreational swimming pools. Various risk factors for cryptosporidiosis in the GCC countries are present, including expatriates, predisposing populations to the infection. Water contamination, imported food, animal contact, and air transmission are also discussed in detail, to address their significant role as a source of infection and, thus, their impact on disease epidemiology in the Gulf countries' populations.
... Giardia duodenalis is one of the commonest intestinal parasites that infects humans worldwide (Yason & Rivera 2007). From 2011 to 2016, 37% of worldwide waterborne protozoa outbreaks were caused by this zoonotic protozoan (Efstratiou et al. 2017). The chlorine-resistance characteristic combined with cysts' ability to permeate filtration units because they are small (6-15 μm) can explain the occurrence of giardiasis epidemics after consumption of treated drinking water in several countries (Smith 1998;Omarova et al. 2018). ...
Article
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Giardia spp. is an intestinal parasite responsible for worldwide disease outbreaks. Guiding researchers and practitioners to choose among current methods for microscopy detection of the infectious forms may be directly beneficial to public health and the environment. This study provides an overall comparison of brightfield (BF), fluorescence and darkfield (DF) microscopies for detecting Giardia duodenalis and Giardia muris cysts, by illustrating micrographs of such protocols applied to purified samples, as well as discussing advantages and constraints based on secondary information and collected data. BF analysis included Lugol's iodine staining. In fluorescence microscopy, samples were processed by immunofluorescence assay (IFA) with DAPI and by standalone DAPI dye. Cyst suspensions were also analysed by DF microscopy using a recently developed low-cost system. The three techniques enabled detecting Giardia spp. cysts, although they did not provide species identification by morphology. The overview of each method points out some relevant aspects to consider when selecting common optical microscopy techniques, and includes challenges and advantages regarding each of them.
... IFM: Immunofluorescence microscopy, RT-PCR: Reverse transcriptase Polymerase Chain Reaction, PCR-RFLP: Restriction fragment length polymorphism, LAMP: Loop-mediated isothermal amplification. et al., 2016a,b;Borchardt et al., 2009;Efstratiou et al., 2017;Gallas--Lindemann et al., 2013a,b). For monitoring the microbiological quality of and setting guidelines for irrigation water, this is particularly critical, as explained in Fig. 1. ...
Article
Contaminated irrigation water is among many potential vehicles of human pathogens to food plants, constituting significant public health risks especially for the fresh produce category. This review discusses some available guidelines or regulations for microbiological safety of irrigation water, and provides a summary of some common methods used for characterizing microbial contamination. The goal of such exploration is to understand some of the considerations that influence formulation of water testing guidelines, describe priority microbial parameters particularly with respect to food safety risks, and attempt to determine what methods are most suitable for their screening. Furthermore, the review discusses factors that influence the potential for microbiologically polluted irrigation water to pose substantial risks of pathogenic contamination to produce items. Some of these factors include type of water source exploited, irrigation methods, other agro ecosystem features/practices, as well as pathogen traits such as die-off rates. Additionally, the review examines factors such as food safety knowledge, other farmer attitudes or inclinations, level of social exposure and financial circumstances that influence adherence to water testing guidelines and other safe water application practices. A thorough understanding of relevant risk metrics for the application and management of irrigation water is necessary for the development of water testing criteria. To determine sampling and analytical approach for water testing, factors such as agricultural practices (which differ among farms and regionally), as well as environmental factors that modulate how water quality may affect the microbiological safety of produce should be considered. Research and technological advancements that can improve testing approach and the determination of target levels for hazard characterization or description for the many different pollution contexts as well as farmer adherence to testing requirements, are desirable.
... Given the significant knowledge gap about groundwater in China, a sound understanding of pathogen concentrations in surface and ground waters remains essential to guiding future environmental and public health research and policy (Bradford and Harvey 2017). However, determining Cryptosporidium and Giardia (and other pathogen) concentrations is expensive and time-consuming, and accurate measurement of ambient concentrations of (oo)cysts is difficult, resulting in a scarcity of observational data, especially from developing countries (Efstratiou et al. 2017). Consequently, modeling is a common approach to increasing insight in this area; for example, by pinpointing high concentration hotspots and elucidating the risks and disease burdens associated with waterborne pathogens Limaheluw et al. 2019). ...
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Cryptosporidium and Giardia (major causes of diarrhea) are widely distributed in Chinese source waters and threaten human health. A new spatially explicit GloWPa-TGR-Crypt-Giar C1 model is presented to simultaneously estimate mean monthly (oo)cyst concentrations in surface and ground waters in the Three Gorges Reservoir (TGR) watershed. A quantitative risk assessment of protozoal infections considered different source waters, transmission pathways, regions, susceptible subpopulations, and drinking water treatments. Monthly mean Cryptosporidium oocyst and Giardia cyst concentrations ranged between 0.5–19.3 oocysts/10 L and 0.2–5.0 cysts/10 L in surface water, respectively, and 0.007–0.3 oocysts/10 L and 0.002–0. 2 cysts/10 L in groundwater. The cumulative disease burdens attributable to cryptosporidiosis and giardiasis were, respectively, 5.77×10⁻⁵ DALYs (disability-adjusted life years/person/year) and 4.63×10⁻⁶ DALYs in urban areas, and 6.35×10⁻⁴ DALYs and 8.84×10⁻⁵ DALYs in rural areas, which were much higher than the reference risk level recommended by the World Health Organization (10-6\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${10}^{-6}$$\end{document} DALYs). The annual burden associated with consuming surface water was calculated to be 3.84×10⁻⁴ DALYs for Cryptosporidium and 5.10×10-5\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$5.10\times {10}^{-5}$$\end{document} DALYs for Giardia, whereas consuming groundwater entailed the lower burdens (1.26×10⁻⁵ and 3.50×10⁻⁶ DALYs, respectively). Most DALYs were a consequence of consumption of directly supplied surface water. Fifty percent of the health burden was carried by immunodeficiency with HIV. Children (0–4 years) were more likely to have an individual disease burden than adults (15–64 years). Males were more susceptible than females. Improving sanitation through adequate ozone and microfiltration treatment should be considered when attempting to reduce disease burden. Sensitivity analysis highlighted the importance of reducing (oo)cyst loads to protect the watershed. The methodology and results described will help in evaluating and reducing the burden of protozoal infection associated with surface and ground waters in the TGR and similar watersheds. Graphical Abstract
... Giardia is a major cause of waterborne [22] and foodborne [23] outbreaks of enteric disease in industrialized nations, and genotyping strains in outbreak situations is of epidemiological relevance. In this study, we show that 15 isolates from an outbreak in Italy all shared the same MLST, which was not found in any other isolate included in this study. ...
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Background The flagellated parasite Giardia duodenalis is a major and global cause of diarrhoeal disease. Eight genetically very distinct groups, known as assemblages A to H, have been recognized in the G. duodenalis species complex, two of which (assemblages A and B) infect humans and other mammalian hosts. Informative typing schemes are essential to understand transmission pathways, characterize outbreaks and trace zoonotic transmission. In this study, we evaluated a published multi-locus sequence typing (MLST) scheme for G. duodenalis assemblage A, which is based on six polymorphic markers. Methods We genotyped 60 human-derived and 11 animal-derived G. duodenalis isolates collected in Europe and on other continents based on the published protocol. After retrieving previously published genotyping data and excluding isolates whose sequences showed allelic sequence heterozygosity, we analysed a dataset comprising 146 isolates. Results We identified novel variants at five of the six markers and identified 78 distinct MLST types in the overall dataset. Phylogenetic interpretation of typing data confirmed that sub-assemblage AII only comprises human-derived isolates, whereas sub-assemblage AI comprises all animal-derived isolates and a few human-derived isolates, suggesting limited zoonotic transmission. Within sub-assemblage AII, isolates from two outbreaks, which occurred in Sweden and Italy, respectively, had unique and distinct MLST types. Population genetic analysis showed a lack of clustering by geographical origin of the isolates. Conclusion The MLST scheme evaluated provides sufficient discriminatory power for epidemiological studies of G. duodenalis assemblage A. Graphical Abstract
... can develop malnutrition and cognitive impairments in addition to clinical illness [2]. In industrialized countries, waterborne outbreaks of cryptosporidiosis are common [3]. Among the more than 40 named Cryptosporidium species, Cryptosporidium parvum is the main species for cryptosporidiosis in farm animals and one of the two dominant species in humans [4]. ...
