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Evaluation of the Xpert™ MRSA Assay on the GeneXpert Real-Time PCR 1
Platform for Rapid Detection of MRSA from Screening Specimens 2
3
Angela S. Rossney1,2*, Celine M. Herra3, Gráinne I. Brennan4, Pamela M. Morgan1, 4
Brian O’Connell1,2,4 5
6
National MRSA Reference Laboratory, St. James's Hospital, James's St., Dublin 8, 7
Ireland1. 8
Department of Clinical Microbiology, University of Dublin, Trinity College, St. 9
James’s Hospital, James’s St., Dublin 8, Ireland2. 10
School of Biological Sciences, Dublin Institute of Technology, Kevin St., Dublin 8, 11
Ireland3 12
Department of Microbiology, St. James's Hospital, James's St., Dublin 8, Ireland4. 13
14
Running Title: Xpert™ MRSA Assay for Rapid Detection of MRSA 15
Key Words: MRSA, Rapid Detection, Real-time PCR, Screening Specimens 16
17
*, Corresponding Author 18
Mailing Address: National MRSA Reference Laboratory, St. James's Hospital, James's 19
St., Dublin 8, Ireland. 20
Telephone: + 353 1 410 3662 21
Fax: + 353 1 410 3666 22
E-mail: arossney@stjames.ie 23
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Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
J. Clin. Microbiol. doi:10.1128/JCM.02487-07
JCM Accepts, published online ahead of print on 6 August 2008
2
ABSTRACT 24
The need for rapid methods to accurately detect methicillin-resistant 25
Staphylococcus aureus (MRSA) is widely acknowledged and a number of molecular 26
assays are commercially available. This study evaluated the Xpert™ MRSA assay which 27
is run on the GeneXpert real-time PCR platform (Cepheid) for use in a clinical 28
laboratory. The following parameters were investigated: 1) limit of detection (LoD) with 29
four MRSA strains, 2) ability to detect isolates of MRSA from a collection representative 30
of MRSA in Ireland since 1974 (n = 114) and ability to detect control strains with 31
SCCmec types IVa (IV.1.1.1), IVb (IV.2.1.1), IVc (IV.3.1.1), IVd (IV.4.1.1), V (V.1.1.1), 32
VT and VI, and 3) performance in a clinical trial with swabs from nose, throat and 33
groin/perineum sites from 204 patients where results were compared with direct and 34
enrichment culture. The average LoD of the four test strains was 610 CFU/ml (equivalent 35
to 58 CFU/swab). All 114 MRSA isolates and seven control strains tested were detected. 36
Sensitivity, specificity, positive and negative predictive values for clinical specimens 37
from all sites investigated was 90%, 97%, 86% and 98%, respectively, but throat 38
specimens yielded poor sensitivity (75%). Sensitivity, specificity, positive and negative 39
predictive values for nasal specimens was 95%, 98%, 90% and 99%, respectively. 40
Overall, the assay was rapid and easy to perform but performance might be enhanced by 41
the inclusion of an ‘equivocal’ interpretive category based on analysis of all available 42
amplification data. 43
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INTRODUCTION 44
45
Rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) with 46
early implementation of appropriate intervention has been reported to reduce prevalence 47
of MRSA especially in critical care areas (2, 6, 10). In 2004, Huletsky et al. described a 48
novel real-time PCR assay targeting DNA sequences in the region of the open reading 49
frame orfX where the staphylococcal cassette chromosome mec (SCCmec) integrates into 50
the S. aureus chromosome (8). SCCmec carries the resistance determinant mecA which 51
encodes methicillin resistance and exhibits at least six different structural types and 52
numerous subtypes (9). To detect MRSA with all SCCmec types known in 2004, the 53
assay used five forward primers designed to target sequences within SCCmec whilst a 54
sixth reverse primer and three probes were specific for orfX (8). Unlike earlier 55
amplification techniques investigating mecA, nuc and/or fem genes, this assay could 56
distinguish between MRSA and mixtures of methicillin-susceptible S. aureus (MSSA) 57
and methicillin-resistant coagulase-negative staphylococci (MR-CNS) (4, 8). The assay 58
became commercially available as the IDI-MRSA assay and is currently marketed as 59
BD GeneOhm MRSA Assay. 60
In 2007, a new real-time PCR MRSA assay also targeting DNA sequences in the 61
chromosomal orfX − SCCmec junction became available. This assay is the Xpert™ 62
MRSA kit (Cepheid, Sunnyvale, CA, U.S.A.) and it is used in conjunction with the 63
GeneXpert real-time PCR platform (Cepheid). In the Xpert™ MRSA assay, DNA from 64
specimens is extracted, amplified and detected in separate chambers of single-use 65
