Detection of enteric viruses, Giardia and Cryptosporidium in two different types of drinking water treatment facilities

In this study, two types of drinking water treatment facilities (two conventional drinking water treatment plants (DWTPs) and two compact units (Cus)) were compared referring to their production capacity. Water samples were collected from three main points: (a) different water treatment steps (b) washings of sand filters and (c) distribution system at different distances from the water treatment plants. Both viruses and protozoa were concentrated from each water sample by adsorption and accumulation on the same nitrocellulose membrane filters (0.45 microm pore size). Enteroviruses were detected by plaque infectivity assay in BGM cells and HAV, HEV and Norovirus were detected by RT-PCR. Giardia and Cryptosporidium were detected by conventional staining methods and PCR. The results revealed that enterovirus load at the intake ranged between 10-15 PFU/L for the two compact units and between 4.5 and 75 PFU/L for the two conventional DWTPs. The virus load in distribution system of the first type DWTPs at 1 km from the plant was the same as that of the intake. Viruses in the other type of treatment plants CUs at 1, 5 and 7 km, were much reduced. Investigation of raw water sediments of the two DWTPs showed enterovirus counts between 12 and 17.5 PFU/L. Virus count was reduced in sand of filters after washing. Giardia cysts were equally detected by microscopy and PCR in only intake samples of EL-Hawamdia CU (33.3%) and Meet Fares DWTP (50%). Cryptosporidium oocysts were equally detected by microscopy and PCR in intake samples of Abo EL-Nomros CU (100%), EL-Hawamdia CU (66.7%) and Fowa DWTP (50%). At Meet Fares DWTP three positive intake samples for Cryptosporidium were detected by PCR, compared with only two positive samples by microscopy. Giardia cysts and Cryptosporidium oocysts were detected in raw water sediment and sand of filters before washing. Only one sample from Meet Fares DWTP sand of filters after washing was positive for both Giardia and Cryptosporidium. It can be concluded that the poor microbial quality of the water may be due to improper operational skills and management of the various water treatment plants (especially at the two high capacity treatment plants).     

Impact of bathers on levels of Cryptosporidium parvum oocysts and Giardia lamblia cysts in recreational beach waters

Recreational beach water samples collected on weekends and weekdays during 11 consecutive summer weeks were tested for potentially viable Cryptosporidium parvum oocysts and Giardia lamblia cysts using the multiplexed fluorescence in situ hybridization (FISH) method. The levels of oocysts and cysts on weekends were significantly higher than on the weekdays (P<0.01). Concentrations of oocysts in weekend samples (n=27) ranged from 2 to 42 oocysts/L (mean: 13.7 oocysts/L), and cyst concentration ranged from 0 to 33 cysts/L (mean: 9.1 cysts/L). For the samples collected on weekdays (n=33), the highest oocyst concentration was 7 oocysts/L (mean: 1.5 oocysts/L), and the highest cyst concentration was 4 cysts/L (mean: 0.6 cysts/L). The values of water turbidity were significantly higher on weekends than on weekdays, and were correlated with the number of bathers and concentration of C. parvum oocysts and G. lamblia cysts (P<0.04). The study demonstrated positive relationships between number of bathers and levels of waterborne C. parvum oocysts and G. lamblia cysts in recreational beach water. It is essential to test recreational waters for Cryptosporidium and Giardia when numbers of bathers are greatest, or limit the number of bathers in a recreational beach area.     

Recovery and detection of Cryptosporidium parvum oocysts from water samples using continuous flow centrifugation

