A common variant of staphylococcal cassette chromosome mec type IVa in isolates from Copenhagen, Denmark, is not detected by the BD GeneOhm methicillin-resistant Staphylococcus aureus assay.
ABSTRACT Rapid tests for detection of methicillin-resistant Staphylococcus aureus (MRSA) carriage are important to limit the transmission of MRSA in the health care setting. We evaluated the performance of the BD GeneOhm MRSA real-time PCR assay using a diverse collection of MRSA isolates, mainly from Copenhagen, Denmark, but also including international isolates, e.g., USA100-1100. Pure cultures of 349 MRSA isolates representing variants of staphylococcal cassette chromosome mec (SCCmec) types I to V and 103 different staphylococcal protein A (spa) types were tested. In addition, 53 methicillin-susceptible Staphylococcus aureus isolates were included as negative controls. Forty-four MRSA isolates were undetectable; of these, 95% harbored SCCmec type IVa, and these included the most-common clone in Copenhagen, spa t024-sequence type 8-IVa. The false-negative MRSA isolates were tested with new primers (analyte-specific reagent [ASR] BD GeneOhm MRSA assay) supplied by Becton Dickinson (BD). The ASR BD GeneOhm MRSA assay detected 42 of the 44 isolates that were false negative in the BD GeneOhm MRSA assay. Combining the BD GeneOhm MRSA assay with the ASR BD GeneOhm MRSA assay greatly improved the results, with only two MRSA isolates being false negative. The BD GeneOhm MRSA assay alone is not adequate for MRSA detection in Copenhagen, Denmark, as more than one-third of our MRSA isolates would not be detected. We recommend that the BD GeneOhm MRSA assay be evaluated against the local MRSA diversity before being established as a standard assay, and due to the constant evolution of SCCmec cassettes, a continuous global surveillance is advisable in order to update the assay as necessary.
- SourceAvailable from: Lorena C C Fehlberg01/2014;
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ABSTRACT: Clinical laboratories are increasingly using molecular tests for methicillin-resistant Staphylococcus aureus (MRSA) screening. However, primers have to be targeted to a variable chromosomal region, the staphylococcal cassette chromosome mec (SCCmec). We initially screened 726 MRSA isolates from a single UK hospital trust by recombinase polymerase amplification (RPA), a novel, isothermal alternative to PCR. Undetected isolates were further characterised using multilocus sequence, spa typing and whole genome sequencing. 96% of our tested phenotypically MRSA isolates contained one of the six orfX-SCCmec junctions our RPA test and commercially available molecular tests target. However 30 isolates could not be detected. Sequencing of 24 of these isolates demonstrated recombinations within the SCCmec element with novel insertions that interfered with the RPA, preventing identification as MRSA. This result suggests that clinical laboratories cannot rely solely upon molecular assays to reliably detect all methicillin-resistance. The presence of significant recombinations in the SCCmec element, where the majority of assays target their primers, suggests that there will continue to be isolates that escape identification. We caution that dependence on amplification-based molecular assays will continue to result in failure to diagnose a small proportion (∼4%) of MRSA isolates, unless the true level of SCCmec natural diversity is determined by whole genome sequencing of a large collection of MRSA isolates.PLoS ONE 01/2014; 9(6):e101419. · 3.53 Impact Factor
- 07/2014; 35(7):772-796.
JOURNAL OF CLINICAL MICROBIOLOGY, May 2009, p. 1524–1527
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 47, No. 5
A Common Variant of Staphylococcal Cassette Chromosome mec
Type IVa in Isolates from Copenhagen, Denmark, Is Not
Detected by the BD GeneOhm Methicillin-Resistant
Staphylococcus aureus Assay?
Mette Damkjaer Bartels,1* Kit Boye,1Susanne Mie Rohde,1Anders Rhod Larsen,2
Herbert Torfs,3Peggy Bouchy,4Robert Skov,2and Henrik Westh1,5
Department of Clinical Microbiology, Hvidovre Hospital, Hvidovre, Denmark1; Staphylococcus Laboratory, Statens Serum Institut,
Copenhagen, Denmark2; Becton Dickinson, Erembodegem, Belgium3; Becton Dickinson, Quebec, Canada4; and
Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark5
Received 10 November 2008/Returned for modification 20 February 2009/Accepted 9 March 2009
transmission of MRSA in the health care setting. We evaluated the performance of the BD GeneOhm MRSA
real-time PCR assay using a diverse collection of MRSA isolates, mainly from Copenhagen, Denmark, but also
including international isolates, e.g., USA100-1100. Pure cultures of 349 MRSA isolates representing variants of
staphylococcal cassette chromosome mec (SCCmec) types I to V and 103 different staphylococcal protein A (spa)
types were tested. In addition, 53 methicillin-susceptible Staphylococcus aureus isolates were included as negative
controls. Forty-four MRSA isolates were undetectable; of these, 95% harbored SCCmec type IVa, and these included
the most-common clone in Copenhagen, spa t024-sequence type 8-IVa. The false-negative MRSA isolates were tested
with new primers (analyte-specific reagent [ASR] BD GeneOhm MRSA assay) supplied by Becton Dickinson (BD).
