Detection of enteroviruses in treated drinking water

This study deals with the routine monitoring of drinking water for the presence of enteroviruses, over a period of 1 year. A rapid and simple method was employed for the simultaneous detection and typing of enteroviruses in large-volume water samples. This included an integrated cell culture/nested PCR approach, followed by restriction enzyme analysis. The two drinking water supplies studied were derived from acceptable quality surface water sources using treatment processes, which conform to international specifications for the production of safe drinking water. Enteroviruses (predominantly coxsackie B viruses) were detected in 11% and 16% of the drinking water samples from two treatment plants, respectively. This study confirms that acceptable water quality indicators do not necessarily reflect the virus content of drinking water.     

Occurrence of enteric viruses and faecal indicators in submarine groundwater discharges in the coastal environment of the Mexican Caribbean

The occurrence of faecal microorganisms such as total coliforms (TCs), Escherichia coli (E. coli), coliphages (somatic and F+ specific) and the genomes of faecal viruses: human adenovirus (HAdV), norovirus (NoV) genogroup I (GI) and II (GII), enterovirus (EV), aichi virus (AiV) and Pepper mild mottle virus (PMMoV), was explored in submarine groundwater discharges (SGDs) in the Mexican Caribbean. Ultra-filtrated water samples were subjected to nucleic acid extraction and real-time quantitative polymerase chain reaction (PCR). Results showed the presence of TC bacteria at all sites, whereas the detection of E. coli occurred only during the dry season. Coliphages occurred during the rainy and dry seasons, with concentrations ranging from 10 to 2560 plaque forming units (PFU/100 ml). HAdV and AiV were detected in 50% and 41% of the samples, at concentrations ranging from 10² to 10⁴ genome copies per litre (GC/L) and from 10² to 10⁶ GC/L, respectively. NoV GI and II were detected in 25% and 16% of the samples, at concentrations of 10¹ GC/L and 10²–10³ GC/L, respectively. PMMoV was detected in 50% of the samples at concentrations ranging from 10¹ to 10³ GC/L. The EV genomes were not detected. These findings demonstrate that faecal microorganisms can be transported through SGDs in the Mexican Caribbean, potentially contributing to human health risks for recreation.     

Survival of infectious Poliovirus-1 in river water compared to the persistence of somatic coliphages, thermotolerant coliforms and Poliovirus-1 genome

The microbiological quality of water is currently assessed by search for fecal bacteria indicators. There is, however, a body of knowledge demonstrating that bacterial indicators are less resistant to environmental factors than human pathogenic viruses and therefore underestimate the viral risk. As river water is often used as a resource for drinking water production, it is particularly important to obtain a valid estimation of the health hazard, including specific viral risk. This work was conducted to compare the survival of infectious Poliovirus-1 used as a pathogenic virus model to the persistence of, on the one hand, thermotolerant coliforms commonly used as indicators and on the other hand, to somatic coliphages and Poliovirus-1 genome considered as potential indicators. We studied the behavior of infectious Poliovirus-1 and the three (potential) indicators of viral contamination in river water at three different temperatures (4 degrees C,18 degrees C and 25 degrees C). This experiment was performed twice with river water sampled at two different periods, once in winter and once in summer. Our results showed that the survival of thermotolerant coliforms can be 1.5-fold lower than infectious Poliovirus-1. In contrast, under all our experimental conditions, somatic coliphages and Poliovirus-1 genome persisted longer than infectious Poliovirus-1, surviving, respectively, 2-6-fold and about 2-fold longer than infectious Poliovirus-1. According to our results exclusively based on survival capacity, somatic coliphages and viral genome, unlike thermotolerant coliforms appear to be better indicators of viral contamination in river water. Moreover, the disappearance of viral genome is well-correlated to that one of infectious virus irrespective of the conditions tested.     

Detection of enteroviruses in untreated and treated drinking water supplies in South Africa