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The protozoan pathogen Cryptosporidium parvum infects intestinal epithelial cells and causes diarrhea in humans and young animals. Among the more than 20 genes encoding insulinase-like metalloproteinases (INS), two are paralogs with high sequence identity. In this study, one of them, INS-16 encoded by the cgd3_4270 gene, was expressed and characterized in a comparative study of its sibling, INS-15 encoded by the cgd3_4260 gene. A full-length INS-16 protein and its active domain I were expressed in Escherichia coli, and antibodies against the domain I and an INS-16-specific peptide were produced in rabbits. In the analysis of the crude extract of oocysts, a ~60 kDa fragment of INS-16 rather than the full protein was recognized by polyclonal antibodies against the specific peptide, indicating that INS-16 undergoes proteolytic cleavage before maturation. The expression of the ins-16 gene peaked at the invasion phase of in vitro C. parvum culture, with the documented expression of the protein in both sporozoites and merozoites. Localization studies with antibodies showed significant differences in the distribution of the native INS-15 and INS-16 proteins in sporozoites and merozoites. INS-16 was identified as a dense granule protein in sporozoites and macrogamonts but was mostly expressed at the apical end of merozoites. We screened 48 candidate INS-16 inhibitors from the molecular docking of INS-16. Among them, two inhibited the growth of C. parvum in vitro (EC50 = 1.058 µM and 2.089 µM). The results of this study suggest that INS-16 may have important roles in the development of C. parvum and could be a valid target for the development of effective treatments.
... Cryptosporidium spp., is an obligatory intracellular protozoan of intestinal, respiratory, and gall bladder epithelia that infect several domestic and wild animals species and humans [1]. It is considered a signi cant public health concern due to its potential transmission through human food and drinks causing outbreak in newborns of humans and animals [2,3]. e main most frequent Cryptosporidium spp. ...
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e present study was conducted to elucidate the prevalence of Cryptosporidium bovis in suckling and weaned cattle calves (Bubalus bubalis) from di erent governorates in northern, middle, and southern Egypt, such as Behera, Meno a, Qaliubiya, Assiut, and Sohag; result revealed that from the overall examined fecal samples (n 825), the overall prevalence was 7.27%, the highest signi cant infection rate was in young suckling calves less than one month (8.2%), and seasonally, winter season has the highest signi cant level (11.24%), but sex and locality were of no signi cant e ect on the prevalence of infection in this study. Gene sequencing and phylogenetic analysis of the 18SSU-rRNA gene of the local bovine isolate were performed, and it was found that C. bovis genotype was highly similar to human isolate, which provoke the zoonotic transmission of bovine isolate to humans and identi ed as a potential source for human cryptosporidiosis infection in Egypt.
... Cryptosporidium spp., is an obligatory intracellular protozoan of intestinal, respiratory, and gall bladder epithelia that infect several domestic and wild animals species and humans [1]. It is considered a signi cant public health concern due to its potential transmission through human food and drinks causing outbreak in newborns of humans and animals [2,3]. e main most frequent Cryptosporidium spp. ...
Article
Full-text available
e present study was conducted to elucidate the prevalence of Cryptosporidium bovis in suckling and weaned cattle calves (Bubalus bubalis) from di erent governorates in northern, middle, and southern Egypt, such as Behera, Meno a, Qaliubiya, Assiut, and Sohag; result revealed that from the overall examined fecal samples (n 825), the overall prevalence was 7.27%, the highest signi cant infection rate was in young suckling calves less than one month (8.2%), and seasonally, winter season has the highest signi cant level (11.24%), but sex and locality were of no signi cant e ect on the prevalence of infection in this study. Gene sequencing and phylogenetic analysis of the 18SSU-rRNA gene of the local bovine isolate were performed, and it was found that C. bovis genotype was highly similar to human isolate, which provoke the zoonotic transmission of bovine isolate to humans and identi ed as a potential source for human cryptosporidiosis infection in Egypt.
... Concerning the detection of protozoa, it is important to emphasize that Cryptosporidium is ubiquitous in water samples; nevertheless, public health authorities are concerned about the human infective species as C. parvum and C. hominis [32,33]. Those species were not analyzed in water samples from the Bay. ...
Article
This study aimed to fast screen the microbiological contamination of recreational waters using a TaqMan Array Card (TAC), a multiplexed platform designed for the simultaneous detection of 35 enteropathogens. Surface and deep marine water samples were concentrated by skimmed milk flocculation and processed for nucleic acid extraction protocol using QIAamp Fast DNA Stool Mini Kit. Twelve microorganisms and parasites, including bacteria (n = 6), protozoa (4), and viruses (2), were detected in 85.7% (24/28) of samples. Campylobacter (82.1%), Cryptosporidium (39.3%), and adenovirus (14.3%) were the most detected pathogens. Neither fungi nor helminths were detected. A spatial pollution profile of microbiological contamination was observed in the area. Methodologies for simultaneous detection of multiple pathogens, such as TAC, can assist decision-makers by providing a quick assessment of the microbiological water quality in areas used for recreational purposes, which in many cases are in accordance with the bacteriological indicators.
... However, high heterogenicity was obtained (I 2 = 85.4%, p < 0.001) even though sampling strategies were adopted from standardized protocols for other waterborne parasites such as Cryptosporidium spp. and G. duodenalis [144]. As expected, similar results were found when analyzing heterogeneity by Cochran's chi-squared (Q = 6679.21 ...
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Toxoplasma gondii is a major foodborne pathogen capable of infecting all warm-blooded animals, including humans. Although oocyst-associated toxoplasmosis outbreaks have been documented, the relevance of the environmental transmission route remains poorly investigated. Thus, we carried out an extensive systematic review on T. gondii oocyst contamination of soil, water, fresh produce, and mollusk bivalves, following the PRISMA guidelines. Studies published up to the end of 2020 were searched for in public databases and screened. The reference sections of the selected articles were examined to identify additional studies. A total of 102 out of 3201 articles were selected: 34 articles focused on soil, 40 focused on water, 23 focused on fresh produce (vegetables/fruits), and 21 focused on bivalve mollusks. Toxoplasma gondii oocysts were found in all matrices worldwide, with detection rates ranging from 0.09% (1/1109) to 100% (8/8) using bioassay or PCR-based detection methods. There was a high heterogeneity (I2 = 98.9%), which was influenced by both the sampling strategy (e.g., sampling site and sample type, sample composition, sample origin, season, number of samples, cat presence) and methodology (recovery and detection methods). Harmonized approaches are needed for the detection of T. gondii in different environmental matrices in order to obtain robust and comparable results.
... Giardia is a waterborne parasite, and its transmission occurs mainly through the fecal-oral route (Efstratiou et al., 2017). In general, studies in developed and developing countries have examined the prevalence and assemblage distribution of Giardia in stool samples of humans and animals that live together (Godínez-Galaz et al., 2019;Lalle et al., 2005;Marangi, Berrilli, Otranto, and Giangaspero, 2010;Rehbein et al., 2019;Volotão et al., 2007). ...
Article
A total of 90 stool samples were collected from dogs, referred to a dog shelter and a veterinary clinic plus 395 stool samples obtained from pet dog owners and shelter keepers, as well as individuals referred to a medical laboratory as controls were collected in Shahryar district, Tehran, Iran. Stool samples were parasitologically examined and the positive G. lamblia isolates were tested with Nested-PCR/sequencing for the tpi, gdh, and bg genes and HRM real-time PCR. Microscopical examination revealed 20 (22.2%) and 34 (8.6%) Giardia-positive samples from dogs and humans, respectively. Regarding HRM real-time PCR, the prevalence of assemblages A and B in humans was 55.8% and 14.7%, respectively. In addition, 14.7% of samples were mix assemblages. HRM real-time PCR detected most of microscopically-positive samples in comparison to PCR/sequencing in both humans and dogs. The high prevalence of assemblages A and B in dogs signified the importance of a same source for infection between dogs and humans.
... Cryptosporidium sparked great public health interest after the large human waterborne outbreaks in Milwaukee in 1993 and rapidly was recognized as one of the most serious waterborne pathogens to date (MacKenzie et al. 1994). The outbreaks of cryptosporidiosis have been and continuously reported in several countries (Karanis et al. 2007;Baldursson & Karanis 2011;Efstratiou et al. 2017). Domestic animals, livestock, wildlife, and humans are potential reservoirs that contribute to the contamination of food, surface waters, and the environment by Cryptosporidium spp. ...
