evaluation of carbapenemase screening and confirmation...
TRANSCRIPT
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Evaluation of carbapenemase screening and confirmation tests in 1
Enterobacteriaceae and development of a practical diagnostic algorithm 2
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Florian P. Maurer1, Claudio Castelberg
1, Chantal Quiblier
1, Guido V. 4
Bloemberg1, Michael Hombach
1,‡ 5
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1) Institut für Medizinische Mikrobiologie, Universität Zürich, 8006 Zürich, Schweiz 7
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Running title: Diagnostic algorithm for carbapenemase detection 9
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Keywords: meropenem, imipenem, ertapenem, ESBL, AmpC, Carba NP, antibiotic 11
resistance 12
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‡Corresponding author: 14
Michael Hombach, M.D. 15
Institut für Medizinische Mikrobiologie 16
Universität Zürich 17
Gloriastr. 30/32 18
8006 Zürich 19
Switzerland 20
Phone: 0041 44 634 27 00 21
Fax: 0041 634 49 06 22
Email: [email protected] 23
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JCM Accepts, published online ahead of print on 29 October 2014J. Clin. Microbiol. doi:10.1128/JCM.01692-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Abstract 30
Reliable identification of carbapenemase producing Enterobacteriaceae is 31
necessary to limit their spread. This study aimed at developing a diagnostic flow-32
chart suitable for implementation in different types of clinical laboratories using 33
phenotypic screening and confirmation tests. In total, 334 clinical Enterobacteriaceae 34
isolates genetically characterized with respect to carbapenemase, extended-spectrum-35
beta-lactamase (ESBL), and AmpC genes were analyzed. 142/334 isolates (42.2%) 36
were suspicious for carbapenemase production, i.e. intermediate or resistant to 37
ertapenem AND/OR meropenem AND/OR imipenem according to EUCAST clinical 38
breakpoints (CBPs). A group of 193/334 isolates (57.8%) showing susceptibility to 39
ertapenem AND meropenem AND imipenem was considered as negative control 40
group for this study. CLSI and EUCAST carbapenem CBPs and the new EUCAST 41
MEM screening cut-off were evaluated as screening parameters. ETP, MEM and IPM 42
+/- aminophenylboronic acid (APBA) or EDTA combined-disc tests (CDTs), and the 43
Carba NP-II test were evaluated as confirmation assays. EUCAST temocillin cut-offs 44
were evaluated for OXA-48 detection. The EUCAST MEM screening cut-off (< 25 45
mm) showed a sensitivity of 100%. The ETP APBA-CDT on Muller-Hinton agar 46
containing cloxacillin (MH-CLX) displayed 100% sensitivity and specificity for class 47
A carbapenemase confirmation. ETP and MEM EDTA-CDTs showed 100% 48
sensitivity and specificity for class B carbapenemases. Temocillin diameters/MIC 49
testing on MH-CLX was highly specific for OXA-48 producers. The overall 50
sensitivity, specificity, PPV, and NPV of the Carba NP-II test were 78.9%, 100%, 51
100%, and 98.7%, respectively. Combining the EUCAST MEM carbapenemase-52
screening cut-off (< 25 mm), ETP (or MEM) APBA- and EDTA-CDTs, and 53
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temocillin disk diffusion on MH-CLX agar promises excellent performance for 54
carbapenemase detection. 55
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Introduction 56
In recent years, the emergence of diverse carbapenemases in Enterobacteriaceae has 57
become a major challenge for healthcare systems (1). Carbapenemase producing 58
bacterial isolates pose a severe clinical problem as non-susceptibility to beta-lactams is 59
frequently accompanied by co-resistance to additional drug classes, e.g. 60
aminoglycosides or quinolones (2, 3). As a consequence, treatment options for 61
carbapenemase producers are alarmingly limited and often drugs displaying significant 62
side effects need to be administered as a last resort (4). 63
β-lactamases are classified according to their functional properties and molecular 64
structure by Ambler and Bush (5, 6). Some of these enzymes also display hydrolytic 65
activity towards carbapenems, e.g. Klebsiella pneumoniae carbapenemase (KPC, 66
Ambler/Bush class A), the New Delhi metallo-β-lactamase (NDM-1), VIM, and GIM 67
type enzymes (all Ambler/Bush class B), or OXA-48 (Ambler/Bush class D). A key 68
characteristic used for discriminating enzymes belonging to different Ambler/Bush 69
classes is the responsiveness to specific inhibitors: Class A enzymes are inhibited by 70
clavulanic and aminophenylboronic acid (APBA), class B enzymes are inhibited by 71
EDTA, and class D enzymes do not respond to any inhibitors used in β-lactamase 72
diagnostics (5, 6). 73
KPC enzymes were first detected in the USA in 1996 and have subsequently spread 74
