Genotyping of single Cryptosporidium oocysts isolated from sewage and river water.

The study was designed to genotype individual Cryptosporidium oocysts using an 18S rRNA gene-based semi-nested PCR and direct sequencing procedure. Positive PCR amplification was observed in all single C. parvum HNJ-1 oocyst samples tested. Semi-nested PCR and direct sequencing was applied to Cryptosporidium oocysts isolated from sewage and river water. The procedure could genotype 54% of FITC-stained single oocysts isolated from sewage and 32% from river water. The predominant genotype in both sewage and river water was C. parvum genotype 1, accounting for 33 and 25%, respectively, of all the FITC-stained intact Cryptosporidium oocysts present.     

上海市浦东给水厂两虫去除研究

采用EPA1623法(免疫磁分离及荧光染色法)对上海市几家典型的给水厂的水源水、出厂水、管道水中贾第鞭毛虫和隐孢子虫的分布状况进行了测定分析,并对给水厂净水过程中不同操作单元对两虫的去除效果进行了研究.研究表明:上海浦东给水厂水源水中存在贾第鞭毛虫与隐孢子虫,但其密度分别维持在0～6个/10 L和0～8个/10 L的低水平,而出厂水与管道水中均未检出两虫;给水厂净水过程中,混凝沉淀对两虫具有明显的去除效果.     

Evaluation of activated sludge treatment and the efficiency of the disinfection of Giardia species cysts and Cryptosporidium oocysts by UV at a sludge treatment plant in Campinas, south-east Brazil.

Among many waterborne diseases the giardiasis and cryptosporidiosis are of particular public health interest, because Giardia cysts and Cryptosporidium oocysts can persist for long periods in the environment, and both pathogenic protozoa have been implicated as the cause of many outbreaks of gastroenteritis in the last 25 years. In order to evaluate the efficiency of cysts and oocysts' removal by the activated sludge process, and by UV reactor in inactivating cysts and oocysts in one wastewater treatment plant (WWTP) of Campinas, three sampling points were selected for study: (1) influent, (2) treated effluent without UV disinfection and (3) treated effluent with UV disinfection. Giardia spp. cysts prevailed with higher density in the three different sample types. Cryptosporidium spp. oocysts were observed in only two samples of influent and just one sample of treated sewage with UV disinfection. In the animal infectivity assay for Giardia spp, one mouse of the UV treated group revealed trophozoites in intestinal scrapings. The results of the present study indicate that treatment by activated sludge process delivered a reduction of 98.9% of cysts and 99.7% of oocysts and UV disinfection was not completely efficient regarding the inactivation of Giardia cysts in the case of the WWTP studied.     

Aerobic bacterial spores as process indicators for protozoa cysts in water treatment plants.

The possibility of using aerobic spores as indicators (surrogates) of water treatment efficiency for the removal of Giardia cysts and Cryptosporidium oocysts was evaluated in a water treatment plant that supplies the Barcelona area of Spain. The water treatment consists of pre-chlorination, flocculation-sedimentation, double filtration (sand and granular activated carbon, GAC) with intermediate ozonation and post-chlorination. Aerobic spores significantly increased after GAC filtration, which indicated an active propagation of aerobic spore-formers. However, anaerobic (Clostridium) spores could be a good surrogate for Cryptosporidium oocysts, especially if their detection in samples at low concentrations was improved.     

Determining potential indicators of Cryptosporidium oocysts throughout the wastewater treatment process