... Cryptosporidium sparked great public health interest after the large human waterborne outbreaks in Milwaukee in 1993 and rapidly was recognized as one of the most serious waterborne pathogens to date (Mac Kenzie et al. 1994). The outbreaks of cryptosporidiosis have been and continuously reported in several countries (Karanis et al. 2007;Baldursson & Karanis 2011;Efstratiou et al. 2017). Domestic animals, livestock, wildlife, and humans are potential reservoirs that contribute to the contamination of food, surface waters, and the environment by Cryptosporidium spp. ...
Article
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Cryptosporidium is an intracellular protozoan parasite, globally distributed and capable of infecting various vertebrate species, including humans as well as domestic and wild animals. Cryptosporidium is increasingly gaining attention as a human and an animal pathogen mainly due to its dominant involvement in worldwide waterborne outbreaks. The present paper reviews the current knowledge and understanding of Cryptosporidium spp. in terrestrial and water animals in Azerbaijan. HIGHLIGHTS First review on knowledge and understanding of Cryptosporidium spp. in terrestrial and water animals in Azerbaijan.; First review zoonotic Cryptosporidium species in Azerbaijan.; Crypto livestock, wildlife, amphibians, birds, and mammals.; An extensive report based on a comparative analysis of morphometric parameters of Crypto oocysts.; High prevalence rates in domestic and wild animals.;
... However, applications are infeasible in some countries due to the high cost and technical and analytical complexity. Efstratiou et al. (2017b) observed that since 2001, Method 1623 has been adopted in almost one third of the publications on Cryptosporidium and Giardia monitoring in water worldwide. However, only 5% of these publications come from Latin American and African countries. ...
Article
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This study evaluated the technology of detection of Giardia spp. cysts and Cryptosporidium spp. oocysts in environmental matrices obtained after water treatment on a bench scale. Calcium carbonate flocculation with immunomagnetic separation was the selected method to quantify the protozoa, and the importance of the number of acid dissociations in the immunomagnetic separation was assessed. When adding the third acid dissociation, an increase of 71% ± 6 in floated residue and 31.9% ± 28.7 in filter backwash water in cyst recovery was observed, while in oocyst recovery, a non-significant increase was detected. In the filtered water, this increased dissociation was important in the protozoa recovery with increases greater than 33%. The results showed that there is a strong interaction of these target organisms with the magnetic microspheres, since protozoa were still recovered in the third acid dissociation and some of them were still adhered to the magnetic microspheres.
... Efstratiou, Ongerth and Karanis (2017a) reported that, in general, Giardia spp. cysts achieve greater recovery than Cryptosporidium spp. ...
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The protozoa Cryptosporidium spp. and Giardia spp. are etiological agents responsible for the transmission of gastroenteritis, mainly due to the consumption of contaminated water. Their (oo)cysts are resistant to adverse environmental conditions, as well as to most conventional water treatment processes. Thus, the objective of this work was to evaluate the occurrence of (oo)cysts of these protozoans in surface water collected for human consumption in the state of Goiás, Brazil. Fifteen samples of raw water were collected to assess the occurrence of (oo)cysts of the protozoa using the Membrane Filtration method, in addition to turbidity and pH analyses. Recovery rates in tests with ultrapure water reached the USEPA (2012) criteria for Giardia (78.1% ± 0%) and for Cryptosporidium (60.6% ± 32.6%); however, recovery in raw water was lower due to turbidity. All samples (n = 15) were positive for Cryptosporidium spp. oocysts, with a maximum concentration of 250 oocysts/L, demonstrating that these protozoa are disseminated in the aquatic environment of the state of Goiás and pose a risk to public health. Due to the use of water sources for public consumption, it is recommended that public authorities and sanitation companies act to preserve and maintain water courses, carry out periodic monitoring of treatment plants that supply the Cerrado, Santana and São Manoel streams and improve existing treatment technologies. The results did not allow to infer whether animal load and grazing area promote an increase in contamination of the lotic aquatic systems.
... Cryptosporidium sparked great public health interest after the large human waterborne outbreak in Milwaukee in 1993 and rapidly was recognized as one of the most serious waterborne pathogens [9]. Outbreaks of cryptosporidiosis have been and continue to be reported in several countries [10][11][12]. Domestic animals, livestock, wildlife, and humans are potential reservoirs that contribute to the contamination of food, surface waters and the environment by Cryptosporidium spp. ...
Article
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Cryptosporidium is an intracellular protozoan parasite and is increasingly gaining attention as a human and an animal pathogen, mainly due to its predominant involvement in worldwide waterborne outbreaks. This paper reviews the current knowledge and understanding of Cryptosporidium spp. in terrestrial and aquatic animals in Azerbaijan. The diagnosis of cryptosporidiosis relies on the identification of oocysts in faecal samples released by the infected host. Stool specimens were processed using the modified acid-fast staining method (Ziehl-Neelsen) and microscopically examined for Cryptosporidium oocysts. Thirteen species of Cryptosporidium (C. fragile, C. ducismarci, C. serpentis, C. varani, C. baileyi, C. meleagridis, C. muris, C. parvum, C. ubiquitum, C. andersoni, C. bovis, C. hominis, C. suis) from amphibians, reptiles, birds and mammals have been identified as a result of studies conducted between 1987 and 2019 on the structural features of Cryptosporidium oocysts in Azerbaijan territory.
... As for propidium iodide incorporation by the protozoan infective forms, the percentage of viable (oo)cysts did not significantly vary throughout the treatment (p > 0.05; Student's t-test), as illustrated in Figure 2. This confirms the need for research that includes viability assessment as a secondary goal to cyst and oocyst detection [55] within sanitary monitoring, as those remaining in the samples may still be viable. ...
Article
Municipal wastewater is a source of pathogenic protozoan (oo)cysts and may play a significant role in spreading waterborne diseases. This scenario becomes more critical as treated sewage from municipal wastewater treatment plants (WWTP) is discharged into springs, which are often used for water supply, irrigation, recreation and, further downstream, indirect potable reuse, quite common in Brazil. This study aimed to elucidate, regarding microbiological quality, the performance of a full-scale WWTP, consisting of preliminary treatment, upflow anaerobic sludge blanket (UASB) reactor, activated sludge system and ultraviolet (UV) radiation disinfection. Pathogenic protozoa (Giardia spp. cysts and Cryptosporidium spp. oocysts), as well as microbiological indicators (Escherichia coli and Clostridium perfringens), were evaluated in terms of their removal. In addition, (oo)cyst viability and fluorescence reduction were assessed. By using the data obtained from this research, the prevalence of infection estimated for the population served by the WWTP was between 7.4 and 14.8% for giardiasis, and between 0.055 and 0.11% for cryptosporidiosis.
... Moreover, Utaaker et al. [25] reported that Cryptosporidium and Giardia (with lower chance) could remain viable on lettuce leaves and increased the chance of contamination in consumers. Although the transmission of Cryptosporidium and Giardia via fecal-contaminated drinking water was well-studied [81][82][83][84][85][86][87][88], it seems that irrigation with raw/treated wastewater, and fertilizing the vegetable farms by either night soil or raw manure can also increase the risk of contamination of crops with enteric pathogens such as Cryptosporidium and Giardia [22,24,89]. ...
Article
Cryptosporidium and Giardia are two major protozoa reported from vegetables and environment. The prevalence of these parasites supposes to be different regarding the climate zones. This review aimed to evaluate the prevalence of Cryptosporidium and Giardia in vegetables according to the major climate zones in Iran. The results showed pooled prevalence 7% (95% CI: 2%, 14%) and 4% (95% CI: 3%, 6%) for Cryptosporidium spp., and Giardia spp., respectively. The prevalence of Giardia spp. in mountain, desert and semi-desert, and Mediterranean regions was 4% (95% CI: 2%, 6%), 5% (95% CI: 3%, 8%) and 7% (95% CI: 1%, 18%), respectively. Cryptosporidium spp. was reported 8% (95% CI: 0%, 65%), 6% (95% CI: 0%, 18%) and 4% (95% CI: 0%, 77%) from mountain, desert and semi-desert, and Mediterranean climate zones, respectively. This review suggests the higher prevalence of Giardia and Cryptosporidium in Mediterranean and mountain regions, respectively.
... Cryptosporidium spp. and Giardia duodenalis are the most common parasitic protozoans responsible for zoonotic diarrhoeal diseases in many countries that are transmitted via water and food contamination (Ahmed and Karanis 2018;Efstratiou et al. 2017;Karanis et al. 2007a;Plutzer and Karanis 2009;Ryan 2010;Ryan et al. 2018). In particular, immunocompromised humans are at a higher risk of contracting Cryptosporidium and Giardia infections, which can develop into a very serious and even life-threatening diarrhoeal disease. ...