worldwide (7). In Europe, KPC is endemic in Italy, Greece, Poland, and northwestern 75
England (7). In Central Europe, France, and Spain other carbapenemases are reported 76
more frequently. NDM-1 is endemic in India, Bangladesh, and Pakistan. In Europe, 77
most NDM-1 are being isolated in Great Britain (8). OXA-48 is endemic in Turkey 78
and Morocco, but is increasingly reported from other European countries mostly in 79
repatriated patients (8, 9). Scandinavian countries, the Netherlands, and other 80
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countries such as Switzerland generally report low prevalence rates for all 81
carbapenemases. Thus, rapid and reliable detection of carbapenemases is desirable in 82
order to limit the spread of these enzymes. 83
Detection of carbapenemase producing bacteria comprises carrier screening and 84
detection of carbapenemase production in routine antimicrobial susceptibility testing 85
(AST). While chromogenic media are often used for carrier screening, laboratory 86
strategies for β-lactamase detection in routine AST consist of a screening and a 87
confirmation step (10-14). 88
A variety of phenotypic and molecular, commercially available and in-house 89
laboratory tests have been described for carbapenemase detection. Molecular 90
techniques comprise end point and real-time PCRs as well as microarray techniques 91
(15-17). Critical diameters/MICs of ertapenem (ETP), meropenem (MEM), and 92
imipenem (IPM), and automated microdilution expert systems have been evaluated as 93
screening methods (14, 18-20). For carbapenemase confirmation, the modified Hodge 94
test is recommended by CLSI and various commercial and in-house combined disk 95
tests (CDTs) using boronic acid derivatives and EDTA/dipicolinic acid as specific 96
inhibitors have been described (13, 19-25). In 2014, EUCAST published new 97
guidelines for the detection of resistance mechanisms including carbapenemases, in 98
which a CDT is recommended for carbapenemase confirmation (14, 22, 25). Recently, 99
Nordmann et al. described a new inhibitor-based biochemical assay for carbapenemase 100
detection, the Carba NP test, which has been published in two versions: The Carba 101
NP-I assay provides a positive or negative result (“carbapenemase detected/not 102
detected”) whereas the Carba NP-II test has been designed to also discriminate 103
between carbapenemase classes A, B, and D (26-29). Apart from the original 104
publications, few studies have systematically evaluated the Carba NP-I test for 105
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Enterobacteriaceae, and both the originally published protocol and modified versions 106
were used. Reported sensitivities varied between 72.5% and 100%, whereas specificity 107
generally was reported to be 100% (30-33). Except for its original description the 108
Carba NP-II assay has been systematically evaluated for Pseudomonas aeruginosa 109
only (30, 31, 34-36). 110
Several issues of carbapenemase detection remain challenging: i) Enterobacteriaceae 111
overexpressing AmpC β-lactamases in combination with reduced cell-wall 112
permeability compromise the specificity of APBA-CDTs as the inhibitor (APBA) 113
affects both AmpC and carbapenemases (37-44); ii) Detection of OXA-48 and related 114
enzymes remains problematic as no specific inhibitor is available. Temocillin-115
resistance was suggested as an indicator for OXA-48 production, but not for OXA-48 116
confirmation (14, 25, 31, 45, 46). 117
This study aimed at developing a modular diagnostic flow-chart suitable for all types 118
of clinical laboratories, which integrates various phenotypic screening and 119
confirmation tests for highly sensitive and specific carbapenemase detection. 120
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Materials and Methods 131
Bacterial isolates. In total, 334 non-duplicate clinical isolates recovered in our 132
laboratory from 2009 until 2014 were included in the study (Table 1). All isolates were 133
genetically characterized for the presence of ESBL (TEM-ESBL, SHV-ESBL, and 134
CTX-M types), plasmid-encoded AmpCs, chromosomal ampC promoter/attenuator 135
mutations leading to overexpression (Escherichia coli only), and for the presence of 136
carbapenemases (16, 47, 48). 142/334 isolates (42.2%) were considered suspicious 137
for carbapenemase production due to non-susceptibility to ertapenem AND/OR 138
meropenem AND/OR imipenem (intermediate or resistant zone diameters according to 139
EUCAST CBPs), whereas 193/334 isolates (57.8%) considered non-suspicious for 140
carbapenemase production (susceptible to ertapenem AND meropenem AND 141
imipenem) served as a negative control group. 142
Genetic detection of carbapenemase, ESBL and ampC genes. Total DNA was 143
