Persistence of endogenous RNA biomarkers of SARS-CoV-2 and PMMoV in raw wastewater: Impact of temperature and implications for wastewater-based epidemiology.

Understanding the persistence of SARS-CoV-2 biomarkers in wastewater should guide wastewater-based epidemiology users in selecting best RNA biomarkers for reliable detection of the virus during current and future waves of the pandemic. In the present study, the persistence of endogenous SARS-CoV-2 were assessed during one month for six different RNA biomarkers and for the pepper mild mottle virus (PMMoV) at three different temperatures (4, 12 and 20 °C) in one wastewater sample. All SARS-CoV-2 RNA biomarkers were consistently detected during 6 days at 4° and differences in signal persistence among RNA biomarkers were mostly observed at 20 °C with N biomarkers being globally more persistent than RdRP, E and ORF1ab ones. SARS-CoV-2 signal persistence further decreased in a temperature dependent manner. At 12 and 20 °C, RNA biomarker losses of 1-log10 occurred on average after 6 and 4 days, and led to a complete signal loss after 13 and 6 days, respectively. Besides the effect of temperature, SARS-CoV-2 RNA signals were more persistent in the particulate phase compared to the aqueous one. Finally, PMMoV RNA signal was highly persistent in both phases and significantly differed from that of SARS-CoV-2 biomarkers. We further provide a detailed overview of the latest literature on SARS-CoV-2 and PMMoV decay rates in sewage matrices.

Bacteriophages pass through candle-shaped porous ceramic filters: Application for the collection of viruses in soil water.

Despite the ubiquity of viruses in soils, their diversity in soil water has not been explored, mainly due to the difficulty of collecting them. In hydrology, soil water is usually collected using porous candles. This study proposes using these porous candles as a new tool for sampling viruses in soil water to analyze their passage through the ceramic part of the candles. The recovery of the viruses was determined after filtration under laboratory conditions using three model bacteriophages (MS2, ΦX174, and Φ6) and Escherichia coli, at neutral and acidic pH. Then, a field experiment was carried out where soil water filtration and viral identification by metagenomic shotgun were performed. At neutral pH, all bacteriophages tested successfully passed through the porous candles during the filtration process, with reductions of 0.02 log, 0.16 log, and 0.55 log for MS2 ΦX174 and Φ6, respectively. At pH 4.4, the passage of MS2 was not affected while ΦX174 underwent a slight reduction in recovery, probably caused by adsorption onto the filter material. Regarding the application of the porous candles in the field, the results obtained allowed the successful recovery of viruses, exposing porous candles as a new method suitable for the collection of viruses from soil water in the context of the study of viral communities.

Soil pH, Calcium Content and Bacteria as Major Factors Responsible for the Distribution of the Known Fraction of the DNA Bacteriophage Populations in Soils of Luxembourg.

Bacteriophages participate in soil life by influencing bacterial community structure and function, biogeochemical cycling and horizontal gene transfer. Despite their great abundance, diversity, and importance in microbial processes, they remain little explored in environmental studies. The influence of abiotic factors on the persistence of bacteriophages is now recognized; however, it has been mainly studied under experimental conditions. This study aimed to determine whether the abiotic factors well-known to influence bacteriophage persistence also control the natural distribution of the known DNA bacteriophage populations. To this end, soil from eight study sites including forests and grasslands located in the Attert River basin (Grand Duchy of Luxembourg) were sampled, covering different soil and land cover characteristics. Shotgun metagenomics, reference-based bioinformatics and statistical analyses allowed characterising the diversity of known DNA bacteriophage and bacterial communities. After combining soil properties with the identified DNA bacteriophage populations, our in-situ study highlighted the influence of pH and calcium cations on the diversity of the known fraction of the soil DNA bacteriophages. More interestingly, significant relationships were established between bacteriophage and bacterial populations. This study provides new insights into the importance of abiotic and biotic factors in the distribution of DNA bacteriophages and the natural ecology of terrestrial bacteriophages.

