Real-time evaluation of an optimized real-time PCR assay versus Brilliance chromogenic MRSA agar for the detection of meticillin-resistant

Abstract

A total of 1204 meticillin-resistant Staphylococcus aureus (MRSA) screens (3340 individual swabs) were tested to evaluate a staphylococcal cassette chromosome mec (SCCmec) real-time PCR. In total, 148 (12.3 %) of the screens were MRSA-positive, where 146 (12.1 %) were MRSA-positive by the SCCmec real-time PCR assay. In contrast, 128 (10.6 %) screens were MRSA-positive by culture. One hundred and twenty-six (10.5 %) of the screens were positive by both culture and PCR. Twenty of the 1204 screens (1.66 %) were negative by culture but positive by PCR; these samples were sequenced. In 14 of the cases, a homology search confirmed the sequence as SCCmec, indicating that these samples could be considered true positives. Two of the 1204 (0.2 %) screens were positive by culture and negative by PCR. The mean turnaround time (TAT) for PCR-negative swabs was 6 h 12 min and for PCR-positive swabs was 6 h 48 min. In comparison, for culture-negative swabs the mean TAT was 29 h 30 min and for culture-positive swabs was 69 h. The cost per swab for routine culture was £0.41 (€0.48) and that of the real-time PCR assay was £2.35 (€2.75). This optimized, in-house, inexpensive, real-time PCR test maintained a very high sensitivity and specificity when evaluated under real-time laboratory conditions. The TAT of this real-time PCR assay was substantially lower than that of chromogenic culture. It was also maintained throughout the entire process, which can be taken as an indirect measure of test performance. This study showed that implementation of a molecular test can be achieved with limited resources in a standard microbiology laboratory.

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Real-time evaluation of an optimized real-time PCR
assay versus Brilliance chromogenic MRSA agar
for the detection of meticillin-resistant
Staphylococcus aureus from clinical specimens
J. Danial,
1
M. Noel,
1
K. E. Templeton,
1
F. Cameron,
1
F. Mathewson,
1
M. Smith
1
and J. A. Cepeda
1,2
Correspondence
J. Danial
janathan.danial@luht.scot.nhs.uk
Received 22 September 2010
Accepted 22 November 2010
1
Department of Microbiology, Royal Infirmary of Edinburgh, Edinburgh, UK
2
Department of Microbiology, Basingstoke and North Hampshire Hospital, Basingstoke, UK
A total of 1204 meticillin-resistant Staphylococcus aureus (MRSA) screens (3340 individual
swabs) were tested to evaluate a staphylococcal cassette chromosome mec (SCCmec) real-time
PCR. In total, 148 (12.3 %) of the screens were MRSA-positive, where 146 (12.1 %) were
MRSA-positive by the SCCmec real-time PCR assay. In contrast, 128 (10.6 %) screens were
MRSA-positive by culture. One hundred and twenty-six (10.5 %) of the screens were positive by
both culture and PCR. Twenty of the 1204 screens (1.66 %) were negative by culture but positive
by PCR; these samples were sequenced. In 14 of the cases, a homology search confirmed the
sequence as SCCmec, indicating that these samples could be considered true positives. Two of
the 1204 (0.2 %) screens were positive by culture and negative by PCR. The mean turnaround
time (TAT) for PCR-negative swabs was 6 h 12 min and for PCR-positive swabs was 6 h 48 min.
In comparison, for culture-negative swabs the mean TAT was 29 h 30 min and for culture-positive
swabs was 69 h. The cost per swab for routine culture was £0.41 (J0.48) and that of the real-
time PCR assay was £2.35 (J2.75). This optimized, in-house, inexpensive, real-time PCR test
maintained a very high sensitivity and specificity when evaluated under real-time laboratory
conditions. The TAT of this real-time PCR assay was substantially lower than that of chromogenic
culture. It was also maintained throughout the entire process, which can be taken as an indirect
measure of test performance. This study showed that implementation of a molecular test can be
achieved with limited resources in a standard microbiology laboratory.
INTRODUCTION
Meticillin-resistant Staphylococcus aureus (MRSA) remains
a leading cause of healthcare-acquired infection and affects
the most vulnerable patients with significant morbidity and
mortality (Harbarth et al., 1998; Cosgrove et al., 2003;
Salgado et al., 2003; Cooper et al., 2004; Francois et al.,
2007). Despite the lack of convincing evidence (Coia et al.,
2006), it is now accepted that a major aspect of controlling
the spread of MRSA is the prompt identification of patients
at risk of MRSA carriage (Chaix et al., 1999; Cepeda et al.,
2005; Malde et al., 2006; Cunningham et al., 2007).
