TEXT

Top Abstract Text References

Staphylococci are the most frequently isolated microorganisms from blood cultures. A major differentiation is made between Staphylococcus aureus and coagulase-negative staphylococci (CoNS). The isolation of S. aureus usually (>90% of the time) represents true infection and serious clinical disease with a high associated mortality (10). Nosocomial S. aureus bacteremia, for example, was associated with a mortality rate of 18% (15). S. aureus bacteremia therefore requires prompt institution of antimicrobial therapy. In contrast, CoNS, although potentially of clinical importance and increasingly identified as important nosocomial pathogens, are often (85% of the time) found to be contaminants (2, 10, 14). Rapid identification of staphylococci in blood cultures is therefore an aid that improves clinical decision making.

The presence of gram-positive cocci in clusters in a Gram-stained smear of a blood culture indicates the presence of staphylococci, but at this stage, no distinction can be made between the species (1). A subculture from the blood culture broth onto solid agar medium, requiring overnight incubation, is necessary to obtain colonies for identification by conventional methods. Conventional methods for identification of S. aureus are based on the demonstration of coagulase production using the tube coagulase test, production of heat-stable DNase, or bound coagulase (clumping factor) in combination with several other products specific for S. aureus (e.g., protein A) by immunological tests (5).

Several of these methods have been applied for direct identification of S. aureus from blood cultures. The tube coagulase test is reported to be sensitive and very specific, but results may differ when rabbit plasma from different manufacturers is used (6, 12). A variety of immunologic tests have been evaluated for this purpose, and although overall specificity has been excellent, a wide range of sensitivities have been reported (6, 9, 11, 12). Different results have been reported with the thermostable-endonuclease test. It has been shown that the test is extremely medium dependent (4, 12).

The RAPIDEC Staph system (bioMérieux, Marcy-l'Etoile, France) is a biochemical test that detects the production of an aurease enzyme specific for S. aureus. Aurease is a proteolytic enzyme of coagulation that reacts with prothrombin to form a complex called staphylothrombin. Staphylothrombin cleaves a fluorescent peptide present in the test, thereby releasing a peptide and a fluorescent radical. Previous studies have shown that the RAPIDEC Staph is a sensitive and specific test for the detection of S. aureus in Vital system (8), BACTEC NR-660, and Oxoid SIGNAL system blood cultures (7). Recently Speers et al. (12) compared Staphaurex Plus, the tube coagulase test, the thermostable-endonuclease test, and RAPIDEC Staph with the BACTEC 9000 Blood Culture System. The RAPIDEC Staph was the most reliable test, but they performed the test in accordance with a protocol requiring two centrifugation steps, which made it relatively time-consuming.

In the present study, a modified specimen-processing protocol for the RAPIDEC Staph system was compared to the manufacturer's protocol to determine whether the modified procedure, which is easier to perform, could be used to differentiate S. aureus from CoNS in positive blood cultures.

Blood cultures collected from patients at St. Elisabeth Hospital, Tilburg; St. Ignatius Hospital and Hospital de Baronie, Breda; University Hospital Dijkzigt, Rotterdam; and St. Franciscus Hospital, Roosendaal, were analyzed over a 3-month period. All centers made use of the BACTEC 9000 Blood Culture System (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.). Positive blood cultures were examined by Gram stain, and if gram-positive cocci in clusters were present, the first blood culture from a patient that was identified as positive was analyzed by conventional methods and by RAPIDEC Staph performed in accordance with the manufacturer's protocol and in accordance with a modified protocol to differentiate S. aureus from CoNS. The conventional method consisted of subculture onto a blood agar plate and incubation for 18 to 24 h at 35°C. Subcultured isolates were identified by a latex agglutination test (Staphaurex Plus; Murex Diagnostics Ltd., Dartford, England), by detection of free coagulase by the tube coagulase test with rabbit plasma (5), and by detection of DNase (DNase agar; Oxoid Unipath Ltd., Basingstoke, England). If all three tests were positive, the isolate was considered S. aureus. If all three tests were negative, the isolate was considered to be a CoNS. If the results of the tests were discordant, an APIStaph (bioMérieux) and an AccuProbe culture identification test, a DNA probe assay directed against rRNA (Gen-Probe; San Diego, Calif.) were performed. The result of the AccuProbe was considered to be the "gold standard."

