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The aim of this study was to explore the temporal relationship between the consumption of different antibiotics, alcohol-based hand disinfection and the incidence of nosocomial bacterial strains producing extended-spectrum β-lactamases (ESBLs).Time-series analysis was performed based on monthly data available from January 2005 to October 2007. The incidence of nosocomial ESBL (cases/1000 patient-days) was regressed on the different antibiotic agents and the volume of alcohol-based hand rub orders. Antibiotic consumption was defined as monthly defined daily doses (DDD)/1000 patient-days, while alcohol-based hand rub was quantified in litres/1000 patient-days.The multivariate analysis showed that using alcohol-based hand rub for hand disinfection had a significant influence on the ESBL incidence (P=0.002). A higher volume of alcohol-based hand rub use was subsequently associated with a lower incidence of ESBL-producing strains. Additionally, the model showed that temporal increase in the use of third-generation cephalosporins (P=0.022) and fluoroquinolones (P=0.001) is, after a time lag of up to 3 months, followed by temporal variations in the incidence of nosocomial ESBLs. Furthermore, the incidence of patients admitted with ESBL was also shown to have an influence on the incidence of nosocomial ESBLs (P < 0.001). The final model explained 75% of the monthly variations in the incidence of nosocomial ESBLs.The analysis identifies selective pressure caused by the use of different antimicrobial agents as a driving factor in the emergence and spread of ESBLs. Furthermore, the study confirms that hand disinfection is key to the prevention of nosocomial ESBLs.