extracted from bacterial colonies after growth on sheep blood agar medium using the 144
InstaGene Matrix (Bio-Rad, Reinach, Switzerland). Genetic detection of 145
carbapenemase genes was done by performing a carbapenemase multiplex PCR (16). 146
For variant analysis OXA-48 genes were amplified with primers described(49). PCR 147
amplicons were sequenced using PCR primers and sequences analyzed using GenBank 148
and DNASTAR Lasergene software (DNASTAR Inc., Madison, Wisconsin USA). The 149
AID ESBL line probe assay (AID Autoimmun Diagnostika GmbH, Germany) was 150
used for the detection of ESBL genes (50). Bacterial isolates were genetically 151
characterized for the presence of plasmid-mediated AmpC type β-lactamase genes by 152
multiplex PCR (51). Chromosomal ampC promoter mutations of E. coli isolates were 153
analyzed as described previously (52). 154
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Susceptibility testing. Disk diffusion susceptibility testing was done according to 155
EUCAST recommendations (53). Antibiotic disks and Mueller-Hinton (MH) agar were 156
obtained from Becton Dickinson, Franklin Lakes, NJ. Cloxacillin supplemented 157
Mueller-Hinton (MH-CLX) agar was obtained from Axonlab AG, Baden, Switzerland. 158
Zone diameters were recorded using the Sirweb/Sirscan system (i2a, Montpellier, 159
France). Minimal inhibitory concentrations (MICs) were determined by gradient 160
diffusion (Etest, bioMérieux, Marcy L’Etoile, France) according to the manufacturer´s 161
instructions. 162
Combined-disk tests (CDTs) for carbapenemase detection. CDTs were performed 163
as described elsewhere (19, 24). Sets of two disks each containing IPM (10 μg), MEM 164
(10 μg), or ETP (10 μg, all Becton Dickinson) were placed onto MH (EDTA-CDT) or 165
both MH and MH-CLX (APBA-CDT) plates inoculated with a sample of the tested 166
isolate (0.5 McFarland turbidity standard). Immediately after placing the disks onto the 167
agar, 10 μL of a 29.2-mg/mL (0.1 M) EDTA solution (EDTA-CDT), or 10 μL of a 30-168
mg/mL APBA solution (APBA-CDT) were added to one of the two carbapenem disks 169
in each set. Plates were incubated at 35°C for 16 to 20 hours, and zone diameters were 170
recorded using the Sirweb/Sirscan system (i2a). Disc diameter differences of ≥ 5 mm 171
between the APBA-free and APBA-containing discs or between the EDTA-free and 172
EDTA-containing discs were considered indicative for production of a class A 173
carbapenemases and class B carbapenemase, respectively. 174
Carba NP-II test. The Carba NP-II test was performed and interpreted as described 175
(26). Reactions were read after 0, 30, 60 and 120 minutes of incubation. Color changes 176
from red to yellow-orange were interpreted as follows: wells 2 and 4, positive (Ambler 177
class A carbapenemase); wells 2 and 3, positive (Ambler class B carbapenemase); 178
wells 2, 3 and 4: positive (probably Ambler class D carbapenemase); no well, 179
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carbapenemase negative; all wells, test not interpretable. The Carba NP-II test was 180
performed by experienced personal, and all discrepant results were additionally 181
repeated at least 3 times. 182
Software. All calculations were done using the IBM SPSS statistics software version 183
20 (IBM Corporation, Armonk, NY) and the Microsoft Excel 2010 software (Microsoft 184
Corporation, Redmond, WA). 185
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Results 186
Evaluation of screening parameters for carbapenemase production 187
The EUCAST non-susceptible ETP CBP (< 25 mm), and the EUCAST 188
recommended carbapenemase MEM screening cut-off (< 25 mm) for carbapenemase 189
production displayed highest sensitivity of all evaluated cut-offs (100%, Table 2). ETP, 190
however, had a lower specificity (62.5%) than MEM (90.7%, Table 2). The ETP non-191
susceptible CLSI CBP (< 22 mm) and the non-susceptible CLSI CBP for MEM (< 23 192
mm) displayed lower sensitivity (95.5% for both compounds, Table 2). The IPM non-193
susceptible EUCAST CBP (< 22 mm) had the lowest sensitivity (81.8%), whereas the 194
non-susceptible CLSI IPM CBP (23 mm) had a sensitivity of 90.9%. 195
Performance of carbapenemase confirmation tests 196
Combined-disc tests (CDTs) 197
The ETP APBA-CDT on MH-CLX agar displayed highest sensitivity and NPV for 198
class A carbapenemase detection (100%, Table 2). Specificity of 100% was found for 199
the ETP APBA-CDT, the IPM APBA-CDT, and the MEM APBA-CDT on MH-CLX, 200
whereas the same CDTs on conventional MH agar showed a specificity of 96.9%, 201
99.4%, and 96.6%, respectively (Table 2). 9/10 false-positive ETP APBA-CDTs on 202
conventional MH agar occurred in species with chromosomal AmpC (6 Enterobacter 203
cloacae, 1 Enterobacter aerogenes, and 2 Hafnia alvei). 9/11 false-positive MEM 204
