Reductions in annual rainfall in some regions and increased human consumption have caused a shortage of water resources at the global level. The recycling of treated wastewaters has been suggested for certain domestic, industrial, and agricultural activities. The importance of microbiological and parasitological criteria for recycled water has been repeatedly emphasized. Among water-borne pathogens, protozoa of the genera Giardia and Cryptosporidium are known to be highly resistant to water treatment procedures and to cause outbreaks through contaminated raw or treated water. We conducted an investigation in four wastewater treatment plants in Italy by sampling wastewater at each stage of the treatment process over the course of 1 year. The presence of the parasites was assessed by immunofluorescence with monoclonal antibodies. While Cryptosporidium oocysts were rarely observed, Giardia cysts were detected in all samples throughout the year, with peaks observed in autumn and winter. The overall removal efficiency of cysts in the treatment plants ranged from 87.0 to 98.4%. The removal efficiency in the number of cysts was significantly higher when the secondary treatment consisted of active oxidation with O 2 and sedimentation instead of activated sludge and sedimentation (94.5% versus 72.1 to 88.0%; P ‫؍‬ 0.05, analysis of variance). To characterize the cysts at the molecular level, the ␤-giardin gene was PCR amplified, and the products were sequenced or analyzed by restriction. Cysts were typed as assemblage A or B, both of which are human pathogens, stressing the potential risk associated with the reuse of wastewater.

Aims: The persistent circulation of hepatitis A virus (HAV) in the Mediterranean area suggests the need for monitoring its presence in the environment. A reverse transcriptasepolymerase chain reaction (RT-PCR) was used to detect the presence of HAV in several consecutive raw sewage and ®nal ef¯uent samples, collected over an 8-month period from an activated sludge treatment plant in southern Italy. Methods and Results: Two distinct puri®cation protocols, either based on antigencapture with monoclonal antibody (AC) or RNA extraction, were compared. The possible in¯uence of the antibody used in the AC phase was evaluated in preliminary experiments on HAV-spiked samples, using two different monoclonal antibodies. Hepatitis A virus RNA was detected in all but one sewage environmental sample examined. The contemporary presence of enteroviruses, reoviruses and phages was observed, while HAV growth in cell culture was hampered. Conclusions: The RT-PCR technique was con®rmed to be a valuable tool for the rapid monitoring of HAV in sewage samples. In addition, this study demonstrated that application of different sample puri®cation methods can result in different levels of sensitivity of the assay and that, in the antigen-capture method, the choice of antibody can have a crucial role. Signi®cance and Impact of the Study: This work underlines the need for technical uniformity in environmental studies from different laboratories for a correct and useful comparison of the results.

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