In the present study, raw, mesophilic anaerobic, and thermophilic anaerobic sludge were analysed to evaluate whether the pathogen content limits established in the "Proposal for a Directive of the European Parliament and of the Council on spreading of sludge on land" were satisfied. Salmonella spp., Escherichia coil, and Clostridium perfringens spores were cultivated and pathogenity genes invA and cpa PCR-amplified. Thermophilic anaerobic digestion produced Class A biosolids by eliminating E. coli and Salmonella spp. but did not accomplished the microbial requirements of the future Directive due to the presence of C perfringens spores (9.6 x 10(4) CFUs mL(-1)). Hence, the final goal of this work was to propose a two-stage process capable of removing the spores of C perfringens to obtain an advanced treated sludge that could be land-applied with no environmental risks. The first stage of the process suggested in this study involved the mesophilic anaerobic digestion of the sludge while the second stage of operation consisted of an aerobic or anaerobic thermophilic digestion. (C) 2013 Elsevier B.V. All rights reserved.