Enteric viruses have been detected in many drinking water supplies all over the world. A meaningful number of these supplies were treated and disinfected according to internationally acceptable methods. In addition, counts of bacterial indicators (coliform bacteria and heterotrophic plate count organisms) in these water supplies were within limits generally recommended for treated drinking water and these findings have been supported by epidemiological data on infections associated with drinking water. The shortcomings of conventional treatment methods and indicator organisms to confirm the absence of enteric viruses from drinking water, was generally ascribed to the exceptional resistance of these viruses. In this study, the prevalence of enteroviruses detected from July 2000 to June 2002 in sewage, river-, borehole-, spring- and dam water as well as drinking water supplies treated and disinfected according to international specifications for the production of safe drinking water was analysed. A glass wool adsorption-elution technique was used to recover viruses from 10--20 l of sewage as well as environmental water samples, in the case of drinking water from more than 100 l. Recovered enteroviruses were inoculated onto two cell culture types (BGM and PLC/PRF/5 cells) for amplification of viral RNA with nested-PCR being used to detect the amplified viral RNA. Results from the study demonstrated the presence of enteroviruses in 42.5% of sewage and in 18.7% of treated drinking water samples. Furthermore, enteroviruses were detected in 28.5% of river water, in 26.7% of dam/spring water and in 25.3% of borehole water samples. The high prevalence of coxsackie B viruses found in this study suggested, that a potential health risk and a burden of disease constituted by these viruses might be meaningful. These findings indicated that strategies, other than end-point analysis of treated and disinfected drinking water supplies, may be required to ensure the production of drinking water that does not exceed acceptable health risks. More reliable approaches to ensure acceptable safety of drinking water supplies may be based on control by multiple-barrier principles from catchment to tap using hazard assessment and critical control point (HACCP) principles.     

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).     

Evaluation of pepper mild mottle virus, human picobirnavirus and Torque teno virus as indicators of fecal contamination in river water

A reliable indicator is needed to predict and reduce the risk of infection associated with fecal contamination of surface water. Since Pepper mild mottle virus (PMMoV), human picobirnaviruses (hPBV) and Torque teno virus (TTV) have been detected at substantial levels in human feces, we explored whether detection of nucleic acids of these viruses is a suitable indicator of fecal contamination in river water. From September 2008 to December 2009, water samples (n = 111) were collected from the Ruhr and Rhine rivers and from the influents and effluents of a wastewater plant (n = 12). Quantitative real time (RT-) PCR was used to determine the abundance of PMMoV, hPBV, and TTV in comparison to human adenoviruses (HAdV) and human polyomaviruses (HPyV) that are frequently detected in surface water and were previously proposed as indicators. While PMMoV was detected in all river water samples, the other viruses were detected less frequently. The concentration of the studied viruses in positive river water ranged from 5 × 10¹ to 1.07 × 10⁶ genome equivalents per liter (gen.equ./l). All wastewater samples were positive for PMMoV, HAdV and HPyV, while TTV and hPBV were detected in 6/12 and 3/12 of samples, respectively. To determine if PMMoV is specific to human-derived fecal waste, fecal samples from human (n = 20) and animal (n = 53) were also tested. In contrast to the ubiquity of PMMoV in human feces (19/20) the virus was only detected at low concentration in a minority of the animal fecal samples tested (7/15 from chicken, 1/10 from Geese and 1/6 from cows). Therefore, in this setting TTV and hPBV do not seem to be suitable indicators of fecal contamination in water. Whereas, the high excretion level and dissemination of PMMoV in human sewage and river water suggest that PMMoV could be a promising indicator of fecal pollution in surface water.     

Relationship between F-specific RNA phage genogroups, faecal pollution indicators and human adenoviruses in river water

Recent studies have shown the increasing interest of F-specific RNA phage genotyping to identify major sources of faecal contamination in waters. This study, conducted in a river located in an urbanized watershed with recognized anthropogenic influences, was aimed at evaluating the relevance of direct phage genotyping by real-time RT-PCR. One hundred percent of positive results were obtained with a 5 mL aliquot of river water (n = 31). Phage distribution was modified after cultivation, since the ratio of the two most abundant genogroups (II and I) reached 1.51 log₁₀ by direct RT-PCR-based method versus 0.30 log₁₀ after cultivation (n = 8). For the first time, positive correlations between the concentrations of genogroup II, bacterial indicators and human adenoviruses were observed, which may indicate a human faecal pollution. No correlation between genogroups II and I has been revealed. The concentration of genogroup I was only correlated with water turbidity, suggesting an animal pollution coming from upstream after rainfall events. Among the microbiological parameters studied, only genogroup II/genogroup I ratio shows variations occurring in the major sources of faecal pollution.     

Coliphages as an indicator of faecal pollution in water. Its relationship with indicator and pathogenic microorganisms

The study was designed to test the proposal that Escherichia coli specific bacteriophages might serve as universal faecal pollution indicators in water. A highly specific, sensitive and rapid technique for the detection and quantification of these virus particles was developed. The numerical relationship between E. coli and its parasitic phages was investigated in three different aqueous ecosystems such as sea water in the vicinity of sewage outfalls, river water contaminated by domestic and industrial sewage discharges, and estuarine waters, and found to be very close. In addition, the results obtained indicate that the coliphages are good indicators of the presence of the pathogenic microorganisms studied. In nearly all the water samples tested, the results suggest that coliphages are better indicators of faecal pollution than the classical indicator systems currently employed.     