Model-based assessment of COVID-19 epidemic dynamics by wastewater analysis.

Continuous surveillance of COVID-19 diffusion remains crucial to control its diffusion and to anticipate infection waves. Detecting viral RNA load in wastewater samples has been suggested as an effective approach for epidemic monitoring and the development of an effective warning system. However, its quantitative link to the epidemic status and the stages of outbreak is still elusive. Modelling is thus crucial to address these challenges. In this study, we present a novel mechanistic model-based approach to reconstruct the complete epidemic dynamics from SARS-CoV-2 viral load in wastewater. Our approach integrates noisy wastewater data and daily case numbers into a dynamical epidemiological model. As demonstrated for various regions and sampling protocols, it quantifies the case numbers, provides epidemic indicators and accurately infers future epidemic trends. Following its quantitative analysis, we also provide recommendations for wastewater data standards and for their use as warning indicators against new infection waves. In situations of reduced testing capacity, our modelling approach can enhance the surveillance of wastewater for early epidemic prediction and robust and cost-effective real-time monitoring of local COVID-19 dynamics.

Making Waves: Collaboration in the time of SARS-CoV-2 - rapid development of an international co-operation and wastewater surveillance database to support public health decision-making.

The presence of SARS-CoV-2 RNA in wastewater was first reported in March 2020. Over the subsequent months, the potential for wastewater surveillance to contribute to COVID-19 mitigation programmes has been the focus of intense national and international research activities, gaining the attention of policy makers and the public. As a new application of an established methodology, focused collaboration between public health practitioners and wastewater researchers is essential to developing a common understanding on how, when and where the outputs of this non-invasive community-level approach can deliver actionable outcomes for public health authorities. Within this context, the NORMAN SCORE "SARS-CoV-2 in sewage" database provides a platform for rapid, open access data sharing, validated by the uploading of 276 data sets from nine countries to-date. Through offering direct access to underpinning meta-data sets (and describing its use in data interpretation), the NORMAN SCORE database is a resource for the development of recommendations on minimum data requirements for wastewater pathogen surveillance. It is also a tool to engage public health practitioners in discussions on use of the approach, providing an opportunity to build mutual understanding of the demand and supply for data and facilitate the translation of this promising research application into public health practice.

Influence of physico-chemical characteristics of sediment on the in situ spatial distribution of F-specific RNA phages in the riverbed.

Riverbed sediment is commonly described as an enteric virus reservoir and thought to play an important role in water column contamination, especially during rainfall events. Although the occurrence and fate of faecal-derived viruses are fairly well characterized in water, little information is available on their presence as their interactions with sediment. This study aimed at determining the main environmental factors responsible for the presence of enteric viruses in riverbed sediment. Using a combination of microbiological and physico-chemical analyses of freshly field-sampled sediments, we demonstrated their contamination by faecal phages. The in situ spatial distribution of phages in sediment was mainly driven by sediment composition. A preferential phage accumulation occurred along one bank of the river, where the quantity of fine sands and clay particles smaller than 0.2 mm was the highest. Additionally, a mineralogical analysis revealed the influence of the heterogeneous presence of virus sorbents such as quartz, calcite, carbonates and iron-bearing phases (goethite) on the phage spatial pattern. A more precise knowledge of the composition of riverbed sediment is therefore useful for predicting preferential areas of enteric virus accumulation and should allow more accurate microbial risk assessment when using surface water for drinking water production or recreational activities.

Detection of Human Enteric Viruses in French Polynesian Wastewaters, Environmental Waters and Giant Clams.