Increasing numbers of hospitals in the UK will be expected
to perform MRSA screening of all elective hospital
admissions and emergency admissions in the near future
(Department of Health in England, 2008; Keshtgar et al.,
2008). However, there is a possibility that, even if adequate
infection control precautions are in place, the delay in
obtaining results from screening swabs will allow trans-
mission of MRSA from colonized patients to occur before
carriage has been detected.
Screening using faster methods such as nucleic acid
amplification can produce results within 2–4 h directly
from clinical samples (Jeyaratnam et al., 2008; Renwick
et al., 2008). However, the reality is that various external
factors such as sample collection, transport, reception,
documentation and reporting significantly increase the real
reporting time, making the target of same-day reporting
difficult to achieve (Harbarth et al., 1998; Jeyaratnam et al.,
2008; Aldeyab et al., 2009). Commercially available
methods have limitations, including detecting meticillin-
sensitive S. aureus, high rates of inhibition (Huletsky et al.,
2004; Conterno et al., 2007; Harbarth et al., 2008; Keshtgar
et al., 2008; Robicsek et al., 2008) and no proven ability to
Abbreviations: MRSA, meticillin-resistant Staphylococcus aureus; NHS,
National Health Service; NPV, negative predictive value; PPV, positive
predictive value; SCCmec, staphylococcal cassette chromosome mec;
TAT, turnaround time.
Journal of Medical Microbiology (2011), 60, 323–328 DOI 10.1099/jmm.0.025288-0
025288 G2011 SGM Printed in Great Britain 323

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test pooled samples (Rossney et al., 2008; Gro
¨bner et al.,
2009; Kobayashi et al., 2009). Commercial assays are also
inflexible to changes when alternative primers are required.
The general perception among standard diagnostic labora-
tories in the National Health Service (NHS) is that
implementation of molecular techniques is too technically
demanding and unaffordable (Conterno et al., 2007;
Robicsek et al., 2008).
In this study, an in-house real-time PCR for the detection
of MRSA is described, which overcomes the technical
problems observed with comparable commercially avail-
able methods, namely showing implementation of the assay
into routine diagnostics to maintain robust performance,
reporting consistent results from pooled samples, the
feasibility of implementing a molecular test in a standard
microbiology laboratory, development of quality-assurance
schemes to maintain performance and the cost-effective-
ness of reporting the assay in routine practice.
METHODS
Patients and samples. All the screening swabs sent to the Royal
Infirmary of Edinburgh, UK, from NHS Lothian over a 1-month
period (11 February 2008 to 12 March 2008) were tested
simultaneously by culture and real-time PCR assay. A routine
MRSA screen in our hospitals comprises a nose, throat, groin and/
or ulcer site swab. All samples were received and labelled for
registration as per routine protocols for MRSA screening. Both
culture and molecular MRSA results were reported electronically via
the hospital information system. The PCR assay MRSA results were
suppressed and not reported until the culture result was available.
Only patients who were routinely screened for MRSA were included.
The study was carried out in full accordance with clinical governance
as stated by NHS Lothian.
Collection and culture of specimens. All specimens were collected
using a Transwab with plain and charcoal medium (Medical Wire &
Equipment). All specimens were transported at room temperature to
the Royal Infirmary of Edinburgh, UK, and tested within 48 h of
collection. The same specimen was used for standard culture and for
PCR. For identification of MRSA, the swabs were streaked directly onto
Brilliance Chromogenic MRSA agar (Oxoid) and cultured for 18 h at
37 uC. Pure colonies were picked and a latex agglutination test for S.
aureus surface antigens was carried out (Pastorex; Biostat) followed by
a DNase test (DNase plate; Oxoid). For new MRSA-positives, antibiotic
susceptibility was determined by VITEK test (bioMe
´rieux) followed by
Etests for cefoxitin resistance and oxacillin resistance. A latex
agglutination test for penicillin-binding protein 2 (PBP29; Oxoid)
was also tested in some cases. Screens that were culture-positive but
PCR-negative were treated as if they were new positives.
MRSA PCR. One technical operator and support worker with no
previous experience in molecular methods was trained over a 1-week
period to conduct all the molecular tests. The screening swabs were
plated for culture and expressed into 1 ml saline (E&O Laboratories),
and samples from individual patients were pooled together, using
200 ml suspension from each of the swabs with a maximum of three
pooled at once. The assay was performed as described by Renwick
et al. (2008) and primers were as described by Huletsky et al. (2004)
with the addition of an internal control as described by Kalpoe et al.
