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Public Health Laboratory Tilburg, St Elisabeth Hospital, Tilburg, The Netherlands
Correspondence
I. H. M. van Loo
ihmvanloo{at}gmail.com
Received 20 October 2006
Accepted 1 December 2006
Abbreviations: MRSA, meticillin-resistant Staphylococcus aureus.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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The routine screening procedure, used in this laboratory, takes at least 72 h to obtain a definitive result. Rapid detection of MRSA is essential to optimize therapy, to minimize patients' discomfort and to reduce costs. Chromogenic media are being developed to detect MRSA strains in one single step. Growth of non-staphylococcal strains is repressed by high salt concentration and enzymic substrate is used to achieve a specific colour reaction to detect S. aureus. Growth of meticillin-susceptible S. aureus strains is repressed by the addition of various combinations of antibiotics (Merlino et al., 2000). Various studies have been performed to evaluate different chromogenic media with clinical samples, two of which included the MRSASelect medium (Bio-Rad) (Becker et al., 2002; Ben Nsira et al., 2006; Blanc et al., 2003; Merlino et al., 2000; Perry et al., 2004; Simor et al., 2001; Stoakes et al., 2006). In this study, we evaluated the performance of a chromogenic MRSA agar, MRSASelect in a low prevalence population. We evaluated the MRSASelect medium by comparing it with our routine screening method on clinical samples. In the period January to March 2005 samples from known MRSA patients or patients at risk for MRSA carriership or contacts of known MRSA carriers were included in this study.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Screening samples. Every sample was cultured on an MRSASelect plate (Bio-Rad) (composition of the medium is proprietary), a Columbia agar plate (Oxoid) supplemented with 5 % sheep blood and 2 µg ciprofloxacin ml–1 (BA-cipro) (Bayer), an MRSA screening agar, containing mannitol salt agar (Oxoid) supplemented with 4 µg oxacillin ml–1 (MSA-oxa) (Sigma), and an enrichment broth containing Mueller–Hinton broth with 6 µg oxacillin ml–1 and 6 µg aztreonam ml–1 (MH-oxa-aztr) (ICN). For each sample the inoculated plates were analysed after 20 h and after 48 h. The enrichment broth was subcultured after 20 h on a Columbia agar plate supplemented with 5 % sheep blood, and analysed after 20 h and after 48 h. The nose, throat and perineum samples from known MRSA patients were put into an enrichment broth separately, while unknown patients' nose, throat and perineum samples were pooled in one enrichment broth. If the pooled enrichment broth was positive the three samples were counted once. For the calculation of the sensitivity and specificity the samples from the solid media and the non-pooled enrichment broth were used.
Testing of colonies on MRSASelect medium. Presumptive MRSA colonies on the MRSASelect agar were pink/mauve and were tested with a latex agglutination test (Staphaurex plus, Murex Diagnostics) and subcultured on a Columbia agar plate supplemented with 5 % sheep blood. If colony morphology was suspect for S. aureus and the latex agglutination test was positive further identification was performed (see below).
A total of 205 white colonies were tested with a latex agglutination test and subcultured on a Columbia agar plate supplemented with 5 % sheep blood. Latex agglutination and catalase tests (Isenberg, 2004) were performed to exclude growth of S. aureus.
Confirmation of MRSA. Presumptive MRSA colonies (any colony on the Columbia agar plate and the MRSA screening agar, and pink/mauve colonies on the MRSASelect plate) were identified by latex agglutination test, coagulase tube test (Isenberg, 2004), DNase (Isenberg, 2004), growth on a Mueller–Hinton plate supplemented with 6 µg oxacillin ml–1, growth on a Mueller–Hinton plate supplemented with 2 % NaCl, AccuProbe (Gen-Probe) and identification with the Vitek2 system (bioMérieux).
Susceptibility testing was performed by Vitek2 system. Isolates resistantfor oxacillin, quinolones or aminoglycosides were further evaluated, including disc diffusion of oxacillin (1 µg) and meticillin (5 µg) (Sigma) on a Mueller–Hinton plate supplemented with 2 % NaCl, disc diffusion of ciprofloxacin (5 µg), ofloxacin (5 µg), mupirocin (5 µg), gentamicin (10 µg) and tobramycin (10 µg) (Sigma) on a Mueller–Hinton plate and an E-test for oxacillin (AB Biodisk) on a Mueller–Hinton plate supplemented with 2 % NaCl. Final confirmation was performed by mecA gene PCR (Del Vecchio et al., 1995).
