Use of Meta-Analysis to Assess the Effect of Conventional Water Treatment Methods on the Prevalence of Cryptosporidium Spp. in Drinking Water

Meta-analysis is defined as the statistical analysis of a collection of analytic results for the purpose of integrating the findings. However, its use in environmental and agricultural health protection has been minor in research to date. Few meta-analysis studies have been applied to assess factors relating to Cryptosporidium but none have examined the effect of conventional water treatment on oocyst prevalence. The objective of this study was to synthesize past scientific research and analyze the effects of conventional water treatment methods on the prevalence of Cryptosporidium in drinking water using a traditional parametric approach of meta-analysis. Twenty-three different published studies featuring 27 separate water treatment surveys were identified for inclusion in the meta-analysis. The meta-analysis indicated that general conventional water treatment methods reduced Cryptosporidium occurrence in drinking water by an average factor of ～11.82 (95% CI = 2.7–52.5). Results highlight the resistance of the pathogen to such treatment methods and emphasize the parasites standing as a waterborne pathogen of primary concern given the possibility of viable oocyst occurrence in water, post treatment. Consequently, findings accentuate the need for new approaches to the control of Cryptosporidium aimed at integrated catchment management, source protection, and also including new treatment technologies. Until such approaches are actively adopted the potential risk posed to consumers by pathogens in potable water will remain active.     

The use of rotifers in detecting protozoan parasite infections in recreational lakes

Although well-known methods for the detection of intestinal parasitic protozoans in water samples exist, they are insufficiently sensitive, expensive, of little practical value in the routine monitoring of waterborne pathogens and time- and labour-consuming. In the investigation reported here we have assessed Cryptosporidium oocyst detection using both the so-called Method 1623[recommended by the U.S. Environmental Protection Agency (USEPA)] and a direct method involving the determination of oocysts of Cryptosporidium in rotifers as detection tools of surface water contamination by dispersive stages of intestinal protozoans. Rotifers were sampled from three lakes located near the city of Poznan (Poland). To detect the oocysts of Cryptosporidium, we applied the fluorescent in situ hybridisation technique, an immunofluorescent assay and an enzyme immunoassay. Oocysts of Cryptosporidium were detected both in water collected from the lakes and in rotifers. The FISH technique applied to rotifers enabled the detection of biological contamination of surface water through an assessment of the dispersive stages of the parasite and was found to be more sensitive, less time-consuming and cheaper than the method recommended by the USEPA.     

Prevalence of Zoonotic Pathogens from Feral Pigs in Major Public Drinking Water Catchments in Western Australia

Australia has the largest number of wild pigs in the world. Their pronounced impacts on agriculture and biodiversity make the estimated 23 million feral pigs one of Australia’s most important vertebrate pest species. The foraging and wallowing behavior of pigs can markedly increase the turbidity of water supplies, but more importantly, they can transmit and excrete a number of infectious waterborne organisms pathogenic to humans. Their persistence in drinking water catchments also makes them potentially significant reservoirs for zoonotic pathogens. In this study, important protozoan parasite pathogens, such as Giardia, Cryptosporidium, Balantidium, and Entamoeba, were detected from the feces of feral pigs caught in metropolitan drinking water catchment areas. All are potentially important waterborne human pathogens that pose a major threat to drinking water quality. Fortunately, the overall prevalence in feral pigs appears to be relatively low, with ≤13% of pigs detected with parasites. In this study, we combined the findings from the parasitological analysis with the use of 14 highly informative DNA markers to define a series of highly structured populations that indicated very little movement of feral pigs between the populations. The implication of this pattern is that any public health risk may spread very slowly between populations, but may be much higher within watercourses. This study represents an innovative and important new approach to drinking water source protection in Australia.     

