Giardia and Cryptosporidium removal from waste-water by a duckweed (Lemna gibba L.) covered pond

AIMS: To determine the ability of duckweed ponds used to treat domestic waste-water to remove Giardia and Cryptosporidium. METHODS AND RESULTS: The influent and effluent of a pond covered with duckweed with a 6 day retention time was tested for Giardia cysts, Cryptosporidium oocysts, faecal coliforms and coliphage. Giardia cysts and Cryptosporidium oocysts were reduced by 98 and 89%, respectively, total coliforms by 61%, faecal coliforms by 62% and coliphage by 40%. There was a significant correlation between the removal of Giardia cysts and Cryptospordium oocysts by the pond (P < 0.001). Influent turbidity and parasite removal were also significantly correlated (Cryptosporidium and turbidity, P=0.05; Giardia and turbidity, P=0.01). CONCLUSIONS: The larger organisms (parasites) probably settled to the bottom of the pond, while removal of smaller bacteria and coliphages in the pond was not as effective. SIGNIFICANCE AND IMPACT OF THE STUDY: Duckweed ponds may play an important role in wetland systems for reduction of Giardia and Cryptosporidium.     

Fate of bacterial indicators,viruses and protozoan parasites in a wastewater multi-component treatment system

The extent of reduction in selected microrganisms was tested at a multi-component wastewater treatment plant that treats sewage for a potential re-use in agriculture. The aim of the investigation was to evaluate possible reciprocal correlation among the different microrganisms and to compare the removal of two encysted pathogenic protozoa with that of microbial indicators, Clostridium perfringens spores, enteroviruses and bacteriophages. Samples collected included the raw wastewater, the chlorinated effluent and the effluent after an ultraviolet light treatment. All of the raw sewage samples were positive for Cryptosporidium oocysts and Giardia cysts, as well as for the other microorganisms tested but the bacteriophage B40-8. The data obtained confirm the removal efficiency of the entire process for indicator bacteria but also show the low and variable removal efficiency for the other microbial parameters, such as Giardia and Cryptosporidium, enteroviruses and Clostridium perfringens spores. Reciprocal correlation between Cryptosporidium and Giardia (oo)cysts and the other microbial groups was not demonstrated. The results confirm the resistance of Clostridium perfringens spores, enteroviruses and protozoa to chlorination and demonstrate the relative persistence of these organisms in the effluents even during the ultraviolet light treatment. The yields also emphasise the influence of the analytical method for the determination of protozoan parasites.     

Comparative analysis of Cryptosporidium, Giardia and indicator bacteria during sewage sludge hygienization in various composting processes

AIMS: To evaluate the suitability of Clostridium perfringens, Escherichia coli and enterococci as indicator organisms for Cryptosporidium and Giardia in treated sludge. METHODS AND RESULTS: Occurrence of Cryptosporidium oocysts and Giardia cysts, detected and enumerated by direct immunofluorescence microscopy, were compared with counts of indicator bacteria during six different sewage sludge hygienization processes, including closed reactor and open windrow composting, and sludge sanitation by quicklime or peat addition. No statistical correlation existed between the counts of indicator bacteria, Cl. perfringens, E. coli, and enterococci and occurrence of Cryptosporidium or Giardia. In sludge end-products, Giardia cysts were detected more frequently than Cryptosporidium oocysts. SIGNIFICANCE OF THE STUDY: Direct analysis is the best method to confirm the presence of (oo)cysts in sludge.     

Waterborne Giardia cysts and Cryptosporidium oocysts in the Yukon, Canada.