Article
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Cryptosporidium and Giardia are well-known parasitic protozoans responsible for waterborne and foodborne diarrhoeal diseases. However, data are not available on market vegetables contaminated with Cryptosporidium and Giardia in China. In the present study, 642 different vegetable samples were collected from Xining City street vendors in the Qinghai Province to study the Cryptosporidium and Giardia contamination rates via PCR and sequence analyses. Cryptosporidium spp. and Giardia duodenalis were detected in 16 (2.5%) and 73 (11.4%) samples, respectively. Two species of Cryptosporidium, C. parvum (n = 11) and C. andersoni (n = 5), were identified. G. duodenalis assemblage B was identified in almost all positive samples (n = 72), except one sample that contained G. duodenalis assemblage E. We report on the rate of Cryptosporidium and Giardia contamination in vegetables for the first time from the Qinghai Tibetan Plateau Area (QTPA) in China.
Article
Laboratory procedures performed in water treatment studies frequently require the characterization of (oo)cyst suspensions. Standard methods commonly used are laborious, expensive and time-consuming, besides requiring well-trained personnel to prepare samples with fluorescent staining and perform analysis under fluorescence microscopy. In this study, an easy cost-effective in situ microscope was assessed to acquire images of Giardia cysts directly from agitated suspensions without using any chemical labels or sample preparation steps. An image analysis algorithm analyzes the acquired images, and automatically enumerates and provides morphological information of cysts within 10 min. The proposed system was evaluated at different cyst concentrations, achieving a limit of detection of ~30 cysts/mL. The proposed system overcomes cost, time and labor demands by standard methods and has the potential to be an alternative technique for the characterization of Giardia cyst suspensions in resource-limited facilities, since it is independent of experts and free of consumables.
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Pathogenic protozoa endanger human health and challenge water treatment, especially during outbreaks in developing countries. For instance, Giardia spp. cysts can recirculate in the filter backwash water (FBW), reinforcing the relevance of their detection protocol in negligible matrices. This study aimed to detect Giardia spp. cysts in the FBW by direct centrifugation (DC) and immunomagnetic separation (IMS) with the addition of the detergent dispersion solution ICN 7X at 1.0%. To do this, cyst suspensions were inoculated into FBW samples (14 NTU), which was generated in bench-scale drinking-water treatment with high-turbidity study water (112 NTU). Furthermore, the DC+ICN 7X method was compared with the calcium carbonate flocculation. For instance, the DC+ICN 7X method provided cleaner microscope slides and minor damage to the cyst walls. The commercial suspension of Giardia lamblia had an adequate recovery rate (19.5%). However, the recovery rate of the EasySeed® suspension was 7.8%, which was below the required range by Method 1623.1 (above 8%). High costs and low efficiencies challenge several methods for detecting Giardia spp. cysts. Therefore, future studies should develop and improve detection protocols, especially for complex matrices. Detecting cysts in water treatment residues is crucial for addressing current sanitation issues in developing countries. HIGHLIGHTS Adding the detergent dispersion solution ICN 7X improved the direct centrifugation (DC) method for concentrating cysts.; After the immunomagnetic separation, the DC+ICN 7X protocol presented clean reading wells and minor damage to the cyst walls.; Cyst recoveries were 19.5 and 7.8% for the commercial Giardia lamblia and EasySeed® suspensions, respectively.; Detection protocols for Giardia spp. cysts showed low efficiency and high costs.;
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Giardiasis is an intestinal disease caused by the parasite protozoan Giardia intestinalis. For more than five decades, the treatment of this disease has been based on compounds such as nitroimidazoles and benzimidazoles. The parasite's adverse effects and therapeutic failure are largely recognized. Therefore, it is necessary to develop new forms of chemotherapy treatment against giardiasis. Lysine deacetylases (KDACs), which remove an acetyl group from lysine residues in histone and non-histone proteins as tubulin, are found in the Giardia genome and can become an interesting option for giardiasis treatment. In the present study, we evaluated the effects of 4-[(10H-phenothiazin-10-yl)methyl]-N-hydroxybenzamide, a new class I/II KDAC inhibitor, on G. intestinalis growth, cytoskeleton, and ultrastructure organization. This compound decreased parasite proliferation and viability and displayed an IC50 value of 179 nM. Scanning electron microscopy revealed the presence of protrusions on the cell surface after treatment. In addition, the vacuoles containing concentric membranous lamella and glycogen granules were observed in treated trophozoites. The cell membrane appeared deformed just above these vacuoles. Alterations on the microtubular cytoskeleton of the parasite were not observed after drug exposure. The number of diving cells with incomplete cytokinesis increased after treatment, indicating that the compound can interfere in the late steps of cell division. Our results indicate that 4-[(10H-phenothiazin-10-yl)methyl]-N-hydroxybenzamide should be explored to develop new therapeutic compounds for treating giardiasis.
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Waterborne diseases are a relevant concern for public health systems since commonly applied treatment techniques may not remove all water contaminants. Cryptosporidium spp. oocysts are an issue in water treatment plants due to their reduced size and resistance to the disinfection process (e.g., chlorination). Moreover, oocysts retained on the filter media might recirculate amongst the filter backwash water (FBW). This study aimed to detect Cryptosporidium parvum oocysts artificially inoculated on the FBW and evaluate the ozone treatment performance. A synthetic FBW underwent three concentration methods followed by immunomagnetic separation: calcium carbonate flocculation, direct centrifugation, and direct centrifugation with the 7X ICN dispersion solution. The latter method was selected as it presented less interference on oocyst viability (37.2% reduction) and higher recovery (22.1%) on preliminary assays. The recovery for the commercial suspensions was 15.4 ± 3.3%, although the analytical quality performed with EasySeed® suspension obtained a recovery of 2.8 ± 0.8%. These limitations and methodologies for protozoan detection are challenging due to low recoveries, especially in complex matrices. Finally, FBW ozonation was performed on a pilot scale, and the propidium iodide dye indicated oocyst viability decreased after treatment. Oocyst inactivation was 2.83 log and 3.44 log for dosages of 7.5 mg O3 L⁻¹ for 10 min (i.e., 75 mg min L⁻¹) and 10 mg O3 L⁻¹ for 5 min (i.e., 50 mg min L⁻¹), respectively. Disinfection is a crucial pathway for addressing outbreak scenarios, and ozone treatment should be further studied.
Article
Cryptosporidium spp. is one of the prime agents of infectious diarrhea. Cryptosporidium spp. has been gaining awareness as a pathogen of public health importance in India and other developing countries. Owing to the nature of multiple transmission routes such as person-to-person, animal-to-person, waterborne and foodborne, the epidemiology of cryptosporidiosis in humans is not well known. A deeper understanding of the pathogenesis may lead to better diagnosis and better treatment of the condition. Asymptomatic human and animal transmission illustrates that the spread of infection through the environment is a more plausible explanation, waterborne transmission in particular. The disease burden is underestimated and its global impact is yet to be quantified due to the lack of country-specific estimates. Assessment of the disease itself has been crucial since the morphological indistinguishability, differences in distribution and transmission, and variations in the genotypes.
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Giardia duodenalis and Cryptosporidium spp. are two of the most prominent aetiological agents of waterborne diseases. Therefore, efficient and affordable methodologies for identifying and quantifying these parasites in water are increasingly necessary. USEPA Method 1623.1 is a widely used and validated protocol for detecting these parasites in water samples. It consists of a concentration step, followed by parasite purification and visualization by immunofluorescence microscopy. Although efficient, this method has a high cost particularly due to the immunomagnetic separation (IMS) step, which is most needed with complex and highly contaminated samples. Based on this, the present study aimed to determine whether it is possible to maintain the efficiency of Method 1623.1 while reducing the amount of beads per reaction, using as a matrix the challenge water recommended by the World Health Organization. As for Giardia cysts, a satisfactory recovery efficiency (RE) was obtained using 50% less IMS beads. This was evaluated both with a commercial cyst suspension (56.1% recovery) and an analytical quality assessment (47.5% recovery). Although RE rates obtained for Cryptosporidium parvum did not meet Method 1623.1 criteria in any of the experimental conditions tested, results presented in this paper indicated the relevance of the described adaptations, even in challenge water. HIGHLIGHTS The high cost of current protozoa detection methods limits their widespread use in limited settings.; Immunomagnetic separation improves detection by cleaning the sample.; Recovery efficiency is maintained for Giardia duodenalis with 50% less beads.; Organisms adhering to beads after dissociation may impact recovery levels.;
Article
Giardia duodenalis is a protozoan parasite infecting the upper intestinal tract of humans, as well as domestic and wild animals worldwide. Transmission of giardiasis occurs through the faecal-oral route, and may be either direct (i.e., person-to-person, animal-to-animal or zoonotic) or indirect (i.e., waterborne or foodborne). While asymptomatic infections are common in both humans and animals, a wide range of enteric symptoms have been reported, along with extra-intestinal and post-infectious complications. A definitive diagnosis of giardiasis is generally made by detection of cysts in stool specimens through microscopical examination of wet mounts, or through the use of permanent or fluorescent antibody stains. More recently, molecular methods have become popular for diagnosis and for testing environmental samples. Symptomatic giardiasis is often treated to reduce the duration of symptoms, to prevent complications, and to minimize transmission of the parasite to other hosts. Direct faecal-oral transmission of giardiasis can be largely controlled thorough improved hygiene and sanitation. In the case of waterborne transmission, a multiple barrier approach, including limiting access of people and animals to watersheds and reservoirs, and treatment using flocculation, filtration and disinfection, is necessary to minimize the risk. Since foodborne transmission is often associated with the consumption of fresh produce, a number of control measures can be taken during pre- and post-harvest, as well as at the food handler/consumer level to minimize the risk of contamination, or for removing or inactivating parasites. Good husbandry and farm management practices are important in controlling the spread of giardiasis in livestock and companion animals.