Lack of wastewater treatment efficiency causes receiving seawaters and bivalve molluscan shellfish to become contaminated, which can lead to public health issues. Six wastewater samples, five seawater samples and three batches of giant clams from Tahiti (French Polynesia) were investigated for the presence of enteric viruses, but also if present, for the diversity, infectivity and integrity of human adenoviruses (HAdV). Enteroviruses (EV), sapoviruses (SaV) and human polyomaviruses (HPyV) were detected in all wastewater samples. In decreasing frequency, noroviruses (NoV) GII and HAdV, rotaviruses (RoV), astroviruses (AsV), NoV GI and finally hepatitis E viruses (HEV) were also observed. Nine types of infectious HAdV were identified. HPyV and EV were found in 80% of seawater samples, NoV GII in 60%, HAdV and SaV in 40% and AsV and RoV in 20%. NoV GI and HEV were not detected in seawater. Intact and infectious HAdV-41 were detected in one of the two seawater samples that gave a positive qPCR result. Hepatitis A viruses were never detected in any water types. Analysis of transcriptomic data from giant clams revealed homologues of fucosyltransferases (FUT genes) involved in ligand biosynthesis that strongly bind to certain NoV strains, supporting the giant clams ability to bioaccumulate NoV. This was confirmed by the presence of NoV GII in one of the three batches of giant clams placed in a contaminated marine area. Overall, all sample types were positive for at least one type of virus, some of which were infectious and therefore likely to cause public health concerns.

Interactions of infectious F-specific RNA bacteriophages with suspended matter and sediment: Towards an understanding of FRNAPH distribution in a river water system.

The association of viruses with settling particles is certainly a major process controlling the spread of viral pollution in surface water and sediment. To better understand the viral distribution in a river system, the behavior of F-specific RNA bacteriophages (FRNAPHs) was investigated in relationship with the suspended solids and sediment. The partitioning of phage particles (free or associated with solids) in surface water and the attachment capabilities of eight distinct strains of phages to sediment were studied in lab experiments. In situ observations were also performed with the genotyping of 166 individual plaques of FRNAPHs isolated from surface water and sediment. The results reported here demonstrate a variation of the status of infectious phages as a function of the hydro-climatological conditions. Phage-solid association seems to mainly occur during the peak of rainfall-runoff events but also to a certain extent during the recession phase compared to low flow conditions. The transfer of phages from the water column to sediment may occur at this time. Furthermore, the ability of FRNAPHs to interact with sediment was established for six strains out of eight, belonging to genogroups II, III and IV. A similar dynamic was observed for strains within a same genogroup despite different intensity of attachment and inactivation rates for strains of genogroups III and IV. The latter results match the in situ observations in the water and sediment compartments of the studied area. Infectious FRNAPH genogroup II was more abundant in sediment than in surface water. Its capability to sorb to sediment and its higher persistence in the environment compared to genogroups III and IV were the two main explanations. Together, lab and in situ experiments produce an overall vision of the mechanisms governing FRNAPH distribution among the water column and riverbed sediment.

In Situ Dynamics of F-Specific RNA Bacteriophages in a Small River: New Way to Assess Viral Propagation in Water Quality Studies.

The occurrence and propagation of enteric viruses in rivers constitute a major public health issue. However, little information is available on the in situ transport and spread of viruses in surface water. In this study, an original in situ experimental approach using the residence time of the river water mass was developed to accurately follow the propagation of F-specific RNA bacteriophages (FRNAPHs) along a 3-km studied river. Surface water and sediment of 9 sampling campaigns were collected and analyzed using both infectivity and RT-qPCR assays. In parallel, some physico-chemical variables such as flow rate, water temperature, conductivity and total suspended solids were measured to investigate the impact of environmental conditions on phage propagation. For campaigns with low flow rate and high temperature, the results highlight a decrease of infectious phage concentration along the river, which was successfully modelled according to a first-order negative exponential decay. The monitoring of infectious FRNAPHs belonging mainly to the genogroup II was confirmed with direct phage genotyping and total phage particle quantification. Reported k decay coefficients according to exponential models allowed for the determination of the actual in situ distance and time necessary for removing 90 % of infectious phage particles. This present work provides a new way to assess the true in situ viral propagation along a small river. These findings can be highly useful in water quality and risk assessment studies to determine the viral contamination spread from a point contamination source to the nearest recreational areas.