Several outbreaks of waterborne giardiasis have occurred in southern Canada, but nothing has been reported from the Canadian North. The objective of this study was to collect information relevant to waterborne giardiasis and cryptosporidiosis in the Yukon including epidemiological data and analyses of water, sewage, and animal fecal samples. Remote, pristine water samples were found to be contaminated with Giardia cysts (7 of 22 or 32%) but not with Cryptosporidium oocysts. Giardia cysts were found in 21% (13 of 61) of animal scats, but no Cryptosporidium oocysts were observed (small sample size). Whitehorse's drinking water was episodically contaminated with Giardia cysts (7 of 42 or 17%) and Cryptosporidium oocysts (2 of 42 or 5%). Neither were found in Dawson City's water supply. The only water treatment in the Yukon is chlorination, but contact times and free chlorine residuals are often too low to provide adequate protection by disinfection. Raw sewage samples from the five largest population centers in the Yukon contained 26 to 3,022 Giardia cysts and 0 to 74 Cryptosporidium oocysts per liter. Treated sewage from Whitehorse contained fewer Giardia cysts but more Cryptosporidium oocysts on average. Both were detected in Lake Laberge, downstream of Whitehorse, which has a history of fecal coliform contamination. Daily monitoring of raw sewage from the suburbs of Whitehorse showed a summertime peak of Giardia cysts and occasional Cryptosporidium oocysts after springtime contamination of drinking water. Despite this evidence, epidemiological data for the Yukon showed an endemic infection rate of only 0.1% for giardiasis (cryptosporidiosis is not notifiable).(ABSTRACT TRUNCATED AT 250 WORDS)     

Simultaneous detection of Cryptosporidium parvum and Giardia in sewage sludge by IC-PCR

AIMS: The aim of this study was to develop a method based on immunomagnetic capture and polymerase chain reaction (IC-PCR assay) for detection of Cryptosporidium parvum and Giardia intestinalis in sewage sludge. METHODS AND RESULTS: The detection limit of the IC-PCR assay for both organisms was 625 oocysts and cysts ml(-1). By hybridization of PCR products the sensitivity could be increased to 125 oocysts and cysts ml(-1). Forty-four sludge samples from 12 wastewater treatment plants were examined. The samples positive for Giardia (9 out of 44) were from eight wastewater plants and the C. parvum genotype 2 samples (3 out of 44) originated from different sewage works. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: IC-PCR offers the possibility to distinguish between Cryptosporidium and Giardia genotypes. This assay can be used to monitor the presence of these organisms in a community and to determine contamination of sludge used as soil amendment.     

Fate of Cryptosporidium oocysts, Giardia cysts, and microbial indicators during wastewater treatment and anaerobic sludge digestion.

The extent of reduction in selected microorganisms was tested during both aerobic wastewater treatment and anaerobic digestion of sludge at the wastewater treatment plant in Ottawa to compare the removal of two encysted pathogenic protozoa with that of microbial indicators. Samples collected included the raw wastewater, the primary effluent, the treated wastewater, the mixed sludge, the decanted liquor, and the cake. All of the raw sewage samples were positive for Cryptosporidium oocysts and Giardia cysts, as well as for the other microorganisms tested. During aerobic wastewater treatment (excluding the anaerobic sludge digestion), Cryptosporidium and Giardia were reduced by 2.96 log10 and 1.40 log10, respectively. Clostridium perfringens spores, Clostridium perfringens total counts, somatic coliphages, and heterotrophic bacteria were reduced by approximately 0.89 log10, 0.96 log10, 1.58 log10, and 2.02 log10, respectively. All of the other microorganisms were reduced by at least 3.53 log10. Sludge samples from the plant were found to contain variable densities of microorganisms. Variability in microbial concentrations was sometimes great between samples, stressing the importance of collecting a large number of samples over a long period of time. In all cases, the bacterial concentrations in the cake (dewatered biosolids) samples were high even if reductions in numbers were observed with some bacteria. During anaerobic sludge digestion, no statistically significant reduction was observed for Clostridium perfringens, Enterococcus sp., Cryptosporidium oocysts, and Giardia cysts. A 1-2 log10 reduction was observed with fecal coliforms and heterotrophic bacteria. However, the method utilized to detect the protozoan parasites does not differentiate between viable and nonviable organisms. On the other hand, total coliforms and somatic coliphages were reduced by 0.35 log10 and 0.09 log10, respectively. These results demonstrate the relative persistence of the protozoa in sewage sludge during wastewater treatment.     

Giardia and Cryptosporidium spp. in filtered drinking water supplies.

