Comparison of MRSASelect Agar, CHROMagar Methicillin-Resistant Staphylococcus aureus (MRSA) Medium, and Xpert MRSA PCR for detection of MRSA in Nares: diagnostic accuracy for surveillance samples with various bacterial densities.
ABSTRACT Rapid laboratory methods provide optimal support for active surveillance efforts to screen for methicillin-resistant Staphylococcus aureus (MRSA). Most laboratories struggle to determine the optimal use of resources, considering options to balance cost, speed, and diagnostic accuracy. To assess the performance of common methods, the first comparison of MRSASelect agar (MS) and CHROMagar MRSA (CA), with and without broth enrichment followed by a 24-h subculture to MS, was performed. Results were compared to those of the Xpert MRSA assay. For direct culture methods, the agreement between MS and CA was 98.8%. At 18 h, direct MS identified 93% of all positive samples from direct culture and 84% of those identified by the Xpert MRSA. For Trypticase soy broth-enriched MS culture, incubated overnight and then subcultured for an additional 24 h, the agreement with Xpert MRSA was 96%. The agreement between direct MS and Xpert MRSA was 100% when semiquantitative culture revealed a bacterial density of 2+ or greater; however, discrepancies between culture and Xpert MRSA arose for MRSA bacterial densities of 1+ or less, indicating low density as a common cause of false-negative culture results. Since 1+ or less was established as the most common MRSA carrier state, broth enrichment or PCR may be critical for the identification of all MRSA carriers who may be reservoirs for transmission. In this active-surveillance convenience sample, the use of broth enrichment followed by subculture to MS offered a low-cost but sensitive method for MRSA screening, with performance similar to that of Xpert MRSA PCR.
- SourceAvailable from: Olivier Denis[Show abstract] [Hide abstract]
ABSTRACT: Cluster randomised crossover trial with seven wards randomly allocated to intervention or control arm.PLoS ONE 01/2014; 9(5):e96310. · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: A meticillin-resistant Staphylococcus aureus (MRSA) polymerase chain reaction assay (Xpert(®) MRSA, Cepheid) was assessed for point-of-care testing (POCT) used by healthcare assistants in an orthopaedic pre-admission clinic and on a vascular ward to reduce turnaround time. POCT results were compared with the routine swabs taken for culture. The POCT assay was easy to use, the turnaround time for negative results was greatly reduced, and sensitivity was 75.0% in the pre-admission clinic and 80.0% in the vascular ward. There were 27 POCT-positive/culture-negative results, but there was no evidence of MRSA infection or colonization in these patients for at least a year post procedure. POCT for MRSA colonization performed beyond the laboratory has important advantages over laboratory-based methods and should be explored further.Journal of Hospital Infection 04/2014; · 2.78 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Background: The Xpert® CT/NG (Cepheid Sunnyvale, CA) is a rapid, fully automated real-time polymerase chain reaction test that simultaneously detects Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG).It has high sensitivity and specificity, but also includes a Specimen Adequacy Control (SAC). SAC controls for false negative results by confirming adequate patient sample and appropriate testing conditions. SAC is quantified by its cycle threshold (Ct ), the number of cycles required to detect the presence of a single copy human gene. A lower SAC indicates an earlier Ct and more human cellular material detected. Our objectives were to describe the frequency and distribution of SAC Ct values and observe any correlations with detected infections. Methods: Urine samples from 1382 HIV-1-infected pregnant women, collected at the time of labor/delivery underwent Xpert® CT/NG testing. Mean SAC Ct values and standard deviation (SD) were calculated. Student’s t-test was used to compare mean SAC Ct values to a reference of urine samples negative for CT and NG. Results: The urine CT positivity was 17.9% (248/1382) and NG, 4.6% (63/1382). The mean SAC Ct value in urine from women without CT or NG was 28.09 (SD: 4.12) and higher than the mean SAC Ct value for CT positive specimens (27.29, SD: 3.84(P=.0054)), NG positive specimens (26.23, SD: 3.09(P<.0001)), and specimens positive for both CT and NG (26.41, SD: 3.01(P=.0027)). Conclusion: Lower SAC Ct values were significantly associated with chlamydial and gonococcal infections. Further studies should be conducted to determine the utility of SAC Ct values for identifying the presence of increased human cellular material and infection.Journal of Microbiology & Experimentation. 08/2014; 1(4):26.
JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2009, p. 3933–3936
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 47, No. 12
Comparison of MRSASelect Agar, CHROMagar Methicillin-Resistant
Staphylococcus aureus (MRSA) Medium, and Xpert MRSA PCR for
Detection of MRSA in Nares: Diagnostic Accuracy for Surveillance
Samples with Various Bacterial Densities?