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This study uncovered the prevalence, harboured species, and subtype diversity of Cryptosporidium species in river water and its sediment from the Apies River in South Africa. Cryptosporidium spp. concentrations in freshwater and its sediment were determined using Ziehl-Neelsen staining and quantitative Polymerase Chain Reaction (qPCR) techniques. Next-generation sequencing (NGS) targeting the 60 kDa glycoprotein (gp60) gene of Cryptosporidium spp. was performed to reveal the species, subtype families and subtypes harboured in freshwater and its sediment. Although the results revealed that water samples had a higher prevalence (30%) compared with sediment (28%), the number of observable Cryptosporidium spp. oocysts in sediment samples (ranging from 4.90 to 5.81 log10 oocysts per 1 Liter) was higher than that of river water samples (ranging from 4.60 to 5.58 log10 oocysts per 1 L) using Ziehl-Neelsen staining. The 18S ribosomal ribonucleic acid (rRNA) gene copy of Cryptosporidium in riverbed sediments ranged from 6.03 to 7.65 log10, whereas in river water, it was found to be between 4.20 and 6.79 log10. Subtyping results showed that in riverbed sediments, Cryptosporidium parvum accounted for 40.72% of sequences, followed by Cryptosporidium hominis with 23.64%, Cryptosporidium cuniculus with 7.10%, Cryptosporidium meleagridis with 4.44% and the least was Cryptosporidium wrairi with 2.59%. A considerable percentage of reads in riverbed sediment (21.25%) was not assigned to any subtype. River water samples had 45.63% of sequences assigned to C. parvum, followed by 30.32% to C. hominis, 17.99% to C. meleagridis and 5.88% to C. cuniculus. The data obtained are concerning, as Cryptosporidium spp. have intrinsic resistance to water treatment processes and low infectious doses, which can pose a risk to human health due to the various uses of water (for human consumption, leisure, and reuse).
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Cryptosporidium spp. and Giardia duodenalis are two waterborne protozoan parasites that can cause diarrhea. Human and animal feces in surface water are a major source of these pathogens. This paper presents a GloWPa-TGR-Crypto model that estimates Cryptosporidium and G. duodenalis emissions from human and animal feces in the Three Gorges Reservoir (TGR), and uses scenario analysis to predict the effects of sanitation, urbanization, and population growth on oocyst and cyst emissions for 2050. Our model estimated annual emissions of 1.6 × 10 ¹⁵ oocysts and 2.1 × 10 ¹⁵ cysts from human and animal feces, respectively. Humans were the largest contributors of oocysts and cysts, followed by pigs and poultry. Cities were hot-spots for human emissions, while districts with high livestock populations accounted for the highest animal emissions. Our model was the most sensitive to oocyst excretion rates. The results indicated that 74% and 87% of total emissions came from urban areas and humans, respectively, and 86% of total human emissions were produced by the urban population. The scenario analysis showed a potential decrease in oocyst and cyst emissions with improvements in urbanization, sanitation, wastewater treatment, and manure management, regardless of population increase. Our model can further contribute to the understanding of environmental pathways, the risk assessment of Cryptosporidium and Giardia pollution, and effective prevention and control strategies that can reduce the outbreak of waterborne diseases in the TGR and other similar watersheds.
Chapter
While this book focuses on emerging methods for the detection of waterborne pathogens, this chapter is devoted to sample processing which plays a key role in the ultimate success of any detection technology. Many of the techniques discussed in the following chapters process μL to a few mL, whereas it may be necessary to sample hundreds of mL to thousands of litres. Large sample volumes are required to gain a more representative sample of a large water volume as well as to increase the likelihood of detecting pathogens present at very low concentrations. Sampling of a large volume over a period of time will to some extent compensate for some spatial and temporal variations in pathogen distribution. Additionally, the concentration aspect of sample processing is necessary to bring the pathogen concentration into the detection limit of monitoring methods. Furthermore, while some techniques would detect single pathogens, the time required for a single pathogen in a large volume of water to reach the detection area/surface could be prohibitively long. Sample processing is also important for isolation of the pathogen of interest and for the removal of interferents which could disturb the detection process. The nature of problematic interferents varies with the final monitoring technique employed and therefore different sample processing methods may be required depending on the full scheme of detection.
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Cysts and (oo)cysts are the infective forms of parasitic protozoa, as Giardia and Cryptosporidium, which are widespread and associated to worldwide waterborne diseases outbreaks. These microorganisms pose a challenge to public health, as they are resistant to conventional disinfection methods, which make them important parameters when evaluating inactivation efficiency. However, when (oo)cysts are targets, it is challenging to infer inactivation efficacy, as it may require infectivity tests that are not often an option for laboratory routine analysis. In this scene, (oo)cyst viability based on induced excystation, membrane integrity and enzyme activity evaluated by dye inclusion and/or exclusion, as well as fluorescence reduction consist on microscopy-based techniques that may be options to estimate inactivation in the environmental context. This scoping review presents applications, advantages and limitations of these methodologies for viability assessment, in order to shed light on the (oo)cyst viability topic and provide insight strategies for choosing protocols in the environmental and sanitation field, in laboratory applications and novel research.
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Cryptosporidium spp. is one of the most important waterborne pathogens worldwide and a leading cause of mortality from waterborne gastrointestinal diseases. Detection of Cryptosporidium spp. in water can be very challenging due to their low numbers and the complexity of the water matrix. This review describes the biology of Cryptosporidium spp. and current methods used in their detection with a focus on C. parvum and C. hominis. Among the methods discussed and compared are microscopy, immunology-based methods using monoclonal antibodies, molecular methods including PCR-based assays, and emerging aptamer-based methods. These methods have different capabilities and limitations, but one common challenge is the need for better sensitivity and specificity, particularly in the presence of contaminants. The application of DNA aptamers in the detection of Cryptosporidium spp. oocysts show promise in overcoming these challenges, and there will likely be significant developments in aptamer-based sensors in the near future.
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We report a field-portable and cost-effective imaging flow cytometer that uses deep learning to accurately detect Giardia lamblia cysts in water samples at a volumetric throughput of 100 mL/h. This flow cytometer uses lensfree color holographic imaging to capture and reconstruct phase and intensity images of microscopic objects in a continuously flowing sample, and automatically identifies Giardia lamblia cysts in real-time without the use of any labels or fluorophores. The imaging flow cytometer is housed in an environmentally-sealed enclosure with dimensions of 19 cm × 19 cm × 16 cm and weighs 1.6 kg. We demonstrate that this portable imaging flow cytometer coupled to a laptop computer can detect and quantify, in real-time, low levels of Giardia contamination (e.g., <10 cysts per 50 mL) in both freshwater and seawater samples. The field-portable and label-free nature of this method has the potential to allow rapid and automated screening of drinking water supplies in resource limited settings in order to detect waterborne parasites and monitor the integrity of the filters used for water treatment.
