Cryptosporidium Oocysts In Water Research Articles

Cell Culture Infectivity to Assess Chlorine Disinfection of Cryptosporidium Oocysts in Water.

This chapter provides a detailed protocol to assess disinfection efficacy of chlorine against Cryptosporidium oocysts including the core chlorine disinfection assay, the in vitro cell culture infectivity assay, and microscopy analysis and data interpretation.

Constructed wetlands – Are they safe in reducing protozoan parasites?

Constructed wetlands have been promoted in recent literature for use in rural communities in developed as well as in developing countries as an appropriate technology to be handled with low operational maintenance costs. Within a joint project supported by BMBF (Project No O2WA0107 and No 02WA0108) research was done concerning the sanitation effect of constructed wetlands on wastewater effluents. This article will focus on the detection and the removal of cysts of Cryptosporidium parvum and Giarda lamblia, those being the most frequently identified pathogenic protozoan parasites worldwide with increasing medical and economical consequences. Two plants, one installed in 2000 as a pilot plant at Langenreichenbach near Leipzig (Saxony, Germany), the other one in routine operation since 1993 in a training center at the town of Belzig (Brandenburg, Germany) were tested for three years. Detection methods from the US EPA (ICR Protozoan Method for Detecting Giardia Cysts and Cryptosporidium Oocysts in Water by a Fluorescent Antibody Procedure (EPA/814-B-95-003;US EPA 1995) were employed in order to assess protozoal and bacterial reduction in the wastewater passing through different combinations of filter beds and fillings. Removal of cysts of Cryptosporidium and Giardia spp. turned out to be a 2 log reduction in all plants. The most effective structural element was a two-stage combination of filter beds leading to the highest removal efficiency both for the protozoan and the bacterial indicator organisms. Also, washed sand (0-2mm grain size) in the filter bed proved to be most effective filter material; the planted reed (phragmites spp.) or willow (salix spp.), however, turned out to be of minor importance for the filtering activity.

Cryptosporidium propidium monoazide-PCR, a molecular biology-based technique for genotyping of viable Cryptosporidium oocysts.

Cryptosporidium is an important waterborne protozoan parasite that can cause severe diarrhea and death in the immunocompromised. The current methods used to monitor for Cryptosporidium oocysts in water are the microscopy-based USEPA methods 1622 and 1623. These methods assess total levels of oocysts in source waters, but do not determine oocyst viability or genotype. Recently, propidium monoazide (PMA) has been used in conjunction with molecular diagnostic tools to identify species and assess the viability of bacteria. The goal of this study was the development of a Cryptosporidium PMA-PCR (CryptoPMA-PCR) assay that includes PMA treatment prior to PCR analysis in order to prevent the amplification of DNA from dead oocysts. The results demonstrated that PMA penetrates only dead oocysts and blocks amplification of their DNA. The CryptoPMA-PCR assay can also specifically detect live oocysts within a mixed population of live and dead oocysts. More importantly, live oocysts, not dead oocysts, were detected in raw waste or surface water samples spiked with Cryptosporidium oocysts. This proof-of-concept study is the first to demonstrate the use of PMA for pre-PCR treatment of Cryptosporidium oocysts. The CryptoPMA-PCR assay is an attractive approach to specifically detect and genotype viable Cryptosporidium oocysts in the water, which is critical for human health risk assessment.

Fluorescence resonance energy transfer (FRET)-based specific labeling of Cryptosporidium oocysts for detection in environmental samples.

Accurate detection and quantification of Cryptosporidium oocysts in water are a challenge to the water industry. This article demonstrates a way to fluorescently label Cryptosporidium oocysts, based on fluorescence resonance energy transfer (FRET). Labeled oocysts can then be applied to environmental waters and their movement followed by flow cytometric detection and enumeration of the FRET-labeled oocysts, as demonstrated here with environmental water samples. Cryptosporidium oocysts were labeled with three fluorochromes, FITC, Texas red, and Cy7, that through FRET yielded a Stokes shift of approximately 272 nm with excitation from a standard argon laser emitting at 488 nm. Defined flow cytometric settings and gatings were used to select FITC/green (530-nm), Texas red/red (650-nm), and Cy7/infrared (780-nm) fluorescing particles with light scatter properties similar to oocysts. Water concentrates were seeded with 10 tri-labeled oocysts and were analyzed using flow cytometry. Unseeded water concentrates were also analyzed. Analysis of unseeded water concentrates detected no autofluorescent particle similar to the labeled oocysts. Labeled oocysts were detected successfully with up to 85% recovery in water concentrates spiked with 10 tri-labeled oocysts. Low numbers of FRET-labeled oocysts can be quantified and clearly distinguished from autofluorescing background in environmental water concentrates.

