Direct Identification of Staphylococcus aureus from Positive Blood Culture Bottles

Abstract

Fluorescence in situ hybridization (FISH) using peptide nucleic acid (PNA) probes targetingIdentification of Staphylococcus aureus in blood cultures begins with presumptive identification of gram-positive cocci in clusters (GPCC) in the Gram-stained smear of blood culture bottles that signal a positive result, whereas final identification must await subculture and overnight incubation (1,8). This delayed identification often results in empirical antibiotic therapy administered to patients with GPCC-positive blood cultures, although the majority of GPCC-positive blood cultures are later identified as coagulase-negative staphylococci (CoNS), such as Staphylococcus epidermidis, a common blood culture contaminant. Direct identification of S. aureus in GPCC-positive blood culture bottles may provide important diagnostic information, which would allow the selection of an appropriate course of treatment in a timely manner.Immunological, tube coagulase, and stable-endonuclease methods routinely used for identification of S. aureus following subculture have been applied directly to GPCC-positive blood culture bottles, but with variable sensitivities and specificities (6, 9, 10). In addition, molecular techniques, such as hybridization protection (3), fluorescence in situ hybridization (FISH) (5), and PCR (2) have been described for identification of S. aureus directly from positive blood cultures. In general, all studies have been performed on just a single blood culture medium and, therefore, do not address the potential for interference from different blood culture media, which in fact might explain the variable results. Furthermore, none of the studies involve blood culture media supplemented with charcoal, such as that used in the FAN BacT/Alert medium (bioMerieux, Hazelwood, Mo.), or resins, such as that in the BACTEC Plus medium (Becton Dickinson, Sparks, Md.). Those supplements may interfere with assays for direct identification of positive blood culture organisms.FISH with a peptide nucleic acid (PNA) probe to target 16S rRNA of S. aureus is a novel method for the rapid and specific identification of S. aureus directly from GPCC-positive blood cultures. S. aureus PNA FISH had a 97% sensitivity and 100% specificity compared to conventional methods when tested with the BacT/Alert blood culture system and FAN medium (7). The aim of the present study was to perform a blinded comparison of S. aureus PNA FISH with conventional methodology on blood cultures representing the ESP medium (Trek Diagnostic Systems, Inc., Westlake, Ohio), BACTEC medium (Becton Dickinson), and BacT/Alert medium (bioMerieux). Routine positive blood culture bottles in which gram-positive cocci in clusters were observed in Gram-stained smears were randomly collected at each of eight clinical microbiology laboratories. For the study, one smear for future testing was prepared from each blood culture shortly after the Gramstaining results were known. The smears are stable at room temperature for several weeks and were collected over a 1-to 2-week pe...

Evaluation of Three Rapid Methods for the Direct Identification of Staphylococcus aureus from Positive Blood Cultures

“…Only one FN result was observed. This performance is comparable to that previously reported with ESP bottles (12). Likewise, similar performance has been noted using BacT/Alert FAN media (bioMerieux) (11) and BACTEC bottles with and without resins (12).…”

“…This assay yields results of S. aureus or non-S. aureus. The protocol has been previously described (11,12). Briefly, 1 drop of the blood culture broth was added to a slide containing a drop of fixation solution.…”

Evaluation of the BinaxNOW Staphylococcus aureus Test for Rapid Identification of Gram-Positive Cocci from VersaTREK Blood Culture Bottles

“…A number of laboratory testing procedures have been used to rapidly differentiate CoNS from S. aureus soon after Gram stain reading. These include nonmolecular methods as well as newer molecular methods that can also determine methicillin susceptibility (2)(3)(4)(5)(6). Our laboratory has used a direct tube coagulase test (DTCT) for many years to rapidly identify S. aureus from blood culture bottles.…”

Direct Identification of Gram-Positive Cocci from Routine Blood Cultures by Using AccuProbe Tests

“…However, rapid identification of S. aureus from blood culture by using the LightCycler system has already been reported (18), and the use of real-time fluorescence PCR for rapid identification of bacteria from blood culture may increase in the future. Fluorescent in situ hybridization (FISH) (8,15,16), as well as the hybridization protection assay (2), have also been shown to be quite reasonable methods for direct identification of microorganisms from blood.…”