Continuous flow centrifugation (CFC) was used in conjunction with immunomagnetic separation (IMS) and immunofluorescence microscopy (IFA) and nested PCR to recover and detect oocysts of Cryptosporidium parvum and cysts of Giardia intestinalis from 10L volumes of source water samples. Using a spiking dose of 100 oocysts, nine of 10 runs were positive by IFA, with a mean recovery of 4.4+/-2.27 oocysts; when another 10 runs were analyzed using nested PCR to the TRAP C-1 and Cp41 genes, nine of 10 were positive with both PCR assays. When the spiking dose was reduced to 10 oocysts in 10L, 10 of 12 runs were positive by IFA, with a mean oocyst recovery of 3.25+/-3.25 oocysts. When 10 cysts of Giardia intestinalis were co-spiked with oocysts into 10L of source water, five of seven runs were positive, with a mean cyst recovery of x=0.85+/-0.7. When 10 oocysts (enumerated using a fluorescence activated cell sorter) were spiked into 10L volumes of tap water, one of 10 runs was positive, with one oocyst detected. For the majority of the source water samples, turbidities of the source water samples ranged from 1.1 to 22 NTU, but exceeded 100 NTU for some samples collected when sediment was disturbed. The turbidities of pellets recovered using CFC and resuspended in 10 mL of water were very high (exceeding 500 NTU for the source water-derived pellets and 100 NTU for the tap water-derived pellets). While not as efficient as existing capsule-filtration based methods (i.e., US EPA methods 1622/1623), CFC and IMS may provide a more rapid and economical alternative for isolation of C. parvum oocysts from highly turbid water samples containing small quantities of oocysts.     

Prevalence of Cryptosporidium oocysts and giardia cysts in the drinking water supply in Japan

A one-year monitoring of Cryptosporidium oocysts and Giardia cysts was conducted at a water purification plant. A total of 13 samples of 50 L river source water and 26 samples of 2,000 L-filtered water, treated by coagulation flocculation, sedimentation and rapid filtration, were tested. Prior to conducting a survey of a water purification plant, we developed a method for concentrating Cryptosporidium oocysts from a large volume of raw or filtered water using a hollow fiber ultrafiltration (UF) membrane, and this procedure was adapted to survey a water purification plant. Cryptosporidium oocysts were detected in all of the 13 raw water samples. The geometric mean concentration was 40 oocysts 100 L. Giardia cysts were detected in 12 of 13 raw water samples (92%) and the geometric mean concentration was 17 cysts/100 L. Probability distributions of both Cryptosporidium oocyst and Giardia cyst concentration in raw water were nearly lognormal. In filtered water samples, Cryptosporidium oocysts were detected in 9 of the 26 samples (35%) with the geometric mean concentration of 1.2 oocysts /1,000 L and Giardia cysts in 3 samples (12%) with 0.8 cysts/1,000 L. The estimated log10 removal efficiency of Cryptosporidium oocysts and Giardia cysts by rapid-sand filtration was 2.47 and 2.53, respectively. Empty particles were removed at a higher log10 than intact oocysts and cysts. The efficiency of particle removal in the rapid sand filtration process tends to be reduced under cold-water conditions. Close management is necessary in the winter when the water temperature is low.     

Occurrence and persistence of enteroviruses, noroviruses and F-specific RNA phages in natural wastewater biofilms

Enteroviruses and noroviruses are pathogenic viruses excreted by infected individuals. Discharged in wastewaters, some of these viruses can be captured by biofilms. In the present study, we assessed the occurrence and persistence of these viruses in wastewaters and in corresponding biofilms. Natural wastewaters and biofilms were analyzed monthly from January to July using real-time RT-PCR. Enterovirus RNA was detected in wastewater in June while norovirus RNA was detected from January to March. In contrast, biofilm analysis revealed the presence of both enterovirus and norovirus genomes throughout the study period. For instance, enterovirus and norovirus genogroups (GG) I and II were detected in 50, 46 and 37% of the biofilm samples, respectively (n = 24). In a laboratory experiment, persistence of norovirus GGI RNA (quantified using molecular techniques) and F-specific bacteriophages (quantified using both culture and molecular techniques) was assessed in wastewater and corresponding naturally-contaminated biofilms at both 4 and 20 °C. The concentrations of viral genomes (norovirus GGI and F-specific RNA phage) were very stable in biofilms. Indeed, no significant decrease was observed during the persistence experiment that lasted 49 days. Furthermore, regardless of our experimental conditions, viral genome and infectious F-specific bacteriophages persisted longer in biofilm than in wastewater. According to our results, wastewater biofilms may contribute to the persistence and dispersal of pathogenic viruses outside of epidemic periods.     