Detection and differentiation of Cryptosporidium oocysts in water by PCR-RFLP.

Consumption of contaminated water has been implicated as a major source of Cryptosporidium infection in various outbreak investigations and case control studies. Surveys conducted in various regions of the United States demonstrated the presence of Cryptosporidium oocysts in 67-100% of wastewaters, 24-100% of surface waters, and 17-26.8% of drinking waters. The identity and human infective potential of these waterborne oocysts are not known, although it is likely that not all oocysts are from human-infecting Cryptosporidium species. Likewise, the source of the oocyst contamination is also not fully clear. Farm animals and human sewage discharge are generally considered to be the major sources of surface water contamination with C. parvum. Because Cryptosporidium infection is common in wildlife, it is conceivable that wildlife can also be a source for Cryptosporidium oocysts in waters. The presence of host-adapted Cryptosporidium spp. and genotypes makes it possible to develop molecular tools to assess the human infection potential and source of Cryptosporidium oocysts in water.Currently, the identification of Cryptosporidium oocysts in environmental samples is largely made by the use of immunofluorescent assay (IFA) after concentration processes (Environmental Protection Agency [EPA] recommended information collection rule [ICR] method or method 1622/1623 or similar techniques). Because IFA detects oocysts from all Cryptosporidium parasites, the species distribution of Cryptosporidium parasites in environmental samples cannot be assessed. Although many surface water samples contain Cryptosporidium oocysts, it is unlikely that all these oocysts are from human-pathogenic species or genotypes, because only five genotypes of Cryptosporidium parasites (the C. parvum human and bovine genotypes, C. meleagridis, C. canis, and C. felis) are responsible for most human infections. Information on the source of C. parvum contamination is necessary for effective evaluation and selection of management practices for reducing C. parvum contamination of surface water and the risk of cryptosporidiosis. Thus, identification of oocysts to species and genotype levels is of public health importance.

Evaluation of an alternative IMS dissociation procedure for use with Method 1622: detection of Cryptosporidium in water

U.S.EPA Methods 1622 and 1623 are used to detect and quantify Cryptosporidium oocysts in water. The protocol consists of filtration, immunomagnetic separation (IMS), staining with a fluorescent antibody, and microscopic analysis. Microscopic analysis includes detection by fluorescent antibody and confirmation by the demonstration of 1–4 sporozoites or nuclei after staining with 4′,6-diamidino-2-phenyl indole dihydrochloride (DAPI). The purpose of this study was to evaluate a new IMS dissociation, a 10-min incubation at 80 °C. Heat dissociation improved the average oocyst recovery from 41% to 71% in seeded reagent water, and from 10% to 51% in seeded river samples. The average DAPI confirmation rate improved from 49% to 93% in reagent water, and from 48% to 73% in river samples. This modification improved both oocyst recovery and confirmation.

An immunomagnetic separation–real-time PCR method for quantification of Cryptosporidium parvum in water samples

The protozoan parasite Cryptosporidium parvum is known to occur widely in both raw and drinking water and is the cause of waterborne outbreaks of gastroenteritis throughout the world. The routinely used method for the detection of Cryptosporidium oocysts in water is based on an immunofluorescence assay (IFA). It is both time-consuming and nonspecific for the human pathogenic species C. parvum. We have developed a TaqMan polymerase chain reaction (PCR) test that accurately quantifies C. parvum oocysts in treated and untreated water samples. The protocol consisted of the following successive steps: Envirochek® capsule filtration, immunomagnetic separation (IMS), thermal lysis followed by DNA purification using Nanosep® centrifugal devices and, finally, real-time PCR using fluorescent TaqMan technology. Quantification was accomplished by comparing the fluorescence signals obtained from test samples with those from standard dilutions of C. parvum oocysts. This IMS–real-time PCR assay permits rapid and reliable quantification over six orders of magnitude, with a detection limit of five oocysts for purified oocyst solutions and eight oocysts for spiked water samples. Replicate samples of spiked tap water and Seine River water samples (with approximately 78 and 775 oocysts) were tested. C. parvum oocyst recoveries, which ranged from 47.4% to 99% and from 39.1% to 68.3%, respectively, were significantly higher and less variable than those reported using the traditional US Environmental Protection Agency (USEPA) method 1622. This new molecular method offers a rapid, sensitive and specific alternative for C. parvum oocyst quantification in water.