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Land use/Land cover (LULC) associated with Cryptosporidium sp. and Giardia sp. quantification and distribution can provide identification of the environmental circulation patterns of these parasites. The aim of this research was to relate the occurrence and circulation of these parasites to the LULC watershed with poor sanitation infrastructure and livestock as important economic activity. The study involved 11 municipalities in the state of São Paulo, located in southeastern Brazil. Sampling was carried out at the catchment sites of each water supply on a monthly basis, starting in December 2014 and lasting until November 2015, totalizing 128 samples. Protozoans were quantified according to the 1623.1 US. EPA Method. For watershed delimitation, the hydrographic network was extracted from the hydrology tool of ArcGIS 10.1. The frequency of occurrence of these pathogens and the high concentrations were evidenced in the municipality with the largest urban area (16.2%) and intense livestock activity (39%) near the catchment site. The municipality that showed the lowest frequency of occurrence presented the smallest urban area (0.87%) and absence of livestock activity near the catchment site. The high concentration of pathogens suggests a correlation between the impact on water supply networks and river basin degradation caused by urban activity and livestock.
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Giardia and Cryptosporidium are pathogenic protozoa often present in the environment in their infective form(cysts and oocysts). These parasites are very resistant to disinfection, which makes them important target organisms in environmental quality monitoring and sanitation. Viability assessment provides an interpretation of cell inactivation, and it can be evaluated by membrane integrity as well as enzyme activity, usingdifferent staining methods. These are straightforward and adequate to laboratories that lack infrastructure for molecular-based technologies or animal infectivity tests. This study investigated simultaneous staining by a commercial live/dead kit, in order to assess viability of Cryptosporidium parvum oocysts and Giardia muris cysts, comparing it to propidium iodide (PI) incorporation, a common stain applied in viability estimation. Results suggested that,although the central hypothesis of one-panel visualization (α=0.05) was met, simultaneous staining impaired (oo)cyst detection by immunofluorescence assay (IFA), which was found to be essential to enumeration, as the live/dead test led to poor (oo)cyst labeling or a 10-fold lower recovery when carried out concomitantly to IFA. As for the viability assessment itself, although red dye uptake occurred as expected by dead or weakened organisms, neitherlive G. muris cysts orC. parvum oocysts present any green fluorescence by esterase metabolism. This may have been caused by low enzyme activity in the infective form and/or wall thickness of these parasites. The results do not exclude the possibility of simultaneous fluorescence staining for protozoa, but it is a starting point for a broader analysis, that may consider, for instance, different incubation conditions.
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With funding from the European Commission, a consortium of members of the European Water Research Institutes is carrying out a programme of work with the objective of optimising and standardising a method for determining the presence in water of (oo)cysts of Cryptosporidium and Giardia. Each of the stages of the conventional analysis procedure (initial concentration, recoveryand identification and enumeration) are being investigated and the relative merits of existing and new methods are being assessed. Newly developed filters (Envirochek and Filta-Max) have been shown to be more efficient for initial recovery of (oo)cysts from water than the previously used Cuno cartridge filters. In addition, for the analysis of raw waters, flocculationwith ferric sulphate has been shown to give recoveries similar to the Envirochek and Filta Max. Modern purification systems such as immunomagnetic separation have also been assessed and found to offer some advantages over flotation although optimisation of the latter has brought improved efficiency. Preliminary assessment of solid phase cytometry has indicated that this technique could offer significant time savings compared to conventional microscopic counting. The results of the study will be used to propose a revised standard method to CEN.
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In this study, hollow-fiber ultrafiltration (UF) was assessed for recovery of Escherichia coli, Clostridium perfringens spores, Cryptosporidium parvum oocysts, echovirus 1, and bacteriophages MS2 and ΦX174 from ground and surface waters. Microbes were seeded into twenty-two 50-L water samples that were collected from the Southeastern United States and concentrated to ∼500 mL by UF. Secondary concentration was performed for C. parvum by centrifugation followed by immunomagnetic separation. Secondary concentration for viruses was performed using centrifugal ultrafilters or polyethylene glycol precipitation. Nine water quality parameters were measured in each water sample to determine whether water quality data correlated with UF and secondary concentration recovery efficiencies. Average UF recovery efficiencies were 66%-95% for the six enteric microbes. Average recovery efficiencies for the secondary concentration methods were 35%-95% for C. parvum and the viruses. Overall, measured water quality parameters were not significantly associated with UF recovery efficiencies. However, recovery of ΦX174 was negatively correlated with turbidity. The recovery data demonstrate that UF can be an effective method for concentrating diverse microbes from ground and surface waters. This study highlights the utility of tangential-flow hollow fiber ultrafiltration for recovery of bacteria, viruses, and parasites from large volume environmental water samples.
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Cryptosporidium spp. oocyst recovery in water and milk samples was evaluated. Samples were inoculated with a suspension of 1.2×107Cryptosporidium spp. oocysts and submitted to centrifugal flotation, using different solutions (sucrose, NaCl, MgSO4, ZnSO4, AlSO4, NH4SO4 40% and NH4SO4 80%). Centrifugation of the samples was carried out in two stages for concentration using two methods that differed in the order in which the saturated solutions were used, namely only in the first stage of method I and only in the second stage of method II. Oocyst identification was performed using the Kinyoun and Koster histochemical staining techniques. Samples analyzed by method I showed different degree of oocyst recovery, namely 10.9% with NaCl and 42.5% with MgSO4 in water and milk samples, while those samples analyzed by method II showed 10.6% with NaCl and 5.3% with sucrose in water and milk, respectively. Histochemical staining methods have no influence on the degree of oocysts recovery. The efficiency of Cryptosporidium spp. oocysts recovery methods depends on the nature and composition of the sample and on the methodology used for oocyst concentration.
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Water samples were collected from four locations on two rivers in Washington State and analyzed by membrane filtration-immunofluorescence assay to establish Cryptosporidium oocyst concentrations. Sampling locations were selected to evaluate effects of watershed character, from pristine mountain to downstream agricultural, on oocyst concentrations. Samples were collected at six biweekly intervals from late June to early September, with two additional sets of five samples taken on separate days (one set taken at bihourly intervals and one set taken simultaneously). Cryptosporidium oocysts were found in 34 of 35 samples at concentrations ranging from about 0.2 to 65 oocysts per liter. Oocyst concentrations were highest early in the sampling period, when they were influenced by postrainfall runoff, and decreased through the summer months. Oocyst concentrations found in ten samples collected on two days (5 samples per day) showed no short-term variations. Oocyst concentrations and oocyst production per square mile (ca. 2.6 km2) of watershed found in water draining a controlled public water supply watershed were the lowest observed. The concentrations and production rates for drainage from an adjacent, comparable, but uncontrolled watershed were nearly 10 times higher. The concentration and production rates of the downstream area influenced by dairy farming were nearly 10 times higher than rates at the upstream stations. The data showed clearly that oocyst concentrations were consistently observed above the detection limit of the analytical method, about 0.1 oocysts per liter; that oocyst concentrations were continuous as opposed to intermittent; and that watershed character and management affected surface water oocyst concentrations significantly.
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Quantitative real-time polymerase chain reaction (qPCR) assays to detect Cryptosporidium oocysts in clinical samples are increasingly being used to diagnose human cryptosporidiosis, but a parallel approach for detecting and identifying Cryptosporidium oocyst contamination in surface water sources has yet to be established for current drinking water quality monitoring practices. It has been proposed that Cryptosporidium qPCR-based assays could be used as viable alternatives to current microscopic-based detection methods to quantify levels of oocysts in drinking water sources; however, data on specificity, analytical sensitivity, and the ability to accurately quantify low levels of oocysts are limited. The purpose of this study was to provide a comprehensive evaluation of TaqMan-based qPCR assays, which were developed for either clinical or environmental investigations, for detecting Cryptosporidium oocyst contamination in water. Ten different qPCR assays, six previously published and four developed in this study were analyzed for specificity and analytical sensitivity. Specificity varied between all ten assays, and in one particular assay, which targeted the Cryptosporidium 18S rRNA gene, successfully detected all Cryptosporidium spp. tested, but also cross-amplified T. gondii, fungi, algae, and dinoflagellates. When evaluating the analytical sensitivity of these qPCR assays, results showed that eight of the assays could reliably detect ten flow-sorted oocysts in reagent water or environmental matrix. This study revealed that while a qPCR-based detection assay can be useful for detecting and differentiating different Cryptosporidium species in environmental samples, it cannot accurately measure low levels of oocysts that are typically found in drinking water sources.
