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In our opinion, there are several possible reasons for the differences in the estimated CDI incidence by hospital size. One reason could be the greater diagnostic yield of the larger hospitals, owing to the use of more sensitive diagnostic tests, as occurred in our study. Another could be the concurrence of two circumstances: the fact that nosocomial CDI is most prevalent in older patients and in patients with more severe infections; and the highest prevalence of this group in larger hospitals, as occurs in the USA. Unfortunately, Fluorouracil the design of this study did not allow us to gather information on the types of patient in each participating hospital. The results of the molecular characterization of Spanish isolates of C.?difficile were quite similar to those obtained in the European survey performed in 2008 [18]. However, ribotype?027, which was found in 5% of episodes in the European study, was not recovered in Spain. Despite the spread SRT1720 clinical trial of this ribotype in most countries in North America and Europe during recent years [23,24], this strain has only been isolated, at the time of the study, in two CDI episodes in Spain, the first in 2007 in a patient transferred from a hospital in England to a hospital in Madrid, and the second in a technician working with the isolate [25]. PCR ribotyping revealed the polyclonal nature of CDI in nearly 5% of UNC2881 episodes. In 2005, van den Berg et?al. [26] found polyclonality in 8.7% of patients with a first episode of CDI diagnosed with PCR ribotyping. In 2009, Wroblewski et?al. [27] found that 13.0% of their episodes had different populations, using binary toxin gene detection by PCR. Tanner et?al. [28] found different multilocus variable-number tandem-repeat analysis profiles in five (12.8%) of 39 specimens containing ribotype?027 strains. In?vitro susceptibility testing revealed no strains resistant to metronidazole. Other authors, including our group, have reported cases of resistance to metronidazole [29�C33]. Resistance to metronidazole is heterogeneous, and is lost during specimen handling. Unfortunately, the methods used in this multicentre study may have favoured the loss of resistance. In summary, CDI was highly underdiagnosed in Spanish hospitals, owing to the use of non-sensitive techniques or the lack of clinical suspicion, particularly in patients with community-acquired infection and in patients aged