Risk assessment for drinking water production: assessing the potential risk due to the presence of Cryptosporidium oocysts in water

This paper presents an approach for assessing the risk of producing non-compliant drinking water (i.e. one of the quality parameters exceeds the standards fixed by legislation), taking into account the quality parameters of raw water and the process line of the treatment plant (technology, different failure mode and corresponding failure rate). Firstly, nominal and degraded modes of each step of the treatment line are analysed, in order to obtain transfer functions (which give output concentration of parameters in function of the input concentration) for each step of the treatment and each quality parameter, in nominal and degraded functioning. The transfer function of the whole treatment process can thereby be obtained by combination of transfer function of each step, and failure conditions of the whole treatment process and corresponding degraded global transfer function could be determined. Secondly, an inversion of both global function (nominal and degraded) permits to estimate probability for the resource to exceed thresholds fixed by regulation (in that case, a scenario of non-compliant drinking water exists), and to obtain a compliant water availability. Finally, this paper presents a software tool realised to evaluate the risk of non-compliant produced water, using the described methodology. Finally, an approach of risk assessment for Cryptosporidium is also presented. This method allows to identification and puts priorities for utilities presenting the highest risk.

Influence of ionic strength and pH on hydrophobicity and zeta potential of Giardia and Cryptosporidium

Abstract Surface charge and hydrophobicity of microorganisms are involved in interfacial interaction of cells, such as flocculation and adhesion. The calculated zeta potentials of Giardia cysts and Cryptosporidium oocysts in waters at neutral pH were on average −17 and −38 mV, respectively. The isoelectric points of cysts and oocysts were estimated as pH 2.2 and pH 3.3, respectively. The hydrophobicities of cysts and oocysts, obtained from the microbial adherence to hydrocarbons (MATH) test, were determined by initial removal rate. Although the initial removal rates of oocysts were larger than those of cysts in all trials, both demonstrated marked hydrophobicity. The hydrophobicities were significantly increased with decreasing pH for both protozoa.

Evaluation of USEPA Method 1622 for detection of Cryptosporidium oocysts in stream waters

To improve surveillance for Cryptosporidium oocysts in water, the US Environmental Protection Agency developed method 1622, which consists of filtration, concentration, immunomagnetic separation, fluorescent antibody and 4, 6‐diamidino‐2‐phenylindole (DAPI) counter‐staining, and microscopic evaluation. Two filters were compared for analysis of 11 stream water samples collected throughout the United States. Replicate 10‐L stream water samples (unspiked and spiked with 100–250 oocysts) were tested to evaluate matrix effects. Oocyst recoveries from the stream water samples averaged 22% (standard deviation [SD] = ±17%) with a membrane disk and 12% (SD = ±6%) with a capsule filter. Oocyst recoveries from reagent water precision and recovery samples averaged 39% (SD = ±13%) with a membrane disk and 47% (SD = ±19%) with a capsule filter. These results demonstrate that Cryptosporidium oocysts can be recovered from stream waters using method 1622, but recoveries are lower than those from reagent‐grade water. This research also evaluated concentrations of indicator bacteria in the stream water samples. Because few samples were oocyst‐positive, relationships between detections of oocysts and concentrations of indicator organisms could not be determined.

Detection of Cryptosporidium oocysts in water: techniques for generating precise recovery data.

