The aim of this study was to select one or several virus extraction techniques that enable simultaneous detection of enterovirus genomes and infectious particles in different types of urban sludge. Eight techniques were compared by using 16 different liquid and solid sludge samples. The numbers of infectious enteroviruses in cell cultures were determined by using the most-probable-number method. The enterovirus genome was quantified by a single-tube reverse transcription-PCR using TaqMan technology. The results were statistically analyzed by Friedman's test, a nonparametric test for analysis of randomized block data using only ranks in terms of extraction technique efficiency. Two techniques seemed to yield higher viral titers as determined by simultaneous detection by cell culture and PCR. The first involved a 10% beef extract solution at pH 9 and sonication; the second involved a 0.3 M NaCl-7% beef extract solution at pH 7.5 followed by Freon treatment. In solid sludge, no significant differences were observed among the eight techniques tested. Both of the best techniques can be used for simultaneous detection of infectious enterovirus particles and genomes in any type of urban sludge.The methods used to detect enteroviruses in environmental samples are of two general kinds, those based on cell culture infectivity and those in which molecular detection methods are used, such as PCR followed by nucleic acid hybridization (13). Environmental samples, especially urban sludge, contain numerous organic and inorganic compounds (humic acids, polyphenols, heavy metals) which are toxic and cause lysis in cell cultures. These compounds are also liable to form complexes with nucleic acids and inhibit amplification enzymes (9,15,18). The results of cell culture analysis and PCR therefore depend on the efficacy with which the viral extraction technique used removes such compounds. The aim of this study was to select one or several of eight previously described viral extraction techniques which would allow simultaneous cell culture and reverse transcription (RT)-PCR analyses for quantification of enteroviruses in sludge samples. We hoped to identify a screening method applicable on a large scale which was based on a real-time genomic quantification technique and allowed confirmation of infectivity with the same viral sludge concentrate. The efficiency of elution was evaluated by counting infectious enteroviruses (most-probable-number cytopathogenic units [MPNCU]/10 g of dry matter) and quantifying enterovirus genomes (number of copies per 10 g of dry matter) by a fluorogenic RT-PCR method developed in our laboratory (14). MATERIALS AND METHODSResidual sludge. Four types of sludge (16 samples) were obtained from two wastewater treatment plants in Lorraine (Nancy and Metz, France). At the Nancy plant, biological sludge produced during treatment of wastewater undergoes mesophilic anaerobic digestion at 37 to 38°C for 15 to 20 days, while at the Metz site sludge is also thickened, dehydrated, and packed. Primary sludge was obta...

Treatments applied to sludge in order to stabilise and dehydrate them may give notable inactivation of microorganisms. This is observed when sludge is exposed either to high temperature or drastic pH when residual sludge is limed. The control of virological, parasitological and bacteriological sludge quality by detecting pathogenic microorganisms is slow and too expensive to be commonly practised. Thus, it is possible to replace pathogenic microorganisms detection by that of contamination indicators. The aim of this study was to determine the influence of liming on the behaviour of pathogenic microorganisms detected in urban sludge. The detection of Salmonella and helminth eggs was carried out in liquid sludge (2-3% dryness) and solid sludge (23% dryness) with added lime (0-45% weight/dry weight) and stored for 24 h-46 weeks. The results showed that liming modified some characteristics such as temperature, dryness and pH of the sludge. It appeared that, whatever the percentage of added lime, the temperature of liquid sludge did not change while it increased by about 9 degrees C when 30-45% lime was added to solid sludge. In the same way, the dryness of liquid sludge did not change during the liming, whereas the dryness of 45% limed solid sludge increased from 23% to 31%. Finally, 15%, 30% and 45% liming gave a pH of at least 10, 11.5 and 12, respectively, although the pH increase depended on the sludge type. The efficiency of liming was considered to be related to the pH and not to the percentage of added lime. Three factors determined the efficiency of pathogen elimination: (a) the pH reached by the sludge, (b) the period of liming activity and (c) the dryness of the sludge. Salmonella were eliminated from liquid sludge in 24 h at pH 10.7 and from solid sludge in 24 h at pH 10.0. Viable helminth egg concentration decreased to 3 eggs/10 g DM in liquid sludge in 14 d at pH 11.9 and 60 d at pH 11.6. In solid sludge, egg reduction was achieved in 24 h at pH 12.5, 7 d at pH 12.0 and 14 d at pH 11.5. From this study, it appeared that liming resulted in a much better microbiological quality of liquid sludge if its pH was maintained at 11.6 over 60 d or at pH 11.9 for 14 d. Solid sludge needed to be maintained at pH 11.5 for 14 d, pH 12.0 for 7 d or pH 12.5 for 24 h to achieve similar results.

The aim of this work was to determine the effect of liming and composting on the fate of three bacteriophages (somatic coliphages, F-RNA phages, Bacteroides fragilis phages) considered as potential indicators of viral contamination. It was shown that the three bacteriophages studied exhibited variable densities in sludge. Somatic coliphages were most abundant (10(4) to 10(5) x 10 g(-1) DM) then F-RNA bacteriophages (10(2) to 10(4) x 10 g(-1) DM) and Bacteroides fragilis phages (10(1) to 10(2) x 10 g(-1) DM). The efficacy of liming was found to be pH dependent but also sludge dependent. The pH allowing 99% elimination of somatic coliphage is close to 9 for solid sludges and close to 13.5 for liquid sludges. For composting, our findings clearly demonstrated that phage inactivation is very clearly temperature-dependent. For temperatures reaching 70 degrees, there is a 5 log reduction in somatic coliphages while for temperature in the 50-55 degrees C range, the drop off is only 2 log. Considering the efficacy of the treatment methods, it is clear that the well-established industrial procedures that reach temperatures in the 60-70 degrees C range totally inactivate all 3 phages tested and present in sludge before composting.