disposable cartridges which contain freeze-dried beads with all reagents required for the 66
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real-time process. Sample preparation time is minimal and the PCR reaction time is 75 67
minutes. The assay is validated for use with nasal specimens taken on Copan swabs with 68
Stuart’s liquid transport medium from patients at risk for colonisation with MRSA and 69
has a quoted limit of detection (LoD) of 80 CFU per swab (1). 70
The present study evaluated the Xpert™ MRSA assay for use in a clinical 71
laboratory. The following parameters were investigated: 1) LoD, 2) ability to detect 72
isolates of MRSA in a collection representative of MRSA in Ireland since 1974 and also 73
ability to detect isolates carrying recently described SCCmec elements and 3) 74
performance in a clinical trial with swabs from nose, throat and groin/perineum sites from 75
204 patients. 76
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MATERIALS AND METHODS 77
78
Limit of Detection (LoD) Assays. LoD assays of the Xpert™ MRSA kit were performed 79
using four MRSA isolates. These isolates exhibited a range of oxacillin MICs and were 80
representative of strains recovered in Ireland as described previously (18). LoDs were 81
determined on 10-fold dilutions of suspensions of each strain (from 105 to 101) adsorbed 82
onto Copan Stuart’s liquid transport medium double swabs (Copan 139C; Copan Italia 83
SPA, Brescia, Italy) (Copan swabs). Colony counts (CFU/ml) of each dilution were 84
determined by spiral plating onto brain heart infusion (BHI) agar (Oxoid CM375; Oxoid 85
Ltd., Basingstoke, U.K.) using a WASP spiral plater (Don Whitley Scientific Ltd., 86
Shipley, England). LoDs were determined for all four isolates in pure culture and in the 87
presence of a mixed ‘cocktail’ of bacteria consisting of 10 MSSA isolates (which were 88
mecA-negative by in-house conventional PCR), five MR-CNS and single isolates of 89
Moraxella catarrhalis, Escherichia coli and Candida species (18). Each component of 90
the ‘cocktail’ was tested with the Xpert™ MRSA kit prior to use. The bacterial 91
suspensions were also adsorbed onto Transwabs™ with Amies’ clear transport medium 92
[MW170; Medical Wire and Equipment Company, Corsham, England] (Transwabs) for 93
subsequent culture. 94
One Copan swab from each dilution was investigated for the presence of MRSA 95
using the Xpert™ MRSA kit on a GeneXpert DX System Version 1.2 (Cepheid, 96
Sunnyvale, CA, U.S.A.) according to the manufacturer’s instructions. The second Copan 97
swab was reserved for repeat testing if required. 98
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The corresponding Transwab was cultured onto Columbia agar (LabM Lab1; 99
International Diagnostics Group plc, Bury, U.K.) containing 7% (w/v) horse blood (BA), 100
MRSA-Select chromogenic agar (CA) (Bio-Rad 63747; Bio-Rad Life Science Group, 101
Marnes La Coquette, France) and into 4 ml tryptic soy broth (TSB, BD 211825; Becton 102
Dickinson) containing 6.5% NaCl (TSB). After 18 h incubation at 35°C, enrichment 103
broths (10-µl volumes) were subcultured onto BA and CA. All isolates from enriched and 104
direct culture were identified as S. aureus and confirmed as MRSA as described 105
previously (18). 106
To express the LoD value as CFU/swab, the volume adsorbed by either Copan 107
swabs or Transwabs was determined by measuring the difference in weight of bijoux of 108
saline before and after adsorbance for 10 seconds by each of 10 Copan swabs and 10 109
Transwabs. The average of the 10 replicate readings was taken as the average volume 110
adsorbed by each swab type. 111
LoDs were recorded as the lowest concentration to give positive results by the 112
Xpert™ MRSA kit or by culture. The Xpert™ MRSA assay defines a specimen as 113
MRSA-positive if the MRSA target has a cycle threshold (Ct) value within the valid 114
range (≤36 cycles). The LoDs for both culture methods were calculated on the basis of 115
growth of >1 colony on a single culture medium or ≥1 colony on more than one medium 116
and were expressed as the average of the counts obtained with the four isolates. 117
118
MRSA Isolates and MRSA Control Strains. To ensure the kit could detect MRSA 119
strains prevalent in Ireland, 114 MRSA isolates representative of MRSA recovered in 120
Ireland were investigated (isolate details are shown in Table 1) (20). In addition, seven 121
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control MRSA strains representing isolates carrying SCCmec IV subtypes IVa (IV.1.1.1), 122