Removal of Giardia and Cryptosporidium in drinking water treatment: a pilot-scale study

Giardia and Cryptosporidium have emerged as waterborne pathogens of concern for public health. The aim of this study is to examine both parasites in the water samples taken from three pilot-scale plant processes located in southern Taiwan, to upgrade the current facilities. Three processes include: conventional process without prechlorination (Process 1), conventional process plus ozonation and pellet softening (Process 2), and integrated membrane process (MF plus NF) followed conventional process (Process 3). The detection methods of both parasites are modified from USEPA Methods 1622 and 1623. Results indicated that coagulation, sedimentation and filtration removed the most percentage of both protozoan parasites. The pre-ozonation step can destruct both parasites, especially for Giardia cysts. The microfiltration systems can intercept Giardia cysts and Cryptosporidium oocysts completely. A significant correlation between water turbidity and Cryptosporidium oocysts was found in this study. The similar results were also found between three kinds of particles (phi=3-5,5-8 and 8-10 microm) and Cryptosporidium oocysts.     

Quantification of pathogenic microorganisms and microbial indicators in three wastewater reclamation and managed aquifer recharge facilities in Europe

Managed Aquifer Recharge (MAR) is becoming an attractive option for water storage in water reuse processes as it provides an additional treatment barrier to improve recharged water quality and buffers seasonal variations of water supply and demand. To achieve a better understanding about the level of pathogenic microorganisms and their relation with microbial indicators in these systems, five waterborne pathogens and four microbial indicators were monitored over one year in three European MAR sites operated with reclaimed wastewater. Giardia and Cryptosporidium (oo)cysts were found in 63.2 and 36.7% of the samples respectively. Salmonella spp. and helminth eggs were more rarely detected (16.3% and 12.5% of the samples respectively) and Campylobacter cells were only found in 2% of samples. At the Belgian site advanced tertiary treatment technology prior to soil aquifer treatment (SAT) produced effluent of drinking water quality, with no presence of the analysed pathogens. At the Spanish and Italian sites amelioration of microbiological water quality was observed between the MAR injectant and the recovered water. In particular Giardia levels decreased from 0.24-6.14 cysts/L to 0-0.01 cysts/L and from 0.4-6.2 cysts/L to 0-0.07 cysts/L in the Spanish and Italian sites respectively. Salmonella gene copies and Giardia cysts were however found in the water for final use and/or the recovered groundwater water at the two sites. Significant positive Spearman correlations (p < 0.05, r_s range: 0.45-0.95) were obtained, in all the three sites, between Giardia cysts and the most resistant microbial markers, Clostridium spores and bacteriophages.     

UASB/MBR组合工艺处理抗生素废水的研究

采用UASB/MBR组合工艺处理抗生素废水,考察了其处理效果及影响因素.结果表明,当UASB的水力停留时间(HRT)为13 h、MBR的HRT为7.5 h时,系统对COD的去除效果最好,在进水COD为1 000～9 000 mg/L的条件下,出水COD可降至199 mg/L,对COD的总去除率可达80%以上.     

某市贾第鞭毛虫和隐孢子虫污染现状

利用免疫荧光分析技术对南方某市饮用水源水、自来水厂出水和污水处理厂进、出水中贾第鞭毛虫和隐孢子虫(两虫)的污染状况进行了调查,并就自来水厂常规处理工艺对两虫的去除特性进行了研究.结果显示:该市饮用水源水中的两虫密度分别为2～120个/100L和0～24个/100 L,自来水厂出水分别为0～12个/1000 L和0～8个/1000 L,污水处理厂进水中的两虫密度分别为7 200～18 300个/L和69～1 210个/L,二级处理出水的分别为6～153个/L和1～46个/L;混凝沉淀和过滤对两虫有较好的去除效果.     

Monitoring of Cryptosporidium and Giardia river contamination in Paris area

This study evaluates the protozoan contamination of river waters, which are used for drinking water in Paris and its surrounding area (about 615,000 m(3) per day in total, including 300,000 m(3) for Paris area). Twenty litre samples of Seine and Marne Rivers were collected over 30 months and analyzed for Cryptosporidium oocysts and Giardia cysts detection according to standard national or international methods. Cryptosporidium oocysts and Giardia cysts were found, respectively, in 45.7% and 93.8% of a total of 162 river samples, with occasional high concentration peaks. A significant seasonal pattern was observed, with positive samples for Cryptosporidium more frequent in autumn than spring, summer and winter, and positive samples for Giardia less frequent in summer. Counts of enterococci and rainfalls were significantly associated with Giardia concentration but not Cryptosporidium. Other faecal bacteria were not correlated with monitored protozoan. Marne seems to contribute mainly to the parasitic contamination observed in Seine. Based on seasonal pattern and rainfall correlation, we hypothesize that the origin of contamination is agricultural practices and possible dysfunction of sewage treatment plants during periods of heavy rainfalls. High concentrations of protozoa found at the entry of drinking water plants justify the use of efficient water treatment methods. Treatment performances must be regularly monitored to ensure efficient disinfection according to the French regulations.     

