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Neonatal herpes simplex virus (HSV) infection can cause significant morbidity and mortality but can be difficult to identify, particularly in neonates without vesicular rash.To determine the unique clinical and laboratory features of neonates with and without HSV infection admitted to Texas Children's Hospital during a 14-year period.An historic case-control study of all hospitalized neonates with laboratory-confirmed HSV infection and a restricted sample (ratio 1:4) of HSV test-negative hospitalized neonates. Univariate and multivariate analyses were performed to identify clinical and laboratory factors associated with neonatal HSV infection.Forty cases and 160 comparison subjects were identified. The following factors were associated with neonatal HSV infection by univariate analysis: maternal primary HSV infection, maternal fever, vaginal delivery, prematurity, postnatal HSV contact, vesicular rash, hypothermia, lethargy, seizures, severe respiratory distress, hepatosplenomegaly, thrombocytopenia, elevated hepatic enzymes, and cerebrospinal fluid (CSF) pleocyosis and proteinosis. Factors not associated with neonatal HSV infection were fever, total peripheral white blood cell count, and red blood cells in the CSF. For neonates presenting without vesicular rash, maternal fever, respiratory distress requiring mechanical ventilation, and CSF pleocytosis were independently associated with HSV infection.Inclusion of the newly appreciated features of maternal fever, respiratory distress, and thrombocytopenia might improve the detection of neonatal HSV infection. Clinical and laboratory factors typically associated with neonatal HSV infection were confirmed to be maternal primary HSV infection, vaginal delivery, prematurity, neonatal seizures, vesicular rash, elevated hepatic enzymes, and CSF pleocytosis.