D. M. Wolk,1,2* J. L. Marx,1L. Dominguez,2D. Driscoll,1and R. B. Schifman1
Southern Arizona VA Health Care System, Tucson, Arizona,1and The University of Arizona/BIO5 Institute, Tucson, Arizona2
Received 25 March 2009/Returned for modification 20 May 2009/Accepted 6 October 2009
Rapid laboratory methods provide optimal support for active surveillance efforts to screen for methicillin-
resistant Staphylococcus aureus (MRSA). Most laboratories struggle to determine the optimal use of resources,
considering options to balance cost, speed, and diagnostic accuracy. To assess the performance of common
methods, the first comparison of MRSASelect agar (MS) and CHROMagar MRSA (CA), with and without
broth enrichment followed by a 24-h subculture to MS, was performed. Results were compared to those of the
Xpert MRSA assay. For direct culture methods, the agreement between MS and CA was 98.8%. At 18 h, direct
MS identified 93% of all positive samples from direct culture and 84% of those identified by the Xpert MRSA.
For Trypticase soy broth-enriched MS culture, incubated overnight and then subcultured for an additional
24 h, the agreement with Xpert MRSA was 96%. The agreement between direct MS and Xpert MRSA was 100%
when semiquantitative culture revealed a bacterial density of 2? or greater; however, discrepancies between
culture and Xpert MRSA arose for MRSA bacterial densities of 1? or less, indicating low density as a common
cause of false-negative culture results. Since 1? or less was established as the most common MRSA carrier
state, broth enrichment or PCR may be critical for the identification of all MRSA carriers who may be
reservoirs for transmission. In this active-surveillance convenience sample, the use of broth enrichment
followed by subculture to MS offered a low-cost but sensitive method for MRSA screening, with performance
similar to that of Xpert MRSA PCR.
The spread of methicillin-resistant Staphylococcus aureus
(MRSA) is a major concern in healthcare settings because human
“carriers” can spread MRSA to others, resulting in increased
morbidity, mortality, and costs (13, 14, 17). One strategy to
help prevent and control MRSA infections is the use of active
surveillance cultures to screen patients for nasal carriage of
MRSA, a practice coupled with appropriate barrier precau-
tions for colonized or infected patients. Active surveillance
programs are growing in number in the United States (10, 20,
23, 24), despite differences in opinions about the practice and
reported gaps in the literature (19). Therefore, clinical micro-
biology laboratories must respond to provide support for active
surveillance screening methods.
There is continued debate about the practicality and cost
benefit of different MRSA detection methods. Typically, agar
culture or PCR methods are used (4); however, additional
workload and costs cause laboratories to struggle with resource
allocation issues, which often depend on the interplay between
assay accuracy, turnaround time, cost per test, and workforce
availability. While PCR results can be available in as little as
2 h and are known to provide excellent sensitivity and speci-
ficity for MRSA screening (4, 25, 28), PCR methods are more
costly than culture methods (6). Alternately, selective and dif-
ferential chromogenic agars have gained popularity because of
lower cost and mid-range speed. Because of their resistance to
antimicrobials in the agar, MRSA colonies grow and subse-
quently produce distinct color changes caused by the cleavage
of a chromogenic substrate by a specific enzyme of S. aureus.
Results can be achieved in as little as 18 to 24 h of incubation,
depending on the agar used (1, 4, 5, 15, 27; BBL CHROMagar
MRSA package insert no. 8012632, 2006 [Becton Dickinson
and Company]; MRSASelect package insert nos. 63747 and
63749, 2007 [Bio-Rad Laboratories]). Although less costly, di-
rect agar cultures are relatively insensitive in comparison to PCR
and broth enrichment cultures. Broth enrichment cultures can
improve sensitivity in comparison to that of nonenriched (direct)
cultures (2, 9, 16, 21); however, enrichment delays results and
adds to workload and costs. Thus, a discussion of the advantages
and limitations of each methodological approach become critical.
Decisions about the type of screening method used must be made
based on the performance of the methods and resources available
for each health care system. Clearly, the most sensitive screening
our clinical practices.
Because of the critical balance between speed, accuracy, cost,
and hands-on time associated with each screening method, we
designed and evaluated a broth enrichment algorithm that
relies on the use of chromogenic agar as the subculture me-
dium for broth enrichment in order to maximize both the
speed and sensitivity of agar-based approaches. We compared
the method’s performance to that of a PCR reference method,
the Cepheid Xpert MRSA assay.