Giardia and Cryptosporidium levels were determined by using a combined immunofluorescence test for filtered drinking water samples collected from 66 surface water treatment plants in 14 states and 1 Canadian province. Giardia cysts were detected in 17% of the 83 filtered water effluents. Cryptosporidium oocysts, were observed in 27% of the drinking water samples. Overall, cysts or oocysts were found in 39% of the treated effluent samples. Despite the frequent detection of parasites in drinking water, microscopic observations of the cysts and oocysts suggested that most of the organisms were nonviable. Compliance with the filtration criteria outlined by the Surface Water Treatment Rule of the U.S. Environmental Protection Agency did not ensure that treated water was free of cysts and oocysts. The average plant effluent turbidity for sites which were parasite positive was 0.19 nephelometric turbidity units. Of sites that were positive for Giardia or Cryptosporidium spp., 78% would have been able to meet the turbidity regulations of the Surface Water Temperature Rule. Evaluation of the data by using a risk assessment model developed for Giardia spp. showed that 24% of the utilities examined would not meet a 1/10,000 annual risk of Giardia infection. For cold water conditions (0.5 degree C), 46% of the plants would not achieve the 1/10,000 risk level.     

Giardia and Cryptosporidium spp. in filtered drinking water supplies.

Giardia and Cryptosporidium levels were determined by using a combined immunofluorescence test for filtered drinking water samples collected from 66 surface water treatment plants in 14 states and 1 Canadian province. Giardia cysts were detected in 17% of the 83 filtered water effluents. Cryptosporidium oocysts, were observed in 27% of the drinking water samples. Overall, cysts or oocysts were found in 39% of the treated effluent samples. Despite the frequent detection of parasites in drinking water, microscopic observations of the cysts and oocysts suggested that most of the organisms were nonviable. Compliance with the filtration criteria outlined by the Surface Water Treatment Rule of the U.S. Environmental Protection Agency did not ensure that treated water was free of cysts and oocysts. The average plant effluent turbidity for sites which were parasite positive was 0.19 nephelometric turbidity units. Of sites that were positive for Giardia or Cryptosporidium spp., 78% would have been able to meet the turbidity regulations of the Surface Water Temperature Rule. Evaluation of the data by using a risk assessment model developed for Giardia spp. showed that 24% of the utilities examined would not meet a 1/10,000 annual risk of Giardia infection. For cold water conditions (0.5 degree C), 46% of the plants would not achieve the 1/10,000 risk level.     

Use of artificial neural networks to accurately identify Cryptosporidium oocyst and Giardia cyst images

Cryptosporidium parvum and Giardia lamblia are protozoa capable of causing gastrointestinal diseases. Currently, these organisms are identified using immunofluorescent antibody (IFA)-based microscopy, and identification requires trained individuals for final confirmation. Since artificial neural networks (ANN) can provide an automated means of identification, thereby reducing human errors related to misidentification, ANN were developed to identify Cryptosporidium oocyst and Giardia cyst images. Digitized images of C. parvum oocysts and G. lamblia cysts stained with various commercial IFA reagents were used as positive controls. The images were captured using a color digital camera at 400 x (total magnification), processed, and converted into a binary numerical array. A variety of "negative" images were also captured and processed. The ANN were developed using these images and a rigorous training and testing protocol. The Cryptosporidium oocyst ANN were trained with 1,586 images, while Giardia cyst ANN were trained with 2,431 images. After training, the best-performing ANN were selected based on an initial testing performance against 100 images (50 positive and 50 negative images). The networks were validated against previously "unseen" images of 500 Cryptosporidium oocysts (250 positive, 250 negative) and 282 Giardia cysts (232 positive, 50 negative). The selected ANNs correctly identified 91.8 and 99.6% of the Cryptosporidium oocyst and Giardia cyst images, respectively. These results indicate that ANN technology can be an alternate to having trained personnel for detecting these pathogens and can be a boon to underdeveloped regions of the world where there is a chronic shortage of adequately skilled individuals to detect these pathogens.     

Comparison of primers and optimization of PCR conditions for detection of Cryptosporidium parvum and Giardia lamblia in water.

Eight pairs of published PCR primers were evaluated for the specific detection of Cryptosporidium parvum and Giardia lamblia in water. Detection sensitivities ranged from 1 to 10 oocysts or cysts for purified preparations and 5 to 50 oocysts or cysts for seeded environmental water samples. Maximum sensitivity was achieved with two successive rounds of amplification and hybridization, with oligonucleotide probes detected by chemiluminescence. Primer annealing temperatures and MgCl2 concentrations were optimized, and the specificities of the primer pairs were determined with closely related species. Some of the primers were species specific, while others were only genus specific. Multiplex PCR for the simultaneous detection of Cryptosporidium and Giardia was demonstrated with primers amplifying 256- and 163-bp products from the 18S rRNA gene of Cryptosporidium and the heat shock protein gene of Giardia, respectively. The results demonstrate the potential utility of PCR for the detection of pathogenic protozoa in water but emphasize the necessity of continued development.     

