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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...

Traditional susceptibility testing of blood cultures requires overnight incubation in order to obtain isolated colonies. Susceptibility results can be reported up to 24 h sooner by using a bacterial pellet from the blood culture broth. This study evaluated the accuracy of direct susceptibility testing from positive ESP blood culture broths by using Sensititre broth microdilution plates compared to testing with isolated colonies. Practical inclusion criteria were applied to gram-positive organisms to avoid reporting susceptibilities for probable contaminants. All gram-negative organisms were tested directly. An aliquot of the blood culture was centrifuged, and the resulting pellet was used to make a 0.5 McFarland suspension. Microdilution plates were inoculated and interpreted according to the manufacturer's instructions. Colony counts were performed to ensure proper colony density was achieved. A total of 199 patient and seeded blood cultures were evaluated for both essential (within ؎1 twofold dilution) and categorical (sensitive, intermediate, or resistant) agreement. Testing of 93 gram-positive isolates (1,214 antimicrobial agent-organism combinations) yielded 98% essential agreement and categorical error rates of 0.3% minor, no major (false resistance), and 1.7% very major (false susceptibility) errors. For 106 gram-negative isolates (1,828 antimicrobial agent-organism combinations), the essential agreement was 99%. Categorical error rates were 0.5, 0, and 2.0% for minor, major, and very major errors, respectively. Performance was comparable for both gram-positive and gram-negative isolates, as well as for both aerobic and anaerobic media. Using this direct testing methodology, reliable susceptibility results can be reported to physicians 24 h sooner, allowing earlier appropriate modification of antimicrobial therapy.Timely intervention in the treatment of bloodstream infection is of paramount importance to increase the chances of a favorable outcome, since empirical therapy can be modified upon receipt of in vitro antimicrobial susceptibility testing (AST) results. AST results can assist in modifying antimicrobial therapy (13), and investigators have demonstrated decreased mortality with early treatment (10). Further data from outcome-based studies assessing the effect of rapid reporting of susceptibility results have shown a decrease in the number of laboratory tests and procedures ordered (4), decreased length of stay (1), and decreased health care costs and quicker modification of antimicrobial therapy (1,4,23).The standard protocol for identification and susceptibility testing of positive blood cultures involves Gram stain and subculture of blood culture broth onto solid medium and overnight incubation to obtain isolated colonies. These colonies are then used to make a standardized inoculum used for AST. A direct susceptibility testing algorithm from positive ESP (TREK Diagnostic Systems, Cleveland, Ohio) blood culture bottles was designed that circumvents the isolation process and expedites the reporti...

The penicillin binding protein 2a (PBP2a) latex agglutination test using a blood culture pellet was compared to the oxacillin screen agar method using isolated colonies. For blood cultures positive for Staphylococcus aureus (n ‫؍‬ 70), the direct PBP2a test was 18% sensitive and 100% specific. The PBP2a test shows poor sensitivity when used directly with positive blood cultures.The gene product of mecA, an altered penicillin binding protein (PBP2a), is the hallmark of methicillin resistance in staphylococci. The PBP2a latex agglutination test (Oxoid, Hampshire, United Kingdom) is a 20-min phenotypic test that detects PBP2a in isolated colonies (4). The PBP2a assay is faster and less complicated than PCR for mecA and has been shown to be more sensitive than other phenotypic methods, such as the use of oxacillin screen agar (2, 5, 7). Other investigators have demonstrated satisfactory performance of the PBP2a assay using isolated colonies (6,8).Conventional work flow for positive blood cultures includes subculture and overnight incubation to obtain isolated colonies. These colonies are then used to test for methicillin resistance by one of the methods mentioned above. Rapid reporting of identification and susceptibility results has been associated with improved outcomes (1, 4). Two groups reported acceptable performance of the PBP2a assay used directly with positive blood cultures, which reduces the time to result by 24 to 48 h (T. Yamazumi, I. Furuta, T. Maeno, Y. Tsubakimoto, and M. A. Pfaller, Abstr. 102nd Gen. Meet. Am. Soc. Microbiol., abstr. C-99, 2002; L. A. Bassiwa and D. Craft, 103rd Gen. Meet. Am. Soc. Microbiol., abstr. C-86, 2003). The present study sought to confirm these results by using the ESP (TREK Diagnostic Systems, Cleveland, Ohio) blood culture system.Isolates tested by the direct PBP2a assay were those previously determined to be Staphylococcus aureus by direct testing methods routinely used in the laboratory. Isolates giving a positive signal in the ESP blood culture system were subjected to Gram staining, and those identified as gram-positive cocci in clusters underwent a direct tube coagulase test. The tube coagulase test was performed using 5 drops of blood culture broth. The test was read after 4 h of incubation at 35°C, and a positive result indicated the presence of S. aureus. Seventy such isolates from different patients were obtained over a 4-month period.These 70 isolates were subsequently seeded into blood culture bottles, and the bottles were pulled from the ESP instrument when the isolates gave a positive signal. An aliquot of blood culture broth was added to a 7-ml serum separator Vacutainer tube (BD, Franklin Lakes, N.J.). The tube was centrifuged at 1,300 ϫ g for 10 min. The supernatant was discarded, and the bacterial pellet was used as the inoculum for the PBP2a test. Direct testing specifically for susceptibility results by using this preparation has been described previously (3). From this point, the PBP2a assay was performed according to manufacturer instructions. The di...