* Corresponding author. Mailing address: University of Arizona,
Department of Pathology and BIO5 Institute, 1501 N. Campbell Av-
enue, P.O. Box 245059, Tucson, AZ 85724-5059. Phone: (520) 626-
3676. Fax: (520) 694-7329. E-mail: email@example.com.
?Published ahead of print on 14 October 2009.
at UNIVERSITY OF ARIZONA LIBRARY on January 6, 2010
To our knowledge, the study is the first clinical assessment of
Bio-Rad’s MRSASelect agar (MS) and Becton Dickinson’s
BBL CHROMagar MRSA (CA) combined with broth enrich-
ment and compared to the Xpert MRSA assay. It is also the
first study to describe the performance of the methods relative
to the various bacterial densities of MRSA which are found in
active surveillance samples from hospitalized patients. The study
goal was to validate an off-label use of MS as a subculture
medium, in combination with tryptic soy broth (TSB) enrich-
ment, to improve the recovery of MRSA bacteria with mini-
mum impact to workload and cost.
(This information was presented, in part, at the 108th Gen-
eral Meeting of the American Society for Microbiology, Bos-
ton, MA, 1 to 5 June 2008 .)
MATERIALS AND METHODS
Culture. Double swabs of BD BBL CultureSwab liquid Stuart medium swabs
(Becton Dickenson, Sparks, MD) were used to collect samples from nares (n ?
498). Agar inoculation was performed by cross-streaking in four quadrants to
yield 1? to 4? semiquantitative culture results (29). Culture results were eval-
uated according to the manufacturers’ instructions (18 and 24 h for MS and 24
to 48 h for CA). One swab was assigned to agar cultures and subsequent broth
enrichment. The other paired swab was assigned to the PCR test, the Xpert
MRSA assay. Technologists performing culture methods were blinded to the
PCR results. To minimize selection bias, each agar type received preference for
the status of “first agar inoculated” 50% of the time. .
TSB (0.05% NaCl) (Becton Dickenson, Sparks, MD) was selected as the
enrichment medium in order to maximize the ability to support growth of
salt-sensitive MRSA strains (11). After 24 h of incubation in TSB, postenrich-
ment subculture was performed to an MS plate that was incubated for an
additional 24 h (TSB-MS), for a total of 48 h. The use of MS as an adjunct to
broth enrichment is an off-label use of MS. With the TSB-MS approach, MRSA
can be identified without the need for additional biochemical confirmation.
PCR. The Xpert MRSA assay, Cepheid, Inc., Sunnyvale, CA, was performed
directly on swabs according to the manufacturer’s instructions, with one excep-
tion; BD BBL CultureSwab Liquid Stuart’s dual swabs (Becton Dickenson,
Sparks, MD), an acceptable substitute for the COPAN 139C swab, were used for
nasal sample collection and testing (Becton Dickenson).
Study design. A convenience sample of remnant specimens was used. The
sample size for analysis was selected by McNemar’s test to detect a 3% improve-
ment in the sensitivity of either agar, with a target sensitivity defined at 95% in
a targeted sample prevalence of 25% (power ? 0.8, alpha ? 0.05).
Remnant samples were used under oversight from the Human Subject Pro-
tection Program (HSPP) at the University of Arizona, Tucson, AZ, and in
accordance with federal policy governing protection of human subjects and
federal medical privacy standards (26).
MRSA prevalence and bacterial density. In this convenience
sample, the culture-confirmed MRSA prevalence, as deter-
mined by direct culture, was 20.5% for both of the agars tested.
The prevalence was statistically greater for the TSB-MS broth
enrichment method and for the Xpert MRSA, at 22.7% and
26.9%, respectively (P ? 0.05).
Among the 113 positive MRSA samples that were identified
by culture, the following patterns of bacterial density were
observed: in 7% of positive samples, MRSA was recovered
only after broth enrichment; for 31%, the semiquantitative
observation was 1?; for 23%, the observation was 2?; for
16%, it was 3?; and for 23%, it was 4? (Fig. 1). The positive
MRSA samples in this specimen set contained a high percent-
age of MRSA at a density of 1? relative to the other densities.
Agreement between direct culture methods and broth en-
richment. Using McNemar’s test to compare agar performance
from direct inoculation, no overall performance differences
were observed. A total of three discrepant results were ob-
served for each of the two agars. In comparison with each
other, total agar agreement was 98.8% (Table 1). The semi-
quantitative result for all six discrepant results ranged from
one colony to 1?, representing the low end of bacterial density
and, perhaps, the agar’s limit of detection, at which one would
expect random positive and negative results to occur. The
order of inoculation was not observed to bias any of the dis-
crepant results. The TSB-MS culture method identified 7%
more positive nasal samples than direct culture alone (P ?
