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To assess the incidence of late onset (> 72 hours) infection and necrotising enterocolitis (NEC) in very low birthweight (VLBW) infants in two 36 month periods using two hand hygiene protocols: conventional handwashing (HW; first 36 month period); an alcohol hand rub and gloves technique (HR; second 36 month period).VLBW infants admitted to the neonatal intensive care unit during the period December 1993–November 1999 were eligible. A new hand hygiene protocol using alcohol handrub and gloves was introduced in December 1996. Each patient’s case record was reviewed retrospectively by two independent investigators using a standard data collection form. The incidence of NEC and systemic infections, including bacterial or fungal septicaemia, meningitis, and peritonitis, in the two periods were compared.The HW and HR groups contained 161 and 176 VLBW infants respectively. The incidence of late onset systemic infection decreased from 13.5 to 4.8 episodes (including NEC)/1000 patient days after introduction of the HR regimen, representing a 2.8-fold reduction. Similarly, the incidence of Gram positive, Gram negative, and fungal infections decreased 2.5-fold, 2.6-fold, and 7-fold respectively. There was also a significant reduction in the incidence of NEC in the HR group (p < 0.0001). Subgroup analysis revealed that the incidence of methicillin resistant Staphylococcus aureus (MRSA) septicaemia was significantly decreased in the second 36 month period (p = 0.048). The clinical data suggest that infants in the HW group had significantly earlier onset of sepsis (p < 0.05) and required oxygen supplementation for longer (p < 0.05) than those in the HR group. Significantly more VLBW infants were discharged from the neonatal intensive care unit without ever being infected (p < 0.0001), and also significantly fewer infants had more than one episode of infection in the HR group (p < 0.0001).The introduction of the HR protocol was associated with a 2.8-fold reduction in the incidence of late onset systemic infection, and also a significant decrease in the incidence of MRSA septicaemia and NEC in VLBW infants. This decrease in infection rate was maintained throughout the second 36 month period.