APBA-CDTs on conventional MH agar were also found in AmpC positive species, i.e. 205
6 Enterobacter cloacae, 1 Enterobacter aerogenes, 1 Hafnia alvei, and 1 E. coli 206
harboring a CIT type plasmid-encoded AmpC. One K. pneumoniae isolate lacking 207
AmpC or ESBL was borderline positive in both ETP and MEM APBA-CDT on 208
conventional MH (5 mm and 7 mm zone difference, respectively). Another 209
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K. pneumoniae isolate producing an ESBL was borderline positive only in the MEM 210
APBA-CDT on conventional MH (5 mm difference). 211
Both the ETP and the MEM EDTA-CDTs displayed 100% sensitivity and specificity 212
for class B carbapenemase detection, whereas the sensitivity of the IPM EDTA-CDT 213
was significantly lower (70%, Table 2). 214
Carba NP-II test 215
The overall sensitivity, specificity, PPV, and NPV of the Carba NP-II test were 216
78.9%, 100%, 100%, and 98.7%, respectively (Table 2). The test created some reading 217
problems resulting in ambiguous results that were treated as follows: One Enterobacter 218
aerogenes isolate possessing a blaVIM gene gave ambiguous results in terms of class 219
assignment (see isolate 8, Figure 1). After 30 min of incubation the pattern was 220
consistent with a class B carbapenemase, while after 120 min of incubation the pattern 221
was consistent with a class D carbapenemase (e.g. OXA-48).. For calculation of 222
performance parameters this isolate was rated carbapenemase positive (Table 3). Three 223
Klebsiella pneumoniae isolates co-producing OXA-48 and CTX-M ESBL gave 224
inconclusive results (Table 3): the NP-II patterns were negative for carbapenemase 225
production until 60 min of incubation. After 120 min of incubation, the patterns could 226
either still be rated negative or weakly positive for class A carbapenemases (see Figure 227
1, isolates 20, 99, 51, results were reproduced three times with independent 228
preparations); these isolates were excluded from the calculation of performance 229
parameters. In addition, one OXA-48 producing Klebsiella pneumoniae (see isolate 19, 230
Figure 1) and three NDM producing isolates of Providencia rettgeri, Providencia 231
stuartti, and Proteus mirabilis, respectively, gave false-negative results with the NP-II 232
test (see Table 3, isolates 136, 138, and 139, Figure 1). One Enterobacter cloacae 233
isolate producing a GIM (class B) gave an OXA-48-like pattern (class D, see isolate 95, 234
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Figure 1). For the calculation of sensitivity and specificity this isolate was rated 235
carbapenemase positive (Table 3). 236
Temocillin testing on MH-CLX agar 237
Nineteen representative carbapenem non-susceptible isolates were tested for 238
temocillin zone diameters and MICs on MH and MH-CLX agar as indicators for the 239
presence of OXA-48. Five isolates harbored blaOXA-48 genes, nine isolates were 240
blaOXA-48 gene negative but showed overexpression of a chromosomally encoded 241
AmpC, and five isolates harbored ESBL genes (but not blaOXA-48, Table 4). All 242
OXA-48 producers showed high-level temocillin resistance on both MH and MH-CLX 243
agar (median diameter 6 mm, median MIC >1024 mg/L, Table 4). Five out of nine 244
AmpC hyperproducers displayed temocillin zone diameters lower than 11 mm on MH 245
(EUCAST screening cut-off for OXA-48 like enzymes) (14). On MH-CLX, the 246
temocillin median diameter of the AmpC hyperproducers increased by 7 mm 247
(corresponding to a median Etest-determined MIC decrease of 2 dilution steps, Table 248
4), and the five EUCAST OXA-48 screen false-positive isolates became true-negatives. 249
Temocillin median diameters and gradient diffusion MICs of the five ESBL producers 250
were not altered by the use of MH-CLX as compared to conventional MH agar. Median 251
temocillin diameters/MICs were 11 mm and 32 mg/L, respectively, on both media 252
(Table 4). The only false-positive temocillin-based OXA-48 screening result originated 253
from an CTX-M type ESBL-producing Klebsiella pneumoniae isolate displaying 254
temocillin diameters/MICs of 10 mm and 64 mg/L on both MH and MH-CLX agar. 255
Genetic characterization of isolates 256
In total, 23 carbapenemase genes were detected in 22 Enterobacteriaceae isolates: 7 257
blaKPC, 1 blaIMI, 4 blaVIM, 4 blaNDM, 1 blaGIM, and 4 blaOXA-48; 1 isolate co-258
produced VIM and OXA-48 enzymes (Tables 1 and 2). Seventy-eight (23.4%) of the 259
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studied isolates were genetically negative for ESBL, AmpC, and carbapenemases; 178 260
(53.3%) of the isolates produced an AmpC β-lactamase (including those species with 261
chromosomally encoded AmpC, i.e. Enterobacter cloacae, Enterobacter aerogenes, 262
Citrobacter freundii, Hafnia alvei, Morganella morganii, Serratia marcescens, and 263