Monitoring of opiates, cannabinoids and their metabolites in wastewater, surface water and finished water in Catalonia, Spain

The occurrence of several opiates and cannabinoids in wastewaters and surface waters has been investigated. Most of the compounds (8 out of 11) were identified in both influent and effluents of fifteen wastewater treatment plants (WWTPs). Codeine, morphine, EDDP and methadone were detected in almost all samples with median values of 69 ng/L; 63 ng/L; 28 ng/L and 18 ng/L, respectively, whereas the main cannabinoid metabolite THC-COOH presented a median value of 57 ng/L in influents. A rough estimate of heroin and cannabis consumption was performed from the analysis of target urinary metabolites in wastewater influents. Data obtained from influents of rural and urban WWTPs gave 0.07% of heroin consumption (0.67% for the largest urban WWTP) and 4% consumption of cannabinoids, respectively for the population aged between 15 and 64 years old. The presence of opiates and cannabinoids in surface waters used for drinking water production showed the presence of the same compounds identified in wastewater effluents at concentrations up to 76 ng/L for codeine; 31 ng/L for EDDP; 12 ng/L for morphine and 9 ng/L for methadone at the intake of the DWTP. A complete removal of all studied drugs present in surface water was achieved during the potabilization process except for methadone and EDDP (91% and 87% removal, respectively).     

Use of eukaryotic mitochondrial DNA to differentiate human, bovine, porcine and ovine sources in fecally contaminated surface water

A molecular method based on the detection of mitochondrial DNA from various animal species was developed to track the origin of surface water pollutions, and to differentiate human and animal sources. Mitochondrial DNA sequences were used to design PCR primers specific for human, bovine, ovine and porcine DNA using single, multiplex and nested PCR protocols. The primers were tested with DNA extracted from untreated domestic sewage, agricultural soils run-off, swine farm effluents and water from two rivers with known pollution sources. At least one of the four species was detected in most of these samples. The limit of detection in wastewater was 10(3)-10(4) cells L(-1) with a multiplex PCR protocol. This is the first report of a method using eukaryotic genetic DNA to detect and differentiate animal DNA from fecal sources in water. This innovative method is simple and could be used to quickly differentiate sources of pollution in a watershed.     

Universal mitochondrial PCR combined with species-specific dot-blot assay as a source-tracking method of human, bovine, chicken, ovine, and porcine in fecal-contaminated surface water

Mitochondrial (mt) DNA has the potential to be used as an animal-specific genetic marker for source-tracking of fecal contamination in surface waters and groundwaters. In this report, we describe the development of a method to detect in a single assay human, bovine, ovine, porcine and chicken mitochondrial (mt) DNA in water. Consensus nucleic sequences were found between human, bovine, porcine, ovine and chicken mtDNA to design three sets of PCR universal primers. Upon polymerase chain reaction (PCR) with a digoxigenin-labeled nucleotide and the universal primers, species determination was carried out by dot-blotting membranes containing specific oligonucleotides for these five animals. Our method was carried out with three river samples and three wastewater samples, and the results were compared with those obtained by multiple nested PCR with specific primers for these five species. Our results showed that the dot-blot assays were as specific and sensitive as the nested-PCR approach. The proposed method has the advantage that it requires the use of only one PCR per sample and very little amounts of DNA. Finally, it is an alternative to multiplex PCR approach which is less sensitive, and shows the way for the development of DNA arrays for source-tracking of many more animal species in fecal-contaminated water.     

Solid phase extraction coupled to liquid chromatography-tandem mass spectrometry analysis of sulfonamides, tetracyclines, analgesics and hormones in surface water and wastewater in Luxembourg

In the early 1990s different studies highlighted the relationship between pharmaceuticals, human health and the environment. Among the emerging contaminants, antibiotics are obviously of high concern, because of their potential for inducing antibiotic resistance. In addition, natural and synthetic hormones are relevant because of their potential endocrine-disrupting effects on wildlife. This investigation focuses on the analysis of four classes of veterinary and human pharmaceuticals (sulfonamides, tetracyclines, analgesics and hormones) in surface water and wastewater in Luxembourg. The selected eleven pharmaceuticals include four sulfonamides (sulfathiazole, sulfamethoxazole, sulfadimethoxine and sulfamethazine), two tetracyclines (tetracycline and oxytetracycline), two analgesics (ibuprofen and diclofenac), and three hormones (2 naturals, estrone and β-estradiol, and a synthetic one, 17-α-ethinyl estradiol). The most innovative parts of this study are the simultaneous extraction of the above-mentioned pharmaceuticals as well as tracking their behaviour during flood events in a small river catchment. The method includes pre-concentration by solid phase extraction using Oasis® HLB (Hydrophilic Lipophilic Balance) which gave superior results compared to Chromabond® C-18EC, Chromabond® EASY and Bond Elut® PLEXA cartridges, also evaluated in this investigation. The analysis of the investigated pharmaceutical compounds is carried out by high performance liquid chromatography coupled to a triple quadrupole mass spectrometer. The limits of quantification were 1 ng L^− 1, except for β-estradiol (2 ng L^− 1) and 17-α-ethinyl estradiol (6 ng L^− 1). Recovery rates range from 70 to 94%, with relative standard deviations between 4 and 19%. Application of this method to river concentration and flood events revealed high concentrations of ibuprofen (10-4000 ng L^− 1), with highest levels during flood events, while concentrations of estrogens (1-240 ng L^− 1) and sulfonamides (1-20 ng L^− 1) were comparatively low.     