When determining the recovery efficiency of a procedure for the detection of Cryptosporidium or the removal efficiency of a treatment process, it is necessary to accurately enumerate a 'seed dose'. Conventional techniques for this are highly variable and consequently, can result in misleading data. In this study, a flow cytometric method was developed for the production of suspensions of Cryptosporidium oocysts in which the number of organisms could be precisely determined. A Becton Dickinson FACScalibur flow cytometer was employed to produce oocyst suspensions containing 100 oocysts. Analysis of these suspensions resulted in a mean dose of 99.5 oocysts (S.D. = 1.1, %cv = 1.1). These results indicate that the use of such suspensions to seed test systems generates far more accurate data than is presently possible using conventional techniques. In addition, the use of immunomagnetic separation (IMS) for the isolation of oocysts from three different water matrices, after seeding with oocysts counted using flow cytometry, was investigated. The recovery efficiency of the IMS procedure was found to be high, with the percentage recovery of oocysts ranging from 82.3 to 86.3%, and the use of precise numbers of oocysts allowed accurate recovery efficiency data to be generated. A laser scanning instrument (ChemScan RDI) was employed for the rapid detection and enumeration of oocysts after capture using membrane filtration. This technique was found to be faster and easier to perform than conventional epifluorescence microscopy. These findings demonstrate that the ChemScan RDI system may be used as alternative procedure for the routine examination of IMS supernatant fluids for the presence of Cryptosporidium.

Giardia Cyst and Cryptosporidium Oocyst Survival in Water, Soil, and Cattle Feces

AbstractGiardiasis and cryptosporidiosis are gastrointestinal diseases caused by protozoan parasites that may infect domestic animals, wildlife and human beings. The ability of cysts and oocysts of these parasites to persist in the environment was determined because agricultural fecal waste has the potential to contaminate municipal water supplies. The degradation rate and viability of Giardia cysts and Cryptosporidium oocysts in water, cattle (Bos taurus) feces, and soil was evaluated at temperatures of −4, 4, and 25°C for up to 12 wk. Cysts and oocysts were enumerated after staining samples with a specific fluorescent monoclonal antibody and the viability was determined using propidium iodide dye exclusion and mouse infectivity assays. Giardia cysts were noninfective in water, feces, and soil following 1 wk of freezing to −4°C and within 2 wk at 25°C. At 4°C Giardia cysts were infective for 11 wk in water, 7 wk in soil, and 1 wk in cattle feces. Cryptosporidium oocysts were more environmentally resistant. At −4 and 4°C, the oocysts could survive in water and soil for >12 wk but degradation was accelerated at 25°C. Cryptosporidium oocysts also were degraded more rapidly in feces and in soil containing natural microorganisms. Contaminated cattle feces should be distributed on fields during warmer weather and after 12 wk of storage to reduce potential waterborne transmission following heavy runoffs.

水中に存在するＣｒｙｐｔｏｓｐｏｒｉｄｉｕｍオーシストの電子制菌装置による除去と不活化

水中に存在するＣｒｙｐｔｏｓｐｏｒｉｄｉｕｍオーシストの電子制菌装置による除去と不活化

Inactivation of Cryptosporidium oocysts in water by ozonation

Inactivation of Cryptosporidium oocysts in water by ozonation

An in vitro method for detecting infectious Cryptosporidium oocysts with cell culture.

Current assay methods to detect Cryptosporidium oocysts in water are generally not able to evaluate viability or infectivity. A method was developed for low-level detection of infective oocysts by using HCT-8 cells in culture as hosts to C. parvum reproductive stages. The infective foci were detected by labeling intracellular developmental stages of the parasite in an indirect-antibody assay with a primary antibody specific for reproductive stages and a secondary fluorescein isothiocyanate-conjugated antibody. The complete assay was named the focus detection method (FDM). The infectious foci (indicating that at least one of the four sporozoites released from a viable oocyst had infected a cell) were enumerated by epifluorescence microscopy and confirmed under Nomarski differential interference contrast microscopy. Time series experiments demonstrated that the autoreinfective life cycle in host HCT-8 cells began after 12 h of incubation. Through dilution studies, levels as low as one infectious oocyst were detected. The cell culture FDM compared well to other viability assays. Vital stains and excystation demonstrated that oocyst populations less than 1% viable (by vital dyes) and having a low sporozoite yield following excystation could not infect host cells. Until now, the water industry has relied on an oocyst detection method (under an information collection regulation) that is unable to determine viability. The quantifiable results of the cell culture method described demonstrate two important applications: (i) an infectivity assay that may be used in conjunction with current U.S. Environmental Protection Agency-mandated detection methodologies, and (ii) a method to evaluate oocyst infectivity in survival and disinfection studies.

Efficacy of iodine water purification tablets against Cryptosporidium oocysts and Giardia cysts.