Performance of culture methods as a function of incubation
time. At 18 h, direct culture on MS identified 93% of all
positive MRSA samples which were identified after a full 24 h
of incubation on MS. In contrast, direct culture on MS iden-
tified only 84% of all positive MRSA samples which were
identified by TSB-MS. In the comparison between direct cul-
ture methods, examined according to the package inserts, MS
examined at 18 h identified 18% more positive MRSA samples
than CA examined at 24 h. No significant differences were
FIG. 1. Semiquantitative distribution of bacterial density in 113
hospitalized MRSA carriers selected from a convenience sample in a
hospital-wide surveillance initiative. The key lists the data shown by pie
chart segments clockwise from the top.
TABLE 1. Performance of chromogenic agar culture with and
without broth enrichment compared to that of
Xpert MRSA PCRa
MS for 24 h vs CA
for 48 h
MS for 24 h vs TSB
97.1 (91.9–99.0) 99.2 (97.8–99.7)97.1 99.2
90.3 (83.4–94.5) 100 (99.0–100)10097.2
84.3 (77.2–89.5) 100 (98.9 –100)10094.5
aA contingency table depicts the performance of chromogenic agar, broth
enrichment, and Xpert MRSA for a convenience sample of hospitalized patients
(n ? 498). PPV, positive predictive value; NPV, negative predictive value.
bNine PCR-positive/MRSA-negative samples grew only MSSA by TSB en-
richment. Testing for the presence of SCCmec variants was not performed.
3934WOLK ET AL.J. CLIN. MICROBIOL.
at UNIVERSITY OF ARIZONA LIBRARY on January 6, 2010
observed between results from direct inoculation of MS exam-
ined at 24 h and CA examined at 48 h.
Culture agreement with PCR. The total agreement between
MS results and Xpert MRSA results was 94.2% (Table 1). The
positive agreement between direct MS and Xpert MRSA was
84.3%. Discrepancies between MS and Xpert MRSA occurred
exclusively when the semiquantitative MRSA bacterial density
in the nasal sample was determined to be 1? or less. In con-
trast, for all samples with MRSA bacterial densities of 2? or
greater, the total agreement between MS and Xpert MRSA
was 100%. The overall agreement between TSB-MS and Xpert
MRSA was 96%. The positive predictive value and negative
predictive value were 100% and 94.5%, respectively.
Performance of Xpert MRSA. The Xpert MRSA assay cor-
rectly identified all culture-positive samples from all three cul-
ture methods and detected more positive samples than all
three culture methods combined (P ? 0.01). One or more of
the direct culture methods confirmed the positive Xpert MRSA
results in all except 21 of 134 (15.7%) of the PCR-positive
samples. Of the 21 PCR-positive culture-negative samples,
broth enrichment revealed 10 samples from which no bacteria
were cultivated, 2 samples from which coagulase-negative
Staphylococcus sp. were recovered, and 9 samples from which
methicillin-sensitive S. aureus (MSSA) was cultivated. These
results effectively exclude the possibility of substantial bias due
to staphylococcal cassette chromosome mec (SCCmec) vari-
ants (aka MRSA-MSSA) in all but 6.7% (n ? 9 MSSA sam-
ples) of the PCR-positive samples (1.8% of all samples).
One-way analysis of variance of MRSA CT. In order to
further assess the relative MRSA bacterial densities of the
concordant and discordant results, an off-label assessment use
of the Xpert MRSA assay was performed, using cycle thresh-
old (CT) values from the GeneXpert system. First, assessment
of the Xpert MRSA internal control values showed no signif-
icant difference between concordant and discordant samples,
indicating no overall differences in the inhibitory components
of the nares’ matrices. In contrast, MRSA target CTvalues
from concordant samples were statistically lower (25.6; 95%
confidence interval [CI], 24.8 to 26.4) than those from discor-
dant samples (32.2; 95% CI, 30.5 to 33.8) and samples that
were positive in broth only (31.7; 95% CI, 28.7 to 34.7). There-
fore, higher MRSA DNA concentrations were observed in
concordant samples than in discordant samples or samples
with bacterial densities that were detected only by TSB-MS
broth enrichment. This pattern is similar to that observed with
culture methods alone. Two specimens had very low CTvalues,
indicative of high DNA concentrations. These specimens could
suggest SCCmec variants; however, MSSA was not cultivated
from these samples, so it is unlikely that these specimens con-
tained SCCmec variants.