IVb (IV.2.1.1), IVc (IV.3.1.1), IVd (IV.4.1.1), SCCmec V (V.1.1.1), VT and VI were 123
tested (3, 5, 15). Isolates were prepared in saline suspensions at concentrations of ~102 124
CFU/ml above the LoD of the kit (determined in the present study) and adsorbed onto 125
Copan swabs and Transwabs. One swab of each pair of Copan swabs was tested with the 126
Xpert™ MRSA assay and the Transwab was cultured onto BA and CA. Any isolate that 127
was negative with the kit was re-tested in pure culture and at one 10-fold higher 128
concentration. 129
130
Clinical Trial. The clinical trial was undertaken with specimens from patients attending 131
St. James’s Hospital (SJH), a large 936-bed tertiary-referral hospital catering for all 132
specialities except maternity and paediatric services. A difficulty encountered when 133
designing the clinical trial was that specimens processed by the Xpert™ MRSA assay 134
could not be used for subsequent culture because the kit’s elution reagent contains 135
sodium hydroxide and quanidium thiocyanate. Ethical approval was obtained to take 136
duplicate specimens from nose, throat and groin/perineum sites from patients undergoing 137
routine MRSA screening with Copan swabs after specimens on Transwabs had been 138
obtained for routine diagnostic culture. Criteria for routine screening were as described 139
previously (18). 140
Specimens collected with Copan swabs were tested using the Xpert™ MRSA kit 141
according to the manufacturer’s instructions as soon as possible after collection. 142
Specimens were tested in batches of eight using two four-module GeneXpert machines. If 143
not tested immediately, specimens were stored at 4°C. Following processing, all 144
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specimens were stored at 4°C until all molecular and culture testing was complete. 145
Specimens yielding invalid results were re-tested using the second Copan swab. 146
Specimens collected with Transwabs were inoculated onto CA in the diagnostic 147
laboratory and subsequently cultured into salt TSB. After incubation at 35°C for 18 h, 10-148
µl volumes of salt TSB were subcultured onto BA and CA. Growth was identified as 149
MRSA as described previously and the presence of mecA was confirmed by a 150
conventional in-house end-point PCR assay (18). All MRSA isolates were stored at –151
70°C on cryoprotective beads (Protect beads; Technical Service Consultants Limited, 152
Heywood, U.K.). Bacterial growth from BA plates from specimens yielding positive 153
Xpert™ MRSA kit results and negative MRSA culture results were also preserved at –154
70°C on cryoprotective beads. Discrepancies between culture and kit results were 155
investigated by salt enrichment culture of the second Copan swab (if available). 156
MRSA isolates recovered from specimens yielding kit-negative results were 157
prepared in suspensions in saline at concentrations of 105 CFU/ml, adsorbed onto Copan 158
swabs and tested using the Xpert™ MRSA kit. Bacterial growth from BA plates from 159
specimens which tested Xpert™ MRSA kit positive but from which MRSA was not 160
recovered in culture was similarly tested to exclude the possibility that such positive 161
results occurred as a result of MSSA or MR-CNS. 162
163
Control Strains. S. aureus ATCC 25923 and S. aureus ATCC 43300 were used as 164
negative and positive controls, respectively. S. epidermidis ATCC 12228 was also used 165
as a negative control as recommended by the manufacturer. Control isolates were 166
prepared in saline suspensions containing 105 CFU/ml and adsorbed onto Copan swabs 167
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prior to testing. When determining LoDs, positive and negative controls were included 168
with each batch of tests. When MRSA strains were tested, a negative control was 169
included each day. During the clinical trial, a positive control was included once a week 170
and after any batch of specimens where all specimens yielded kit-negative results. S. 171
aureus ATCC 25923 and S. aureus ATCC 43300 were also included as negative and 172
positive controls with each batch of CA plates. 173
174
Statistics. In the clinical trial, sensitivity, specificity, positive and negative predictive 175
values of the Xpert™ MRSA kit were calculated for all specimen types by comparison 176
with direct culture and by comparison with enrichment culture. Because the Xpert™ 177
MRSA kit detects DNA which may come from non-viable MRSA whereas culture 178