Most research on wastewater treatment efficiency compliance focuses on physicochemical and microbial indicators; however, very little emphasis has been placed so far on determining suitable indicator organisms to predict the discharge level of pathogens from treatment plants. In this study, raw wastewater, activated sludge, and the resulting final effluents and biosolids in four municipal wastewater treatment plants (WWTPs A, B, C and D) were seasonally investigated for human-virulent water-borne pathogens Cryptosporidium parvum/hominis and Giardia duodenalis, and microsporidia (e.g. Encephalitozoon hellem, E. intestinalis, and Enterocytozoon bieneusi) between 2008 and 2009. A suite of potential microbial indicators for human-virulent protozoa and microsporidia was also determined. A combination of multiple fluorescent in situ hybridization and immunofluorescent antibody assays were applied to detect Cryptosporidium oocysts, Giardia cysts, and microsporidian spores. Escherichia coli, enterococci and Clostridium perfringens spores were cultivated in selective media. Positive correlations were found between the abundance of enterococci and E. coli and abundance of Cryptosporidium oocysts (r(s) > 0.47, p < 0.01) and Giardia cysts (r(s) > 0.44, p < 0.01) at WWTPs A-D. Cryptosporidium perfringens spores were positively correlated to Cryptosporidium oocysts (r(s) = 0.40, p < 0.01) and Giardia cysts (r(s) = 0.46, p < 0.01). There was a strong positive correlation between abundance of Giardia cysts and that of Cryptosporidium oocysts (r(s) > 0.89, p < 0.01). To sum up, a suite of faecal indicator bacteria can be used as indicators for the presence of Cryptosporidium oocysts and Giardia cysts in these activated-sludge systems (WWTPs A, B and C). Overall, Giardia duodenalis was noted to be the best Cryptosporidium indicator for human health in the community-based influent wastewater and throughout the treatment process.     

Evidence for the existence of Cryptosporidium oocysts as single entities in surface runoff.

There is uncertainty whether Cryptosporidium oocysts attach to particles or to each other under ambient water conditions. Particle size distributions of Cryptosporidium oocyst suspensions were determined over a range of ionic strengths and pHs to determine under those environmental conditions that may promote oocyst aggregation. Cryptosporidium oocysts were shown to only aggregate in high ionic strength solutions (>0.45 M) and remain largely as single entities at ionic strengths and pHs that were likely to be encountered in surface runoff. Similarly, in loam soil suspensions, rather than attaching to the soil particles the majority of oocysts also remained as single entities. Overall, oocysts are expected to remain largely unattached to either themselves or soil particles in overland runoff. This has implications for pathogen transport and modelling since oocysts that are freely suspended are more likely to be transported in runoff to surface waters than if attached to more dense soil/faecal particles.     

Cryptosporidium spp. and Giardia spp. removal by bank filtration at Beberibe River,Brazil

In bank filtration (BF) technology, a production well is pumped near surface water and induces water flow from the river through a porous medium to the well by percolation into the soil. Several physical, chemical, and biological processes occur, providing a natural water treatment along the river banks. An experimental area was installed on the Beberibe River with 2 production wells and 7 monitoring wells. The BF potential in removing pathogenic intestinal parasites and analysis of physical–chemical and bacteriological parameters was evaluated, according to Standard Methods. River–aquifer interaction was characterized by piezometric levels of production wells. Monitoring of the wells was correlated with the water depth of the river and precipitation. Parasite analysis was performed using Hoffman, Pons, and Janer's methods of spontaneous sedimentation, followed by centrifugation and preparation of slides stained with acetic Lugol. Protozoa oocysts were isolated by a modified Ziehl‐Neelsen method, preceded by sedimentation and centrifugation. The pathogenic protozoa found in samples from Beberibe River were Cryptosporidium spp., Giardia spp., Entamoeba histolytica/dispar complex, and Isospora belli. Pathogenic helminths were also detected: Ascaris lumbricoides, Strongyloides stercoralis, and hookworm eggs and larvae, Hymenolepis nana. In water samples from the production wells, no waterborne pathogens were found. The BF pilot project was effective in reducing levels of turbidity and color. Total coliforms and Escherichia coli were absent in the production wells. Piezometric levels of production wells and monitoring wells correlated with water depth of Beberibe River show hydraulic connection between the production wells and river, thus featuring a river–aquifer interaction. The BF pilot system showed potential for reduction or elimination of pathogenic intestinal parasites.     

Direct detection of bacterial faecal indicators in water samples using PCR.