Assessment of Antibacterial Activity of Colebrookia oppositifolia against Waterborne Pathogens Isolated from Drinking Water of the Pothwar Region in Pakistan

An attempt was made to control waterborne pathogens by using medicinal plant extracts. One hundred and twenty-six water samples from filtration plants, tube wells, and water supplies were collected and analyzed for total and faecal Coliform bacteria as well as for total viable count. Results showed that waterborne pathogens were numerous and significantly higher than the World Health Organization's recommended guidelines. The methanolic and aqueous extracts of different parts of Colebrookia oppositifolia (Labiateae) were examined for antibacterial activities in vitro by an agar diffusion method. Antibacterial activity of leaves, shoots, and roots of Colebrookia oppositifolia was assessed against Gram positive and Gram negative bacteria that were isolated and identified from water samples by the API 20E method. Extract of roots showed more antibacterial activities against Staph. aureus and B. cereus var. mycoides, Pseudomonas aeruginosa, Klebsiella pneumonia, and Shigella flexneri at 37°C, than extracts from leaves and shoots. The lowest MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) were observed in roots as compared to shoots and leaves. These results suggest that there is an urgent need for improvement in existing water quality treatment. Secondly, the fruit extract can be practical for protection and to avoid risk of contamination by waterborne pathogens and to promote indigenous solutions for disease-control and environmental management.     

Presence of Cryptosporidium spp. and Giardia duodenalis in Drinking Water Samples in the North of Portugal

Cryptosporidium and Giardia are 2 protozoan parasites responsible for waterborne diseases outbreaks worldwide. In order to assess the prevalence of these protozoans in drinking water samples in the northern part of Portugal and the risk of human infection, we have established a long term program aiming at pinpointing the sources of surface water, drinking water, and environmental contamination, working with the water-supply industry. Total 43 sources of drinking water samples were selected, and a total of 167 samples were analyzed using the Method 1623. Sensitivity assays regarding the genetic characterization by PCR and sequencing of the genes, 18S SSU rRNA, for Cryptosporidium spp. and β,-giardin for G. duodenalis were set in the laboratory. According to the defined criteria, molecular analysis was performed over 4 samples. Environmental stages of the protozoa were detected in 25.7% (43 out of 167) of the water samples, 8.4% (14 out of 167) with cysts of Giardia, 10.2% (17 out of 167) with oocysts of Cryptosporidium and 7.2% (12 out of 167) for both species. The mean concentrations were 0.1-12.7 oocysts of Cryptosporidium spp. per 10 L and 0.1-108.3 cysts of Giardia duodenalis per 10 L. Our results suggest that the efficiency in drinking water plants must be ameliorated in their efficiency in reducing the levels of contamination. We suggest the implementation of systematic monitoring programs for both protozoa. To authors'' knowledge, this is the first report evaluating the concentration of environmental stages of Cryptosporidium and Giardia in drinking water samples in the northern part of Portugal.     

Sources and Species of Cryptosporidium Oocysts in the Wachusett Reservoir Watershed

Understanding the behavior of Cryptosporidium oocysts in the environment is critical to developing improved watershed management practices for protection of the public from waterborne cryptosporidiosis. Analytical methods of improved specificity and sensitivity are essential to this task. We developed a nested PCR-restriction fragment length polymorphism assay that allows detection of a single oocyst in environmental samples and differentiates the human pathogen Cryptosporidium parvum from other Cryptosporidium species. We tested our method on surface water and animal fecal samples from the Wachusett Reservoir watershed in central Massachusetts. We also directly compared results from our method with those from the immunofluorescence microscopy assay recommended in the Information Collection Rule. Our results suggest that immunofluorescence microscopy may not be a reliable indicator of public health risk for waterborne cryptosporidiosis. Molecular and environmental data identify both wildlife and dairy farms as sources of oocysts in the watershed, implicate times of cold water temperatures as high-risk periods for oocyst contamination of surface waters, and suggest that not all oocysts in the environment pose a threat to public health.     