detects viable organisms only, Xpert™ MRSA kit-positive specimens that were culture-179
negative were considered possible ‘true’ positive results if the patient had been 180
previously positive for MRSA within the last two years or was positive at another site. 181
Sensitivity, specificity, positive and negative predictive values were also calculated using 182
these ‘amended’ results for all specimen sites and for each specimen site individually. 183
Further calculations were made when patients were grouped into those who were known 184
to have been previously positive for MRSA and those who were not known to have been 185
previously positive. 186
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RESULTS 187
188
LoD Assays. The average LoD value for the four MRSA test isolates in pure culture 189
with the Xpert™ MRSA kit using Copan swabs was 610 CFU/ml. The average volume 190
adsorbed by Copan swabs was 95 µl/swab; hence the average LoD/swab was 58 CFU. 191
The amplification data obtained with two of these isolates suggested that both isolates 192
were positive at concentrations below 58 CFU/swab but since Ct values of 36.4 and 37.1 193
were obtained, the kit recorded negative results (FIG. 1, Panels A and B). The average 194
LoD values of direct and enrichment culture using Transwabs was 750 CFU/ml and 40 195
CFU/ml, respectively. The average volume adsorbed by Transwabs was 228 µl/swab; 196
hence the average LoDs of direct and enrichment culture were 171 and 9 CFU/swab, 197
respectively. The average LoD of the kit for the four test isolates in the presence of mixed 198
flora was the same as the LoD obtained in pure culture (600 CFU/ml; 57 CFU/swab). 199
During preparation of the mixed culture ‘cocktail’, the MSSA component yielded 200
a kit-positive result. When the ‘cocktail’ was prepared using fresh subcultures of the 201
same 10 MSSA isolates, the ‘cocktail’ tested negative and continued to test kit-negative 202
on the successive days when the LoDs of the kit were determined. However when the 203
‘cocktail’ was re-tested following completion of the LoD assays, the kit again recorded a 204
positive result although the accompanying amplification curve failed to demonstrate 205
evidence of efficient amplification suggesting this could be a false-positive result. 206
Conversely, although the Xpert™ assay always recorded negative results for the MSSA 207
control strain S. aureus ATCC 25923 (FIG. 1, Panel C), on some occasions the 208
amplification data demonstrated evidence of amplification of MRSA target DNA with Ct 209
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values just exceeding the ‘cut-off’ value of 36 cycles (for example, Ct value of 37.3) and 210
accompanying amplification curves showing an exponential rise in fluorescence emission 211
(FIG. 1, Panel D). 212
213
MRSA Isolates and MRSA Control Strains. When designing this study, it was 214
intended that the MRSA isolates and MRSA control strains would be tested in the 215
presence of the mixed ‘cocktail’ of bacteria to mimic normal flora but the inconsistent 216
results with the MSSA component of the ‘cocktail’ suggested that this would be unwise. 217
Isolate suspensions were therefore prepared in pure culture in saline at a concentration of 218
~104 CFU/ml (i.e. ~102 CFU/ml above the LoD determined in the present study). All 219
MRSA isolates except two yielded positive Xpert™ MRSA kit results; one negative 220
isolate was positive on repeat testing and the second negative isolate was positive when 221
re-tested at a higher concentration (105 CFU/ml). All seven control MRSA strains yielded 222
positive results. 223
224
Clinical Trial. Six hundred and twelve specimens from 204 patients were investigated. 225
An overview of the numbers of positive specimens obtained with the Xpert™ MRSA kit, 226
with direct culture, with enrichment culture and with ‘amended’ results is shown in Table 227
2. Using ‘amended’ results, Xpert™ MRSA kit positive specimens that were culture-228
negative were considered to be ‘true’ positive results if the patient had been previously 229
positive for MRSA or was positive at another site. MRSA was detected in 99 specimens 230
(16.2%; 99/612) from 58 patients (28.4%, 58/204) by the Xpert™MRSA kit. Direct 231
culture yielded positive results from 59 specimens (9.7%, 59/606) from 37 patients 232
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(18.3%, 37/202); direct culture results were unavailable for six specimens from two 233
patients. Specimens from 89 specimens (14.5%, 89/612) from 45 patients (22.1%, 234
