Evaluation of Penicillin Binding Protein 2a Latex Agglutination Assay for Identification of Methicillin-Resistant Staphylococcus aureus Directly from Blood Cultures

Abstract

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...

Direct Detection of Methicillin Resistance in Staphylococcus aureus in Blood Culture Broth by Use of a Penicillin Binding Protein 2a Latex Agglutination Test

“…In addition, the PBP-LA test showed identical performance characteristics with aerobic and anaerobic blood culture broth samples. When both aerobic and anaerobic bottles from the same set were positive for S. aureus (n ϭ 40; 21 MRSA and 19 MSSA samples), the PBP-LA results from each bottle were concordant.The observed differences in sensitivity and specificity compared with those reported from studies were likely due to different sample preparation protocols (1,3,13,20). For example, in a previous study showing low sensitivity with the ESP system, bacteria were collected by serum separator tubes, which may not efficiently recover the organisms (3).…”

“…The observed differences in sensitivity and specificity compared with those reported from studies were likely due to different sample preparation protocols (1,3,13,20). For example, in a previous study showing low sensitivity with the ESP system, bacteria were collected by serum separator tubes, which may not efficiently recover the organisms (3).…”

Rapid Differentiation of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus by Flow Cytometry after Brief Antibiotic Exposure

“…These assays would be faster than flow cytometry for determining the presence of methicillin resistance in S. aureus isolated in pure culture on solid media. The sensitivity of latex agglutination is lower with smaller inocula, and PBP2a latex agglutination assays have lower sensitivity when used directly in positive blood culture bottles (3,14). The promise of flow cytometry is that it can detect far fewer microorganisms and that the technology could potentially be developed to be able to identify and determine resistance of microorganisms directly in body fluids.…”

“…Phenotypic testing for susceptibility using automated systems still require at least 4 to 16 h to provide results. Latex agglutinating assays based on detecting the presence of penicillin-binding protein 2a (PBP2a) are rapid tests that can determine the presence of methicillin resistance when bacteria have been isolated in pure culture in the laboratory but have lower sensitivity with smaller numbers of bacteria (2,3).…”

Anticoagulant Carryover May Influence Clot Formation in Direct Tube Coagulase Tests from Blood Cultures

“…More commonly, coagulase-negative staphylococci (CoNS) are isolated, and these are frequently found to be contaminants (14,15). Rapid identification of S. aureus from blood cultures is essential for expeditious patient management, and several reports have described novel phenotypic and molecular methods for this purpose (5,6,11,16). The tube coagulase test (TCT), however, remains the "gold standard" for S. aureus identification (1), and while the direct TCT has been reported to be highly specific, it has often lacked sensitivity when inoculated directly from BCB growing S. aureus (6,13,17).…”