Evaluation of BBL™ CHROMagar™ MRSA for Detection

As presented at the 15th European Congress of Clinical Microbiology
and Infectious Disease (ECCMID), Copenhagen, 2005.
Evaluation of BBL™ CHROMagar™ MRSA for Detection of
Methicillin-Resistant Staphylococcus aureus from Surveillance Swabs
B M WILLEY, T MAZZULLI, A MCGEER, J STRAUSS, A TYLER, K WONG, O IMAS, P AKHAVAN, N KREISWIRTH,
B SKULNICK, S POUTANEN, G SMALL, I EDWARDS, N NELSON, M SKULNICK
Toronto Medical Laboratories and Mount Sinai Hospital Department of Microbiology, Toronto, Ontario, Canada
REVISED ABSTRACT
OBJECTIVE: Highly sensitive and cost effective screening
media that aid in a rapid turnaround-time (TAT) is of prime
importance in the control of MRSA. Good specificity is also
paramount as false positives significantly increase labour and
material costs. This study compares selective CHROMagar MRSA
with 6 µg/mL cefoxitin (Cfox) to commonly used Mannitol Salt
Agar with 4 µg/mL Oxacillin (MSox).
METHODS: A total of 2,561 MRSA surveillance specimens
from 1,313 patients (775 nasal swabs, 733 rectal, 266 wound,
682 nasal/axilla/groin/perineum, 105 other) were planted to
Cfox and MSox. The order of inoculation was alternated every
200 swabs. Cfox were incubated in the dark at 35°C, and both
media were read blinded at 24 and 48 h. Mauve colonies from
the Cfox and yellow from the MSox were tested for capsular
antigens 5 and 8 (Pastorex Staph Plus), tube coagulase and PBP2a
(Denka Seiken) to identify MRSA directly from the medium
when possible. Positive PBP2a reactions were confirmed using
the NCCLS 6 µg/mL Oxacillin Screen Agar.
RESULTS: From the 2,561 swabs, 152 (5.9%) MRSA grew
from at least one medium. 133 (87.5%) grew on Cfox and 104
(68.4%) grew on MSox, while 85 (55.9%) grew on both media.
Of the total 152 MRSA, 19 (12.5%) failed to grow on Cfox and
48 (31.6%) failed on MSox. The sensitivities for Cfox and MSox
were 88.9% and 76%, respectively. Cfox grew 17% of its
133 MRSA at 24 h and 83% at 48 h, while MSox grew 25% of
its 104 MRSA at 24 h and 75% at 48 h. From the 2,407 MRSAnegative specimens, non-S. aureus mauve or yellow colonies
(coagulase negatives, enterococci or gram-neg bacilli) required
work-up on 46 from Cfox and 415 from MSox, resulting in
specificities of 98.1% for Cfox and 85.2% for MSox, respectively.
CONCLUSION: Compared to MSox, the Cfox plates were both
more sensitive and specific (P<.0001). This resulted in fewer
unnecessary work-ups on Cfox for a saving of ~$2 per false
positive. The impact of the breakthrough rate of 16% associated
with MSox in this study represents an extremely significant
potential for cost avoidance to laboratories processing large
numbers of surveillance screens. However, the impact on overall
cost to the laboratory depends on the cost differential between
MSox and Cfox.
INTRODUCTION
Highly sensitive media capable of screening for methicillinresistant Staphylococcus aureus (MRSA) in a wide range of
surveillance specimens is of prime importance in preventing
its spread in the hospital setting. It is imperative that such media
provide a rapid turnaround-time to notification of new
MRSA cases.
In these times of decreased laboratory resources, it is also
imperative that screening media are highly specific and give as few
as possible false positive results. Growth of other organisms
resembling MRSA increases both labour and material costs, as
well as reducing specimen turnaround times. Paradoxically,
as the incidence of MRSA climbs, the associated escalation in
screening costs results in laboratory pressure on Infection Control
Programs to reduce their surveillance.
Traditionally, MRSA screening media have been based on the
less than optimal method of relying on mannitol fermentation and
resistance to oxacillin to separate S. aureus from coagulasenegative stapylococci (CoNS). Unfortunately, in the hospital
setting, CoNS that are both mannitol fermenting and methicillinresistant are highly endemic, and therefore a new approach to
screening is needed.