45/204) were positive by enrichment culture. No specimen was culture-positive by direct 235
culture and negative by salt enrichment culture. When ‘amended’ results were calculated, 236
95 specimens (15.5%; 95/612) from 47 patients (23.0%; 47/204) were deemed positive. 237
Sixteen specimens from 14 patients yielded invalid Xpert™ MRSA kit results on initial 238
testing but on repeat testing only four specimen results remained invalid [throat (n = 3); 239
groin (n = 1)]. 240
MRSA isolates from all but two of the 19 culture-positive kit-negative specimens 241
were kit-positive when tested in pure culture at a concentration of 105 CFU/ml. One of 242
the two negative isolates tested positive when re-tested at 105 CFU/ml; the other 243
remained kit negative when re-tested at both 105 and 106 CFU/ml (FIG. 1, Panel E). 244
Repeat testing of the latter isolate at 106 CFU/ml also yielded a negative kit result but 245
evidence of amplification (with a Ct value of 36.7) was noted on this occasion (FIG. 1, 246
Panel F). This isolate was mecA gene positive by conventional in-house endpoint PCR. 247
Non-MRSA bacterial growth from BA plates from specimens which were kit-248
positive and MRSA culture-negative was also tested by the kit but growth from all these 249
non-MRSA cultures yielded negative results with the kit. 250
251
Statistics. The sensitivity, specificity, positive and negative predictive values of the 252
Xpert™ MRSA kit compared with direct culture, enrichment culture and with ‘amended’ 253
results are shown in Table 3. With nasal swabs, when the ‘amended’ results were 254
considered, the sensitivity of the kit was 95%. In comparison, sensitivity for specimens 255
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from the throat was only 75%. When the Xpert™ MRSA kit results from specimens from 256
all sites were compared with ‘amended’ results, the overall sensitivity, specificity, 257
positive and negative predictive values were 90%, 97%, 86% and 98%, respectively 258
(Table 3). When this analysis was restricted to specimens from patients who were 259
recorded as being previously MRSA-positive, the sensitivity of the kit for nasal 260
specimens was 93% whereas with specimens from patients with no record of being 261
previously positive (i.e. new MRSA patients), the sensitivity for nasal specimens was 262
100%. 263
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DISCUSSION 264
265
Ireland has a serious problem with MRSA and the proportion of MRSA among S. 266
aureus blood culture isolates is among the highest in Europe (13). It has been reported 267
that in areas where MRSA is endemic, the introduction of a full ‘search and destroy’ 268
approach including screening of all patient contacts of all index cases and the institution 269
of precautionary isolation for all such contacts could reduce the prevalence of MRSA to 270
<1% within six years (2). It has also been reported that rapid detection of carriage has an 271
important role to play in such a ‘search and destroy’ strategy (2). A study investigating 272
the value of rapid diagnostic tests (RDT) for MRSA when used for admission screening 273
to a critical care area reported a reduction in the incidence of transmission of MRSA from 274
13.89/1000 patient days to 4/1000 patient days (6). 275
Whilst RDTs may play a major role in reducing the prevalence of MRSA, the 276
chosen method must be sensitive, specific and have good positive and negative predictive 277
values. Failure to recognise a particular strain of MRSA can have very serious 278
consequences as was recently experienced in the Netherlands where phenotypic tests 279
failed to detect a mecA-positive strain with a low oxacillin MIC in one hospital. Over a 280
two-year period this strain spread extensively not only within that hospital but in other 281
hospitals also and it took over a year to control the resulting outbreaks (10). 282
As more molecular RDTs for MRSA become available, the choice of an 283
appropriate assay becomes more difficult. Evaluating new molecular assays is expensive 284
and time-consuming and it is hard to exclude confounding variables even when assays 285
are evaluated at the same time. For example, the present study required duplicate 286
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specimens to allow comparison between the Xpert™ MRSA kit and enrichment culture. 287
Because sampling using the routine Transwab method had to be undertaken prior to 288
sampling with Copan swabs to ensure the routine diagnostic culture protocol was not 289