(2004). When a positive pool was identified, the individual samples
from the pool were reprocessed and analysed by MRSA PCR assay.
Briefly, samples and controls were extracted using a NucliSens
easyMAG system (bioMe
´rieux) (Huletsky et al., 2005; Loens et al.,
2007). Lysis buffer contained phocine herpesvirus (PhoHV) as an
internal control. Saline suspensions were pre-treated with proteinase
K (Qiagen) and extracted according to the manufacturer’s instruc-
tions. Purified nucleic acid was eluted in 110 ml resuspension buffer.
The PCR was performed in a volume of 25 ml, consisting of 10 ml
extracted nucleic acid, 2.5 U HotStarTaq DNA polymerase, 200 mM
each dNTP, 1.5 mM MgCl
2
(final concentration 5 mM; Qiagen),
0.5 mM each staphylococcal cassette chromosome mec (SCCmec)
primer, 0.35 mM SCCmec probe, 0.3 mM each forward and reverse
PhoHV primer and 0.05 mM PhoHV probe. Amplification, detection
and analysis were performed in an ABI 7500 real-time PCR system
(Applied Biosystems) under the following conditions: 1 cycle of 95 uC
for 15 min, followed by 50 cycles of 95 uC for 15 s, 60 uC for 40 s and
72 uC for 30 s.
Reporting and data gathering. Results were reported on the
laboratory information system (APEX; iSOFT) in real time, and the
data gathered for each patient were as follows: age, sex, location,
specimen type, culture result and real-time PCR result (including
cycle threshold value, if positive). The laboratory turnaround time
(TAT) for culture and PCR was calculated from the actual electronic
record of the sample at reception and result authorization held in
APEX. Time from sample collection from patient to reception and
time from authorization to telephoning results (patient receiving
results) were not collected.
Molecular epidemiology and surveillance of mecA variants.
One hundred and sixty-one MRSA isolates from positive blood
cultures collected from 1 July 2007 to 1 July 2008 identified by the
local laboratory using standard identification methods were used to
monitor for SCCmec variation. These isolates are part of an ongoing
epidemiological surveillance programme. These isolates were used
because they are likely to represent circulating strains, particularly
those involved with severe or invasive disease. All isolates were tested
for the presence of the mecA gene with the in-house PCR and
subsequently sequenced.
SCCmec sequencing. Sequence analysis was set up using a BigDye
Terminator Cycle Sequencing kit (Applied Biosystems) according to
the manufacturer’s instructions. The products were analysed on an
ABI 3730 DNA Analyser (Applied Biosystems) and the sequences
were analysed using Molecular Evolutionary Genetics Analysis (MEGA)
software version 4.0 (Tamura et al., 2007). Extracted DNA samples
were amplified using an orfX primer and the same primer was also
used as the sequencing primer. The sequences were aligned and
assigned to their respective SCCmec types.
Statistical analysis. The sensitivity, specificity and positive
predictive value (PPV) and negative predictive value (NPV) of the
MRSA PCR assay were calculated by comparing the results of PCR
with a combination of sequencing of SCCmec and the results of the
standard culture method. A x
2
test was also carried out using the
Microsoft Excel data analysis tool to compare whether the difference
in PCR and culture result was statistically significant (P,0.05).
RESULTS
Clinical validation of the SCCmec real-time PCR
assay
Over the study period, 3340 swabs were received from 1204
patients. In total, 148 (12.3 %) of the screens were MRSA-
positive, where 146 (12.1 %) were MRSA-positive by
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SCCmec real-time PCR assay. In contrast, 128 screens
(10.6 %) were MRSA-positive by culture. One hundred and
twenty-six (10.5 %) of the screens were positive by both
culture and PCR. Twenty of the 1204 (1.66 %) screens were
negative by culture and positive by PCR; these samples
were sequenced. In 14 of the cases, a homology search
confirmed the sequence as SCCmec, indicating that these
samples could be considered true positives and the
sensitivity and specificity adjusted accordingly. Two of
the 1204 (0.2 %) screens were positive by culture but
negative by PCR. As a follow-up, Etests for cefoxitin and
oxacillin resistance and an agglutination test for penicillin-
binding protein were carried out to confirm these two
screens during the study.
Compared with routine culture, the PCR-based assay had a
sensitivity of 98.6 %, a specificity of 99.4 %, and a PPV and
NPV of 95.9 and 99.8 %, respectively (Table 1). The PCR
method was significantly more sensitive (P,0.05) than the
culture method, detecting 0.7 % more MRSA screen
positives within a 1-month period.