A convenience sample of remnant nasal specimens from an
active surveillance initiative in Arizona was used to compare
the performance of culture with MS and CA, with and without
broth enrichment, to that of the Xpert MRSA assay. No sig-
nificant differences were observed between results from direct
inoculation of either MS or CA direct culture plates. TSB-MS
provided the most sensitive culture method, and the results
were not statistically different from the results of PCR. The
TSB-MS method, while slower than PCR, provided a low-cost
method that achieved accuracy similar to that of PCR.
MS offers several advantages over other culture media. Most
MRSA-positive cultures, 93%, were identified on MS after
18 h of incubation. Therefore, adjusting the agar examination
time to accommodate the first MS examination at 18 h may
speed positive results to allow appropriate isolation of MRSA
carriers and identify almost all positive samples with relative
ease. Negative samples from MS can be reincubated to achieve
a total incubation time of 24 to 28 h to maximize recovery.
Alternatively, if one inoculates a broth at the same time as the
direct culture is performed, the broth can be subcultured to an
MS plate to maximize cultivation of MRSA. In contrast, CA
requires 24 to 48 h of incubation to maximize recovery and
requires a confirmatory coagulase test in order to correctly
identify MRSA that grows after 24 h of incubation. Operation-
ally, it is more expedient to use MS as a subculture agar. The
TSB-MS method detected additional carriers, identifying 7%
more positive samples than direct MS culture at 24 h and CA
at 48 h.
Detection by Xpert MRSA identified 15.7% more positive
samples than direct agar culture methods. These “PCR-posi-
tive culture-negative” specimens can create confusion about
laboratory surveillance results. For laboratories using culture
methods for screening, this scenario results in patients who
may go undetected by culture methods but may be able to
transmit MRSA to others. Although this scenario can occur for
a variety of reasons (7, 8, 12), one reason for this phenomenon
is the fact that bacteria may be noncultivable at the time of
sampling or may contain SCCmec variants. In this sample set,
composed of MRSA strains isolated from patients in Arizona,
there was a relatively low percentage (6.7% of positive sam-
ples) in which SCCmec variants could not be excluded. The
presence of SCCmec variants can change and does vary de-
pending on locality (7, 8). While it may be prudent to period-
ically monitor for potential SCCmec variants in each locality,
by dual testing of a proportion of samples with both PCR and
culture, results from this sample set do not appear to warrant
routine culture backup of PCR results, as the percentage of
potential variants appears to be low.
In addition to SCCmec variants, PCR-positive culture-neg-
ative results occur when patients’ nasal samples contain only
low MRSA densities, which can be identified by PCR but are
below the direct culture’s limit of detection. The high percent-
age of cultures containing MRSA in low bacterial densities
(1? or less) supports the premise that high CTvalues, observed
in the discordant samples, correspond to low MRSA density,
the most likely reason for discrepancies. These false-negative
cultures potentially represent unidentified reservoirs of MRSA,
which may be the source of further transmission. Further in-
vestigation of the bacterial densities found in MRSA carriers
may be warranted, as there is limited information to describe
the quantitative differences in carrier state or the densities
required for optimal transmission.
Lastly, while our broth enrichment culture was designed to
optimize recovery of salt-sensitive staphylococci (3, 11), limi-
tations in experimental design exist for S. aureus strains that
may require anaerobic incubation (22) or other special growth
conditions. These scenarios, as well as antibiotic use, may ac-
VOL. 47, 2009ACCURACY OF CULTURE AND PCR FOR MRSA DETECTION3935
at UNIVERSITY OF ARIZONA LIBRARY on January 6, 2010
count for some of the 10 PCR-positive culture-negative sam-
The approach described here uses a rapid selective agar,
which does not require biochemical confirmation, to detect
most of the positive MRSA samples. This approach was de-
signed to identify as many positive results as possible within
18 h and identify the remaining positives with ease, after the
enrichment broth is subcultured to another MS. The approach
may save time and money and provide overall results that are
equivalent to those of PCR methods. Rapid and sensitive ac-
tive surveillance screening methods are critical for supporting
rapid initiation of contact precautions for MRSA carriers. Di-
rect culture to MS, coupled with broth enrichment and sub-
culture, provides a screening method with sensitivity equiva-
lent to that of the PCR method.
The authors thank Bio-Rad, Inc. for contributing MRSASelect agar
plates and for providing educational support. Our thanks also go to
Linda Hilbert for technical editing of the manuscript.
The authors disclose that the MRSASelect agar for this study was
provided by BioRad, Inc.
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