Providencia stuartii, Table 1) (54); 105 (31.4%) of the isolates harbored an ESBL 264
(Table 1). 265
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Discussion 266
Screening parameters for carbapenemases 267
Disk diffusion critical diameters have been reported to display high sensitivity for the 268
detection of carbapenemases (13, 20). This study found 100% sensitivity for the 269
EUCAST critical MEM diameter (< 25 mm) with a comparably high specificity of 270
90.7% (Table 2). ETP screening using the EUCAST non-susceptible CBP (< 25 mm) 271
also showed high sensitivity (100%), but low specificity (62.5%, Table 2). Thus, our 272
results confirm the current EUCAST recommendation (15). CLSI non-susceptible ETP 273
(< 22 mm) and MEM (< 23 mm) CBPs displayed lower sensitivity as compared to the 274
current EUCAST recommendation (95.5%, Table 2). Based on the findings of this 275
study, carbapenemase screening using MEM is recommended, whereas the use of IMP 276
as screening drug is discouraged (IMP sensitivity EUCAST < 22 mm / CLSI < 23 mm 277
81.8% and 90.9%, respectively). Since automated microdilution AST reportedly lacks 278
sensitivity and specificity due to antibiotic panel composition and drug concentrations 279
tested (18, 55), disk diffusion critical MEM diameters promise the best performance for 280
carbapenemase detection among all evaluated techniques. In addition, disk diffusion is 281
cheap, simple, and widely implemented by many laboratories for routine AST. 282
Carbapenemase confirmation tests 283
The modified Hodge test, which is recommended by CLSI for carbapenemase 284
confirmation, is cheap and, in principle, simple to perform (23). However, it displays 285
significant investigator dependence, practical interpretation is technically demanding, 286
the test cannot distinguish between the different carbapenemase classes, and reportedly 287
shows low specificity due to AmpC β-lactamase overproduction and decreased 288
permeability, e.g. caused by porin loss (13, 20, 55). The problem of discriminating 289
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carbapenemase activity from AmpC and impermeability is well known both for species 290
possessing a chromosomal AmpC (e.g. Enterobacter spp., Citrobacter spp., or Hafnia 291
alvei), and for producers of plasmid-encoded AmpC, in particular Klebsiella 292
pneumoniae (39, 44, 56). Even E. coli overproducing AmpC due to mutations in the 293
promoter/attenuator region and/or showing mutations in the active center of the enzyme 294
resulting in an extended–spectrum AmpC (ESAC) phenotype display carbapenem non-295
susceptibility (41, 43). The same pattern accounts for ESBL producers in combination 296
with porin loss (37, 38). AmpC and ESBL production interferes not only with 297
carbapenemase screening, but also with APBA-CDT confirmation for class A 298
carbapenemases (14, 19, 57). False positive results occur as APBA is not only an 299
inhibitor of class A carbapenemases, but also of AmpC β-lactamases. To improve 300
specificity of APBA-CDTs, MEM/CLX disks are used to check for AmpC interference 301
(indirect approach) (13, 14, 20, 22, 25). However, based on the current EUCAST 302
algorithm, class A carbapenemases in isolates co-producing AmpC may be missed as 303
synergy of MEM with both CLX and APBA is interpreted as AmpC and porin loss (14). 304
A recent study found two Enterobacter cloacae isolates overproducing AmpC, but also 305
harboring KPC and NMC-A enzymes that would have been misclassified using this 306
approach (13). Other authors pointed out that MEM-MEM/CLX zone diameter 307
differences are relatively lower in AmpC hyperproducers co-expressing a class A 308
carbapenemase (i.e. mean difference 1 mm) than in AmpC hyperproducers without a 309
class A carbapenemase (mean difference 5 mm) (22). Another study, however, 310
described MEM-MEM/CLX zone diameter differences of 6 to 7 mm and 0-7 mm for 311
AmpC hyperproducing E. cloacae harboring class A carbapenemases and AmpC 312
hyperproducers devoid of carbapenemases, respectively (13). Thus the discriminative 313
power of relative MEM-MEM/CLX diameter differences may be insufficient. In 314
addition, classification based on the relative degree of MEM-MEM/CLX diameter 315
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differences is difficult to standardize and requires significant expertise. The present 316
study on 178 (53.3%) AmpC producing isolates shows that APBA-CDTs performed on 317
MH-CLX agar reliably detect class A carbapenemases with increased specificity 318
(100%) due to suppression of AmpC activity (Table 2). The approach is simple to 319
interpret as it uses a single critical zone diameter difference (5 millimeters), and it can 320