Sites testing positive for MRSA by culture and
PCR
One hundred and twenty-six of the screens were positive by
both PCR and culture for MRSA. Of these, 103 PCR-
positive screens and 93 culture-positive screens were full
screens (combination of three swabs from nose, throat,
groin, wound or other), whilst the rest were partial screens
(two or fewer swabs). By PCR, nose swabs were positive in
88 screens (85 %), but additional positives were obtained
by PCR from other sites: five from the throat (5 %), eight
from the groin, wound or another site (8 %) and two from
a combination of these sites (2 %) (Fig. 1). In contrast, by
culture, nose swabs were positive in 72 screens (77 %),
whilst additional positives were obtained by culture from
the throat (five screens; 5 %), groin or wound (12 screens;
13 %) or a combination of these sites (four screens; 4 %)
(Fig. 2).
Total TATs
The mean TAT for the PCR-positive screen was 6 h 48 min
and for the PCR-negative screen was 6 h 12 min. The mean
TAT for the culture-positive screen was 67 h and for the
culture-negative screen was 27 h 30 min. Therefore, a
negative and a positive assay were approximately 22 and
60 h shorter, respectively, than those for the culture
method.
Cost
The consumables cost for MRSA culture screening was
£0.41 per swab versus £2.35 per swab for the PCR assays,
both incorporating the mean positive rate for swabs and
the concomitant cost of full identification. Comparison of
the projected costs for monthly screening for each of the
methods was £5711 (J6674) and £14 329 (J16 744),
respectively, as presented in Table 2.
Table 1. Breakdown of the screen results: comparison of the
SCCmec real-time PCR assay with routine culture
Where discrepancies between the real-time PCR assay and the culture
method occurred, sequencing of DNA extracts from the culture
screens was used to determine true- or false-positive or negative
culture results, and the sensitivity/specificity was adjusted accord-
ingly.
Culture and SCCmec sequencing
Positive Negative
PCR-positive 140 6
PCR-negative 2 1056
Sensitivity 98.6 %
Specificity 99.4 %
PPV 95.9 %
NPV 99.8 %
18
12
Nose
43
15
8
2
5
Groin/wound/other
Throat
Fig. 1. Venn diagram showing the number of PCR-positive isolates
from various sites.
19
16
Nose
23
14
12
4
5
Groin/wound/other
Throat
Fig. 2. Venn diagram showing the number of culture-positive
isolates from various sites.
Real-time PCR for MRSA screening
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Sequencing results from blood culture samples
The 161 MRSA isolated from blood cultures by a routine
culture method were all mecA PCR-positive. All isolates
were SCCmec PCR-positive. Sequencing showed that there
were 151 SCCmec II or IV (two forms with 254A/C and
263G/T), eight identical SCCmec III, one SCCmec I and
one that did not give convincing sequence data. The
sequence in relation to the reverse primer in SCCmec
showed two strains with one nucleotide difference within
the SCCmec II or IV types, and the primers were fully
compatible with the MRSA PCR.
DISCUSSION
In this study, an in-house PCR assay for MRSA was shown
to perform well in a routine diagnostic service. In order for
an assay to be successful in a routine diagnostic laboratory,
a high level of automation and robust straightforward
protocols are required. Here, a staff member with minimal
molecular skill was trained within a week to fully run the
routine assay. The design of this assay with an internal
control, which was co-extracted and co-amplified, ensured
that there was a robust check for the nucleic acid extraction
procedure and PCR inhibition. Of the 1204 screens tested,
less than 2 % of the screens needed to be repeated due to
internal control failure and they were subsequently
successfully retested from the freeze–thawed extract. The
high throughput of real-time PCR, with automated
extraction and no requirement for extensive post-amp-
lification analysis, also makes this assay plausible to use in
large-scale screening programmes.
The use of all blood culture MRSA isolates allowed
monitoring of the variation in local SCCmec types over
1 year. This process was used as a surveillance tool to
detect the emergence of variants of SCCmec that could
render the PCR ineffective. Here, no significant variants
were detected and the PCR detected all the isolates tested,
so no additional changes of the target primers were
necessary. The addition of a new primer into an in-house
assay would be a small change but would be completely
necessary to enable the assay to continue to improve and
detect significant SCCmec variants. According to
Hiramatsu et al. (2001) and Deurenberg et al. (2007),
MRSA evolution in SCCmec is likely to happen and the
monitoring of MRSA variation is necessary to maintain
confidence in the assay.