Effect of pathogen concentrations on removal of Cryptosporidium and Giardia by conventional drinking water treatment

The presence of waterborne enteric pathogens in municipal water supplies contributes risk to public health. To evaluate the removal of these pathogens in drinking water treatment processes, previous researchers have spiked raw waters with up to 10(6) pathogens/L in order to reliably detect the pathogens in treated water. These spike doses are 6-8 orders of magnitude higher than pathogen concentrations routinely observed in practice. In the present study, experiments were conducted with different sampling methods (i.e., grab versus continuous sampling) and initial pathogen concentrations ranging from 10(1) to 10(6) pathogens/L. Results showed that Cryptosporidium oocyst and Giardia cyst removal across conventional treatment were dependent on initial pathogen concentrations, with lower pathogen removals observed when lower initial pathogen spike doses were used. In addition, higher raw water turbidity appeared to result in higher log removal for both Cryptosporidium oocysts and Giardia cysts.     

Detection of somatic phages, infectious enteroviruses and enterovirus genomes as indicators of human enteric viral pollution in surface water

In the present study, we aimed to determine whether the concentrations of somatic coliphages, infectious enteroviruses or the detection of enterovirus genomes were associated with the detection of human pathogenic viruses in surface water. Four French rivers were sampled monthly or semimonthly for the quantitative detection of somatic coliphages, infectious enteroviruses and the qualitative RT-PCR detection of enterovirus, hepatitis A virus, Norwalk I viruses, Norwalk II viruses, astrovirus and rotavirus genomes over 12 months. All the 68 water samples tested were positive for the quantitative detection of somatic coliphages (range of concentrations: 4 x 10(2) to 1.6 x10(5) FUl(-1)). Infectious enteroviruses were isolated by a cell culture system in only two (3%) of the 68 concentrated water samples tested, whereas enterovirus genomes were detectable in 60 (88%) of the same samples. A positive RT-PCR detection of the genome of hepatitis A virus, Norwalk-like virus genogroup II, astrovirus, rotavirus and Norwalk-like virus genogroup I was demonstrated, respectively, in 1.5% (1/68), 1.5% (1/68), 3% (2/68), 0% and 0% of the 68 concentrated water samples tested. All of these four water samples were positive for the detection of enterovirus genomes, whereas only one of them was positive for the isolation of enteroviruses on cell culture. Moreover, the genomic detection of human pathogenic viruses appeared not to be statistically associated with the concentration levels of somatic coliphages in the 68 concentrated water samples tested (Wilcoxon rank test; P=0.14). Taken together, our findings indicate that the quantitative detection of somatic coliphages and the isolation of enteroviruses on cell culture are not suitable parameters for the control of the viral contamination in surface water, whereas the detection of enterovirus genomes may be useful for predicting the presence of waterborne viruses.     

Contribution of treated wastewater to the contamination of recreational river areas with Cryptosporidium spp. and Giardia duodenalis