Contribution of hydrological data to the understanding of the spatio-temporal dynamics of F-specific RNA bacteriophages in river water during rainfall-runoff events.

Heavy rainfall events were previously reported to bring large amounts of microorganisms in surface water, including viruses. However, little information is available on the origin and transport of viral particles in water during such rain events. In this study, an integrative approach combining microbiological and hydrological measurements was investigated to appreciate the dynamics and origins of F-specific RNA bacteriophage fluxes during two distinct rainfall-runoff events. A high frequency sampling (automatic sampler) was set up to monitor the F-specific RNA bacteriophages fluxes at a fine temporal scale during the whole course of the rainfall-runoff events. A total of 276 rainfall-runoff samples were collected and analysed using both infectivity and RT-qPCR assays. The results highlight an increase of 2.5 log10 and 1.8 log10 of infectious F-specific RNA bacteriophage fluxes in parallel of an increase of the water flow levels for both events. Faecal pollution was characterised as being mainly from anthropic origin with a significant flux of phage particles belonging to the genogroup II. At the temporal scale, two successive distinct waves of phage pollution were established and identified through the hydrological measurements. The first arrival of phages in the water column was likely to be linked to the resuspension of riverbed sediments that was responsible for a high input of genogroup II. Surface runoff contributed further to the second input of phages, and more particularly of genogroup I. In addition, an important contribution of infectious phage particles has been highlighted. These findings imply the existence of a close relationship between the risk for human health and the viral contamination of flood water.

Fine-Scale Spatial Heterogeneity in the Distribution of Waterborne Protozoa in a Drinking Water Reservoir.

BACKGROUND: The occurrence of faecal pathogens in drinking water resources constitutes a threat to the supply of safe drinking water, even in industrialized nations. To efficiently assess and monitor the risk posed by these pathogens, sampling deserves careful design, based on preliminary knowledge on their distribution dynamics in water. For the protozoan pathogens Cryptosporidium and Giardia, only little is known about their spatial distribution within drinking water supplies, especially at fine scale. METHODS: Two-dimensional distribution maps were generated by sampling cross-sections at meter resolution in two different zones of a drinking water reservoir. Samples were analysed for protozoan pathogens as well as for E. coli, turbidity and physico-chemical parameters. RESULTS: Parasites displayed heterogeneous distribution patterns, as reflected by significant (oo)cyst density gradients along reservoir depth. Spatial correlations between parasites and E. coli were observed near the reservoir inlet but were absent in the downstream lacustrine zone. Measurements of surface and subsurface flow velocities suggest a role of local hydrodynamics on these spatial patterns. CONCLUSION: This fine-scale spatial study emphasizes the importance of sampling design (site, depth and position on the reservoir) for the acquisition of representative parasite data and for optimization of microbial risk assessment and monitoring. Such spatial information should prove useful to the modelling of pathogen transport dynamics in drinking water supplies.

Human Adenovirus Diversity in Water Samples Using a Next-Generation Amplicon Sequencing Approach.

This study aims to establish a straightforward and original workflow for high-throughput typing of human adenoviruses (HAdVs) in environmental samples. Occurrence of HAdVs in water is well documented worldwide, but data on diversity of HAdV types circulating in water are scarcely available. Here, the characterisation of viral particles was performed by determination of amplicon sequences using a next-generation sequencing (NGS) approach. Adenoviral DNA was either directly isolated from wastewater or river water concentrates or after a cell culture passage. Genome amplification targeted a hyper variable region of the hexon gene, allowing the discrimination of the 54 human adenoviral types described until now. After read generation on the benchtop MiSeq platform (Illumina), data were analysed using the Mothur software for identification of all HAdV species and types simultaneously present in a unique sample. NGS results showed a relatively wide HAdV diversity of up to six types in one sample, whereas Sanger sequencing always only retrieved the dominant one. Detected types included HAdV-1, HAdV-2, HAdV-3, HAdV-6, HAdV-12, HAdV-31, HAdV-40 and HAdV-41, HAdV-41 being the most abundant in tested samples. In addition, the influence of the cell line (A549 vs 293A cells) on the infectious HAdV typing results was clearly determined. The 293A appeared to be the most suitable cell line allowing the detection of a larger diversity of infectious HAdVs and reflecting a more realistic initial species distribution than using the A549 cells. These findings demonstrated the feasibility of amplicon sequencing NGS approach to identify viruses in complex environmental water samples.