The RAPIDEC Staph was performed in accordance with the manufacturer's protocol and in accordance with the modified protocol (Fig. 1). In accordance with the manufacturer's protocol, 2 ml broth from a positive blood culture bottle was added to 2 ml of distilled water and centrifuged at 1,000 × g for 10 min to lyse the erythrocytes and produce a bacterial pellet. The pellet was then resuspended in approximately 250 µl of distilled water to get an inoculum equivalent to a 4 McFarland standard, and 50 µl of this suspension was added to cupules 0 and 1 (the negative control and aurease test cups, respectively). The strip was incubated for 2 h at 35°C and then read under UV light (365 nm). The test was considered positive when the fluorescence visible in cup 1 was more than that in cup 0. In accordance with the modified protocol, 2 ml of broth was centrifuged at 100 × g for 5 min to sediment the erythrocytes and 50 µl of the supernatant was directly added to cupules 0 and 1. The rest of the procedure was identical to that recommended by the manufacturer.

View larger version (16K): [in this window] [in a new window]   FIG. 1.   RAPIDEC Staph protocols. Please note: 3,000 rpm is equivalent to 1,000 × g and 600 rpm is equivalent to 100 × g.

A total of 129 patients had positive blood cultures with gram-positive cocci in clusters. Forty-two were S. aureus, 85 were CoNS, and 2 were Micrococcus spp. The RAPIDEC Staph performed in accordance with the manufacturer's protocol correctly detected 38 of the 42 S. aureus isolates (sensitivity, 90.5%). Two CoNS gave a false-positive signal (specificity, 97.7%); one was identified as S. epidermidis, and one was identified as S. caprae. The RAPIDEC Staph performed in accordance with the modified protocol detected all of the 42 S. aureus isolates correctly (sensitivity, 100%). Three CoNS gave a false-positive signal (specificity, 96.6%); one was identified as S. epidermidis, one was identified as S. caprae, and one was not further identified. The positive predictive values for the manufacturer's protocol and the modified protocol were 95.0 and 93.3%, respectively. The negative predictive values were 95.5 and 100%, respectively (Table 1).

Rapid bacterial identification and susceptibility testing in the microbiology laboratory can have a major impact on the care and disease outcome of hospitalized patients with infections (3). It has been shown that information provided by rapid direct tests is significantly more likely to result in initiation of antibiotic therapy, a change to more effective therapy, or a change to less expensive therapy than the routine method (13).

Our modified protocol is easier to perform than the manufacturer's protocol. To add distilled water to the blood culture broth and to resuspend the bacterial pellet obtained after centrifugation and adjust this to an inoculum size equivalent to a 4 McFarland standard is no longer necessary. Furthermore, the results of our study show that the RAPIDEC Staph performed in accordance with our easier, modified protocol is a reliable test for the direct identification of S. aureus from blood cultures. Specificity with both protocols was not 100%. Two specimens gave false-positive results with both the manufacturer's protocol and the modified protocol; one specimen gave a false-positive result only with the modified protocol. Excessive hemolysis could have been the cause of this false-positive result (12). With the modified protocol, there were no false-negative results; with the manufacturer's protocol, however, there were four false-negative results.

We perform the RAPIDEC Staph in accordance with our modified protocol on all positive blood cultures containing gram-positive cocci in clusters. If the RAPIDEC Staph is negative, the clinician is told that the blood culture is positive for gram-positive cocci that are most likely to be CoNS. Depending on the clinical symptoms and condition of the patient, appropriate therapy is advised while awaiting the results of additional tests. If the RAPIDEC Staph is positive, the clinician is warned that the patient probably has an infection with S. aureus. The final identification depends on the results of conventional tests.

In conclusion, with our modification, the RAPIDEC Staph is an easy, rapid, and reliable test for screening of BACTEC blood culture bottles containing gram-positive cocci in clusters for the presence of S. aureus. Results of the test can be used to optimize antibiotic therapy in severely ill patients.