Evaluation of five chromogenic agar media and the ROSCO Rapid CARB Screen kit for the

JCM Accepts, published online ahead of print on 29 October 2014 J. Clin. Microbiol. doi:10.1128/jcm.02068-14 Copyright 2014, American Society for Microbiology. All Rights Reserved. 1 2 Evaluation of five chromogenic agar media and the ROSCO Rapid CARB Screen kit for the detection and confirmation of carbapenemase production in Gram-negative bacilli 3 4 5 Patricia J. Simner, a,b # Matthew W. Gilmour, a,b Pat DeGagne, a Kim Nichol, a and James A. Karlowsky a,b 6 7 8 Diagnostic Services Manitoba, Winnipeg, Manitoba, Canada a ; University of Manitoba, Department of Medical Microbiology and Infectious Diseases, Winnipeg, Manitoba, Canada b 9 10 Running Title: Detection of carbapenemase-producing Gram-negatives 11 12 # Address correspondence to Patricia J. Simner, psimner@dsmanitoba.ca 1

13 Abstract: 14 15 16 17 18 19 20 21 22 23 24 An efficient workflow to screen for and confirm carbapenemase-producing Gram-negative bacilli was developed by evaluating five chromogenic screening agar media and two confirmatory assays, the Rapid CARB Screen(Rosco Diagnostica A/S, Tasstrup, Denmark) test and the modified Hodge test. A panel of 150 isolates was used, including 49 carbapenemaseproducing isolates representing a variety of β-lactamase enzyme classes. Evaluation of analytical performance, assay cost and turnaround time indicated that the preferred workflow (screening test followed by confirmatory testing) was chromid CARBA agar media (biomémieux, Marcy-l Etoile, France) followed by the Rapid CARB Screen test, yielding a combined sensitivity of 89.8% and a specificity of 100%. As an optional component of the workflow, determination of carbapenemase gene class via molecular means could be performed subsequent to confirmatory testing. 25 2

26 Introduction: 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 The worldwide dissemination of carbapenemase-producing Gram-negative bacilli (CPGNB) is a significant clinical and public health concern (10, 14). Rapid detection of these antibiotic resistant pathogens by the clinical microbiology laboratory is of utmost importance to control nosocomial spread and initiate appropriate antimicrobial therapy. Chromogenic media containing a carbapenem are convenient tools for screening and rapid detection of carbapenem resistant Gram-negative bacilli (GNB). However, growth on a chromogenic media simply signifies carbapenem resistance (e.g., an AmpC producer with porin loss may grow) and does not confirm the production of a carbapenemase. Recently, a novel rapid chromogenic test based on the hydrolysis of imipenem, the CarbaNP test, and a commercial version of the CarbaNP test, the Rapid CARB screen (RCS), have been described (3, 5, 11-13, 16). The purpose of the current study was to evaluate screening and confirmation methods, that when paired, would provide a streamlined workflow to detect CPGNB in the clinical microbiology laboratory. Firstly, five different chromogenic media were evaluated to select a sensitive media type to screen for CPGNB. Secondly, the RCS test (Rosco Diagnostica A/S, Tasstrup, Denmark) was evaluated to confirm carbapenemase production through a method comparison to the current gold standard modified Hodge (MH) test (1). A combined workflow approach was designed to achieve the maximum sensitivity and specificity for the detection of CPGNB, with consideration for cost and turnaround time of testing. Lastly, upon finding a CPGNB, it may be of value in some circumstances to determine the carbapenemase gene class, and therefore an established multiplex PCR was also incorporated as an optional endpoint to this workflow. 3