compromised by the clinical trial, it is possible that less material was available on the 290
Copan swabs for detection by the Xpert™ MRSA assay. In contrast, in an earlier study 291
evaluating the IDI-MRSA™ kit on the SmartCycler platform, swabs were cultured by salt 292
enrichment after they had been used with the kit (18). In that evaluation however, the 293
LoD of salt enrichment culture (using Transwabs) was 240 CFU/ml (55 CFU/swab) 294
whereas the LoD of salt enrichment culture (using Transwabs) in the present study was 9 295
CFU/swab indicating that the Xpert™ MRSA kit was subjected to a more rigorous 296
evaluation. A possible explanation for the lower LoD of salt enrichment culture in the 297
present study is that the volume of medium used for salt enrichment was 4 ml whereas 298
the protocol in the IDI-MRSA™ kit evaluation used 1-ml volumes and enrichment 299
culture was performed after material on the swab was removed for testing with the IDI-300
MRSA™ kit. Interestingly the inclusion of enrichment culture increased the number of 301
culture-positive specimens by 23% in the earlier study and by 51% (30/59) in the present 302
study (18). 303
The Xpert™ MRSA assay demonstrated an average LoD (58 CFU/swab) three-304
fold lower than direct culture (171 CFU/swab) but six-fold higher than enrichment 305
culture (9 CFU/swab). In comparison, the earlier IDI-MRSA™ kit evaluation reported an 306
average LoD value of 2000 CFU/ml (equivalent to 190 CFU/swab) which was similar to 307
LoD of direct culture but 10-fold higher than the LoD of enrichment culture (18). The 308
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LoDs of direct culture using Transwabs was comparable in both studies; 171 CFU/swab 309
in the present study and 800 CFU/ml (182 CFU/swab) in the earlier study (18). 310
The Xpert™ MRSA assay detected all MRSA isolates investigated including 311
control isolates exhibiting SCCmec V, VT and VI (3, 5, 15). Although not investigated 312
here, both Xpert™ MRSA assay and IDI-MRSA™ are reported to lack sensitivity when 313
detecting the pulsed field gel electrophoresis non-typeable MRSA strain associated with 314
pigs in the Netherlands (17, 21). 315
In the clinical trial, the Xpert™ MRSA assay exhibited sensitivities of 95% and 316
97% for detection of MRSA from nasal and groin/perineum sites but had difficulty 317
detecting MRSA DNA from throat specimens (sensitivity 75%). Interestingly although 318
the previous evaluation of the IDI-MRSA™ kit reported lower sensitivities for nose and 319
groin/perineum specimens (90% and 88%, respectively), specimens from the throat 320
yielded a higher sensitivity (89%) (18). 321
A major difficulty evaluating molecular assays for detection of MRSA from 322
clinical specimens is defining ‘true’ positive and negative specimens. In the present 323
study, data were analysed by direct comparison with culture but to overcome the problem 324
of considering all kit-positive culture-negative specimens as false-positive kit results, kit 325
performance was analysed using ‘amended’ results where kit-positive culture-negative 326
results from patients who were previously positive for MRSA were regarded as ‘true’ 327
positive results. Using these ‘amended’ results for comparison, the sensitivities and 328
specificities of direct culture, enrichment culture and the Xpert™ MRSA assay were 329
60.6% and 99.6%, 84.2% and 98.3% and 89.5% and 93.3%, respectively. However this 330
comparison is not completely valid and may overestimate the sensitivity of the assay and 331
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underestimate that of culture because culture-negative results from patients who have 332
undergone successful treatment for MRSA should not be considered false-negative 333
culture results. 334
Despite the encouraging findings of the present study, the overall performance of 335
the Xpert MRSA assay might be improved if result interpretation took account of all 336
available assay data. The manufacturer’s interpretation is based on the application of a Ct 337
‘cut-of’ value of 36 cycles with specimens being recorded as MRSA-positive if the Ct is 338
≤36 and MRSA-negative if the Ct is >36, regardless of the presence or absence of 339
evidence of amplification (the former being demonstrated by an amplification curve 340
indicating an exponential rise in fluorescence emission). Data obtained in the present 341
study suggested that this Ct ‘cut-off’ value might have introduced false negative results in 342