Assessment of Giardia and Cryptosporidium spp. as a Microbial Source Tracking Tool for Surface Water: Application in a Mixed-Use Watershed

Knowledge of host specificity, combined with genomic sequencing of Giardia and Cryptosporidium spp., has demonstrated a microbial source tracking (MST) utility for these common waterborne microbes. To explore the source attribution potential of these pathogens, water samples were collected in a mixed rural-urban watershed in the Township of Langley, in southwestern British Columbia (BC), Canada, over a 2-year period. Cryptosporidium was detected in 63% of surface water samples at concentrations ranging from no positive detection (NPD) to 20,600 oocysts per 100 liters. Giardia was detected in 86% of surface water samples at concentrations ranging from NPD to 3,800 cysts per 100 liters of water. Sequencing at the 18S rRNA locus revealed that 50% of Cryptosporidium samples and 98% of Giardia samples contained species/genotypes (Cryptosporidium) or assemblages (Giardia) that are capable of infecting humans, based on current knowledge of host specificity and taxonomy. Cryptosporidium genotyping data were more promising for source tracking potential, due to the greater number of host-adapted (i.e., narrow-host-range) species/genotypes compared to Giardia, since 98% of Giardia isolates were zoonotic and the potential host could not be predicted. This report highlights the benefits of parasite genomic sequencing to complement Method 1623 (U.S. Environmental Protection Agency) and shows that Cryptosporidium subtyping for MST purposes is superior to the use of Giardia subtyping, based on better detection limits for Cryptosporidium-positive samples than for Giardia-positive samples and on greater host specificity among Cryptosporidium species. These additional tools could be used for risk assessment in public health and watershed management decisions.     

Assessment of Enteric Pathogen Shedding by Bathers during Recreational Activity and its Impact on Water Quality

An assessment was made to determine the potential loading of enteric pathogenic protozoa and viruses into drinking water supply reservoirs by body contact recreation. These and other organisms of fecal origin are shed from the body during bathing. A literature review was conducted on the concentration of selected enteric viruses and protozoa during infection, the incidence of these infections, and duration of excretion. In addition, from existing literature, the amount of fecal material released during bathing was estimated from the shedding of fecal coliforms by bathers. The mean amount of fecal material shed per bather was estimated at 0.14[emsp4 ]gram. The concentration of protozoan parasites (Giardia or Cryptosporidium) in feces of infected persons can range from 10⁵ to 10⁷ per gram and enteric viruses (enteroviruses, adenoviruses, rotavirus) from 10⁵ to 10¹² per gram. From this information, the concentration of enteric pathogens, shed into the water, could be calculated for a group of bathers. This information can be used to model the impact of body contact recreation on water quality in reservoirs used for drinking water supplies. Such information is useful in assessing the required treatment of the water to meet water quality regulations.     

Identification of zoonotic Cryptosporidium and Giardia genotypes infecting animals in Sydney's water catchments

To identify the animal sources for Cryptosporidium and Giardia contamination, we genotyped Cryptosporidium and Giardia spp. in wildlife from Sydney’s water catchments using sequence analysis at the 18S rRNA locus for Cryptosporidium and 18S rRNA and glutamate dehydrogenase (gdh) for Giardia. A total of 564 faecal samples from 16 different host species were analysed. Cryptosporidium was identified in 8.5% (48/564) samples from eight host species and Giardia was identified in 13.8% (78/564) from seven host species. Eight species/genotypes of Cryptosporidium were identified. Five G. duodenalis assemblages were detected including the zoonotic assemblages A and B.     

A Risk Assessment Framework for Waterborne Pathogens and Requirements for Producing a Complete Protocol

The U.S. Environmental Protection Agency (USEPA), Office of Water, is developing a risk assessment protocol for determining microbiological pathogen risks in water (drinking, recreational, waste waters, etc.). This effort has been conducted in collaboration with the International Life Sciences Institute. A microbiological risk assessment framework was prepared and has been peer reviewed in the open literature and vetted at the USEPA and other federal government risk assessment venues. Some goals in development of the Framework were to make it comprehensive, easy to understand and to use, since it is recognized that improvements to the framework structure and instructional material would facilitate its use. The USEPA's Office of Water wishes to develop improved tools, methods, and approaches for conducting the analysis phase for risk assessments and would like to evaluate its efficacy for a broad range of waterborne pathogens in water/wastewater media. Improved understanding of microbiological survival, infectivity, and virulence factors is needed, especially at the genomic and proteomic levels to accurately assess the occurrence and fate of pathogens in water and to predict what intrinsic factors allow pathogens to be invasive and virulent. Development of improved dose-response models (including animal models) for pathogen exposures focusing on the dynamic circumstances of immunity, secondary spread, and sensitive subpopulations, would be useful additions to the Framework. In the future, USEPA may consider establishing comprehensive pathogen risk assessment guidelines that all its program offices can use.     

