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Journal of Clinical Microbiology, November 2001, p. 4149-4151, Vol. 39, No. 11
Department of Microbiology, Sunnybrook and
Women's College Health Sciences Centre, and the University of
Toronto, Toronto, Ontario, Canada
Received 16 January 2001/Returned for modification 26 June
2001/Accepted 12 August 2001
The MRSA-Screen (Denka-Seiken, Tokyo, Japan) latex
agglutination test was evaluated for its ability to detect PBP 2a from 200 clinical isolates of coagulase-negative staphylococci (CoNS; 84 mecA-positive strains and 116 mecA-negative strains) consisting of 108 Staphylococcus epidermidis, 37 S.
saprophyticus, 15 S. haemolyticus, 11 S.
hominis, 10 S. capitis, 10 S.
warneri, and 3 S. lugdunensis species as well as
6 other species of CoNS. The assay was compared with susceptibility
testing with an agar screen plate with oxacillin at 6 µg/ml (OXA6),
by oxacillin disk diffusion (DD), by broth microdilution (BMDIL), by
the E test, and with Vitek GPS-SV and Vitek GPS-107 susceptibility
cards. PCR for the detection of the mecA gene
was used as the "gold standard." The sensitivities and
specificities for the methods evaluated were as follows: MRSA-Screen,
100 and 100%, respectively; OXA6, 100 and 99%, respectively; DD, 98 and 62%, respectively; BMDIL, 100 and 60%, respectively; E test, 100 and 51%, respectively; Vitek GPS-SV susceptibility card, 98 and 87%,
respectively; and Vitek GPS-107 susceptibility card, 100 and 61%,
respectively. The MRSA-Screen test accurately and rapidly detected
oxacillin resistance in CoNS.
Coagulase-negative staphylococci (CoNS) are
recognized as important causes of nosocomial infection, especially in
neonates, those who are immunocompromised, and patients with indwelling prosthetic devices (6). The rate of resistance to
oxacillin among CoNS has been increasing (2), and the
empirical treatment of choice for infections caused by these organisms
is often vancomycin. The rapid and accurate identification of oxacillin
resistance is essential in order to determine the most appropriate
antimicrobial therapy. The major mechanism of oxacillin resistance in
staphylococci is mediated by the production of a modified
penicillin-binding protein, PBP 2a, specified by the mecA
gene. In 1999, the National Committee for Clinical Laboratory Standards
(NCCLS) lowered the breakpoint for determination of oxacillin
resistance in CoNS from A rapid latex agglutination test, the MRSA-Screen
(Denka-Seiken, Tokyo, Japan), has recently been developed in order to
detect the presence of oxacillin resistance mediated by PBP 2a.
Although several studies have evaluated the use of this test for the
detection of oxacillin resistance in Staphylococcus aureus
(8, 18, 19), its accuracy for the detection of oxacillin
resistance in CoNS has not been extensively assessed (5,
9). In the study described here, we evaluated the MRSA-Screen
for its ability to detect PBP 2a in clinical strains of CoNS. In
addition, an evaluation of other test methods, including the oxacillin
agar screen test, disk diffusion, broth microdilution, E test (AB
Biodisk, Solna, Sweden), and tests with Vitek susceptibility cards
(bioMérieux Inc., Hazelwood, Mo.), was also performed; and the
results obtained by those tests were compared to those obtained by PCR
detection of the mecA gene.
A total of 200 clinical isolates of CoNS recovered from blood,
cerebrospinal fluid, urine, and other normally sterile sites were
selected for testing. These included S. epidermidis (108 strains), S. saprophyticus (37 strains), S. haemolyticus (15 strains), S. hominis (11 strains),
S. capitis (10 strains), S. warneri (10 strains),
S. lugdunensis (3 strains), S. simulans (2 strains), and one strain each of S. cohnii, S. sciuri, S. schleiferi, and S. xylosus. All
isolates were identified with the API Staph ID 32 system
(bioMérieux sa, Marcy-l'Étoile, France). The MRSA-Screen test was performed according to the manufacturer's instructions but
with one modification: a large, "heaping" 1-µl loopful of test
organism (approximately 30 to 50 colonies, depending on the colony
size) was used instead of the standard 1-µl loopful. Antimicrobial susceptibility testing of isolates was performed with an oxacillin agar
screen plate (11) prepared in-house (BBL Mueller-Hinton II
agar; Becton Dickinson, Cockeysville, Md.) and by disk diffusion and
broth microdilution in accordance with the recommendations of
NCCLS (13). Oxacillin MIC determination by the E
test and breakpoint MIC testing with Vitek GPS-SV and Vitek GPS-107
susceptibility cards were performed according to the manufacturers'
instructions. Multiplex PCR assays were performed for the simultaneous
detection of the mecA (10) and nucA
(1) gene sequences, as described previously
(8).
Of the 200 isolates tested, 84 were mecA positive and 116 were mecA negative by PCR (Table
1). All strains positive for the mecA gene were detected by the MRSA-Screen latex
agglutination test, and all strains negative for mecA
produced no agglutination. The sensitivities and specificities of the
assays evaluated are summarized in Table
2A. The MRSA-Screen latex agglutination
assay rapidly and accurately detected oxacillin resistance that was mediated by the mecA gene in CoNS, regardless of the
species. Induction of oxacillin resistance was not required in this
study, as was previously reported by Hussain et al. (5).