The ASR BD GeneOhm MRSA assay detected 42 of the 44 isolates that were false negative in the BD GeneOhm
MRSA assay. Combining the BD GeneOhm MRSA assay with the ASR BD GeneOhm MRSA assay greatly
improved the results, with only two MRSA isolates being false negative. The BD GeneOhm MRSA assay alone is not
adequate for MRSA detection in Copenhagen, Denmark, as more than one-third of our MRSA isolates would not
be detected. We recommend that the BD GeneOhm MRSA assay be evaluated against the local MRSA diversity
before being established as a standard assay, and due to the constant evolution of SCCmec cassettes, a continuous
global surveillance is advisable in order to update the assay as necessary.
Methicillin-resistant Staphylococcus aureus (MRSA) is a com-
mon nosocomial pathogen in countries all over the world. In
recent years, community-associated MRSA (CA-MRSA) has be-
come increasingly prevalent and has shown potential to cause
health care-associated bloodstream infections (8, 26). Screening
and isolation of MRSA-positive patients is essential to control the
transmission of MRSA in hospitals (16, 24). However, conven-
tional detection of MRSA by culture takes at least 48 h before a
preliminary result is available, and as patients in many countries
are only isolated when they are recognized as MRSA positive, the
risk of having already transmitted MRSA is high. The real-
time PCR BD GeneOhm MRSA assay (Becton Dickinson
[BD] Diagnostics GeneOhm; San Diego, CA), formerly called
IDI-MRSA, is one of a number of commercial kits for rapid
MRSA detection directly from nasal swabs (7) and is based on
primers developed by Huletsky et al. (18). The forward primers
bind to the J3 region of the staphylococcal cassette chromosome
mec (SCCmec), and the reverse primer binds in the orfX region
that is specific for Staphylococcus aureus. At least seven SCCmec
types are known (types I to VII) (3), and several subtypes, espe-
cially of type IV, have been described (21, 27).
The BD GeneOhm MRSA assay has been tested in a num-
ber of studies (4, 5, 10, 11, 13–15, 22, 23, 25, 29–31). Most
studies screened hospitalized patients, but only two studies
described the SCCmec types of their MRSA isolates (15, 25).
Therefore, it is possible that only a few predominant hospital
clones with the same SCCmec types were tested. In Denmark,
different CA-MRSA clones dominate and MRSA isolates
mainly harbor SCCmec types IV (85%) and V (6%) (2). In-
house testing with the Huletsky primers (18) revealed that they
did not amplify a PCR fragment from our most-common
MRSA clone, spa t024-sequence type 8 (ST8)-IVa. Based on
this finding and with the knowledge of the high number of type
IV subtypes known, we were interested in finding out whether
the BD GeneOhm MRSA assay could detect MRSA isolates
from a collection that included mainly CA-MRSA strains. We
tested 349 MRSA isolates representing variants of SCCmec
types I to V. Furthermore, we chose MRSA isolates of differ-
ent staphylococcal protein A (spa) types to have a broad range
of genetic backgrounds, testing the hypothesis that the same
SCCmec type might have minor differences in different MRSA
lineages and that these differences could be in the primer
regions of the assay.
MATERIALS AND METHODS
Setting. Denmark is a country with 5.5 million inhabitants and has a low
prevalence of MRSA (20). The Department of Clinical Microbiology at Hvidovre
* Corresponding author. Mailing address: Department of Clinical Mi-
crobiology 445, Hvidovre Hospital, Dk-2650 Hvidovre, Denmark. Phone:
4536326349. Fax: 4536323357. E-mail: firstname.lastname@example.org.
?Published ahead of print on 18 March 2009.
Hospital services five hospitals and general practices in the Copenhagen and
Frederiksberg municipalities (600,000 inhabitants). The Danish Reference Lab-
oratory for Staphylococci at Statens Serum Institut, Copenhagen, receives all
MRSA strains isolated in Denmark.
Isolates. Three hundred forty-nine MRSA isolates (327 Danish and 22 inter-
national reference isolates) (Table 1) were included. Isolates of 103 spa types
harboring variants of SCCmec types I to V plus nontypeable (NT) isolates were
chosen. Fifty-three methicillin-susceptible S. aureus (MSSA) isolates (23 spa
types) were included as negative controls. All isolates were tested with a duplex
PCR on a pure culture, detecting the mecA gene and the spa gene. MRSA
isolates had both genes, and MSSA isolates only the spa gene.