Assessing the Effects of Climate Change on Waterborne Microorganisms: Implications for EU and U.S. Water Policy

Despite advances in water treatment, outbreaks of waterborne diseases still occur in developed regions including the United States and Europe Union (EU). Water quality impairments attributable to elevated concentrations of fecal indicator bacteria, and associated with health risk, are also very common. Research suggests that the impact of such microorganisms on public health may be intensified by the effects of climate change. At present, the major regulatory frameworks in these regions (i.e., the US Clean Water Act [CWA] and the EU Water Framework Directive [WFD]), do not explicitly address risks posed by climate change. This article reviews existing U.S. and EU water quality regulatory legislation for robustness to climate change and suggests watershed modeling approaches to inform additional pollution control measures given the likely impacts on microbial fate and transport. Comprehensive analysis of future climate and water quality scenarios may only be achievable through the use of watershed-scale models. Unless adaptation measures are generated and incorporated into water policy, the potential threat posed to humans from exposure to waterborne pathogens may be amplified. Such adaptation measures will assist in achieving the aims of the EU WFD and US CWA and minimize impacts of climate change on microbial water quality.     

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better characterize the prevalence of these pathogens, EPA Method 1623 was developed and used to monitor levels of these organisms in US drinking water supplies ¹². The method has three main parts; the first is the sample concentration in which at least 10 L of raw surface water is filtered. The organisms and trapped debris are then eluted from the filter and centrifuged to further concentrate the sample. The second part of the method uses an immunomagnetic separation procedure where the concentrated water sample is applied to immunomagnetic beads that specifically bind to the Cryptosporidium oocysts and Giardia cysts allowing for specific removal of the parasites from the concentrated debris. These (oo)cysts are then detached from the magnetic beads by an acid dissociation procedure. The final part of the method is the immunofluorescence staining and enumeration where (oo)cysts are applied to a slide, stained, and enumerated by microscopy.Method 1623 has four listed sample concentration systems to capture Cryptosporidium oocysts and Giardia cysts in water: Envirochek filters (Pall Corporation, Ann Arbor, MI), Envirochek HV filters (Pall Corporation), Filta-Max filters (IDEXX, Westbrook, MA), or Continuous Flow Centrifugation (Haemonetics, Braintree, MA). However, Cryptosporidium and Giardia (oo)cyst recoveries have varied greatly depending on the source water matrix and filters used^1,14. A new tangential flow hollow-fiber ultrafiltration (HFUF) system has recently been shown to be more efficient and more robust at recovering Cryptosporidium oocystsand Giardia cysts from various water matrices; moreover, it is less expensive than other capsule filter options and can concentrate multiple pathogens simultaneously^{1-3,5-8,10,11}. In addition, previous studies by Hill and colleagues demonstrated that the HFUF significantly improved Cryptosporidium oocysts recoveries when directly compared with the Envirochek HV filters⁴. Additional modifications to the current methods have also been reported to improve method performance. Replacing the acid dissociation procedure with heat dissociation was shown to be more effective at separating Cryptosporidium from the magnetic beads in some matrices^9,13 .This protocol describes a modified Method 1623 that uses the new HFUF filtration system with the heat dissociation step. The use of HFUF with this modified Method is a less expensive alternative to current EPA Method 1623 filtration options and provides more flexibility by allowing the concentration of multiple organisms.     

Contamination of Groundwater Systems in the US and Canada by Enteric Pathogens, 1990–2013: A Review and Pooled-Analysis

Background

Up to 150 million North Americans currently use a groundwater system as their principal drinking water source. These systems are a potential source of exposure to enteric pathogens, contributing to the burden of waterborne disease. Waterborne disease outbreaks have been associated with US and Canadian groundwater systems over the past two decades. However, to date, this literature has not been reviewed in a comprehensive manner.

