Clostridium difficile is currently one of the most common cause of diarrhea in hospitalized or residents in long term care institutions patients, symptoms of infection ranging from diarrhea to pseudo-membranous colitis and toxic megacolon.The disease is due to the production of enterotoxin A, cytotoxin B or binary toxin. The emergence of hypervirulent new strains showing a tcdC regulatory gene deletion (ribotype 027) has been observed in recent years. Stool or rectal swab specimens were screened using automated real-time multiplex PCR (GeneXpert, Cellbio) to detect the presence of toxins producing (B toxin, binary toxin and tcdC gene deletion) C. difficile. All positive samples were cultured in order to isolate the causing infection strains.The binary toxin and/or tcdC deletion producing strains were genotyped using Multilocus Sequence Typing (MLST). MLST compares the intragenic sequences of seven housekeeping genes and provides a unique combination of alleles; to each combination is then assigned a Sequence Typing (ST). In the period January 2010 – June 2011, 3681 samples from 2234 patients were analyzed. Four hundred seventy-three patients (21.2%) were positive for the presence of C. difficile, 34 (7.2%) of there were also positive for binary toxin and 3 were positive for the tcdC gene deletion (suspected C. difficile NAP027).All the strains having tcdC gene deletion, showes ST3 typing by MLST method.These strains were isolated during the same period. The three patiens were epidemiologically linked: the patient A was hospitalized in the same room of patient B, while patient C was managed by the same team care of patient A.The spread ofsuch C. difficile strains is at the present very low in Italy. Our data confirm the poor prevalence of ribotype NAP027 and the usefulness of a presumptive diagnosis to implement immediately appropriate control measures.