be integrated in one step with ESBL confirmation testing on the same MH-CLX agar 321
plate (48). 322
In the present study, the Carba NP-II showed an overall sensitivity of 78.9% and a 323
NPV of 98.7% (Table 2). Our results closely parallel those of a recent study, which 324
found a sensitivity of 72.5% for the Carba NP-I and a NPV of 69.2%. The difference in 325
NPV is well explained by the different prevalence of carbapenemase producers in the 326
study populations, i.e. 6.6% (n = 22) in this study and >45% (n =145) in the study of 327
Tijet et al. (31). Other authors found higher sensitivities for the Carba NP-I test using 328
different types of protocols (32, 33). Our data confirm ambiguities in the reading of the 329
Carba NP-I/II test in particular for OXA-48 producing isolates that tend to produce 330
inconclusive, or false-negative results (see Figure 1, isolates 19, 20, 51, and 99) (31). If 331
the inconclusive OXA-48 results from Figure 1 would have been rated negative (only a 332
slight color-change was visible after 120 min of incubation), sensitivity would have 333
been 68.2% (Table 2). If rated positive, the three ambiguous OXA-48 results would 334
have been consistent with a class A carbapenemase pattern, most likely due to the 335
simultaneous presence of a CTX-M type ESBL (class A enzyme), which may be 336
responsible for the weak color-change in wells II and IV after 120 min of incubation, 337
and which is inhibited by tazobactam in well III (see Figure 1). False-negative Carba 338
NP results have also been described for mucoid colonies, e.g. of Providencia rettgeri, 339
Providencia stuartii, or Proteus mirabilis isolates (29, 31). Negative results were 340
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attributed to difficulties in protein extraction, species-specific traits, or the influence of 341
the agar type and ion content on the Carba NP test (30, 31, 36). Besides OXA-48 342
producers, false-negative results in the present study also occurred in non-mucoid 343
isolates of Providencia rettgeri, Providencia stuartii, and Proteus mirabilis producing 344
NDM enzymes. All tests for these isolates were repeated three times with the standard 345
protocol and additionally performed using colonies grown on various agar media of 346
different manufacturers, i.e. MH (Becton Dickinson), MH-CLX (Axonlab), Columbia 347
sheep blood, MacConkey (bioMérieux), and Uriselect4 agar (BioRad). Despite reports 348
that the Carba NP I test performed better from Columbia sheep blood and Uriselect4 349
agar results for these isolates remained false-negative for all media types pointing to 350
species-specific issues related to Providencia and Proteus isolates, and a low sensitivity 351
for OXA-48 enzymes (34). Other authors recently found a higher sensitivity and 352
specificity for the detection of OXA-48 (28). In summary, due to the higher NPV, the 353
Carba NP-II test may perform better in a low prevalence environment (i.e. our study) as 354
compared to high prevalence settings such as those investigated by Tijet et al. (31). 355
However, the issues of false-negative OXA-48 producers and species specific false-356
negative results due to the unknown impact of different genetic backgrounds need to be 357
further analyzed. 358
The phenotypic detection of OXA-48-like carbapenemases remains challenging. 359
EUCAST recommends indirect OXA-48 confirmation by decreased zone diameters or 360
increased MICs for temocillin (< 11 mm, and > 32 mg/L, respectively) to exclude 361
ESBLs in combination with porin loss in cases where both APBA-CDT and EDTA-362
CDT are negative (14). Temocillin MICs, however, are not recommended to 363
discriminate AmpC overproduction combined with porin loss from OXA-48 as 364
temocillin MICs are variable in this setting resulting in poor specificity. By suppressing 365
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potential AmpC activity, temocillin disk diffusion testing or MIC determination by a 366
gradient diffusion method on MH-CLX can help to clearly increase specificity of 367
temocillin-based OXA-48 screening without compromising sensitivity (Table 4). 368
In summary, a combination of the EUCAST MEM carbapenemase-screening cut-off 369
(< 25 mm) and ETP (or MEM) APBA- and EDTA-CDTs plus temocillin disk diffusion 370
(or gradient diffusion-based MIC determination) on MH-CLX agar promises excellent 371
performance for carbapenemase detection. The proposed diagnostic flow-chart (Figure 372
2) would have resulted in a sensitivity, specificity, PPV, and NPV of 100% in the study 373
population. This algorithm is simple, easy to use, cost-efficient and applicable in the 374
majority of clinical microbiology laboratories. 375
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Acknowledgments 377
We are grateful to the laboratory technicians of the Institute of Medical 378
