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The objective of this study is to assess the clinical utility of direct fluorescent assay in buffy coat in the diagnosis of Candida sepsis (CS) in neonates.A cross-sectional study was conducted in a Neonatal Intensive Care Unit and 22 neonates with suspected CS were enrolled. Fungus isolation from blood cultures and direct fluorescent tests in buffy coat were performed and validity parameters were estimated.Candida was isolated in 13/22 (59%) blood cultures. The direct fluorescent test was positive in 12/13 and 1/9 cases with positive and negative blood culture as corresponding. Estimated sensitivity, specificity, positive predictive value, negative predictive value, positive likehood ratio and negative likehood ratio were 92%, 89%, 92%, 89%, 8.31 and 0.09, respectively.The direct fluorescent assay in buffy coat might be useful to support early and accurate diagnosis of CS in neonates.