The present study compared the antimicrobial susceptibility testing (AST) results generated by the Automated Incubation and Reading System (ARIS) with custom Sensititre plates (TREK Diagnostic Systems, Cleveland, Ohio) and MicroScan PC10 GP and NUMIC10 GN plates interpreted with the WalkAway-96 system (Dade Behring, West Sacramento, Calif.) for gram-positive (GP) and gram-negative (GN) organisms as part of an in-house validation. A total of 326 isolates (3,689 antimicrobial agent-organism combinations) were evaluated. Sensititre plates were inoculated according to the instructions of the manufacturer with a suspension adjusted to a 0.5 McFarland standard, while the Prompt Inoculation System was used for the MicroScan plates. ARIS and the WalkAway system were used for automated reading of the Sensititre and MicroScan plates, respectively, at 18 to 24 h. The results were analyzed for essential (؎1 twofold dilution) and categorical (sensitive, intermediate, or resistant) agreements. Plates that resulted in ARIS interpretations with major (falsely resistant) or very major (falsely susceptible) errors compared to the results obtained with the WalkAway system were read manually to corroborate instrument readings. Isolates for which very major or major errors were obtained and for which the results were not resolved by manual reading were retested in parallel. Isolates for which very major or major errors were obtained and for which the results were not resolved upon repeat testing were tested by the National Committee for Clinical Laboratory Standards M7-A5 frozen reference microdilution method. Essential agreement was 95.8% for 246 GN isolates. The following categorical error rates were obtained for the GN isolates: 1.3% minor errors, 0% major errors, and 0.4% very major errors. For 95 GP isolates, there was 93.5% essential agreement. Categorical error rates for GP isolates were 0.9% minor errors, 0.6% major errors, and 0.4% very major errors. ARIS-Sensititre is a diagnostic system feasible for use for automated AST in a clinical laboratory.Sensititre broth microdilution antimicrobial susceptibility plates (TREK Diagnostic Systems, Cleveland, Ohio) were introduced in 1980 (4, 7); and 18-to 24-h susceptibility results have been shown to compare favorably to standardized broth microdilution (4, 5, 6, 7, 16), agar dilution (3, 13, 14, 16), and disk diffusion (5, 6, 12, 16) susceptibility results. Favorable results have also been documented with manually and semiautomatically interpreted Sensititre plates in parallel with MicroScan plates (6) with the WalkAway-96 system (Dade Behring, West Sacramento, Calif.) and the Vitek system (bioMerieux, Durham, N.C.) (6) with the Vitek automated instrument (5, 16) compared to the results of the disk diffusion and broth microdilution methods. While manually interpreted Sensititre plates are commonly used in the veterinary, surveillance, research, and pharmaceutical industries, the plates have not been commonly used in the clinical diagnostic laboratory. The antimicrobial susceptibil...