We noted with interest the recent publication by Cavassini et al. (2) evaluating the MRSA-Screen, a latex agglutination kit detecting PBP 2a production by Staphylococcus aureus, thus allowing rapid detection of methicillin resistance. We have recently undertaken a similar study in a geographically distinct location (Sydney, Australia) and wish to share our findings.
A total of 159 clinically significant coagulase-positive and -negative staphylococcal isolates were collected between May and December 1998 from patients at St. Vincent's Hospital, Sydney, and an additional 24 isolates were kindly supplied by Tom Gottlieb, Microbiology Department, Concord Repatriation Hospital, Sydney. All organisms were tested for phenotypic methicillin resistance by a disc diffusion technique (modified Stokes method). The MRSA-Screen latex agglutination test (Denka-Seiken Pty Ltd) was performed according to the manufacturer's instructions. The mecA and 16S rRNA genes were detected by multiplex PCR using published methods (1) to confirm methicillin resistance in staphylococci. The MRSA-Screen and mecA gene detection were performed by investigators blinded to other results for each organism.
The results are summarized in Table 1. One hundred and twenty-one methicillin-resistant S. aureus (MRSA) isolates were confirmed by mecA gene detection and MRSA-Screen latex agglutination test. One phenotypic MRSA was repeatedly negative by mecA gene detection but positive by the MRSA-Screen. Of the 35 organisms reported by phenotypic testing as methicillin-sensitive S. aureus (MSSA), there were discordant results for four isolates. One organism was determined to be MSSA but was positive by the MRSA-Screen test and possessed a mecA gene. Two MSSA isolates demonstrated the mecA gene but were negative by the MRSA-Screen test, and another MSSA was positive by the MRSA-Screen but lacked the mecA gene.
TABLE 1.
Results for 183 staphylococcal isolates
| Method | No. of isolates identified as:
|
|||
|---|---|---|---|---|
| MRSA | MSSA | MRCNS | MSCNS | |
| Disc susceptibility testing | 121 | 35 | 16 | 11 |
| MRSA-Screen | 121 | 2 | 13 | 0 |
| mecA detection | 120 | 3 | 16 | 0 |
The MRSA-Screen kit is not validated for coagulase-negative staphylococci (CNS), although the mechanism of methicillin resistance in the organisms is similar to that in S. aureus. In the original description of this test (3) the authors tested five methicillin-resistant CNS (MRCNS), and the MRSA-Screen was positive for each isolate. As CNS are increasingly important clinical isolates, we performed PCR for the mecA gene and the MRSA-Screen on 27 CNS. No methicillin-sensitive CNS (MSCNS) were positive by the MRSA-Screen test. However, 3 of 16 CNS which possessed the mecA gene were negative by the MRSA-Screen test.
Our results and those of Cavassini et al. suggest that the MRSA-Screen is a rapid and accurate method to detect methicillin resistance in staphylococci. However an abstract was recently published by Wallrauch and Braveny (4) in which they evaluated a latex agglutination test for the detection of PBP 2a (manufacturer not specified) against 38 MRSA isolates and 10 MRCNS. Despite prolonged observation (up to 10 min) 9 of 38 MRSA isolates and 10 of 10 MRCNS failed to agglutinate. The specificity was high (100%) but the sensitivity was only 47% after 3 min of observation, increasing to 76% at 10 min.
It is possible that we studied clonal isolates of MRSA which produced a PBP 2a that was readily detected by the MRSA-Screen. DNA fingerprinting is currently being undertaken on all isolates, but preliminary data suggests that the 121 MRSA isolates consisted of at least six different clones. In addition, Cavassini et al. studied isolates with 60 different pulsed-field gel electrophoresis patterns, suggesting that the MRSA-Screen kit is accurate over a wide range of MRSA clonal variants. An alternative explanation for the poor results of Wallrauch and Braceny may be difficulties with methodology. Positive and negative controls are not provided with the MRSA-Screen kit. We found it essential to include these from our stock isolates, as overreading weak agglutination by mecA gene-negative organisms may lead to false-positive results. In addition, one critical step is the extraction procedure in which boiling for 3 min is recommended. We noted that more reproducible results were obtained when a heating block was substituted for boiling. The heating time is critical for avoiding false-positive results associated with nonspecific agglutination from underheating or false-negative results from overheating.
One important feature of the MRSA-Screen is the rapid turnaround time, which reduced the detection time for methicillin-resistant isolates in our laboratory from 48 to 72 h to approximately 26 h. The test takes approximately 20 min, and organisms can be batched to further reduce the workload.
In summary, we found the MRSA-Screen latex agglutination test to be a simple, rapid, and accurate test to determine methicillin resistance in S. aureus isolates. No false-negative results were noted for isolates which phenotypically expressed methicillin resistance, although two S. aureus isolates possessed the mecA gene but were negative by the MRSA-Screen and methicillin sensitive by phenotypic testing, suggesting repression of the mecA gene and, therefore, the PBP 2a gene product. There was only one false-positive result for an isolate which was negative for the mecA gene and phenotypically methicillin sensitive. The results for CNS suggest that although the MRSA-Screen is not validated for these organisms it is a useful screening test. Further work is needed to confirm this.
REFERENCES
- 1.Bignardi G E, Woodford N, Chapman A, Johnson A P, Speller D C E. Detection of the mecA gene and phenotypic detection of resistance in Staphylococcus aureusisolates with borderline or low level methicillin resistance. J Antimicrobiol Chemother. 1996;37:53–63. doi: 10.1093/jac/37.1.53. [DOI] [PubMed] [Google Scholar]
- 2.Cavassini M, Wenger A, Jaton K, Blanc D S, Bille J. Evaluation of MRSA-Screen, a simple anti-PBP 2a slide latex agglutination kit, for rapid detection of methicillin resistance in Staphylococcus aureus. J Clin Microbiol. 1999;37:1591–1594. doi: 10.1128/jcm.37.5.1591-1594.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Nakatomi Y, Sugiyama J. A rapid latex agglutination assay for the detection of penicillin-binding protein 2′. Microbiol Immunol. 1998;42:739–743. doi: 10.1111/j.1348-0421.1998.tb02347.x. [DOI] [PubMed] [Google Scholar]
- 4.Wallrauch C, Braveny I. Ninth Annual Scientific Meeting of the Society for Healthcare Epidemiology of America. 1999. Evaluation of a latex agglutination test to screen for methicillin resistant Staphylococcus aureus, abstr. M30. [Google Scholar]