both the LoD investigations and during the clinical trial where some tests (n = 14) 343
demonstrated clear evidence of MRSA amplification but Ct values exceeded 36 cycles 344
(FIG. 1 Panels A, B and F). Conversely, false-positive results may have also occurred 345
(with for example, the MSSA component of the mixed culture ‘cocktail’) where Ct values 346
≤36 were obtained and reported as positive but amplification curves failed to demonstrate 347
an exponential rise. The inclusion of an ‘equivocal’ interpretative category for tests 348
where the amplification data of the MRSA target DNA fails to support the Ct ‘cut-off’ 349
result warrants investigation. 350
Aside from the interpretative difficulties associated with the use of the Ct ‘cut-off’ 351
value, findings from the present study also indicate that false-positive results may have 352
occurred due perhaps to lack of specificity of the assay’s proprietary target sequence for 353
MRSA at the SCCmec − orfX junction. There are increasing numbers of reports of SCC 354
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elements that do not contain mecA for example, SCCcap1 in MSSA which integrates into 355
the SCCmec chromosomal attachment site attB at the end of orfX (11, 12, 14). Although 356
the Xpert™ assay recorded negative results for the MSSA control S. aureus ATCC 357
25923, on some occasions the accompanying amplification curves demonstrated evidence 358
of amplification of target DNA (FIG. 1, Panel D) suggesting that like the IDI-MRSA 359
assay, the Xpert™ MRSA kit may have the problem of detecting SCC in MSSA strains 360
such as S. aureus ATCC 25923 which may be why the manufacturer recommends using 361
S. epidermidis ATCC 12228 as the negative control (7). This problem requires further 362
investigation. 363
Whilst the Xpert™ MRSA assay requires more interpretation than currently 364
suggested by the manufacturer, it was found to perform well with nasal and 365
groin/perineum specimens. It is rapid, easy to use and has the advantage of random 366
access (i.e. an urgent specimen can be accommodated because specimens do not need to 367
be tested in batches). Relative to culture, it is expensive and results need to be confirmed 368
by culture. A more extensive prospective study is warranted to determine its role in 369
clinical practice in Ireland. A question that such a prospective clinical study might 370
investigate is the duration of kit-positive results from specimens from patients who have 371
been successfully treated for MRSA and whether there is a period during which 372
previously culture-positive patients should not be screened by molecular methods. These 373
data could inform how kit-positive results from patients who are known to have been 374
previously positive for MRSA should be interpreted. 375
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ACKNOWLEDGEMENTS 376
377
We thank the participant patients, infection control staff (Lisa Fetherstone and Catherine 378
O’Reilly) and the clinical staff in St. James’s Hospital (SJH) for duplicate specimens. 379
Thanks to Mary Kelleher, Surveillance Scientist, Microbiology Dept., SJH for data on 380
diagnostic specimens. We thank Biofact Ireland and Cepheid for providing the 381
GeneXpert machines for the duration of the evaluation and for providing the Xpert™ 382
MRSA kits at an evaluation price. We thank Teruyo Ito (Juntendo University, Tokyo, 383
Japan), Robert Daum (University of Chicago, Chicago, Illinois, USA) and Herminia de 384
Lancastre (Instituto de Technologia Quimica e Biologica, Oeiras, Portugal) for the gift of 385
control strains carrying SCCmec IV subtypes, SCCmec V and SCCmec VI. 386
387
Declaration 388
There are no conflicting interests to declare. 389
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TABLE 1. MRSA isolates used to determine whether the Xpert™ MRSA kit could detect 473
all strains of MRSA prevalent in Ireland (n = 114) 474
MLST SCCmec Antibiogram-Resistogram (AR) typea
ST5b (n = 10) II 07.3b (n = 3); 07.4 (n = 5); 11 (n = 2)
ST5 (n = 1) IV Unfamiliarc (n = 1)
ST8d (n = 11) IV or IVv 43 (n = 8); Unfamiliarc (n = 3d)
ST8 (n = 26) II or IIv 13 (n = 7); 14 (n = 15); New03 (n = 3); 05 (n = 1)
ST12 (n = 1) IV ‘NT’ (n = 1)
ST22 (n = 14) IV 06 (n = 13); ‘NT’ (n = 1)
ST30 (n = 2) IV ‘NT’ (n = 2)
ST34 (n = 1) IV ‘NT’ (n = 1)
ST36 (n = 3) II 07.0 (n = 2); 07.2 (n = 1)
ST45 (n = 1) IV ‘NT’ (n = 1)
ST239 (n = 13) III or IIIv 01 (n = 4); 09 (n = 3); 15 (n = 2); 44 (n = 3); 23 (n = 1)
ST247 (n = 3) IA 22 (n = 2); New02 (n = 1)
ST250 (n = 4) I or Iv 02 (n = 4)
