Evaluation of the LightCycler Staphylococcus M ^GRADE Kits on Positive Blood Cultures That Contained Gram-Positive Cocci in Clusters

Abstract

We evaluated the Roche LightCycler Staphylococcus M GRADE kits to differentiate between Staphylococcus aureus and coagulase-negative staphylococci in blood cultures growing clusters of gram-positive cocci. Testing 100 bottles (36 containing S. aureus), the assay was 100% sensitive and 98.44% specific for S. aureus and 100% sensitive and specific for coagulase-negative staphylococci.Gram-positive cocci in clusters (GPCC) noted on Gram staining of blood cultures are usually either Staphylococcus aureus or coagulase-negative staphylococci (CoNS). While the latter may be considered contaminants, the former is usually considered a true pathogen. It takes an additional day and supplementary testing to differentiate these two organisms by conventional methods. Multiple studies have reported rapid and accurate detection of S. aureus in blood culture bottles growing GPCC, which speak to the feasibility and interest in this approach (1-3, 5, 6, 9, 10). Accurate detection of the presence of all staphylococci in blood culture bottles and simultaneous identification of S. aureus among them using realtime PCR have also been demonstrated (8). This approach identifies CoNS by actually detecting its presence rather than by assuming its presence in the absence of S. aureus in cultures growing GPCC. A limitation to the use of real-time PCR for detection of staphylococci in a clinical laboratory is the expertise necessary to conduct real-time PCR and control for all the variables that might give rise to inconsistencies in the reaction over a period of time. The availability of a standardized kit would ease the process to the point where staphylococcal detection in blood cultures by real-time PCR may be seriously considered for practical application in clinical microbiology laboratories. The purpose of this study was to evaluate the LightCycler Staphylococcus M GRADE kits for the detection and identification of S. aureus and CoNS in blood culture bottles growing GPCC.Aerobic and anaerobic FAN-containing blood culture bottles that gave a positive signal in the BacT/ALERT blood culture system (bioMérieux, Inc., Durham, NC) and revealed the presence of GPCC were studied. A 0.5-ml aliquot of fluid was immediately removed for PCR from the positive blood culture bottle after brief manual agitation. Identification of the bacterium proceeded according to the standard laboratory protocol that included inoculation onto 5% sheep blood agar, incubation at 37°C, and identification based on colony morphology and coagulase test results. The aliquot removed for PCR was processed to remove the charcoal and DNA was prepared by a differential centrifugation and lysis buffer treatment as previously described (9). Charcoal was removed by centrifuging the aliquot at 850 ϫ g for 2 min and discarding the pellet. The supernatant was centrifuged at 11,500 ϫ g for 5 min. The resulting pellet was resuspended in 200 l of a lysis buffer (7) containing 1% Triton X-100, 0.5% Tween 20, 10 mM Tris-HCl (pH 8.0), and 1 mM EDTA and incubated in a screwcap reaction tube...

Differentiation BetweenStaphylococcus AureusandCoagulase-Negative StaphylococcusSpecies by Real-Time PCR Including Detection of Methicillin Resistants in Comparison to Conventional Microbiology Testing

“…The reliability of this test has been established in earlier studies, showing high sensitivity of 95.7–100%, with very good specificity was 90.8–98.44% . Of note, comparison of these studies with our data is inherently biased, because the specimens in our study being collected after the initiation of antibiotic treatment (see study design above).…”

Comparison of four methods for rapid identification of Staphylococcus aureus directly from BACTEC 9240 blood culture system

“…In a study, all isolates were correctly identified with LightCycler kit with a sensitivity and specificity of 100%. [15] In our study, LightCycler Staphylococcus M Grade kit correctly identified S. aureus and CoNS in a mixed culture, which was identified as CoNS with conventional PCR. The advantage of this technique over conventional PCR method is the ability to detect more than one Staphylococcus species in the same sample although there was limited number of mixed cultures in our study.…”

“…In a study, the LightCycler Staphylococcus M Grade kit was evaluated to differentiate S. aureus and CoNS from BacT/ Alert bottles and showed sensitivity and specificity of 100 and 98.44%, respectively. [15] To our knowledge, there is no study with Dry Spot Staphytect Plus and LightCycler Staphylococcus M Grade kit, which was performed directly from BACTEC-9240 system. In this study, we evaluated the performance of TCT, slide agglutination test (Dry Spot Staphytect Plus), conventional PCR and LightCycler Staphylococcus M Grade kit directly from blood culture bottles to achieve rapid identification of S. aureus by using the BACTEC-9240 blood culture system.…”

Rapid testing for methicillin-resistant Staphylococcus aureus: Implications for antimicrobial stewardship

“…Both the LightCycler MRSA and LightCycler Staphylococcus MGRADE tests can produce results in approximately two hours, and their overall sensitivity and specificity have been found to be reasonably good. 9,[32][33][34] The LightCycler Staphylococcus MGRADE test can readily distinguish between S. aureus and coagulase-negative staphylococcal species, which can be useful in antimicrobial stewardship programs, but the use of the assay in combination with the LightCycler MRSA test in order to determine methicillin susceptibility has not been studied.…”