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Background.Patients in the neonatal intensive care unit (NICU) are at high risk for healthcare-associated infections. Variability in reported infection rates among NICUs exists, possibly related to differences in prevention strategies. A better understanding of current prevention practices may help identify prevention gaps and areas for further research.MethodsWe surveyed infection control staff in NICUs reporting to the National Healthcare Safety Network (NHSN) to assess strategies used to prevent methicillin-resistant Staphylococcus aureus (MRSA) transmission and central line–associated bloodstream infections in NICUs.ResultsStaff from 162 of 342 NICUs responded (response rate, 47.3%). Most (92.3%) NICUs use central line insertion and maintenance bundles, but maintenance practices varied, including agents used for antisepsis and frequency of dressing changes. Forty-two percent reported routine screening for MRSA colonization upon admission for all patients. Chlorhexidine gluconate (CHG) use for central line care for at least 1 indication (central line insertion, dressing changes, or port/cap antisepsis) was reported in 82 NICUs (51.3%). Among sixty-five NICUs responding to questions on CHG use restrictions, 46.2% reported no restrictions.ConclusionsOur survey illustrated heterogeneity of CLABSI and MRSA prevention practices and underscores the need for further research to define optimal strategies and evidence-based prevention recommendations for neonates.Infect Control Hosp Epidemiol 2014;35(9):1126-1132