…(n = 6) … 07 (n = 2); New02 (n = 1); 06 (n = 1); SCV (n = 2)
…(n = 18) … PVL-positive community-acquired MRSA (n = 18)
aAR types were determined from the patterns of resistance obtained when isolates were 475
tested by disk diffusion against a panel of 23 antimicobials as described previously (19). 476
b One isolate was a double locus variant of ST5. 477
c Unfamiliar AR pattern. 478
d Two isolates were single locus variants of ST8. 479
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…, not done; MLST, multilocus sequence type; ‘NT’, these AR patterns were designated 480
‘No Type’ (‘NT’) pending the results of DNA macrorestriction digestion analysis; PVL, 481
Panton-Valentine leucocidin; SCV, small colony variant; v, previously reported variants 482
of SCCmec (20) (16). 483
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484
TABLE 2. Overview of results obtained with the Xpert™ MRSA kit and culture from 485
612 specimens 486
Xpert™ MRSA Kit
Positive Negative Invalidb Total
Direct Culturea Positive 52 7 … 59
Negative 46 497 4 547
Total 98 504 4b 606a
Enrichment Culture Positive 70 19 … 89
Negative 29 490 4 523
Total 99 509 4b 612
Amended Resultsc Positive 85 10 …
……
… 95
Negative 14 499 4 517
Total 99 509 4b 612c
aDirect culture results were unavailable on six specimens from two patients. 487
bFour isolates were invalid by the Xpert™ MRSA kit. 488
cAmended results include numbers of specimens that were culture-positive and those that 489
were Xpert™ MRSA kit-positive but culture-negative from previously positive patients 490
or from patients from whom a specimen from another site was positive. 491
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TABLE 3. Sensitivity, specificity, positive and negative predictive values (expressed as a 492
percentage) of the Xpert™ MRSA kit compared with direct and enrichment culture from 493
nose, throat and groin/perineum specimens 494
Xpert™ MRSA Kit
Reference Method Site Sensitivity
Specificity PPV NPV
Direct Culture All 88 92 53 99
Enrichment Culture All 79 94 71 96
Nose 84 92 67 97
Throat 63 98 83 93
Groin / Perineum 92 94 69 99
Amended Resultsa All 90 97 86 98
Nose 95 98 90 99
Throat 75 98 88 96
Groin / Perineum 97 96 80 99
Previously Positive Patientsb
Amended Resultsa All 87 100 100 87
Nose 93 100 100 88
Throat 73 100 100 77
Groin / Perineum 95 100 100 96
Not Previously Positive Patientsc
Amended Resultsa All 96 97 62 100
Nose 100 97 67 100
Throat 83 98 63 99
Groin / Perineum 100 95 59 100
aAmended results: See footnote to Table 2. 495
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bAnalysis restricted to specimens from patients who were previously MRSA-positive. 496
With these specimens, kit-positive culture-negative specimens were also included as true 497
positives because the patients were known to have been previously positive for MRSA. 498
cAnalysis restricted to specimens from patients with no record of being previously 499
MRSA-positive. With these specimens, kit-positive culture-negative specimens were also 500
included as true positives if the patient was positive for MRSA at another site. 501
All, nose, throat and groin/perineum. 502
PPV, positive predictive value; NPV, negative predictive value. 503
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FIG. 1. Amplification data obtained with the Xpert MRSA kit for two of the four 504
MRSA isolates used in limit of detection assays (Panels A and B), two replicates of S. 505
aureus ATCC 25923 (Panels C and D) and two replicates of an MRSA isolate (tested at 506
106 CFU/ml) recovered from a specimen yielding a kit-negative result (Panels E and F). 507
All isolates demonstrated kit-negative results with appropriate amplification of the 508
internal control. The amplification plots in Panels A and B demonstrate an exponential 509
rise in fluorescence emission consistent with amplification of the MRSA target 510
suggesting false-negative kit results. The amplification data shown in Panel C are 511
representative of results obtained with most replicates of S. aureus ATCC 25923 but on 512
some occasions, this methicillin-susceptible S. aureus isolate yielded a plot suggestive of 513
MRSA target amplification (Panel D). The data shown in Panels E and F were obtained 514
from an MRSA isolate tested at a bacterial concentration of 106 CFU/ml on two separate 515
occasions. Whilst both replicates yielded kit-negative results, one replicate demonstrated 516
evidence of MRSA target amplification (Panel F). 517
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