OBJECTIVES
The purpose of this prospective, blinded study was to compare
the commonly used Mannitol Salt agar with 4 mg/L of oxacillin
(Oxoid Inc., Nepean, Canada) to a new chromogenic medium for
the identification of S. aureus and MRSA, namely the BD BBL
CHROMagar MRSA with 6 mg/L of cefoxitin (Becton, Dickinson
and Co., Spark, MD, USA). Currently approved for use with nasal
swabs, this study aimed at evaluating the CHROMagar MRSA
for use with all specimen types received for MRSA surveillance.
MATERIALS AND METHODS
In a low prevalence population of approximately
5%, 2,561 consecutive specimens received for
MRSA surveillance screening were enrolled into the
study. These specimens, mainly swabs, were from
1,313 patients from 11 different healthcare facilities
and included the following specimen types:
775 nasal, 733 rectal, 266 wound, 682 combination
nasal/axilla/groin/perineum, 75 axilla or axilla/groin
or groin, 17 devices, 13 sputa and 1 urine. No
duplicate sample from any patient from the same site
and date was accepted.
On receipt in the study laboratory, specimens
were planted onto Mannitol Salt Oxacillin and
CHROMagar MRSA. To ensure that there was no
bias due to the order of inoculating the plates,
the Mannitol Salt Oxacillin and CHROMagar
MRSA were inoculated in rotation in batches of
200 specimens.
As the CHROMagar is sensitive to light, this
medium was exposed to light only when labeling
and during inoculation and reading. At all other
times, including during incubation, the plates were
held in loosely covered cardboard boxes to protect
them from unnecessary light exposure.
Both Mannitol Salt Oxacillin and CHROMagar
MRSA were incubated at 35°C in ambient air before
being examined at 18–24 h and 36–48 h. Each
media type was read independently by a different
technologist and the results kept blinded.
Identification of yellow colonies from the
Mannitol Salt Oxacillin and mauve colonies from the
CHROMagar MRSA were identified using Pastorex
Staph-Plus (Bio-Rad, Redmond, Washington, USA)
tube coagulase and PBP2a detection for MRSA
(Denka Seiken, Tokyo, Japan). Isolates positive for
PBP2a were confirmed using the NCCLS Oxacillin
6 mg/L screen plate.
Figure 1a.
The BD BBL CHROMagar MRSA containing
6 mg/L of cefoxitin showing a heavy pure
growth of mauve colonies that were identified
as methicillin-resistant Staphylococcus aureus
(MRSA).
1a
Figure 1b.
The CHROMagar MRSA showing the growth of
MRSA (mauve) mixed with coagulase-negative
staphylococci (blue pigmentation). Non-MRSA
may appear dark blue, light blue or white on this
medium, depending on the species, due to the
incorporation of various chromogenic substrates.
1b
Figure 1c.
The Mannitol Salt Agar with 4 mg/L of
oxacillin showing a pure growth of mannitol
fermenting, methicillin-resistant staphylococci
(yellow). Non-mannitol fermenting staphylococci
typically grow as “hot” pink colonies. Yellow
staphylococci, as seen here, require further
testing to determine whether they are
MRSA or a coagulase-negative staphylococcal
species. This lack of differentiation results in
a high “false-positive” rate and increases the
laboratory workload.
1c
RESULTS
A total of 152 MRSA were isolated from the 2,561 specimens
in the study giving a positivity rate of 5.9%. The 152 isolates
came from the following sources: 40 wound, 36 rectal, 36 nasal,
26 combined nasal/axilla/groin/perineum, 9 axilla/groin, 3 devices
and 2 sputa. Of these, 120 were derived from multiple sites and
dates on patients already known to have MRSA. These ranged
from two to eight positive specimens per patient. Thirty-two
patients had a single positive specimen.
From Table 1 it may be seen that CHROMagar MRSA grew
the largest number of MRSA in the study, with 133 (87.5%) of
the 152 total MRSA being identified.
Mannitol Salt Oxacillin was less efficient in isolating MRSA
with only 104 of 152 isolates detected.
Of note, only 85 (55.9%) MRSA grew on both media, thus
demonstrating that while CHROMagar was far more sensitive, it
too missed 19 (12.5%) of the possible 152 MRSA in the study.