Typing. Amplification and sequencing of the spa region were performed on
MRSA and MSSA isolates as previously described (2, 17). Designation of spa
type was conducted by using the Ridom StaphType program. The SCCmec type
was determined with an in-house multiplex PCR (6). The SCCmec multiplex
PCR was also used to screen MSSA isolates that were positive by the BD
GeneOhm MRSA assay. NT isolates were tested in singleton mode with each
primer set from the multiplex PCR. Isolates of SCCmec type IV were subtyped
by the method of Milheirico et al. (21).
BD GeneOhm MRSA assay and ASR BD GeneOhm MRSA assay. Colonies
from pure cultures of all MRSA and MSSA strains were resuspended in sterile
saline (0.85%) to a turbidity of 0.5 McFarland. DNA extraction was performed
by using a BD GeneOhm MRSA lysis kit (BD Diagnostics). The BD GeneOhm
MRSA assay was performed as recommended by the manufacturer, and a pos-
itive and negative control were included in each run. All PCRs were run on a
SmartCycler system (Cepheid, Sunnyvale, CA). Due to a high number of false-
negative MRSA results with the BD GeneOhm MRSA assay, seven of the
false-negative MRSA isolates along with two lysates of each strain, produced
either by using the MRSA lysis kit or by boiling one colony in 250 ?l of sterile
water, were sent to BD. They determined that the MRSA isolates had other
MREJ (SCCmec right-extremity junction) types than those detected by the BD
GeneOhm MRSA assay (18). Therefore, BD developed an investigational real-
time PCR assay (analyte-specific reagent [ASR] BD GeneOhm MRSA assay)
containing new primers. The primer sequences are proprietary.
From the first collection, 139 MRSA and 25 MSSA isolates were selected for
ASR BD GeneOhm MRSA testing, including all MRSA strains that were ini-
tially negative. The DNA lysates from the initial BD GeneOhm MRSA evalua-
tion had been stored at minus 20°C and were reused. PCR amplification products
were analyzed on a 1.5% agarose gel. The band sizes were compared to the band
sizes of the isolates examined by BD. DNA from MRSA isolates that were
negative by both assays was reextracted with the lysis kit and retested with both
Two hundred ninety-five of 349 MRSA isolates (84.5%)
were positive with the BD GeneOhm MRSA assay, including
all international reference isolates. Fifty-four (15.5%) of the
MRSA strains gave false-negative results. BD examined seven
of these isolates. They contained two MREJ types that are not
detected by the BD GeneOhm MRSA assay and led to the
development of the ASR BD GeneOhm MRSA assay. The
ASR BD GeneOhm MRSA assay identified 42 of the 54 false-
negative MRSA isolates as MRSA. After a new DNA extrac-
tion of the remaining 12 false-negative isolates, 10 isolates
were positive in the BD GeneOhm MRSA assay; 1 of these was
also positive by the ASR BD GeneOhm MRSA assay (with an
aberrant gel band size), and 2 remained false negative. The
SCCmec characteristics of all isolates and the results of the BD
GeneOhm MRSA assay are shown in Table 2. The spa and
SCCmec types of the 44 MRSA isolates that were false nega-
tive in the BD GeneOhm MRSA assay are presented in Table
3. The majority of the 44 false-negative MRSA isolates har-
bored SCCmec IVa. Only 5 of 33 t024-ST8-IVa isolates were
detected with the BD GeneOhm MRSA assay, three of them
after the DNA reextraction, and all were positive in a late PCR
Eighty-five MRSA isolates that were positive by the BD
GeneOhm MRSA assay were also tested with the ASR BD
GeneOhm MRSA assay to detect potential overlap of the
assays. Only three isolates were positive, but these had gel
bands of unexpected sizes. Because the primer sequences were
proprietary, the significance of this finding is unknown.
Out of 53 MSSA isolates, 8 (15%) with different spa types
were false positive by the BD GeneOhm MRSA assay, and
none of the 25 MSSA isolates was false positive by the ASR
BD GeneOhm MRSA assay. In one of the false-positive MSSA
isolates (t127), which was only resistant to penicillin, we iden-
tified the insertion sequence-like element IS1272 (confirmed
TABLE 1. The 22 international MRSA strains
spa typeSCCmec typeST/CCb
aThe strains from Finland, France, and the United Kingdom are from the
Harmony collection (9).
bST, sequence type; CC, clonal complex. The ST and CC of the Harmony
collection strains are from Cookson et al. (9), and those of the United States
strains from the Ridom spa server and E-Burst on the MLST website (www.mlst
TABLE 2. Results of BD GeneOhm MRSA assay
Total no. of MRSA isolates349 305a
No. of MSSA isolates538 15
aTen isolates were detected only after repeated DNA extraction from the
VOL. 47, 2009 BD GeneOhm FOR SCCmec IVa MRSA FROM COPENHAGEN1525
by sequencing of the PCR product). One false-positive MSSA
isolate (t843) was resistant to penicillin, erythromycin, and
moxifloxacin, while the remaining six were only resistant to
penicillin (five isolates) or were fully susceptible (one isolate).