Prevalence of Giardia cysts and Cryptosporidium oocysts and characterization of Giardia spp. isolated from drinking water in Canada.

This study was carried out to estimate the prevalence and potential for human infectivity of Giardia cysts in Canadian drinking water supplies. The presence of Cryptosporidium oocysts was also noted, but isolates were not collected for further study. A total of 1,760 raw water samples, treated water samples, and raw sewage samples were collected from 72 municipalities across Canada for analysis, 58 of which treat their water by chlorination alone. Giardia cysts were found in 73% of raw sewage samples, 21% of raw water samples, and 18.2% of treated water samples. There was a trend to higher concentration and more frequent incidence of Giardia cysts in the spring and fall, but positive samples were found in all seasons. Cryptosporidium oocysts were found in 6.1% of raw sewage samples, 4.5% of raw water samples, and 3.5% of treated water samples. Giardia cyst viability was assessed by infecting Mongolian gerbils (Meriones unguiculatus) and by use of a modified propidium iodide dye exclusion test, and the results were not always in agreement. No Cryptosporidium isolates were recovered from gerbils, but 8 of 276 (3%) water samples and 19 of 113 (17%) sewage samples resulted in positive Giardia infections. Most of the water samples contained a low number of cysts, and 12 Giardia isolates were successfully recovered from gerbils and cultured. Biotyping of these isolates by isoenzyme analysis and karyotyping by pulsed-field gel electrophoresis separated the isolates into the same three discrete groups. Karyotyping revealed four or five chromosomal bands ranging in size from 0.9 to 2 Mb, and four of the isolates had the same banding pattern as that of the WB strain. Analysis of the nucleotide sequences of the 16S DNA coding for rRNA divided the isolates into two distinct groups corresponding to the Polish and Belgian designations found by other investigators. The occurrence of these biotypes and karyotypes appeared to be random and was not related to geographic or other factors (e.g., different types were found in both drinking water and sewage from the same community). Biotyping and karyotyping showed that isolates from this study were genetically and biochemically similar to those found elsewhere, including well-described human source strains such as WB. We conclude that potentially human-infective Giardia cysts are commonly found in raw surface waters and sewage in Canada, although cyst viability is frequently low. Cryptosporidium oocysts are less common in Canada. An action level of three to five Giardia cysts per 100 liters in treated drinking water is proposed on the basis of the monitoring data from outbreak situations. This action level is lower than that proposed by Haas and Rose (C. N. Haas and J. B. Rose, J. Am. Water Works Assoc. 87(9):81-84, 1995) for Cryptosporidium spp. (10 to 30 oocysts per 100 liters).     

Recovery of Cryptosporidium oocysts and Giardia cysts from source water concentrates using immunomagnetic separation

Immunomagnetic separation (IMS) procedures for the simultaneous isolation of Cryptosporidium oocysts and Giardia cysts have recently become available. We validated Dynal's GC-Combo IMS kit using source water at three turbidity levels (5000, 500 and 50 nephelometric turbidity units [ntu]) obtained from different geographical locations and spiked with approximately 9--11 (oo)cysts per ml. Mean recoveries of Cryptosporidium oocysts and Giardia cysts in deionized water were 62% and 69%, respectively. In turbid water matrices, mean recoveries of Cryptosporidium oocysts were between 55.9% and 83.1% while mean recoveries of cysts were between 61.1% and 89.6%. Marginally higher recoveries of the heat inactivated (oo)cysts were observed (119.4% Cryptosporidium oocysts and 90.9% Giardia cysts) in deionized water when compared with recoveries of viable (oo)cysts (69.7% Cryptosporidium oocysts and 79% Giardia cysts). Age of (oo)cysts on recoveries using the GC-Combo IMS kit demonstrated no effects up to 20 months old. Recovery of Giardia cysts was consistent for isolates aged up to 8 months (81.4%), however, a significant reduction in recoveries was noted at 20 months age. Recoveries of low levels (5 and 10 (oo)cysts) of Cryptosporidium oocysts and Giardia cysts in deionized water using IMS ranged from 51.3% to 78% and from 47.6% to 90.0%, respectively. Results of this study indicate that Dynal's GC-Combo IMS kit is an efficient technique to separate Cryptosporidium/Giardia from turbid matrices and yields consistent, reproducible recoveries. The use of fresh (recently voided and purified) (oo)cysts, aged (oo)cysts, viable and heat-inactivated (oo)cysts indicated that these parameters do not influence IMS performance.     

