evaluation of carbapenemase screening and confirmation...

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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




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



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



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



25 Hafnia alvei - cAmpC 10 21 32 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



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


OXA-48, CTX-M

D


OXA-48, CTX-M

D


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


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evaluation of carbapenemase screening and confirmation...

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