Municipal and agricultural wastewater contain a variety of microorganisms and in particular enteric viruses. For the reuse of this treated wastewater it is important to ensure the efficiency of purification treatments and disinfection practices, that have often been insufficient to lower the viral load below the risk level. For this reason, for the routine analysis of recycled waters, the research into pathogenic viruses (e.g. HAV) and classical bacterial parameters (E. coli, enterococci and Salmonella) has to be associated with specific viral indicators such as somatic coliphages, adenovirus and TTV. The results of environmental monitoring, carried out in a wastewater treatment plant, showed the presence of adenovirus DNA in 100% of collected samples and TTV DNA in 95% (19/20) of raw sewage and in 85% (17/20) of the exit samples, while HAV was detected only in 2 samples over 40 (5%). The quantitative analysis has revealed an average reduction of 2 log for adenovirus and 1.58 log for TTV. The bacterial indicators were reduced by 1.74 log and 1.99 log respectively for E. coli and enterococci, while for somatic coliphages an average reduction of 2.2 log was observed. No significant correlation was shown between these parameters, confirming their inadequacy for the virological risk assessment. However the results of adenovirus confirm it as the best indicator to evaluate the efficacy of wastewater depuration plant in eliminating viruses.

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The discharge of treated civil wastewater into natural waters or their reuse in industry and agriculture involves virological risks for the exposed population. Although European and Italian regulations do not require routine viral analysis of treated wastewater, a better understanding of viral contamination and resistance to treatments is needed to assess and control such risks. To this end, a wastewater treatment plant was monitored by analysing the sewage at the plant entry and exit points in order to quantify the initial presence and eventual reduction of adenovirus, Torque Teno virus, Hepatitis A virus, rotavirus, enterovirus, norovirus genogroups I and II, somatic coliphages, Escherichia coli and enterococci. The results reveal that treated water may still contain infectious human viruses and thereby represent a potential health hazard. No significant correlations were found between bacterial indicators and the viruses considered, confirming their inadequacy for virological risk assessment, while the best indicators for virus inactivation in recycled waters seem to be adenovirus, followed by somatic coliphages.

In the aim of studying possible relations between viruses detected in clinical specimens and the ones found in different environmental matrices, in the period May 2004 to April 2005, the collection of faecal samples from gastroenteritis cases and the monthly monitoring of raw and treated wastewater, river water, seawater and mussels were carried out. The viruses considered for environmental monitoring were adenovirus, rotavirus, enterovirus, norovirus, hepatitis A virus (HAV) and Torque teno virus (TTV): they were searched for with PCR and RT-PCR and confirmed by gene sequencing. Faecal coliforms and somatic coliphages' counts were also determined. The surveillance of case detected 45 positive faecal samples out of 255 (17.6%) while 35 of 56 environmental samples (62.5%) resulted positive for at least one of the considered viruses. The detection of the same viral strain in the faeces of gastroenteritis cases and in water was possible for adenovirus and rotavirus, which were also predominant in environmental matrices; thus they could be considered as a reference for risk assessment.

Deep groundwater, even if generally protected, could be contaminated by surface or rain water infiltration through soil fractures, septic tanks, cesspits, land irrigation, disposal of wastewater and disposal of muds from depuration systems. The sanitary importance of such possible contamination is related to the different uses of the water and it is at the maximum level when it is intended for human use. Routine microbiological analyses do not consider viruses, only bacterial parameters, as contamination indicators. However, it is known that enteric viruses can survive a long time in deep aquifers and that they may not always be associated with bacterial indicators. The virological analysis of waters intended for drinking use is provided only as an occasional control exercised at the discretion of the sanitary authority. Technological difficulties with obtaining data about groundwater viral contamination led to a study to devise rapid and efficient methods for their detection and the application of these methods to samples from different sources. Four acid nucleic extraction techniques have been tested (classic proteinase K- phenol/chloroform, QIAamp Viral RNA Kit (Qiagen), SV Total RNA Isolation System (Promega) and NucleoSpin Virus L (Macherey-Nagel). Sensitivity and specificity of RT-PCR protocols for entero- (EV), hepatitis A (HAV) and small round structured (SRSV) viruses have been verified. Deep groundwater samples (100 L) were concentrated (2-step tangential flow ultrafiltration) and the concentrate contaminated with serial 10-fold dilutions of a known titre of poliovirus type 3. Extracted RNA was concentrated (microcon-100) and analysed by RT-PCR using specific EV primers and visualising amplification products by agarose gel electrophoresis. In addition, two different methods of RT-PCR for non-cultivable viruses have been tested: (a) RT-PCR and nested RT-PCR for HAV and (b) RT-PCR with generic primers and RT-PCR with specific primers for SRSV. Different specificity tests have been carried out in the presence of some of the commoner microorganisms. The most efficient, sensitive and specific protocols were used to test 35 x 100L deep groundwater samples. Sample concentrates were split with one part treated with chloroform and analysed by cell culture (BGM and Frp/3, derived from FrHK/4, cells) and the other tested by RT-PCR for HAV, EV and SRSV. Results demonstrated the high efficiency of the classic and QIAamp methods. Microcon-100 did not increase the sensitivity of the technique used. The highest sensitivity was observed for RT-PCR with specific primers for SRSV and for nested RT-PCR for HAV. One sample showed a cytopathic effect, not confirmed at the third subculture, while the RT-PCR allowed the detection of echovirus 7. Cell culture did not allow detection of the majority of the enteric viruses while PCR gave sensitive, specific and rapid detection of a range of agents in the same samples. Even if it was impossible to fix a virological quality standard, it would be necessary to find a viral indicator ...