The presence of enteric pathogens in water resources represents a serious risk for public health. Therefore, their precise detection, and especially detection of E. coli, which is obviously regarded as the main indicator of faecal contamination of water, is an essential step in ensuring bacterial safety of water. Numerous PCR protocols for detection of E. coli have been published to date. They are usually based on amplification of regions derived from lacZ (beta-D-galactosidase) and uidA (beta-D-glucuronidase) gene sequences. However, these methods are not universal enough for precise detection of all E. coli strains found in water samples. We developed a novel triplex PCR method for detection of E. coli in which cyd gene coding for cytochrome bd complex was co-amplified along with lacZ and uidA genes. Our triplex PCR approach significantly increases the specificity and reliability of E. coli detection in water samples. This approach allowed us to distinguish Shigella flexneri from E. coli. In addition, we were able to detect even non-coliform Klebsiella and Raoutella spp., some of which can also cause infections to humans.     

Evaluation of the purification capacity of nine portable, small-scale water purification devices.

A test was performed to evaluate the microbial and chemical purification capacity of nine portable, small-scale water purification filter devices with production capacity less than 100 L/h. The devices were tested for simultaneous removal capacity of bacteria (cultured Escherichia coli, Clostridium perfringens, Klebsiella pneumoniae and Enterobacter cloacae), enteric protozoans (formalin-stored Cryptosporidium parvum oocysts), viral markers (F-RNA bacteriophages) and microcystins produced by toxic cyanobacterial cultures. In general, the devices tested were able to remove bacterial contaminants by 3.6-6.9 log10 units from raw water. Those devices based only on filtration through pores 0.2-0.4 microm or larger failed in viral and chemical purification. Only one device, based on reverse osmosis, was capable of removing F-RNA phages at concentrations under the detection limit and microcystins by 2.5 log10. The present study emphasised the need for evaluation tests of water purification devices from the public safety and HACCP (Hazard Analysis and Critical Control Point) points of view. Simultaneous testing for various pathogenic/indicator microbes and microcystins was shown to be a useful and practical way to obtain essential data on actual purification capacity of commercial small-scale drinking-water filters.     

Influence of raw water storage on Giardia,Cryptosporidium and nematodes

A watershed derived from a disused gravel-quarry has been studied for the relocation of the catchment area of Turin surface water treatment plants. The improvement of river water quality as a consequence of shortterm storage has been investigated, focusing the attention on three problem organisms, namely the parasitic protozoans Giardia spp, and Cryptosporidium spp. and the free-living Nematodes, which could be considered indicators of healthy compliance and product agreeability respectively.     

Cryptosporidium monitoring system at a water treatment plant, based on waterborne risk assessment.

The water volume required for daily monitoring of Cryptosporidium (which can statistically ensure an annual risk of infection below 10(-4)), was assessed by evaluating the applicability of the Poisson lognormal (PLN) distribution in microbial risk assessment. PLN showed as good a fit to the observed data as to the negative binomial distribution. From the estimated PLN distributions for the source and finished water, the efficacy of the oocyst removal by the conventional water treatment process was estimated to follow log-normal distribution (median = 3.16 log10, 95% CI = 4.27-2.05 log10). The 365 consecutive negative results of daily monitoring for 180 L of finished water were found to be statistically equivalent to the annual risk of infection below 10(-4). This research also suggested the possibility of applying a qualitative detection method, such as CC-PCR, as a routine monitoring method for the quantitative risk management.     

A fast stir bar sorptive extraction method for the analysis of geosmin and 2-methylisoborneol in source and drinking water.

The presence of unpleasant taste and odour in drinking water is an ongoing aesthetic concern for water providers worldwide. The need for a sensitive and robust method capable of analysis in both natural and treated waters is essential for early detection of taste and odour events. The purpose of this study was to develop and optimise a fast stir bar sorptive extraction (SBSE) method for the analysis of geosmin and 2-methylisoborneol (MIB) in both natural water and drinking water. Limits of detection with the optimised fast method (45 min extraction time at 60 degrees C using 24 microL stir bars) were 1.1 ng/L for geosmin and 4.2 ng/L for MIB. Relative standard deviations at the detection limits were under 17% for both compounds. Use of multiple stir bars can be used to decrease the detection limits further. The use of 25% NaCl and 5% methanol sample modifiers decreased the experimental recoveries. Likewise, addition of 1 mg/L and 1.5 mg/L NaOCI decreased the recoveries and this effect was not reversed by addition of 10% thiosulphate. The optimised method was used to measure geosmin concentrations in treated and untreated drinking water. MIB concentrations were below the detection limits in these waters.     