Samples of the influent and final effluent from 12 wastewater treatment plants from Galicia (NW, Spain) were analyzed for the presence of Cryptosporidium spp. oocysts and Giardia duodenalis cysts. All of the plants discharge effluent to a hydrographic basin in which there are numerous recreational areas and fluvial beaches. The samples (25-50 liters) were collected in spring, summer, autumn and winter of 2007. A total of 96 samples were analyzed using techniques included in the US Environmental Protection Agency Method 1623. To identify the genotypes present, the following genes were amplified and sequenced: 18S SSU rRNA (Cryptosporidium spp.) and beta-giardina (G. duodenalis). Both parasites were detected in influent and effluent samples from all treatment plants (100%) throughout the year, and G. duodenalis always outnumbered Cryptosporidium spp. The mean concentration of G. duodenalis per liter of influent was significantly higher (P<0.05) than the mean concentration of Cryptosporidium spp. per liter of influent. The mean concentrations of parasites in influent samples ranged from 6 to 350 Cryptosporidium spp. oocysts per liter and from 89 to 8305 G. duodenalis cysts per liter. In final treated effluent, the mean concentration of parasites ranged from 2 to 390 Cryptosporidium spp. oocysts per liter and from 79 to 2469 G. duodenalis cysts per liter. The distribution of results per season revealed that in all plants, the highest number of (oo)cysts were detected in spring and summer. Cryptosporidium parvum, Cryptosporidium andersoni, Cryptosporidium hominis and assemblages A-I, A-II, E of G. duodenalis were detected. The risk of contamination of water courses by Cryptosporidium spp. and G. duodenalis is therefore considerable. It is important that wastewater treatment authorities reconsider the relevance of the levels of contamination by both parasites in wastewater, and develop adequate countermeasures.     

Release of infectious human enteric viruses by full-scale wastewater utilities

In the United States, infectious human enteric viruses are introduced daily into the environment through the discharge of treated water and the digested sludge (biosolids). In this study, a total of 30 wastewater and 6 biosolids samples were analyzed over five months (May-September 2008-2009) from five full-scale wastewater treatment plants (WWTPs) in Michigan using real-time PCR and cell culture assays. Samples were collected from four different locations at each WWTP (influent, pre-disinfection, post-disinfection and biosolids) using the 1MDS electropositive cartridge filter. Adenovirus (HAdV), enterovirus (EV) and norovirus genogroup II (NoV GGII) were detected in 100%, 67% and 10%, respectively of the wastewater samples using real-time PCR. Cytopathic effect (CPE) was present in 100% of the cell culture samples for influent, pre- and post-disinfection and biosolids with an average log concentration of 4.1 (2.9-4.7, range) 1.1 (0.0-2.3, range) and 0.5 (0.0-1.6, range) MPN/100 L and 2.1 (0.5-4.1) viruses/g, respectively. A significant log reduction in infectious viruses throughout the wastewater treatment process was observed at an average 4.2 (1.9-5.0, range) log units. A significant difference (p-value <0.05) was observed using real-time PCR data for HAdV but not for EV (p-value >0.05) removal in MBR as compared to conventional treatment. MBR treatment was able to achieve an additional 2 and 0.5 log reduction of HAdV and EV, respectively. This study has demonstrated the release of infectious enteric viruses in the final effluent and biosolids of wastewater treatment into the environment.     

Settling velocity of Cryptosporidium parvum and Giardia lamblia

Understanding transport behavior of Cryptosporidium parvum oocysts and Giardia lamblia cysts (together referred to as (oo)cysts) in overland flow is important for beneficial uses of receiving water bodies. Like sediment, (oo)cysts are subjected to deposition once they are present in overland flow or low flow environments like reservoirs, wetlands and sedimentation basins. The objectives of this paper are to present the theory and experiment to determine the free settling velocity (v(s)) of (oo)cysts and to compare experimental settling velocities to estimates using Stokes' law. A settling experiment was designed to quantify the v(s) of (oo)cysts in an aqueous column. C. parvum oocysts used were spherical with average diameter (+/-1SD) of 6.6+/-1.1 microm. G. lamblia cysts were oval shaped (average eccentricity = 1.48+/-0.19) with average size of 11.8 +/-1.3 microm. Average densities were 1009 kg m(-3) for C. parvum oocysts and 1013 kg m(-3) for G. lamblia cysts. Observed experimental settling velocities are 0.27 microm s(-1) and 0.67 microm s(-1) for C. parvum and G. lamblia, respectively. Estimated average settling velocities using Stokes' law were 0.36 microm s(-1) for C. parvum and 0.84 microm s(-1) for G. lamblia. R-squared values of the observations from the settling experiments with the Stokes' law estimation are 0.87 and 0.88 for G. lamblia and C. parvum, respectively. Our results suggest that Stokes' law can be used to estimate settling velocities of (oo)cysts. Qualitatively, the low settling velocities indicate that (oo)cysts will very slowly settle out of suspension.     