Spatial and temporal distribution of Cryptosporidium and Giardia in a drinking water resource: implications for monitoring and risk assessment.

Because of their significant public health impact, waterborne Cryptosporidium and Giardia have been monitored in surface water in order to assess microbial quality of water bodies used for drinking water production and/or for recreational purposes. In this context, sampling strategy is of key importance and should be representative enough to appropriately assess the related microbial risk. This, however, requires sound knowledge on the behaviour of both pathogens in water. In the present study, the spatial and temporal distribution of Cryptosporidium and Giardia was explored in the rural Upper-Sûre watershed used for drinking water production in Luxembourg. By subdividing it into three compartments including (i) sub-catchments, (ii) the Sûre River fed by the sub-catchments and (iii) the Upper-Sûre reservoir fed by the Sûre River, parasite distribution was assessed using sampling designs adapted to the hydro-dynamic characteristics of the respective compartments. Results highlighted the high spatial and temporal variability in parasite distribution at watershed scale, as well as the prevalence of Giardia over Cryptosporidium. Besides land use features and catchment characteristics, hydro-climatology appeared to be a major driver of parasite behaviour in the watershed. It introduced a seasonal trend in their occurrence, highest densities being detected during the wet season. Peaks of contamination triggered out by rainfall-induced runoff were further observed in the three compartments. In the Sûre River, Cryptosporidium and Giardia fluxes peaked at 10(9) and 10(10) (oo)cysts.d(-1), respectively, and were discharged into the drinking water reservoir, where they underwent a 2 to 3 log10 removal rate. Despite this, parasite fluxes entering the drinking water treatment plant were still high (10(6) to 10(7) (oo)cysts.d(-1)) and stressed on the need for improved watershed management upstream the water treatment barrier. The catchment-wide analysis described here constitutes a valuable tool for assessment of catchment microbial dynamics, especially within the framework of Water Safety Plans.

Development of a quantitative immunocapture real-time PCR assay for detecting structurally intact adenoviral particles in water.

Development of rapid, sensitive and specific methods for detection of infectious enteric viruses in water is challenging but crucial for gaining reliable information for risk assessment. An immunocapture real-time PCR (IC-qPCR) was designed to detect jointly the two major viral particle components, i.e. the capsid protein and the viral genome. Targeting both constituents helps circumventing the technical limits of cell culture approaches and the inability of PCR based methods to predict the infectious status. Two waterborne pathogenic virus models, human adenovirus types 2 and 41, were chosen for this study. IC-qPCR showed a detection limit of 10MPNCU/reaction with a dynamic range from 10(2) to 10(6)MPNCU/reaction. Sensitivity was thus 100-fold higher compared to ELISA-based capture employing the same anti-hexon antibodies. After optimisation, application on environmental water samples was validated, and specificity towards the targeted virus types was obtained through the qPCR step. Heat-treated pure samples as well as surface water samples brought evidence that this method achieves detection of encapsidated viral genomes while excluding free viral genome amplification. As a consequence, adenovirus concentrations estimated by IC-qPCR were below those calculated by direct qPCR. The results demonstrate that the IC-qPCR method is a sensitive and rapid tool for detecting, in a single-tube assay, structurally intact and thus potentially infectious viral particles in environmental samples.