Development of a sensitive polymerase chain reaction method for the detection of Toxoplasma gondii in water.

Toxoplasma gondii is becoming a potential threat for public water supplies worldwide, as demonstrated by the occurrence of waterborne toxoplasmosis outbreaks in developing countries as well as industrialised countries. The aim of the present study was to develop a sensitive molecular approach (PCR) for the detection of Toxoplasma oocysts in water. Sporulated and unsporulated T. gondii oocysts (strains DX and AHC1) were isolated from faeces of laboratory-infected cats. After purification and enumeration, oocysts were spiked into 1 -L water replicates and concentrated using centrifugation, Al2(SO4)3 or Fe2(SO4)3 flocculation. DNA was extracted from the concentrated pellets, and a universal primer and a T. gondii-specific primer were selected to amplify a region at the small subunit ribosomal RNA gene. A theoretical detection limit of 0.1 oocysts was achieved for samples that had been concentrated using centrifugation or Al2(SO4)3 flocculation. No PCR products were generated for samples that had been pre-treated using Fe2(SO4)3 flocculation. The final target would be the development of a complete technique able to work as a diagnostic tool for the detection of Toxoplasma in environmental and drinking water.     

Fate and transport of faecal contamination microbial indicators, pathogenic protozoa and Campylobacter in the artificially recharged fractured aquifer of Salento, Italy.

The study investigates the fate and transport of microorganisms introduced by artificial groundwater recharge at the Nardò fractured aquifer in Salento, Italy. Microbial indicators of faecal contamination, parasitic protozoa (Giardia and Cryptosporidium) and pathogenic bacteria (Campylobacter spp.), were monitored into injected water and groundwater to test the efficiency of the "natural disinfection" into the fractured aquifer. A remarkable decrease of microbial indicators and pathogens was observed suggesting that pathogens removal or inactivation may be possible during water flow in fractured aquifer. The recently described PNA probe CJE195 (Lehtola et al. 2005) was utilised for the rapid and specific detection of Campylobacter spp. by fluorescence in situ hybridization (FISH) after enrichment. FISH results were consistent with those of traditional cultural method (ISO 17995) applied in parallel: time required for Campylobacter identification was reduced of 4 days.     

Inactivation of enteric microbes in water by electro-chemical oxidant from brine (NaCl) and free chlorine.

Oxidant solutions of mostly free chlorine can be electrochemically produced on-site from brine (NaCl) solution and used to disinfect water at the household or community level. In this study electrochemical oxidant (ECO) from brine and free chlorine were evaluated under laboratory conditions for inactivation of test microbes. Purified suspensions of Escherichia coli, the rugose strain of Vibrio cholerae, Clostridium perfringens spores, MS2 coliphage and Cryptosporidium parvum oocysts were treated with 2 mg/L or 5 mg/L solutions of ECO or free chlorine at 5 degrees C and 25 degrees C and pH 6, 8, and 10 (pH 7 and 25 degrees C only for C. parvum oocysts) for contact times <60 min. Under nearly all conditions, inactivation kinetics were more rapid for E. coli, V. cholerae, C. perfringens spores and MS2 coliphage with ECO than with free chlorine. ECO reduced E. coli, V. cholerae and MS2 by >4 log10 within 30 min and C. perfringens spores by >2 log10 within 10 min at pH 8 and 25 degrees C. Contrary to previous results, however, C. parvum oocysts were not inactivated by ECO, and the reasons for this difference are uncertain. The on-site electrolytic generation of oxidants from brine provided a convenient and inexpensive disinfectant containing free chlorine that was effective against many enteric microbes, for the treatment of household and community drinking-water supplies worldwide. However, the effectiveness of such oxidants for inactivating C. parvum oocysts was variable and sometimes ineffective.     