Methods and Principal Findings

A combined review and pooled-analysis approach was used to investigate groundwater contamination in Canada and the US from 1990 to 2013; fifty-five studies met eligibility criteria. Four study types were identified. It was found that study location affects study design, sample rate and studied pathogen category. Approximately 15% (316/2210) of samples from Canadian and US groundwater sources were positive for enteric pathogens, with no difference observed based on system type. Knowledge gaps exist, particularly in exposure assessment for attributing disease to groundwater supplies. Furthermore, there is a lack of consistency in risk factor reporting (local hydrogeology, well type, well use, etc). The widespread use of fecal indicator organisms in reported studies does not inform the assessment of human health risks associated with groundwater supplies.

Conclusions

This review illustrates how groundwater study design and location are critical for subsequent data interpretation and use. Knowledge gaps exist related to data on bacterial, viral and protozoan pathogen prevalence in Canadian and US groundwater systems, as well as a need for standardized approaches for reporting study design and results. Fecal indicators are examined as a surrogate for health risk assessments; caution is advised in their widespread use. Study findings may be useful during suspected waterborne outbreaks linked with a groundwater supply to identify the likely etiological agent and potential transport pathway.     

Phenotypic and genotypic characterization of Cryptosporidium species and isolates

Recent outbreaks of cryptosporidiosis from contaminated water supplies have led to a need for the detection of Cryptosporidium oocysts from various hosts and contaminating sources. The presence of nonpathogenic species or strains of Cryptosporidium is important for diagnostic purposes as there is a potential for false-positive detection of pathogenic parasites. The present review focuses on phenotypic differences and recent advances in genotypic analyses of the genus Cryptosporidium with an emphasis on detecting various isolates and identifying differences in Cryptosporidium parvum and other species in this genus. The information currently available demonstrates important patterns in DNA sequences of Cryptosporidium, and our understanding of macro- and microevolutionary patterns has increased in recent years. However, current knowledge of Cryptosporidium genetic diversity is far from complete, and the large amount of both phenotypic and genotypic data has led to problems in our understanding of the systematics of this genus. Journal of Industrial Microbiology & Biotechnology (2001) 26, 95–106. Received 18 March 2000/ Accepted in revised form 13 August 2000     

Spatiotemporal Analysis of Cryptosporidium Species/Genotypes and Relationships with Other Zoonotic Pathogens in Surface Water from Mixed-Use Watersheds

Nearly 690 raw surface water samples were collected during a 6-year period from multiple watersheds in the South Nation River basin, Ontario, Canada. Cryptosporidium oocysts in water samples were enumerated, sequenced, and genotyped by detailed phylogenetic analysis. The resulting species and genotypes were assigned to broad, known host and human infection risk classes. Wildlife/unknown, livestock, avian, and human host classes occurred in 21, 13, 3, and <1% of sampled surface waters, respectively. Cryptosporidium andersoni was the most commonly detected livestock species, while muskrat I and II genotypes were the most dominant wildlife genotypes. The presence of Giardia spp., Salmonella spp., Campylobacter spp., and Escherichia coli O157:H7 was evaluated in all water samples. The greatest significant odds ratios (odds of pathogen presence when host class is present/odds of pathogen presence when host class is absent) for Giardia spp., Campylobacter spp., and Salmonella spp. in water were associated, respectively, with livestock (odds ratio of 3.1), avian (4.3), and livestock (9.3) host classes. Classification and regression tree analyses (CART) were used to group generalized host and human infection risk classes on the basis of a broad range of environmental and land use variables while tracking cooccurrence of zoonotic pathogens in these groupings. The occurrence of livestock-associated Cryptosporidium was most strongly related to agricultural water pollution in the fall (conditions also associated with elevated odds ratios of other zoonotic pathogens occurring in water in relation to all sampling conditions), whereas wildlife/unknown sources of Cryptosporidium were geospatially associated with smaller watercourses where urban/rural development was relatively lower. Conditions that support wildlife may not necessarily increase overall human infection risks associated with Cryptosporidium since most Cryptosporidium genotypes classed as wildlife in this study (e.g., muskrat I and II genotype) do not pose significant infection risks to humans. Consequently, from a human health perspective, land use practices in agricultural watersheds that create opportunities for wildlife to flourish should not be rejected solely on the basis of their potential to increase relative proportions of wildlife fecal contamination in surface water. The present study suggests that mitigating livestock fecal pollution in surface water in this region would likely reduce human infection risks associated with Cryptosporidium and other zoonotic pathogens.     