Influence of land use and watershed characteristics on protozoa contamination in a potential drinking water resources reservoir

Relative changes in the microbial quality of Lake Texoma, on the border of Texas and Oklahoma, were investigated by monitoring protozoan pathogens, fecal indicators, and factors influencing the intensity of the microbiological contamination of surface water reservoirs. The watershed serves rural agricultural communities active in cattle ranching, recreation, and is a potential drinking water source. A total of 193 surface water samples were tested over a 27-month period to determine levels of parasite contamination. The overall occurrence of Cryptosporidium oocysts was higher in both frequency and concentration than Giardia cysts. Cryptosporidium oocysts were found in 99% and Giardia cysts in 87% of the samples. Although Cryptosporidium and Giardia occurrence were significantly but not strongly correlated, all other correlation coefficients including turbidity and total dissolved solids were non-significant. Statistically supportable seasonal variations were found suggesting that Cryptosporidium and Giardia were higher in summer and fall than in other seasons of the year. While Cryptosporidium levels were correlated with rainfall, this was not the case with Giardia. The maximum numbers for both protozoan parasites were detected from a site impacted by cattle ranching during calving season. Restriction fragment length polymorphism analysis was used for confirmation of Cryptosporidium in surface waters influenced by agricultural discharges. As we had expected, oocysts were of the bovine type indicating that the Cryptosporidium parvum detected in surface waters perhaps came from cattle living in the watershed.     

Cryptosporidium spp. and Giardia duodenalis in two areas of Galicia (NW Spain)

The aim of the present study was to investigate the environmental dispersal of Cryptosporidium spp. and Giardia duodenalis in two distinct areas (coastal and inland) in Galicia (NW Spain). Faecal samples were collected from healthy asymptomatic domestic (cows and sheep) and wild animals (deer and wild boars) in the selected areas. In each of the selected areas, samples of untreated water (influent) and of treated water (final effluent) were collected from each of the 12 drinking water treatments plants (DWTPs) and 12 wastewater treatment plants (WTPs) under study. Analysis of a single sample from each of the 635 (coastal) and 851 (inland) domestic and wild animals selected at random revealed that the prevalences of cryptosporidiosis and giardiosis in coastal area were 9.2% and 15.9% respectively, and in inland area, 13.7% and 26.7% respectively. In the coastal area, Cryptosporidium spp. oocysts were detected in influent and effluent samples from 2/12 (16.6%) DWTPs and 8/12 (66.6%) WTPs, while G. duodenalis cysts were detected in influent and effluent samples from 3/12 (25.0%) DWTPs and 12/12 (100%) WTPs. The concentrations were notably higher in WTPs; the mean parasite concentrations in the final treated effluent were 10 oocysts per litre and 137.8 cysts per litre for Cryptosporidium and Giardia, respectively. The mean concentration of G. duodenalis cysts per litre was significantly higher (P < 0.05) than the mean concentration of Cryptosporidium spp. oocysts per litre in both the influent and the effluent samples from all the treatment plants. In the coastal area, C. parvum, C. hominis and G. duodenalis assemblages A (I and II) and E were most repeatedly detected. In the inland area, C. parvum, C. andersoni and G. duodenalis assemblages A (I and II), B and E were most frequently identified.     

Inactivation of Escherichia coli by ozone under bench-scale plug flow and full-scale hydraulic conditions

To determine the disinfection efficacy of ozonation, water companies can apply several disinfection calculation methods. The goal of this study was to evaluate the use of the T10 and continuous stirred tank reactor (CSTR) method to extrapolate inactivation rates of ozone sensitive microorganisms observed in laboratory tests to full-scale ozonation in drinking water treatment. The inactivation efficacy of the ozonation at the Amsterdam water treatment works was assessed by determining Escherichia coli concentrations in large volume samples before and after ozonation over a period of 1 year. The inactivation of dosed E. coli WR1 was tested in a bench-scale dissolved ozone plug flow reactor (DOPFR) on the same feed water as the full-scale ozonation in which a concentrated ozone solution in Milli-Q water was dosed. Applying the T10 method on the inactivation rates observed in the DOPFR strongly overestimated the inactivation capacity of the full-scale ozonation. The expected inactivation based on the CSTR method (LT2ESWTR) approached the observed inactivation at full-scale. Therefore, the CSTR method should be preferred to calculate inactivation of ozone sensitive organisms such as E. coli, viruses, Giardia and Campylobacter by full-scale ozonation.