在线镉柱还原-流动注射法测定水样中硝酸盐氮实验

建立了在线镉柱还原测定水样中硝酸盐氮的流动注射光度法,并考察了反应管长度、注样体积、泵流量等实验条件对结果的影响。经过优化实验条件,获得该方法的线性范围为0~50 mg/L,相关系数r=0.999,检出限为0.034 mg/L,相对标准偏差为0.65%(n=11)。实验结果表明,该方法线性范围宽,试剂消耗量少、测定快速、灵敏,抗干扰能力强,实际水样的加标回收率均在98.8%~105.0%之内,适宜于现场即时监测。     

Using ultraviolet light for disinfection of finished water.

Ultraviolet light is now recognised to be very effective for inactivation of Cryptosporidium parvum oocysts; however, its application for disinfection of finished water necessitates validation of UV reactors prior to their installation. Although reactor performance will likely be assessed using non-pathogenic microorganisms as biodosimetry surrogates, it would be prudent for the water industry to simultaneously measure Cryptosporidium oocysts inactivation in controlled bench-scale studies using the water matrix intended for disinfection. The likelihood of that occurring is dependent upon the availability of infectivity measurement procedures that are more user-friendly than the mouse infectivity assays currently used. This study describes a modified cell culture procedure that would enable reliable measurement of changes in oocysts' infectivity following their UV treatment. Also, a number of different biodosimetry surrogates were examined and one selected for comparing the UV doses delivered between bench-scale and full-scale biodosimetry studies. Impacts of UV disinfection on production of disinfection byproducts, effects of lamp ageing on effectiveness of disinfection and the costs associated with employing this technology were also examined.     

Endemic Cryptosporidium infection and drinking water source: a systematic review and meta-analyses.

Cryptosporidium is a well-known cause of diarrhoea in humans. Little is known about risk factors associated with endemic cryptosporidiosis, which constitutes the majority of cases. We carried out meta-analyses to verify if drinking water is also associated with endemic infection and to assess the magnitude of the associations. The global meta-analysis suggests that there is an increased risk of Cryptosporidium infection among unsafe water users (OR 1.40 [1.15, 1.72]). Studies were stratified, according to the exposure to different sources of safe drinking water, due to the heterogeneity presented. The consumption of non-well and unboiled water was associated with an increased chance of endemic cryptosporidiosis, though only the latter was significant (OR 1.45 [0.95, 2.20]; OR 1.61 [1.09, 2.38]). Drinking non-bottled water did not present a risk factor associated with endemic cryptosporidiosis (OR 0.87 [0.72, 1.05]). These meta-analyses present results that could be useful to clarify the epidemiology of Cryptosporidium. We recommend that other risk factors could also be studied by this approach.     

Disinfection of sludge with high pathogenic content using silver and other compounds.

A physicochemical sludge with high microbial content (10(2)-10(4) FPU/g TS bacteriophages, 10(6)-10(7) MPN/g TS faecal coliforms, 10(4) MPN/g TS Salmonella spp., 10(4) MPN/g TS Shigella spp., 10(3) MPN/g TS Pseudomonas aeruginosa, 10(2) MPN/g TS Vibrio cholerae, 10(2)-10(3) cysts/g TS Giardia sp., 10(2)-10(4) oocyts/g TS Cryptosporidium sp., 168-215 viable helminth ova/g TS) was disinfected using silver, silver-copper, and silver-copper plus a synergistic agent (SA). Twenty milligrams Ag/g TS inactivated 4.8 log of faecal coliforms in 1 h; however, 40 mg Ag/g TS are needed to reduce helminth ova viability from 84 to 38.4% in the same period of time. Combinations of Ag-Cu (60:600 mg Ag-Cu/g TS) and Ag-SA (60:24 mg Ag-SA/g TS) inactivated 7.8 log of faecal coliforms and around 90% of helminth ova in 60 min. To produce USEPA class A biosolids, 10:100:8 and 5:50:13.3 mg Ag-Cu-SA/gTS are needed. Bacterial regrowth was not observed for all conditions producing <1000 MPN/gTS faecal coliforms, suggesting a residual disinfection effect. Recommended doses to produce class A biosolids inactivated 2-4 log of bacteriophages, 4 log of Salmonella spp., 4 log of Shigella spp., 3 log of Pseudomonas aeruginosa, 2 log of Vibrio cholerae, 87-99.9% of Giardia sp., and 75-99.9% of Cryptosporidium sp.