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Background.We describe our experience using a real-time polymerase chain reaction (PCR) assay for methicillin-resistant Staphylococcus aureus (MRSA) during a period of active surveillance in the neonatal intensive care unit (NICU) from March 2007 until November 2007.Objective.TO compare PCR with bacterial culture methods and find the screening algorithm that most successfully ensures appropriate isolation of colonized patients.Methods.Patients in the NICU were screened for MRSA on admission and weekly thereafter until discharge. Healthcare workers (HCWs) were also screened as part of an outbreak investigation. A total of 599 individuals were screened for MRSA with both a PCR assay and selective bacterial culture. Strain typing was performed on all MRSA isolates to determine clonal relatedness.Results.Twenty-one of 435 infants (4.8%) screened positive for MRSA with the PCR assay. Only 11 patients (52.4%) had concomitant bacterial cultures positive for MRSA. Compared to bacterial culture, the PCR assay had a sensitivity of 100% and a specificity of 97.6%, with a positive predictive value (PPV) of 52.4%. Infants that tested positive for MRSA by both culture and PCR were more likely to have a positive PCR assay result when retested than were those who tested positive by PCR alone (80% vs 20%; P = .02). Strain typing of MRSA isolates identified a common clone in only 2 colonized infants.Conclusion.Our data show that, in our neonatal population, the reproducibility of PCR assay results for culture-negative patients was low compared with the reproducibility of results for culture-positive Patients. Furthermore, the low PPV suggests that for nearly half of individuals who were PCR-positive, the result was falsely positive, which argues against the use of PCR assays alone for MRSA screening in the NICU.