A number of Danish MRSA isolates were not detected by
the BD GeneOhm MRSA assay. Investigations at BD revealed
that they harbored MREJ types different from the ones in-
cluded in the BD GeneOhm MRSA assay. By using new prim-
ers, the investigational ASR BD GeneOhm MRSA assay de-
tected 42 of the 44 MRSA isolates that gave false-negative
results with the BD GeneOhm MRSA assay. The main issue
with the BD GeneOhm MRSA assay when used for isolates
from Copenhagen was the lack of sensitivity in detecting iso-
lates of SCCmec type IVa. The type IVa cassette is common in
CA-MRSA and is found in the most-abundant MRSA clone
in Copenhagen—the t024-ST8-IVa clone. The t024-ST8-IVa
clone has mainly affected people in nursing homes and has
caused small outbreaks in local hospitals. In our area, it ac-
counts for 32% of all MRSA isolates found between 2003 and
2007. While 5 of the 33 t024-ST8-IVa isolates were detected by
the BD GeneOhm MRSA assay, this was only with weak sig-
nals near the limit of detection. Since the PCR was run on pure
cultures, it is unlikely that they would have been detected from
nasal swabs. In our selection of isolates, the BD GeneOhm
MRSA assay detected 60% of the isolates harboring SCCmec
IVa, including USA300, while 40% were undetectable. The
subtyping of type IV is mainly based on differences in the J1
region (21), whereas the primers from BD GeneOhm MRSA
amplify from the J3 region across the origin of replication
(orfX). This shows that in type IVa, the J3 region exhibits some
variability. Two isolates, t690-IVa and t688-V, were negative by
both assays. Both patients from whom these isolates were ob-
tained had recently traveled in Egypt, and it is currently un-
known why these isolates were not detected.
Eight of 53 MSSA isolates (15%) gave false-positive results.
This false-positive rate is much higher than the rate of 4.6%
found by Huletsky et al. (18). However, relatively high frequen-
cies of false-positive results have been reported by others (11,
23). High false-positive rates could be a problem in countries
with a low prevalence of MRSA, as this would result in low
positive predictive values. Remnants of the SCCmec cassette
have been identified in some MSSA strains and could explain
false-positive results by PCR (12, 28). It is of interest whether
this is the cause in a country with a low prevalence of MRSA,
and it needs to be addressed in further studies. In one MSSA
isolate (t127), we found the insertion sequence-like element
IS1272 that is present in SCCmec type I and type IV and could
indicate that this isolate has a remnant of an SCCmec cassette.
However, IS1272 has been found in coagulase-negative staph-
ylococci, MRSA and MSSA, and may have been disseminated
irrespective of the presence of mec DNA (19).
Though the BD GeneOhm lysis kit has been reported to be
superior to five other extraction methods (1), 10 MRSA iso-
lates (3%) were only detected by the BD GeneOhm MRSA
assay after repeated DNA extraction. While this might be due
to a handling error in our laboratory, the internal control
(which is added together with the PCR reagents) was detected
in all 10 cases after the first round of extraction. These samples
would have been classified as MRSA negative in a clinical
setting. False-negative results possibly due to extraction prob-
lems have been reported by others (25).
In a screening situation with the goal of preventing nosoco-
mial MRSA infections, a high negative predictive value of a
MRSA screening assay is important. In Denmark, the preva-
lence of MRSA is low and we do not expect to find many
MRSA carriers. Hospitalized MRSA patients are always iso-
lated, and patients at risk of being MRSA carriers are kept in
isolation until a negative MRSA test result is obtained. A
false-negative result would release these patients from isola-
tion and could start a MRSA outbreak.
The local diversity of CA-MRSA strains makes the current
version of the BD GeneOhm MRSA assay inadequate for
screening in Copenhagen. The main problem with the test was
that most of the type IVa cassettes of our major MRSA clone
(t024-ST8-IVa) were undetected. New primers not yet commer-
cialized can detect these strains, but the ASR BD GeneOhm
MRSA assay would be an add-on assay to the BD GeneOhm
MRSA assay, increasing the laboratory work and costs. Based
on the findings in this study, we recommend that the BD
GeneOhm MRSA assay be evaluated against the local MRSA
diversity before being established as a standard assay. Due to
the rapid evolution of SCCmec in CA-MRSA, we recommend
that the assay’s usefulness be continuously monitored.
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assay false-negative MRSA isolates
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Total 44 2064
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