Real-time PCR for quantification of Giardia and Cryptosporidium in environmental water samples and sewage

The protozoan pathogens Giardia lamblia and Cryptosporidium parvum are major causes of waterborne enteric disease throughout the world. Improved detection methods that are very sensitive and rapid are urgently needed. This is especially the case for analysis of environmental water samples in which the densities of Giardia and Cryptosporidium are very low. Primers and TaqMan probes based on the beta-giardin gene of G. lamblia and the COWP gene of C. parvum were developed and used to detect DNA concentrations over a range of 7 orders of magnitude. It was possible to detect DNA to the equivalent of a single cyst of G. lamblia and one oocyst of C. parvum. A multiplex real-time PCR (qPCR) assay for simultaneous detection of G. lamblia and C. parvum resulted in comparable levels of detection. Comparison of DNA extraction methodologies to maximize DNA yield from cysts and oocysts determined that a combination of freeze-thaw, sonication, and purification using the DNeasy kit (Qiagen) provided a highly efficient method. Sampling of four environmental water bodies revealed variation in qPCR inhibitors in 2-liter concentrates. A methodology for dealing with qPCR inhibitors that involved the use of Chelex 100 and PVP 360 was developed. It was possible to detect and quantify G. lamblia in sewage using qPCR when applying the procedure for extraction of DNA from 1-liter sewage samples. Numbers obtained from the qPCR assay were comparable to those obtained with immunofluorescence microscopy. The qPCR analysis revealed both assemblage A and assemblage B genotypes of G. lamblia in the sewage. No Cryptosporidium was detected in these samples by either method.     

Occurrence of Cryptosporidium and Giardia genotypes and subtypes in raw and treated water in Portugal

Aims:  Waterborne outbreaks of diarrhoeal illness reported worldwide are mostly associated with Cryptosporidium spp. and Giardia spp. Their presence in aquatic systems makes it essential to develop preventive strategies for water and food safety. This study was undertaken to monitor the presence of Cryptosporidium and Giardia in a total of 175 water samples, including raw and treated water from both surface and ground sources in Portugal.
Methods and Results:  The samples were processed according to USEPA Method 1623 for immunomagnetic separation (IMS) of Cryptosporidium oocysts and Giardia cysts, followed by detection of oocysts/cysts by immunofluorecence (IFA) microscopy, PCR-based techniques were done on all water samples collected. Out of 175 samples, 81 (46·3%) were positive for Cryptosporidium and 67 (38·3%) for Giardia by IFA. Cryptosporidium spp. and G. duodenalis genotypes were identified by PCR in 37 (21·7%) and 9 (5·1%) water samples, respectively. C. parvum was the most common species (78·9%), followed by C. hominis (13·2%), C. andersoni (5·3%), and C. muris (2·6%). Subtype IdA15 was identified in all C. hominis -positive water samples. S ubtyping revealed the presence of C. parvum subtypes IIaA15G2R1, IIaA16G2R1 and IIdA17G1. Giardia duodenalis subtype A1 was identified.
Conclusions:  The results of the present study suggest that Cryptosporidium spp. and Giardia spp. were widely distributed in source water and treated water in Portugal. Moreover, the results obtained indicate a high occurrence of human-pathogenic Cryptosporidium genotypes and subtypes in raw and treated water samples.
Significance and Impact of the Study:  Thus, water can be a potential vehicle in the transmission of cryptosporidiosis, and giardiasis of humans and animals in Portugal.     