Microbiology, University of Zurich for their dedicated help, and to Erik C. Böttger and 379
Reinhard Zbinden for valuable discussions. 380
381
Funding 382
This work was supported by the University of Zurich. 383
384
Transparency declaration 385
All authors: No conflicts of interest to declare. 386
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Tables and Figures 593
594
Table 1: Species identification and beta-lactamase genotypes of studied isolates. 595
Species N % ESBL, AmpC,
Carbapenemase
negative AmpC ESBL
Carbapenemases
KPC IMI VIM NDM GIM OXA-48
Escherichia coli 5 1.5 + +
26 7.8 +
34 10.2 +
45 13.5 +
1 0.3 +
total 111 33.3
Enterobacter cloacae 59 17.7 NA +
15 4.5 NA + +
1 0.3 NA + +
2 0.6 NA + +
1 0.3 NA + +
total 78 23.4
Klebsiella pneumoniae 24 7.2 +
22 6.6 +
13 3.9 +
2 0.6 + +
2 0.6 + +
4 1.2 +
1 0.3 +
3 0.9 + +
1 0.3 +
1 0.3 +
total 73 21.9
Enterobacter aerogenes 11 3.3 NA +
4 1.2 NA + +
1 0.3 NA + +
total 16 4.8
Klebsiella oxytoca 6 1.8 +
4 1.2 +
6 1.8 +
total 16 4.8
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596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 NA, not applicable, for species naturally harboring chromosomally-encoded AmpC beta-lactamases 646 647 648 649 650 651 652 653 654
Table 1 continued
Species N % ESBL, AmpC,
Carbapenemase
negative AmpC ESBL
Carbapenemases
KPC IMI VIM NDM GIM OXA-48
Citrobacter freundii 1 0.3 NA + +
3 0.9 NA + +
10 3.0 NA +
total 14 4.2
Hafnia alvei 5 1.5 NA +
1 0.3 NA + +
total 6 1.8
Proteus mirabilis 1 0.3 + +
1 0.3 + +
2 0.6 +
total 4 1.2
Morganella morganii 1 0.3 NA + +
2 0.6 NA +
total 3 0.9
Serratia marcescens 3 0.9 NA +
Citrobacter koseri 2 0.6 +
Salmonella spp. 2 0.6 +
Providencia rettgeri 1 0.3 + +
Providencia stuartii 1 0.3 NA + +
Enterobacter sp. 1 0.3 NA +
Pantoea spp. 1 0.3 +
Citrobacter spp. 1 0.3 NA +
Serratia spp. 1 0.3 NA +
Total 334 100 78 178 105 7 1 5 4 1 5
Genotypes (%) 100 23.4 53.3 31.4 2.1 0.3 1.5 1.2 0.3 1.5
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655
Table 2: Performance parameters of screening and confirmation assays and the proposed 656
diagnostic flow chart (see Figure 2). 657
658 659
TP, true-positive; FP, false-positive; TN, true-negative; FN, false-negative; MEM, meropenem; ETP, 660
ertapenem; CDT, combined-disk test; APBA, aminophenylboronic acid; MH-CLX agar, Muller Hinton 661
agar supplemented with cloxacillin; MH agar, Muller Hinton agar without cloxacillin. 662
1 inconclusives were excluded from the calculation; 663
2 inconclusives rated negative. 664
665
Parameter TP
(N) FP
(N) TN
(N) FN
(N) Total
(N) Sensitivity
(%) Specificity
(%) PPV
(%) NPV
(%)
Screening cut-offs / CBPs
MEM Screen EUCAST (< 25 mm) 22 29 283 0 334 100.0 90.7 43.1 100.0
ETP EUCAST I/R (< 25 mm) 22 117 195 0 334 100.0 62.5 15.8 100.0
IPM EUCAST I/R (< 22 mm) 18 16 296 4 334 81.8 94.9 52.9 98.7
MEM EUCAST I/R (< 22 mm) 20 18 294 2 334 90.9 94.2 52.6 99.3
ETP CLSI I/R (< 22 mm) 21 68 244 1 334 95.5 78.2 23.6 99.6
IPM CLSI I/R (< 23 mm) 20 19 293 2 334 90.9 93.9 51.3 99.3
MEM CLSI I/R (< 23 mm) 21 20 292 1 334 95.5 93.6 51.2 99.7
CDTs
ETP-BA MH 6 10 316 2 334 75.0 96.9 37.5 99.4
IPM-BA MH 6 2 324 2 334 75.0 99.4 75.0 99.4
MEM-BA MH 7 11 315 1 334 87.5 96.6 38.9 99.7
ETP-BA MH-CLX 8 0 326 0 334 100.0 100.0 100.0 100.0
IPM-BA MH-CLX 6 0 326 2 334 75.0 100.0 100.0 99.4
MEM-BA MH-CLX 7 0 326 1 334 87.5 100.0 100.0 99.7
ETP-EDTA MH 10 0 324 0 334 100.0 100.0 100.0 100.0
IPM-EDTA MH 7 0 324 3 334 70.0 100.0 100.0 99.1
MEM-EDTA MH 10 0 324 0 334 100.0 100.0 100.0 100.0
Carba NP-II1 15 0 312 4 331 78.9 100.0 100.0 98.7
Carba NP-II2 15 0 312 7 334 68.2 100.0 100.0 97.8
Proposed algorithm 22 0 312 0 334 100.0 100.0 100.0 100.0
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666
Table 3: Carbapenemase-positive isolates with characteristics and confirmation test results. 667
Isolate
number Species AmpC ESBL Carbapenemase type Carbapenemase class NP-II
CDTs (Δ mm)
BA on MH BA on MH-CLX EDTA on MH
ETP IMI MEM ETP IMI MEM ETP IMI MEM
7 Klebsiella pneumoniae - - KPC A + 7 5 8 8 6 10 0 0 0
29 Klebsiella pneumoniae - SHV-ESBL KPC A + 6 5 5 11 7 11 0 1 0
31 Klebsiella pneumoniae - - KPC A + 7 11 7 9 8 9 0 0 0
35 Klebsiella pneumoniae - - KPC A + 4 5 8 7 5 6 0 0 1
37 Klebsiella pneumoniae - - KPC A + 7 7 9 8 4 9 0 0 0
40 Enterobacter cloacae cAmpC - IMI A + 11 13 13 11 8 10 2 1 1
55 Escherichia coli - - KPC A + 4 2 6 7 3 6 2 0 0
137 Klebsiella pneumoniae - CTX-M KPC A + 6 4 4 5 5 2 0 3 0
8 Enterobacter aerogenes cAmpC - VIM B + 0 0 0 0 0 0 7 5 8
9 Klebsiella pneumoniae - - NDM B + 0 0 0 2 0 0 16 7 13
17 Enterobacter cloacae cAmpC - VIM B + 0 0 0 3 0 0 5 3 5
70 Citrobacter freundii cAmpC - VIM B + 0 0 0 0 0 0 5 4 7
82 Klebsiella pneumoniae - - VIM B + 0 0 0 0 0 1 15 17 21
95 Enterobacter cloacae cAmpC - GIM-1 B + 0 0 0 2 0 2 10 3 10