The ability to control water-borne diseases is critical for soldiers, hikers, and others who may need to drink directly from an outdoor source. Water-borne protozoan parasites that are specifically of concern are Giardia and Cryptosporidium because of their resistance to halogen disinfection. The purpose of this study was to determine the effectiveness of iodine tablets against Giardia and Cryptosporidium under general- and worst-case water conditions that might be found in the field. Giardia cysts and Cryptosporidium oocysts were exposed to iodine according to manufacturer's instructions (two tablets/L = 13-18 mg/L for 20 minutes). This dose inactivated 3-log10 of Giardia in general-case water and pH 9. In worst-case water, however, only about 35% of cysts were inactivated at pH 5. Fifty minutes were required to achieve a 3-log10 reduction at pH 5. Cryptosporidium oocysts were more difficult to inactivate. Only 10% were inactivated after a 20-minute exposure to iodine according to manufacturer's instructions; even after 240 minutes of exposure to iodine only 66-81% oocysts were inactivated. These data strongly suggest that iodine disinfection is not effective in inactivating Cryptosporidium oocysts in water. Because this organism is common in all surface waters, it is recommended that another method of treatment be used before ingestion.

Environmental methods for Cryptosporidium

A number of approaches offer promise for addressing the limitations of current antibody‐based detection methods.This report was prepared by the Working Group on Waterborne Cryptosporidiosis (Technical Task Force E, Developmental Status of Environmental Sampling, Water Testing, and Surrogate Indicators). Methods for detecting Cryptosporidium oocysts in water have centered around microscopic examination of fluorescent antibody‐stained concentrates from large‐volume water samples. The limitations of these antibody‐based methods include the need for experienced analysts, lengthy analytical time, expense, lack of specificity, erratic efficiency, low precision, and difficulty in determining viability. A number of methods, assays, and procedures that have the potential for ameliorating some of these limitations are currently being evaluated. How successful such processes will be remains to be demonstrated by the scientific community.

Comparison of two methods for detection of Giardia cysts and Cryptosporidium oocysts in water

The steps of two immunofluorescent-antibody-based detection methods were evaluated for their efficiencies in detecting Giardia cysts and Cryptosporidium oocysts. The two methods evaluated were the American Society for Testing and Materials proposed test method for Giardia cysts and Cryptosporidium oocysts in low-turbidity water and a procedure employing sampling by membrane filtration, Percoll-Percoll step gradient, and immunofluorescent staining. The membrane filter sampling method was characterized by higher recovery rates in all three types of waters tested: raw surface water, partially treated water from a flocculation basin, and filtered water. Cyst and oocyst recovery efficiencies decreased with increasing water turbidity regardless of the method used. Recoveries of seeded Giardia cysts exceeded those of Cryptosporidium oocysts in all types of water sampled. The sampling step in both methods resulted in the highest loss of seeded cysts and oocysts. Furthermore, much higher recovery efficiencies were obtained when the flotation step was avoided. The membrane filter method, using smaller tubes for flotation, was less time-consuming and cheaper. A serious disadvantage of this method was the lack of confirmation of presumptive cysts and oocysts, leaving the potential for false-positive Giardia and Cryptosporidium counts when cross-reacting algae are present in water samples.

Evaluation of different filtration techniques for the concentration of cryptosporidium oocysts from water

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.

Evaluation of the immunofluorescence procedure for detection of Giardia cysts and Cryptosporidium oocysts in water.

The accurate determination of the presence of Giardia cysts and Cryptosporidium oocysts in surface waters requires a reliable method for the detection and enumeration of these pathogenic organisms. Published methods have usually reported recovery efficiencies of less than 50% for both cysts and oocysts. Typically, the losses are greater for Cryptosporidium oocysts than they are for Giardia cysts. The purpose of this study was to examine procedures used for sample collection, elution, concentration, and clarification to determine when losses of cysts and oocysts occurred during processing. The results showed that major losses of cysts and oocysts occurred during centrifugation and clarification. Depending on the centrifugation force, oocyst losses of as high as 30% occurred for each centrifugation step. A 1.15-specific-gravity Percoll-sucrose gradient was needed to optimize recovery of oocysts from natural water samples. Minor improvements in the procedure could be accomplished by selecting a filter other than the recommended 1-micron-pore-size (nominal-porosity) polypropylene filter.