As may be seen in Table 2, the Mannitol Salt Oxacillin gave
the fastest reportable yield with 26 (25%) of the 104 specimens
growing yellow colonies at 18–24 h. The CHROMagar MRSA
performed less well with only 21 (15.8%) of its 133 isolates
producing mauve colonies by 24 h.
Table 1. Breakdown of yield of MRSA from the 2,651 specimens in the study
Growth on Culture Medium
No. of MRSA (% of total 152 MRSA)
Total Mannitol Salt Oxacillin
104 (68.4)
Total CHROMagar MRSA
133 (87.5)
Both CHROMagar MRSA and Mannitol Salt Oxacillin
85 (55.9)
Only Mannitol Salt Oxacillin
19 (12.5)
Only CHROMagar MRSA
48 (31.6)
Table 3 illustrates the major pitfall of most screening
media for MRSA. Besides the workup of true MRSA, it
was also necessary to work up and rule out an
additional 415 and 46 potential isolates from Mannitol
Salt Oxacillin and CHROMagar MRSA agars,
respectively, that were not MRSA.
On Mannitol Salt Oxacillin, the 415 “breakthrough”
yellow colonies comprised 396 methicillin-resistant
coagulase-negative staphylococci, 17 gram-negative
bacilli, one mixed gram-negative bacilli and enterococcus
and one methicillin-susceptible S. aureus.
On CHROMagar MRSA, 41 of 46 “breakthrough”
mauve colonies were methicillin-resistant coagulasenegative staphylococci and 5 were enterococci and/or
gram-negative bacilli. Although only four specimens
(two rectal, one wound and one nasal/axilla/groin swab)
grew such colonies at 24 h, it shows that it should not be
taken for granted that mauve colonies are MRSA. All
MRSA from new cases should be confirmed.
The sensitivity for Mannitol Salt Oxacillin and
CHROMagar MRSA was 76.0% and 88.9%, and
specificity was 85.3% and 98.1%, respectively. The
Mannitol Salt Oxacillin had a poor positive-predictive
value due to the false positives associated with this type
of medium (see Table 4).
Costing
The false-positive rate for CHROMagar MRSA was
1.8%, significantly less than Mannitol Salt Oxacillin at
16.2% (P<0.0001). This difference is of prime importance
when costing the workup of a yellow versus mauve
colony, where ~CDN$2.00 is factored in for each. In a lab
testing 60,000 specimens annually for MRSA, and where
the positivity rate is ~5%, this would result in
CDN$19,440 being spent on confirmatory tests to rule
out false positives for Mannitol Salt Oxacillin compared
to CDN$2,160 for CHROMagar MRSA. The labour
savings for CHROMagar MRSA would be ~CDN$2,500.
Table 2. Growth of MRSA on each medium at 18–24 h and 36–48 h
Culture Medium
Growth at 18-24 h
Growth at 36-48 h
Mannitol Salt Oxacillin
26/104 (25%)
78/104 (75%)
21/133 (15.8%)
112/133 (84.2%)
CHROMagar MRSA
Table 3. Number of specimens growing false positive colonies per medium
False Positive Colonies*
Culture Medium
At 18-24 h
At 36-48 h
Total number
45
370
415
4 (CNST)
42
46
Mannitol Salt Oxacillin
CHROMagar MRSA
*Yellow colonies on Mannitol Salt Oxacillin or mauve colonies on
CHROMagar MRSA that were not S. aureus
Table 4. Statistical data for culture media performance
Mannitol Salt Oxacillin
CHROMagar MRSA
Sensitivity
Category
76.0%
88.9%
Specificity
85.3%
98.1%
Positive Predictive Value
26.8%
76.8%
Negative Predictive Value
98.0%
99.2%
False Positive Rate
16.2%
1.8%
CONCLUSIONS
■ In this study CHROMagar outperformed Mannitol Salt Oxacillin
agar in all areas except growth at 18-24 h. Sensitivity, specificity
and positive predictive value of CHROMagar MRSA were
all significantly higher than for Mannitol Salt Oxacillin agar
(P<0.0001), proving it to be a suitable medium to replace the
commonly used Mannitol Salt Oxacillin.
LR889