The occurrence of pharmaceuticals, personal care products, endocrine disruptors and illicit drugs in surface water in South Wales, UK

The presence and fate of 56 pharmaceuticals, personal care products, endocrine disruptors and illicit drugs (PPCPs) were investigated in the South Wales region of the UK. Two contrasting rivers: River Taff and River Ely were chosen for this investigation and were monitored for a period of 10 months. The impact of the factors affecting the levels of concentration of PPCPs and illicit drugs in surface water such as surrounding area, proximity to wastewater effluent and weather conditions, mainly rainfall was also investigated. Most PPCPs were frequently found in river water at concentrations reaching single microgL(-1) and their levels depended mainly on the extent of water dilution resulting from rainfall. Discharge of treated wastewater effluent into the river course was found to be the main cause of water contamination with PPCPs. The most frequently detected PPCPs represent the group of pharmaceuticals dispensed at the highest levels in the Welsh community. These were antibacterial drugs (trimethoprim, erythromycin-H(2)O and amoxicillin), anti-inflammatories/analgesics (paracetamol, tramadol, codeine, naproxen, ibuprofen and diclofenac) and antiepileptic drugs (carbamazepine and gabapentin). Only four PPCPs out of 56 (simvastatin, pravastatin, digoxin and digoxigenin) were not quantified over the course of the study. Several PPCPs were found to be both ubiquitous and persistent in the aqueous environment (e.g. erythromycin-H(2)O, codeine, carbamazepine, gabapentin and valsartan). The calculated average daily loads of PPCPs indicated that in total almost 6 kg of studied PPCPs are discharged daily into the studied rivers. The illicit drugs studied were found in rivers at low levels of ng L(-1). Average daily loads of amphetamine, cocaine and its main metabolite benzoylecgonine were as follows: 8, 1.2 and 39 gday(-1), respectively. Their frequent occurrence in surface water is primarily associated with their high illegal usage and is strongly associated with the discharge of insufficiently treated wastewater effluent.     

Assessing the efficacy of pre-harvest,chlorine-based sanitizers against human pathogen indicator microorganisms and Phytophthora capsici in non-recycled surface irrigation water

Many factors must be considered in order to develop and implement treatment systems to improve the microbial quality of surface water and prevent the accidental introduction of plant and human pathogens into vegetable crops. The efficacy of chlorine gas (Cl_2(g)) and chlorine dioxide (ClO₂) injection systems in combination with rapid sand filtration (RSF) was evaluated in killing fecal indicator microorganisms in irrigation water in a vegetable-intensive production area. The efficacy of ClO₂ and Cl_2(g) was variable throughout the distribution systems and coliform bacteria never dropped below levels required by the United States Environmental Protection Agency for recreational waters. Sampling date and sampling point had a significant effect on the abundance of coliforms in Cl_2(g)- and ClO₂-treated water. Sampling date and sampling point also had a significant effect on the abundance of generic Escherichia coli in Cl_2(g) treated water but only sampling point was significant in ClO₂ treated water. Although the waterborne plant pathogen Phytophthora capsici was detected in five different sources of surface irrigation water using baiting and P. capsici-specific PCR, in vitro studies indicated that ClO₂ at concentrations similar to those used to treat irrigation water did not reduce mycelial growth or direct germination of P. capsici sporangia and reduced zoospore populations by less than 50%. This study concludes that injection of ClO₂ and Cl_2(g) into surface water prior to rapid sand filtration is inadequate in reducing fecal indicator microorganism populations and ClO₂ ineffectively kills infectious propagules of P. capsici. Additional research is needed to design a system that effectively targets and significantly reduces both plant and human pathogens that are present in surface irrigation water. A model for a multiple barrier approach to treating surface water for irrigation is proposed.