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Journal of Clinical Microbiology, November 1999, p. 3783-3784, Vol. 37, No. 11
0095-1137/99/$04.00+0
LETTERS TO THE EDITOR
Further Evaluation of the MRSA-Screen Kit for Rapid
Detection of Methicillin Resistance
 |
LETTER |
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.
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.,
N. Woodford,
A. Chapman,
A. P. Johnson, and D. C. E. Speller.
1996.
Detection of the mecA gene and phenotypic detection of resistance in Staphylococcus aureus isolates with borderline or low level methicillin resistance.
J. Antimicrobiol. Chemother.
37:53-63[Abstract/Free Full Text].
|
| 2.
|
Cavassini, M.,
A. Wenger,
K. Jaton,
D. S. Blanc, and J. Bille.
1999.
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.
37:1591-1594[Abstract/Free Full Text].
|
| 3.
|
Nakatomi, Y., and J. Sugiyama.
1998.
A rapid latex agglutination assay for the detection of penicillin-binding protein 2'.
Microbiol. Immunol.
42:739-743[Medline].
|
| 4.
|
Wallrauch, C., and I. Braveny.
1999.
Evaluation of a latex agglutination test to screen for methicillin resistant Staphylococcus aureus, abstr. M30.
In
Ninth Annual Scientific Meeting of the Society for Healthcare Epidemiology of America.
|
| | | | |
Deborah J. E. Marriott
Thomas Karagiannis
John L. Harkness
Department of Microbiology
St. Vincent's Hospital
Darlinghurst, NSW 2010 Australia
|
| | | | |
Phillip Kearney
Department of Molecular Biology
St. Vincent's Hospital
Darlinghurst, NSW 2010 Australia
|
| |
AUTHORS' REPLY |
The letter of Marriott et al. is interesting and presents new data on
the performance of the recently released latex agglutination test
designed to rapidly detect the PBP 2a linked to methicillin resistance
in S. aureus. These authors obtained sensitivity and specificity values (100 and 94%, respectively, according to their definition of methicillin resistance) similar to ours, using a collection of 156 S. aureus clinical isolates from
Australia. While confirming the usefulness of this test, they made two
interesting observations. The first one relies on the necessity of
strictly respecting the recommended procedure (inoculum, boiling
temperature and duration, reading time of the agglutination) and of
adding positive and negative control strains not provided by the
manufacturer. When complying with these technical points, one can also
obtain the same excellent sensitivity and specificity at the bench
during normal daily activity. After the completion of our study
(1) we have introduced this test in our routine work, where
we encounter methicillin resistance in about 2% of our S. aureus clinical isolates. Rather than doing an MRSA-Screen test
for all S. aureus isolates, we restrict its use to the
strains presenting resistance to several antibiotics and/or reduced
susceptibility to ceftriaxone (diameter of zone of inhibition 
20 mm)
by the National Committee for Clinical Laboratory Standards recommended
disk diffusion test.
Out of a total of 154 fresh clinical isolates tested, the concordance
between MRSA-Screen test and PCR results for the mecA gene
reached 99.4% (31 isolates positive by both methods, 122 isolates
negative by both methods, and 1 isolate MRSA-Screen negative and
PCR-positive for the mecA gene).
The second interesting comment of Marriott et al. concerns the
performance of the test with CNS. It is important to mention first that
this test has not been designed or validated for species of
staphylococci other than S. aureus. However, because of the analogy of the mechanisms of resistance to methicillin in S. aureus and in CNS, we tested it, as did the Australian authors,
against a series of 48 clinically significant CNS isolates belonging to various species. Our results differed somewhat from those obtained by
Marriott et al. Out of 26 mecA gene-positive CNS isolates, between 13 and 23 were positive by the MRSA-Screen test when tested on
three separate occasions (giving a sensitivity varying from 50 to
88%). In contrast to MRSA isolates, the agglutination reaction was
often weak and poorly reproducible with CNS but was generally more
pronounced when the time of mixing before reading was prolonged to 10 to 15 min. The specificity was also lower (59 to 86%) for MRCNS than
for MRSA, mainly due to many false, weakly positive agglutination
reactions. Thus, albeit the reason is not straightforward, this test
should not be used to determine the resistance to methicillin in
organisms other than S. aureus.
Two articles on this test have recently been published (2,
3).
| |
REFERENCE |
| 1.
|
Cavassini, M.,
A. Wenger,
K. Jaton,
D. S. Blanc, and J. Bille.
1999.
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.
37:1591-1594.
|
| 2.
|
van Leeuwen, W. B.,
C. van Pelt,
A. Luijendijk,
H. A. Verbrugh, and W. H. F. Goessens.
1999.
Rapid detection of methicillin resistance in Staphylococcus aureus isolates by the MRSA-Screen latex agglutination test.
J. Clin. Microbiol.
37:3029-3030[Abstract/Free Full Text].
|
| 3.
|
van Griethuysen, A.,
M. Pouw,
N. van Leeuwen,
M. Heck,
P. Willemse,
A. Buiting, and J. Kluytmans.
1999.
Rapid slide latex agglutination test for detection of methicillin resistance in Staphylococcus aureus.
J. Clin. MIcrobiol.
37:2789-2792[Abstract/Free Full Text].
|
| | | | |
M. Cavassini
A. Wenger
K. Jaton
J. Bille
Institut de Microbiologie
Centre Hospitalier Universitaire Vaudois
1011 Lausanne, Switzerland
|
| | | | |
D. S. Blanc
Division Autonome de Médecine Préventive Hospitalière
Centre Hospitalier Universitaire Vaudois
1011 Lausanne, Switzerland
|
Journal of Clinical Microbiology, November 1999, p. 3783-3784, Vol. 37, No. 11
0095-1137/99/$04.00+0
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