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Water samples from three pristine rivers in the Pacific Northwest were analyzed for Giardia cyst presence and concentration using a membrane-filtration-immunofluorescence-assay procedure. A total of 222 samples were collected either monthly or bimonthly over a nine-month period from 17 sampling stations on three rivers and 12 tributaries. Cyst recovery efficiency was monitored using samples seeded with cysts at levels ranging from 0.5 to 50 cysts/L. The recovery efficiency of the procedure averaged 21.8 percent ± 6 percent through 26 sets of samples. Giardia cysts were found in 94 (43 percent) of the samples. The corresponding cyst concentrations calculated from the recovery efficiency and the sample volume ranged from 0.1 to 5.2 cysts/L. The distribution of cyst concentrations in positive samples was lognormal. Both the magnitude of cyst concentrations (as indicated by the mean value) and the variability (as indicated by the slope of the distributions) differed among the three rivers. No statistically supportable seasonal variations were found. The principal conclusion was that Giardia cysts appear to be continuously present, though at low concentrations, even in relatively pristine rivers.
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Beginning in 2006 a USA Federal regulation required public water suppliers using surface water serving more than 10,000 population to analyse for Cryptosporidium at least 24 consecutive monthly samples from each surface water source. In July of 2012, the USEPA released the resulting data consisting of ca. 45,000 records. No Cryptosporidium were found in 93% of samples and no Cryptosporidium were found in any samples analysed from over half of 1670 locations sampled. Never-the-less at 250 locations in every region of the US Cryptosporidium were found in sufficient numbers of samples to provide a picture of their occurrence nation-wide. Data from about 100 sites reporting the highest numbers were examined in detail. Although analysis of matrix spikes was required for quality control results do not permit estimating organism concentrations. The data reported at each of the individual sample locations were analysed in the form of cumulative probability distributions to describe key risk-related features of median level and variability. Taken as a whole, the data describe a spectrum of median Cryptosporidium occurrence in surface waters of the USA ranging from ca. 0.005 oocysts/L to ca. 0.5 oocysts/L. The variability at individual sites ranged from ca. 1 to 15 r.s.d. Based on the LT2 positive data, comparison to measurements other water quality parameters, and independent means of estimating organism production from watersheds reported in the literature the hypothesis is offered that Cryptosporidium may be found in surface water anywhere world-wide continuously and within the spectrum defined above.
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We evaluated the occurrence of Cryptosporidium and Giardia in the Lake Baroon catchment, Queensland, Australia. A total of 132 water samples were collected from 6 sites as well as from raw influent and treated effluent from a local sewage treatment plant (STP), over a 12-month period. Three new sites close to the entrance of the lake were added following initial assessment The prevalence of faecal indicator organisms, E coli and enterococci was also examined. There was no clear relationship between the level of indicator organisms, rainfall or the detection of Cryptosporidium and Giardia for most sites. Of the five sites showing levels of contamination with Cryptosporidium, only three coincided with elevated rainfall. The highest level of Cryptosporidium oocysts detected in the raw influent from the STP, coincided with the occurrence of an outbreak of cryptosporicliosis in the local area but no Cryptosporidium was detected in surface water samples during that period. our results indicate that while the human contribution of Cryptosporidium is minimal, the high level of faecal indicator bacteria in surface water was not always associated with the presence Cryptosporidium in this catchment.
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Bacterial indicators are used to indicate increased health risk from pathogens and to make beach closure and advisory decisions; however, beaches are seldom monitored for the pathogens themselves. Studies of sources and types of pathogens at beaches are needed to improve estimates of swimming-associated health risks. It would be advantageous and cost-effective, especially for studies conducted on a regional scale, to use a method that can simultaneously filter and concentrate all classes of pathogens from the large volumes of water needed to detect pathogens. In seven recovery experiments, stock cultures of viruses and protozoa were seeded into 10-liter lake water samples, and concentrations of naturally occurring bacterial indicators were used to determine recoveries. For the five filtration methods tested, the highest median recoveries were as follows: glass wool for adenovirus (4.7%); NanoCeram for enterovirus (14.5%) and MS2 coliphage (84%); continuous-flow centrifugation (CFC) plus Virocap (CFC+ViroCap) for Escherichia coli (68.3%) and Cryptosporidium (54%); automatic ultrafiltration (UF) for norovirus GII (2.4%); and dead-end UF for Enterococcus faecalis (80.5%), avian influenza virus (0.02%), and Giardia (57%). In evaluating filter performance in terms of both recovery and variability, the automatic UF resulted in the highest recovery while maintaining low variability for all nine microorganisms. The automatic UF was used to demonstrate that filtration can be scaled up to field deployment and the collection of 200-liter lake water samples.
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A total of 262 water-related samples were analysed for the presence of Cryptosporidium spp. oocysts, of which 39.3% were positive, over a twelve month period. Similar data were obtained for their occurrence in samples of both raw and treated water from Scotland, with 40.5% of raw water and 40.1% of treated water being positive. Fewer oocysts were detected in the summer than in the autumn, winter or spring. In a 12 month survey of water used for abstraction, oocysts were detected at various times throughout the year in both raw and treated water. A series of conventional and fluorogenic dyes was used, in conjunction with a fluorescent labelled monoclonal antibody, in an attempt to assess their potential fox imprpving the identification of oocysts in water-related samples. In addition, preliminary data regarding the possible use of fluorogenic vital dyes as indicators of in vitro excystation and viability are presented.
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Modifications of the existing methods for the isolation of Giardia spp. cysts from water enabled up to 33% of seeded cysts to be recovered. Using this method, 263 environmental samples were analysed for the presence of cysts, of which 34.4% were positive. All sewage effluents, 85% of recreational waters, 46% of raw potable water samples and 22% of treated water samples contained cysts (range 0.14 - 359/litre). A higher percentage of positive samples occurred in the summer and autumn in raw water, whereas the levels of cysts in treated waters showed little seasonal variation. A fluorogenic vital dye assay, which compares favourably with in vitro excystation, has been developed, and using this assay, viable cysts wire detected in 33% of the positive raw water samples and 42% of the positive treated water samples.
Article
During a period of 9 months (May 1995-January 1996), various environmental sources were investigated for the presence of Giardia and Cryptosporidium in Israel. Out of 15 samples from five streams, 12 were positive for Cryptosporidium (80%) with an average concentration of 0.04–1.9 oocysts/l and 8 were positive for Giardia (53.3%) with 0.05–0.78 cysts/l. Two springs were also tested and found positive for Cryptosporidium and (0.54 oocysts/l) only. The main drinking water reservoir of Israel, Lake Kineret, was also sampled 6 times at two sites. Cryptosporidium was isolated in 4/6 samples (66.6%) at an average concentration of 0.3–1.09 oocysts/l while Giardia was present in 5/6 samples (83.3%), 0.135–16.2 cysts/l. Drinking water entering a filtration pilot plant was also tested and found positive for Cryptosporidium in 23/35 samples (0–317 oocysts/l) and 8/35 for Giardia (0–16.7 cysts/l). In order to evaluate some potential inputs of contamination of the drinking water sources, two possible contributors were tested: domestic sewage and cowshed effluents. In sewage effluents, 3/3 samples were positive for both Cryptosporidium and Giardia. The oocysts and cysts were present at 8.3–8.05/l and 5–27.3/l respectively. In cowshed effluents, 1/6 samples was positive for Cryptosporidium at a high concentration (3,630 oocysts/l) but no Giardia cysts were found. The levels of Cryptosporidium and Giardia oocysts and cysts isolated from these environmental samples may present a public health hazard although no major outbreaks have so far been reported in Israel.
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A prototype immunomagnetic separation (IMS) technique was tested in five laboratories, which undertake routine analysis of water samples for Cryptosporidium oocysts, by comparing the recovery efficiency of the IMS technique with techniques in current use (the “Blue Book” Standing Committee of Analysts, SCA, method and flow cytometry, FCM). In very low turbidity samples (clean waters) of both 1ml and 10ml volumes the IMS demonstrated significantly better results than both SCA and FCM methods. Not only were higher oocyst recovery efficiencies reported but variation in recovery efficiency was reduced and fewer negative results were reported from oocyst-seeded samples than with the other two techniques. In trials with clean water or low turbidity water, FCM was the technique which most consistently reported negative results in oocyst-seeded samples and for clean water this difference was found to be statistically significant. When the water sample was turbid the recovery efficiency of the IMS technique diminished. The results suggest that the IMS technique is affected to different extents by different material constituents in water concentrates and that FCM is apparently less affected by interfering particulate matter. Despite the potential difficulties with the IMS method with turbid water samples, the results from these trials indicate that this technique would be a very useful addition to the armoury of methods for the concentration of oocysts from water samples and was considered by the trial participants to be simple, user-friendly and applicable to the processing of multiple samples simultaneously.