This may have been because we used a larger inoculum (a "heaping"
loopful). This modification was made on the basis of previous
experience with the MRSA-Screen test and testing of S. aureus isolates (8). This did not result in any loss
of specificity.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.11.4149-4151.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Evaluation of a Latex Agglutination Test
(MRSA-Screen) for Detection of Oxacillin Resistance in
Coagulase-Negative Staphylococci
ABSTRACT
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4 to 0.5 µg/ml (12) in order
to more accurately reflect mecA-mediated resistance. In
addition, the use of the oxacillin agar screen plate (Mueller-Hinton
agar with 6 µg of oxacillin per ml supplemented with 4% NaCl)
was no longer recommended by NCCLS for the detection of
oxacillin resistance in CoNS, as reports in the literature had
indicated that this method may not be able to detect
mecA-mediated oxacillin resistance in these strains
(15, 17).
TABLE 1.
Results of oxacillin broth microdilution and
mecA PCR for 200 isolates of coagulase-negative
staphylococci
TABLE 2.
Sensitivities, specificities, and positive and negative
predictive values for tests evaluated in comparison with PCR for
detection of the mecA gene for oxacillin resistance and for
tests evaluated in comparison with PCR without inclusion of 37 S. saprophyticus and 3 S. lugdunensis
isolatesa
Current NCCLS susceptibility test breakpoints (14) provide
improved sensitivity for detection of oxacillin resistance in CoNS,
especially S. epidermidis. For all of the isolates of CoNS that were tested in the present evaluation and that were found to have
the mecA gene by PCR, oxacillin MICs were 1
µg/ml. For a total of 18 isolates, including all 3 strains of
S. lugdunensis, several strains of S. saprophyticus, S. warneri, and one strain each of
S. capitis, S. epidermidis, and S. xylosus, oxacillin MICs were 0.5 µg/ml, but the strains did not
have the mecA gene. In addition, for 27 S. saprophyticus isolates, oxacillin MICs were 1 µg/ml, but the
isolates were negative for the mecA gene. Discrepancies
between the results of the PCR assay for the detection of the
mecA gene and the results of the oxacillin broth
microdilution susceptibility test occurred primarily for species of
CoNS other than S. epidermidis (most notably, S. saprophyticus and S. lugdunensis). Mechanisms of
oxacillin resistance other than that mediated by the mecA
gene may be responsible for this observation. Alternatively, these
strains may be susceptible to oxacillin and the present NCCLS
breakpoint guidelines may need to be reevaluated for certain staphylococcal species, such as S. saprophyticus and
S. lugdunensis (4, 17). For this reason, the
sensitivities and specificities of the assays in this evaluation were
recalculated without the inclusion of these two staphylococcal
species (Table 2). In fact, the present NCCLS
guidelines no longer recommend routine susceptibility testing of
S. saprophyticus, as urinary tract infections with this
organism respond well to the concentrations of antimicrobial agents
achieved in urine (14). In the present study, there were two isolates, one S. epidermidis isolate and one S. capitis isolate, for which oxacillin MICs were 4 µg/ml but
that were both mecA negative. These strains may possess an
alternate resistance mechanism, such as other altered
penicillin-binding proteins, as described by Suzuki et al.
(16), but this was not investigated further.
The oxacillin agar screen plate prepared in-house performed well for the detection of methicillin resistance in CoNS when the results were examined after 48 h of incubation. These results are similar to those reported in some previous investigations (7, 20) but not all previous investigations (15). Optimal performance of the oxacillin agar screen plate may depend in part on the source of the Mueller-Hinton agar base (3, 17), as well as on how the medium is prepared, on the inoculum size used, and on the length of incubation (15, 17).
Both the Vitek GPS-SV and Vitek GPS-107 cards were able to accurately detect methicillin resistance in CoNS when S. saprophyticus and S. lugdunensis strains were excluded from the evaluation. The E test had a relatively lower level of specificity (78%), even when these two species were excluded. This may have occurred because the oxacillin MIC for several of the CoNS strains by the E test was 0.38 µg/ml. As this MIC falls between the NCCLS breakpoints for susceptibility and resistance (0.25 and 0.5 µg/ml, respectively), the E test MIC was interpreted as 0.5 µg/ml to allow comparison with NCCLS breakpoint values. As a result, these strains were interpreted by the E test to be resistant, although they lacked the mecA gene and may, in fact, have been susceptible.
In summary, compared to PCR as the "gold standard," the MRSA-Screen
latex agglutination test was able to rapidly and accurately determine
the presence of oxacillin resistance mediated by the mecA
gene in S. epidermidis and most other species of CoNS. Our findings suggest that induction of oxacillin resistance may be unnecessary if a large initial inoculum is used. All of the phenotypic methods evaluated in the present study appeared to perform very well
for the detection of oxacillin resistance. The methods evaluated were
very sensitive, although the E test had a lower level of specificity
than the other phenotypic methods when the results were compared with
those of PCR for the detection of mecA-mediated resistance.
Future studies of oxacillin susceptibility testing for certain species
of CoNS (such as S. lugdunensis) may indicate the need for
reevaluation of the NCCLS breakpoints. Until such breakpoints are
reassessed, the MRSA-Screen latex agglutination test may be considered
an accurate method for confirmation of an oxacillin MIC of 0.5
µg/ml, regardless of the phenotypic method used for susceptibility
testing, for the detection of resistance which is mediated by
the mecA gene.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, Sunnybrook and Women's College Health Sciences Centre, B121-2075 Bayview Ave., Toronto, Ontario, Canada M4N 3M5. Phone: (416) 480-4242. Fax: (416) 480-6845. E-mail: lisa.louie{at}swchsc.on.ca.
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Journal of Clinical Microbiology, November 2001, p. 4149-4151, Vol. 39, No. 11
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.11.4149-4151.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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