New method using sedimentation and immunomagnetic separation for isolation and enumeration of Cryptosporidium parvum oocysts and Giardia lamblia cysts

A new method for the isolation of Cryptosporidium parvum oocysts and Giardia lamblia cysts from biosolid samples has been developed that utilizes sedimentation and immunomagnetic separation. The method was used to recover stained cysts and oocysts (spike organisms) from primary settled sewage sludge, anaerobically digested sewage sludge, and bovine manure. Recovery efficiencies associated with this method were approximately 40 to 60% and were significantly greater than those associated with similar methods based on sucrose flotation (P < 0.001). The recovery efficiency of the sedimentation-based method showed no significant reduction as a result of sample storage for up to 21 days (P > 0.05). Recovery efficiencies were determined by spiking samples with prestained cysts and oocysts, allowing them to be differentiated from those naturally present in the biosolid samples. The prestained cysts and oocysts had been fixed in 5% formalin, and the recovery efficiencies associated with this method may be different from recovery efficiencies for fresh cysts or oocysts.     

Occurrence of Cryptosporidium oocysts and Giardia cysts in sewage in Norway

Samples of sewage influent from 40 sewage treatment works (STW) throughout Norway were examined for Cryptosporidium oocysts and Giardia duodenalis cysts. Both parasites were detected frequently (80% of STW were Cryptosporidium positive; 93% of STW were Giardia positive) and at maximum concentrations of > 20,000 parasites/liter. The data suggest giardiasis is more widespread, and/or occurs with greater infection intensity, than cryptosporidiosis in Norway. STW serving higher person equivalents were more likely to be positive and had higher parasite concentrations. Parasite concentrations were used to estimate the proportion of contributing populations that could be clinically infected. For Cryptosporidium, the highest estimates were up to 5 per 100,000 individuals for two populations in eastern Norway. For Giardia, the highest estimate was 40 infected per 100,000 persons (approximately five times the usual national annual average) contributing to an STW in western Norway. As this population experienced a large waterborne giardiasis outbreak 6 months after sampling, it can be speculated that regular challenge with Giardia may occur here. Most Giardia isolates in sewage influent were assemblage A, although some assemblage B isolates were detected. There was substantial heterogeneity, but most samples contained isolates similar to genotype A3. Removal efficiencies at two STW with secondary treatment processes were estimated to be approximately 50% for Cryptosporidium and > 80% for Giardia. An STW with minimal treatment had negligible removal of both parasites. Many STW in Norway have minimal treatment and discharge effluent into rivers and lakes, thus, risk of contamination of water courses by Cryptosporidium and Giardia is considerable.     

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.     

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.     

Giardia and Cryptosporidium in water: evaluation of two concentration methods and occurrence in wastewater.

Giardia and Cryptosporidium are important agents of water-borne parasitic diseases. In this work we have examined the recovery efficiency of two methods for concentrating Giardia cysts and Cryptosporidium oocysts from water: a membrane filtration method and a crossflow filtration method. Results demonstrated a higher recovery efficiency for crossflow filtration method in comparison to the membrane filtration method. In addition, Giardia cysts and Cryptosporidium oocysts concentration was evaluated in wastewater samples submitted to chemical flocculation or chemical flocculation followed by slow sand filtration. Results showed that slow sand filtration was capable of reducing the number of Giardia cysts, but not of Cryptosporidium oocysts in wastewater.     

An evaluation of methods for the simultaneous detection of Cryptosporidium oocysts and Giardia cysts from water.

Methods for the simultaneous detection of Cryptosporidium parvum oocysts and Giardia cysts from water are described and their relative recovery efficiencies are assessed for seeded samples of both tap and river water. Cartridge filtration, membrane filtration, and calcium carbonate flocculation were evaluated, and steps to optimize the concentration procedures were undertaken. Increasing centrifugation to 5,000 x g, coupled with staining in suspension, was found to increase the overall efficiency of recovery of both cysts and oocysts. Cartridge filtration for both cysts and oocysts was examined by use of 100-liter volumes of both tap and river water. Improvements in recovery were observed for Cryptosporidium oocysts after extra washes of the filters. Calcium carbonate flocculation gave the maximum recovery for both Cryptosporidium oocysts and Giardia cysts and for both water types. A variety of 142-mm membranes was examined by use of 10-liter seeded samples of tap and river water. Cellulose acetate with a 1.2-micron pore size provided the best results for Cryptosporidium oocysts, and cellulose nitrate with a 3.0-micron pore size did so for Giardia cysts.