Pathogen Sources Estimation and Scenario Analysis Using the Soil and Water Assessment Tool (SWAT)

Concerns over water quality in Ireland have increased in recent years, in part due to the more frequent contamination of drinking water by pathogens such as Escherichia coli and Cryptosporidium. The objective of this study was to assess the use of SWAT for pathogen source estimation and to analyze the effects of various source scenarios on pathogen outputs in Irish catchments. Two agricultural catchments in Ireland susceptible to pathogen contamination of source water were the center of the SWAT model development with the primary focus on levels of E. coli in surface water. Model simulations used site and source specific information which was analyzed considering the total E. coli count for the simulation period (Fergus: January 2005–October 2006; Kilshanvey: January 2006–July 2007). Pathogen source estimation identified point sources as the most significant contributors to E. coli output with direct deposition the primary contributor (95%) in Kilshanvey and wastewater treatment plant outflow (89%) the main contributor in the Fergus catchment. A scenario analysis evaluated possible situations that may occur in study locations. The analysis indicated that restriction of livestock access to water sources and improved wastewater treatment would represent effective methods of improving water quality in both catchments.     

Development and evaluation of a real-time PCR assay for quantification of Giardia and Cryptosporidium in sewage samples

Cryptosporidium and Giardia are major causes of diarrheal disease in humans worldwide and are major causes of protozoan waterborne diseases. Two DNA TaqMan PCR-based Giardia and Cryptosporidium methods targeting a 74-bp sequence of the β-giardin Giardia gene and a 151-bp sequence of the COWP Cryptosporidium gene, respectively, were used as models to compare two different LNA/DNA TaqMan probes to improve the detection limit in a real-time PCR assay. The LNA probes were the most sensitive resulting in 0.96 to 1.57 lower C _t values than a DNA Giardia TaqMan probe and 0.56 to 2.21 lower than a DNA Cryptosporidium TaqMan probe. Evaluation of TaqMan Giardia and Cryptosporidium probes with LNA substitutions resulted in real-time PCR curves with an earlier C _t values than conventional DNA TaqMan probes. In conclusion, the LNA probes could be useful for more sensitive detection limits.     

Molecular assessment of bacterial pathogens - a contribution to drinking water safety

Human bacterial pathogens are considered as an increasing threat to drinking water supplies worldwide because of the growing demand of high-quality drinking water and the decreasing quality and quantity of available raw water. Moreover, a negative impact of climate change on freshwater resources is expected. Recent advances in molecular detection technologies for bacterial pathogens in drinking water bear the promise in improving the safety of drinking water supplies by precise detection and identification of the pathogens. More importantly, the array of molecular approaches allows understanding details of infection routes of waterborne diseases, the effects of changes in drinking water treatment, and management of freshwater resources.     

Assessment of Antibacterial Activity of Solanum surrattense Against Waterborne Pathogens Isolated from Surface Drinking Water of the Potohar Region in Pakistan

People dwelling in different cities of the Potohar region, Pakistan, are mostly dependent on surface water for drinking and domestic use. In an attempt to make available potable, safe water, filtration plants were constructed along with dams in the Potohar region. Water samples from these filtration plants were collected and analyzed for total and faecal coliform bacteria as well as for total viable count. Results showed that bacterial indicators of faecal contamination were numerous and significantly greater than World Health Organization recommended guidelines. Accordingly, antibacterial activity of methanolic and aqueous extracts of different parts of Solanum surrattense were assessed in vitro against waterborne pathogens. Fruits exhibited more antibacterial activities at incubation at 37°C than shoots and roots, which showed lowest Minimum Inhibitory Concentration (MIC) and zones of inhibition. These results suggest that plants offer a great potential for purification of drinking water that needs to be explored further because fruit extract of the aforementioned plant can be of practical use against waterborne pathogens.