Article
We have established a simple procedure for the enumeration of Giardia cysts in environmental waters of Hong Kong. The procedure involves concentrating water or sewage samples by the flocculation-percoll/sucrose gradient method followed by a modified tube method using immunofluorescent staining containing fluorescein isothiocyanate labelled anti-Giardia monoclonal antibodies. Enumeration of cysts was carried out using fluorescent microscopy. This procedure has the highest recovery rate (61%) compared with other conventional procedures tested (0-4%). This method was successfully applied to the detection and enumeration of Giardia cysts in water samples collected from two selected rivers of Hong Kong, Shing Mun River and Lam Tsuen River. Preliminary results indicate that water of both rivers has high counts of Giardia cysts.
Article
This study has been conducted, to estimate the distribution of Giardia and Cryptosporidium in German water supplies and the removal efficiency of surface water treatment plants for Giardia and Cryptosporidium by conventional treatment. Water samples from six surface water treatment plants in different parts of Germany were simoultaneously examined for Giardia and Cryptosporidium. Investigations for both parasites were carried out in the period from July 1993 until December 1995. The results confirmed the occurrence of Giardia and Cryptosporidium in surface and raw water, in intermediate steps after treatment, in back wash water, in the first filtrate and in final water. Giardia or Cryptosporidium or both have been found in 76.2% of the investigated raw water sources. The average number of the detected Giardia cysts was 88.2/100 1 (max. 1314/100 1), and the average number of Cryptosporidium oocysts was 116/100 1 (max. 1081/100 1). In the intermediate steps (including flocculation and several steps of filtration), Giardia or Cryptosporidium or both have been found in 33.3% (50/150) of the samples. 14.9% of drinking water samples (7/47) were positive for Giardia (max. 16.8/100 1) and 29.8% (14/47) were positive for Cryptosporidium (max. 20.8/100 1). Overall, Giardia and Cryptosporidium, or both were detected in 38.3% of the drinking water samples. The parasites have been found in nearly all of the investigated backwash water samples. The filtrate of a rapid sand filter was analysed immediately after filter backwashing during the ripening period of the filter. Good elimination results were obtained by optimizing relevant water treatment process, but a low flocculant dose following sudden variation in the raw water quality, causes a breakthrough of Cryptosporidium into the treated water. Although water treatment technologies are effective to remove Giardia and Cryptosporidium, the results clearly show that Giardia and Cryptosporidium evade the filter barries in the absence of visible treatment deficiencies and low turbitidy level, and contaminate final water.
Article
A total of 64 beach water samples with various bacteriological quality (Grades 1 to 4) were analysed for their bacteriological and parasitological contents (E coli and Giardia cysts respectively). Results indicated that Giardia cysts were detected in less than 10% of the Grade 1 beach water samples with E coli concentrations of <24/100mL. For Grades 2, 3 & 4 beach water samples, Giardia cysts were found, respectively, in 85, 50 and 64% of the samples. Except for one beach water sample which had an unusually high concentration of Giardia cysts (23 cysts/L), they were generally present at moderate concentrations (<10 cysts/L) in all other beach water samples. Despite moderate levels of Giardia cysts present in beach water of different grades, the potential health risk faced by swimmers bathing in local beach water needs to be carefully assessed as Giardia is known to have a low infectious dose.
Article
Flow cytometry with fluorescence activated cell sorting (FACS) was used to purify Cryptosporidium (oo)cysts and Giardia cysts from water. With this purification step Cryptosporidium and Giardia were found in a higher percentage of the samples and significantly higher Giardia concentrations were detected in these positive samples. Because FACS removed most of the debris from the concentrated water sample, the microscopic preparations could be examined more rapidly for the presence of (oo)cysts and the morphological characteristics of the (oo)cysts could be interpreted more unambiguously than with the conventional immunofluorescence microscopy method. The use of FACS made it possible to apply PI-staining on environmental samples to determine the fraction of dead (oo)cysts. Sample processing did not appear to influence the PI-staining characteristics of the Cryptosporidium (oo)cysts, but did increase the percentage of PI-positive Giardia cysts. This suggests that this protocol can be used for determining the percentage of dead Cryptosporidium oocysts in environmental samples. Analysis of environmental samples suggests that reservoir storage increases the percentage of PI-positive (dead) oocysts.
Article
The relative performances of different filter matrices were assessed for the concentration of Cryptosporidium oocysts in water. Cartridge filtration methods were examined and compared with membrane filtration and a flocculation method using seeded tap and river water to determine the most efficient and consistent method of sample concentration. From 10L samples, calcium carbonate flocculation gave the most consistently high recovery rate of Cryptosporidium oocysts (tap water mean 72.9%; river water mean 41.9%), followed by membrane filtration using 1.2μm cellulose acetate membranes (tap water mean 37.8%; river water mean 26.6%). For filtration of larger volumes (>100L) of water the Cuno Microwynd II cartridge filter provided the highest recovery rate of oocysts (tap water mean 13.3%; river water mean 10.8%) when compared with the Vokes cartridge and Balston filters. All recoveries fell dramatically when sucrose flotation techniques were applied.
Article
This review provides a comprehensive update of worldwide waterborne parasitic protozoan outbreaks that occurred with reports published between January 2011 and December 2016. At least 381 outbreaks attributed to waterborne transmission of parasitic protozoa were documented during this time period. The nearly half (49%) of reports occurred in New Zealand, 45% in North America and 6% in Europe. The most common etiological agent was Cryptosporidium spp., reported in 63% (240) of the outbreaks, while Giardia spp. was mentioned in 37% (141). No outbreaks attributed to other parasitic protozoa were reported. The distribution of reported outbreaks does not correspond to more broadly available epidemiological data or general knowledge of water and environmental conditions in the reporting countries. Noticeably, developing countries that are probably most affected by such waterborne disease outbreaks still lack reliable surveillance systems, and an international standardization of surveillance and reporting systems has yet to be established.
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Methods to detect protozoa in water samples are expensive and laborious. We evaluated the formalin/ether concentration method to detect Giardia sp, Cryptosporidium sp. and Toxoplasma in water. In order to test the properties of the method, we spiked water samples with different amounts of each protozoa (0, 10 and 50 cysts or oocysts) in a volume of 10 L of water. Immunofluorescence assay was used for detection of Giardia and Cryptosporidium. Toxoplasma oocysts were identified by morphology. The mean percent of recovery in 10 repetitions of the entire method, in 10 samples spiked with ten parasites and read by three different observers, were for Cryptosporidium 71.3 ± 12, for Giardia 63 ± 10 and for Toxoplasma 91.6 ± 9 and the relative standard deviation of the method was of 17.5, 17.2 and 9.8, respectively. Intraobserver variation as measured by intraclass correlation coefficient, was fair for Toxoplasma, moderate for Cryptosporidium and almost perfect for Giardia. The method was then applied in 77 samples of raw and drinkable water in three different plant of water treatment, enabling to identify significant differences in treatment process to reduce the presence of Giardia and Cryptosporidium. In conclusion, the formalin ether method to concentrate protozoa in water is a new alternative for low resources countries, where is urgently need to monitor and follow the presence of theses protozoa in drinkable water.
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Giardia lamblia is one of the most important waterborne pathogenic protozoa. Its occurrence in source and drinking water threatens human health seriously. In this study, four quantitative real-time PCR protocols involving Envirochek filtration, flat membrane filtration, immunomagnetic separation (IMS) and IMS-free separation to detect G. lamblia from surface water were compared. The results showed that acceptable recoveries of 21-28% could be obtained from all of the protocols with 500 cysts spiked into the samples. However, the flat membrane filtration methods were not applicable for its ready being clogged. The Envirochek protocols with or without IMS were comparable with a recovery range of 24-34% when lower volume (5 L) water was tested with different cysts spiked; but for larger volume (15 L), only Envirochek - IMS protocol worked, which suggested that the much cheaper and simpler IMS-free protocol should be an option for clean source water or drinking water, or small volume samples; while the IMS one could be used for contaminated source water with higher turbidity and more PCR inhibitors.
Article
The method for concentration of Cryptosporidium oocysts in large drinking water samples using the Envirocheck capsule has been optimized for the detection of low levels of oocysts. Elution from the filter by contact time and vortex agitation gave 68% oocyst recovery. Centrifugation (1,250 g; 30 min; 4°C) improved recovery to 94% without morphological damage of the oocysts. Increasing the ratio of magnetic beads to sample volume in the IMS procedure led to 69% efficiency. In these conditions, the overall recovery of the procedure was 49% as assessed with low oocysts spike doses in 100 litres tap water samples. The methodology described allows the detection of 0.1 oocyst per litre when 100 litres samples are processed.
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