The design of this assay gave an improved assay
performance with sensitivity, specificity, PPV and NPV of
98.6, 99.4, 95.9 and 99.8 %, respectively; in other studies on
commercial PCR, the reported sensitivity varied between
95 and 97 % (Jonas et al., 2002; Fang & Hedin, 2003;
Francois et al., 2003, 2007; Bishop et al., 2006; Wren et al.,
2006; Gro
¨bner et al., 2009; Kobayashi et al., 2009). One
possible explanation is that this assay included extraction,
the Taqman probe and an internal control, which was co-
extracted and co-amplified, all of which enhanced the
performance of the assay. Overall, the real-time PCR assay
detected 0.7 % more MRSA-positive screens than the
routine standard Brilliance Chromogenic MRSA agar
culture method. It has been suggested that there is a need
for enrichment culture (Fang & Hedin, 2003). However,
the use of enrichment would increase the TAT, and this
PCR method has been shown to perform well without
enrichment (Niesters, 2004; Krishna et al., 2008). Any
laboratory performing tests for MRSA needs to weigh up
the patient benefit in relation to MRSA prevalence and the
benefit of rapid results. In our study, there were two
screens that were not detected by the PCR but were positive
by culture. The most likely explanations include poor
inoculation into the saline broth, a bacterial count on the
swab below the level of detection by PCR or false-positive
results. Sequence analysis of the cultured isolates from
these two screens confirmed that they had the full SCCmec
cassette present and a subsequent re-run of the isolates by
PCR proved that the assay was capable of detecting these
MRSA isolates. This problem could be avoided by using an
improved swab collection system such as the flocked swab
with liquid Amies medium (Chernesky et al., 2006); this
would negate the need to express the swab in a liquid prior
to PCR extraction.
With this improved assay and methodology, we were able
to maintain the TAT in the laboratory to within an 8 h
shift, i.e. within the same working day, whereas culture
always required substantially longer. For the purposes of
this study, TAT was calculated from booking-in time to
authorization, as specimens were collected throughout the
morning and the cut-off was set at 13.30 Monday to Friday.
The TAT could be improved further by reducing delays in
specimen collection from time of hospitalization, transport
from the patient to laboratory and telephoning of results.
In this study, it was found to take 12 h, on average,
whether for PCR or culture. This could be achieved by
developing a platform for testing in specific areas within
the hospital.
Testing of pooled samples proved effective: it had a
significant impact and helped decrease costs to the
laboratory. Screening programmes currently recommend
nasal swabs only; however, the risk is that a significant
number of MRSA carriers will not be identified in time. As
in Fig. 1, 15 cases (five positive only in throat, eight
Table 2. Comparison of the projected cost of screening per
swab
Routine
screening
Real-time
PCR assay
Consumables £0.41 £2.35
Staff £0.78 £0.66
Overheads £0.52 £1.28
Total cost per swab £1.71 £4.29
Total cost for 3340 swabs £5711.40 £14 328.60
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positive only in groin, wound or other, and two positive by
a combination of the two) would have been missed by
screening only nasal swabs. However, additional swabs add
complexity and cost to the laboratory, and the requirement
to process only nose swabs is driven by the need to have a
simple, less costly programme for screening.
Molecular screening is more expensive than culture.
However, the difference in cost for this assay was in
consumables and not in staffing time. The machinery used
is generic to other molecular platforms, which enables
laboratories to use these platforms for a range of PCR
assays. The assay described in this study cost £2.35 (J2.75)
per test, which is considerably less than commercial
options, most of which cost more than £10 (J11.69) per
test. Further study needs to be carried out to see whether
intervention within 1 day has an impact on hospital-wide
costs, number of patients, hospital stay, etc.
Further work would include other improvements to the
assay for automated liquid handling in the laboratory,
liquid swabs and laboratory interface machines so the assay
becomes as simple as pressing one button to obtain results,
as well as reducing the laboratory time for extraction and
PCR to enable test results to be available within 2 h.
This optimized, in-house, real-time PCR test maintained a
very high sensitivity and specificity when evaluated under
real-time laboratory conditions. The TAT of this real-time
PCR assay was substantially lower than that of chromo-
genic culture. It was also maintained throughout the entire
process, which can be taken as an indirect measure of the
test performance. Thus, implementation of such a
molecular test could be achieved with limited resources
in a standard microbiology laboratory.
ACKNOWLEDGEMENTS
This study was funded by an unrestricted grant from the Scottish
Executive. The funding source did not have any role in the study
design, execution, analysis or writing of the manuscript conclusions.
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