Objective: To develop a new protocol for the simultaneous detection and differentiation of Staphylococcus aureus and coagulase-negative staphylococci (CoNS) in blood cultures using fluorescence in situ hybridization (FISH), without cultivation and biotyping. Materials and Methods: Oligonucleotide probes were used to target the variable regions of the 16S rRNA of S. aureus and CoNS, the probes were labeled with fluorochrome Cy3 (red signal) and fluorescein isothiocyanate (green signal), respectively. It is not possible to design one probe that will hybridize to all CoNS species. Therefore, in this study two differentially labeled probes (STA and SAU) were mixed and used to detect and differentiate S. aureus from CoNS rapidly in a single smear. Samples of 189 positive blood cultures with Gram-positive cocci in clusters and 11 Gram-positive cocci in chains or pairs were included. Results: The FISH assay showed 91.8% sensitivity and 100% specificity for S. aureus and 100% sensitivity and 93.3% specificity for CoNS when hybridized with STA and SAU probes. Meanwhile, the assay showed 91.8% sensitivity and 100% specificity for S. aureus and 100% sensitivity and 90% specificity for CoNS, when hybridized with the SAU probe only. Conclusion: Our data showed that the FISH technique was suitable for the simultaneous detection and differentiation of Staphylococcus sp. in blood cultures.

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