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Other studies evaluating MRSASelect medium found a lower number of false-positive samples, 3 (3 %) and 1 (1 %), respectively (Stoakes et al., 2006; Ben Nsira et al., 2006). We found that prolonging incubation from 20 to 48 h increased the yield of confirmed MRSA strains by 38 % (20 strains) (Table 1). Ben Nsira et al. (2006) found an increase of 3 %. Stoakes et al. (2006) did not evaluate MRSASelect medium after 48 h. However, 97 % of the MRSA strains were recovered after 18 h incubation, compared to 79 % after 48 h in our study. For other chromogenic media the yield also increased after prolonged incubation. For example, Perry et al. (2004) found an increased yield of 11 %, 19 % and 20 % for MRSA-ID, CHROMagar MRSA and ORSAB, respectively. Simor et al. (2001) also found an increase in recovery (23 %) of MRSA on ORSAB plates after prolonged incubation.
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). Specificity decreased from 92 % after 20 h incubation to 89 % after 48 h. False positives were enterococci or coagulase-negative staphylococci (Table 1). Ben Nsira et al. (2006) found four (4 %) false positives after 48 h. Compared to other chromogenic media (e.g. ORSAB medium) MRSASelect generated less false-positive results (Becker et al., 2002; Blanc et al., 2003; Simor et al., 2001). Other chromogenic media also show a decrease in specificity after 48 h incubation (Perry et al., 2004). The colony morphology of false positives in our study showed less pigmentation compared to confirmed MRSA strains, an observation that was also noted by Ben Nsira et al. (2006). Similar observations were found in studies evaluating other chromogenic media (Becker et al., 2002; Blanc et al., 2003; Perry et al., 2004; Simor et al., 2001).
In total, 107 white colonies were evaluated, 106 of which were determined as coagulase-negative staphylococci and one of which as meticillin-susceptible S. aureus (Table 1). The number of white colonies doubled after prolonging incubation from 20 to 48 h (Table 1).
We compared MRSASelect medium with the routine screening method (MSA-oxa/BA-cipro+MH-oxa-aztr) (Table 2). Seventy confirmed MRSA isolates were found in eighteen patients (Table 2). Fifty-five MRSA isolates were detected by the screening method and fifty-five by the MRSASelect medium after 48 h incubation. Fifteen MRSA isolates were not detected by the MRSASelect medium and fifteen MRSA isolates were not detected by the routine screening method. Sensitivity of the MRSASelect medium was 78.6 % and specificity was 99.5 %, compared to sensitivity of 78.6 % and specificity of 100 % for the routine screening method. The number of discrepancies between the MRSASelect medium and the routine screening method is unexpectedly high. We cannot explain the lower sensitivity in our study in comparison to the studies of Stoakes et al. (2006) and Nsara et al. (2006). Stoakes et al. (2006) found that 3 from the 111 confirmed MRSA strains were not detected by MRSASelect (sensitivity of 97.3 %) and Ben Nsira et al. (2006) reported 1 missing MRSA isolate (sensitivity 99.8 %). Differences in sampling, in MRSA prevalence and in circulating clones in the different countries may account for differences in sensitivity. The sensitivity we found was similar to that of other chromogenic media evaluated with clinical samples (Perry et al., 2004). To increase the yield we added an enrichment broth (MH-oxa-aztr) in a pilot study. From 225 samples we subcultured the enrichment broth on MRSASelect medium. We found 12 % more MRSA isolates. We have now used the MRSASelect medium in combination with the enrichment broth (MH-oxa-aztr) in our laboratory for more then a year. Thirty percent of all the MRSA isolates were yielded from the enrichment broth, indicating an increase in yield that is higher than in the pilot study.
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In conclusion, in our hands MRSASelect is a useful medium for the detection of MRSA, although in this study it was less sensitive than in other studies. The MRSASelect medium generated a lower number of false positives compared to other chromogenic media. The increase in true-positive MRSA isolates after a prolonged incubation in our study is higher than for other chromogenic media. Optimization of screening was achieved by the addition of an enrichment broth next to primary culture on the MRSASelect medium. In a country with low MRSA prevalence (e.g. The Netherlands) the main advantage of MRSASelect medium compared to the routine screening procedure is that the time to obtain a negative culture result is reduced from 72 to 48 h.
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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