Two-day detection of infectious enteric and non-enteric adenoviruses by improved ICC-qPCR.

In order to provide a more suitable response to public health concerns, we improved the detection of infectious human adenoviruses in water by optimising the commonly used integrated cell culture-PCR method. Risk evaluation studies seek for rapid detection of infectious adenoviruses, including the enteric types 40 and 41 that are considered as the second most common agents of gastroenteritis in children next to rotaviruses. The here-employed 293A cell line used for infectious status assessment showed its ability to multiply adenoviruses including type 41. Two modifications were moreover applied to the workflow for viral detection. The first occurred at the nucleic acid extraction step performed directly on all infected cells, while the second was the application of real-time quantitative PCR as detection tool. All adaptations led to a 3-day reduction of the response delay and an improved sensitivity especially for the enteric adenoviral types. The infectious status of laboratory strain types 2 and 41 was demonstrated by a more than 2-log10 increase in genome quantity. These conclusions were confirmed and reinforced by the analysis of water samples applying the improved assay. Naturally occurring infectious adenoviruses were detected in wastewater and river water, within 2 days. Types belonging to the species human adenoviruses C and type 31 were observed, but the most frequently identified type was 41 (71 % of identified sequences, n = 34). This highlights the usefulness of our method for a wide range of types, and especially for the most prevalent and public health-relevant enteric adenoviruses.

Occurrence, survival, and persistence of human adenoviruses and F-specific RNA phages in raw groundwater.

Detection of specific genetic markers can rapidly identify the presence of enteric viruses in groundwater. However, comparison of stability characteristics between genetic and infectivity markers is necessary to better interpret molecular data. Human adenovirus serotype 2 (HAdV2), in conjunction with MS2 phages or GA phages, was spiked into raw groundwater microcosms. Viral stability was periodically assessed by both infectivity and real-time PCR methods. The results of this yearlong study suggest that adenoviruses have the most stable persistence profile and an ability to survive for a long time in groundwater. According to a linear regression model, infectivity reductions of HAdV2 ranged from 0.0076 log(10)/day (4°C) to 0.0279 log(10)/day (20°C) and were significantly lower than those observed for phages. No adenoviral genome degradation was observed at 4°C, and the reduction was estimated at 0.0036 log(10)/day at 20°C. Occurrence study showed that DNA of human adenoviruses could be observed in groundwater from a confined aquifer (7 of the 60 samples were positive by real-time PCR), while no fecal indicators were detected. In agreement with the persistence of genetic markers, the presence of adenoviral DNA in groundwater may be misleading in term of health risk, especially in the absence of information on the infective status.

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 degrees 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.

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 log(10) by direct RT-PCR-based method versus 0.30 log(10) 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.

Development of real-time RT-PCR methods for specific detection of F-specific RNA bacteriophage genogroups: application to urban raw wastewater.

F-specific RNA bacteriophages have been classified into four genogroups (GI, GII, GIII and GIV). It was suggested that two of these genogroups are more frequent in human excreta (GII and GIII) and the two other (GI and GIV) are specific for animal excreta. Real-time RT-PCR methods using TaqMan MGB probe were developed to detect the four genogroups. Primers and probes of each specific RT-PCR were designed to target all sequenced bacteriophages belonging to one genogroup, without cross-reactivity with other genogroups. These four methods showed detection limits ranging between 0.01 and 10 PFU/mL and PCR efficiencies ranging between 87 and 95%. The newly methods were tested in urban raw wastewater. Genogroups I and II were detected in all samples (n=7); GIII in six samples and GIV was never detected. GI was predominant in one sample, in which the quantity of Cryptosporidium and Giardia was, respectively, three and eight times higher than the mean values. Because GI is mainly observed in animals, it was hypothesized that this increase was due to an animal input. The use of F-specific RNA phage genotyping to estimate the origin of faecal pollution requires appropriate validation. In this context, real-time RT-PCR will undoubtedly be useful.