136 Providencia rettgeri cAmpC - NDM B - 0 0 0 0 0 0 10 19 19
138 Providencia stuartii cAmpC - NDM B - 0 0 0 0 0 0 9 13 12
139 Proteus mirabilis CIT - NDM B - 0 4 0 0 0 0 6 16 6
36 Enterobacter cloacae cAmpC SHV-ESBL VIM B + 0 0 0 3 0 1 5 6 8
20 Klebsiella pneumoniae - CTX-M OXA-48 D inconclusive 0 0 0 3 0 0 0 0 0
51 Klebsiella pneumoniae - CTX-M OXA-48 D inconclusive 0 0 0 2 0 3 0 0 0
99 Klebsiella pneumoniae - CTX-M OXA-48 D inconclusive 0 0 0 2 0 3 0 0 0
19 Klebsiella pneumoniae - - OXA-48 D - 3 0 1 3 0 2 0 0 0
MEM, meropenem; ETP, ertapenem; CDT, combined-disk test; APBA, aminophenylboronic acid; MH-CLX, Muller Hinton agar supplemented with cloxacillin; MH, Muller Hinton 668
agar without cloxacillin; ESBL, extended-spectrum beta-lactamase; cAmpC, chromosomally encoded ampC gene669
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Table 4: Temocillin critical zone diameters and MICs for confirmation of OXA-48-like 670
carbapenemases. 671
672
673
ID, isolate identification number, ESBL, extended-spectrum beta-lactamase, MH-CLX: 674
Muller Hinton agar supplemented with cloxacillin, MH: Muller Hinton agar without 675
cloxacillin, cAmpC, chromosomally-encoded AmpC beta-lactamase676
677
ID Species ESBL AmpC Carbapenemase Temocillin zone (mm) Temocillin MIC (mg/L)
MH MH-CLX MH MH-CLX
19 Klebsiella pneumoniae - - OXA-48 6 6 1024 1024
20 Klebsiella pneumoniae + - OXA-48 6 6 1024 1024
51 Klebsiella pneumoniae + - OXA-48 6 6 1024 1024
99 Klebsiella pneumoniae + - OXA-48 6 6 1024 1024
36 Enterobacter cloacae + cAmpC VIM 6 8 1024 128
16 Hafnia alvei - cAmpC 6 11 128 32
18 Enterobacter cloacae - cAmpC 9 17 32 16
5 Enterobacter cloacae - cAmpC 10 21 32 8
27 Enterobacter cloacae - cAmpC 10 12 32 32
25 Hafnia alvei - cAmpC 10 21 32 4
26 Enterobacter cloacae - cAmpC 11 18 32 8
2 Enterobacter cloacae - cAmpC 12 16 32 16
125 Enterobacter cloacae - cAmpC 14 22 16 4
1 Enterobacter aerogenes - cAmpC 16 20 8 8
39 Klebsiella pneumoniae + - 10 10 64 64
60 Escherichia coli + - 11 11 32 32
38 Proteus mirabilis + - 11 11 32 32
130 Klebsiella pneumoniae + - 14 13 16 16
128 Klebsiella pneumoniae + - 18 17 8 8
median values
OXA-48 positive isolates 6 6 1024 1024
AmpC overexpression 10 18 32 8
ESBL 11 11 32 32
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Figure 1: Discrepant test results of the Carba NP-II test and the carbapenemase genotype 678
679
680
isolate num-ber
species genotype /
Ambler class
Carba NP-II result (examples of replicate testing)
t = 0 minutes t = 30 minutes t = 60 minutes t = 120 minutes
8 Enterobacter aerogenes
VIM B
20 Klebsiella pneumoniae
OXA-48, CTX-M
D
99 Klebsiella pneumoniae
OXA-48, CTX-M
D
51 Klebsiella pneumoniae
OXA-48, CTX-M
D
19 Klebsiella pneumoniae
OXA-48 D
136 Providencia rettgeri
NDM B
138 Providencia stuartii
NDM B
139 Proteus mirabilis
NDM B
95 Enterobacter cloacae
GIM B
interpretation (expected test result)
negative result class A carbapenemase class B carbapenemase class D carbapenemase
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Figure 2: Proposed diagnostic flow chart for carbapenemase detection. 681
682
683
MEM, meropenem; ETP, ertapenem; CDT, combined-disk test; APBA, aminophenylboronic 684
acid; MH-CLX agar, Muller Hinton agar supplemented with cloxacillin; MH agar, Muller 685
Hinton agar without cloxacillin. 1
MEM can be used alternatively with slightly lower sensitivity. 686
2Carbapenem resistance phenotype is most likely due to a combination of AmpC and/or ESBL 687
overexpression and decreased permeability, e,g, due to porin deficiency. 688
Enterobacteriaceae isolates
Inhibition zone diameter MEM < 25mm
No
No carbapenemase
suspicion
Yes
CDT ETP versus ETP/APBA on MH-CLX
agar1
Δ(ETP/APBA – ETP) < 5 mm Δ(ETP/APBA – ETP) ≥ 5 mm
CDT ETP versus ETP/EDTA on MH agar
1 CDT ETP versus ETP/EDTA
on MH agar1
Δ(ETP/EDTA – ETP) < 5 mm
Δ(ETP/EDTA – ETP) ≥ 5 mm
Δ(ETP/EDTA – ETP) < 5 mm
Δ(ETP/EDTA – ETP) ≥ 5 mm
Carbapenemases
class A No
class B No
class D ?
Carbapenemases
class A No
class B Yes
class D ?
Carbapenemases
class A Yes
class B Yes
class D ?
Carbapenemases
class A Yes
class B No
class D ?
Temocillin disk diffusion or MIC on
MH-CLX agar
≥ 11 mm or ≤ 32 mg/L
< 11 mm or >32 mg/L
Oxa-48-like
enzyme unlikely
2
Suspicion for
Oxa-48-like
enzyme
Perform molecular assay for the detection of class D carbapenemases
Initial screening step
Time to result
24 h (regular
antibiogram)
Carbapenemases
excluded for 57.8% of study population
Phenotypic confirmation step
Additional time to
result 24 h (total 48 h)
Carbapenemases
excluded/confirmed for 98.5% of study
population
Genotypic confirmation step
Additional time to
result 24 h (total 72 h)
(1.5% of study population, OXA-48
only)
Carbapenemases
excluded/confirmed for 98.5% of study
population
on July 19, 2018 by guesthttp://jcm
.asm.org/
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