47 Materials and Methods: 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 Bacterial Isolates. A total of 150 isolates were tested including an assortment of 49 isolates of GNB harboring a diverse set of carbapenemase enzyme classes and 101 carbapenemase negative isolates. The 49 carbapenemase producers, a combination of clinical and ATCC strains, were previously molecularly characterized for β-lactamase genes and included 16 KPC producers (bla KPC-2 [n=9], -3 [n=5], -11 [n=1],-12 [n=1] ; Citrobacter freundii, n=2; Citrobacter koseri, n=1; Enterobacter cloacae, n=1; Escherichia coli, n=4; Klebsiella oxytoca, n=1; Klebsiella pneumoniae, n=5; Pseudomonas aeruginosa, n=1; Serratia marcenscens, n=1), 12 VIM producers (bla VIM-1 [n=6],-2 [n=3],-4 [n=2],-5 [n=1] ; C. freundii, n=2; C. koseri, n=1; Enterobacter aerogenes, n=1; E. cloacae, n=1; K. oxytoca, n=1; Proteus mirabilis, n=1; Providencia stuartii, n=1; P. aeruginosa, n=5 ), 4 IMP producers (bla IMP-1,-26 ; E. coli, n=1; K. pneumoniae, n=2; P. aeruginosa, n=1), 9 NDM producers (bla NDM-1 [n=6], -4 [n=1], -5 [n=1], -7 [n=1] ; Acinetobacter baumanii, n=1; E. coli, n=4; K. oxytoca, n=1; K. pneumoniae, n=1; P. mirabilis, n=1; Providencia rettgerii, n=1), 8 OXA producers (bla OXA-24 [n=1],-48 [n=7] ; A. baumanii, n=1; C. koseri, n=1, E. cloacae, n=1; K. pneumoniae, n=3; Morganella morganii, n=1, S. marcescens, n=1) and one SME producer (S. marcescens, n=1). Of note, one E. cloacae isolate possessed both VIM-4 and OXA-48. The 101 carbapenemase-negative isolates included extended-spectrum β-lactamse (ESBL) and AmpC producers (previously molecularly characterized) with and without porin mutations, clinical isolates with elevated carbapenem minimum inhibitory concentrations (and negative for carbapenemases by PCR) and carbapenem susceptible isolates. The isolates were blinded and 75 isolates were distributed to each of the two clinical microbiology laboratory testing sites (Health Sciences Centre and St. Boniface Hospital, Winnipeg, Canada). 4

69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 Chromogenic Media to Screen for CPGNB: The first part of this study involved evaluating five different chromogenic media types to screen for CPGNB. The five chromogenic media evaluated were: 1) Oxoid Brilliance ESBL (Besington, Hants, UK), 2) Oxoid Brilliance CRE, 3) biomérieux chromid TM CARBA (Marcy-l Etoile,France), 4) CHROMAgar Colorex C3Gr (Paris, France) and 5) CHROMagar Colorex KPC. The Brilliance ESBL and Colorex C3Gr media are designed to detect ESBLs, and in this study were evaluated both with and without an ertapenem disk (10 μg) to select for carbapenem resistant isolates. Inoculums for each of the chromogenic media types and a nonselective blood agar (BA) plate were 10 μl (~10 6 CFU/mL) of a 0.5 McFarland standard prepared in sterile saline (0.85%). Brilliance ESBL, Brilliance CRE and chromid CARBA were streaked using the Isoplater (Vista Technology Inc, Edmonton, Alberta, Canada) to mimic standard laboratory practice. The Colorex media C3Gr and Colorex KPC were evaluated as a biplate so the media were streaked manually. Ertapenem disks were placed between the 2 nd and 3 rd quadrant of the Brilliance ESBL and Colorex C3Gr media following streaking of the plate. The plates were incubated for 24 hours at 37 o C and the presence/absence and color of growth were interpreted according to the package inserts. Any isolate on the ESBL chromogenic media with an ertapenem zone of inhibition of 27 mm were considered putative carbapenemase producers and would be considered for further evaluation using a confirmatory method. The use of the 27mm cut-off was established in a previous study using an ertapenem disk (10 μg) to screen for carbapenem resistant GNB on MacConkey media (6). 5

90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 Phenotypic Confirmatory Asssays: In the second part of the study, a method comparison between the MH test and the RCS, was performed on all study isolates, using growth from the BA plate inoculated concurrent with the chromogenic media. The BA plate was added to perform the confirmatory tests the following day since the RCS cannot be performed off the chromogenic media as the color of the colonies interferes with the interpretation of the test. The MH test using meropenem disks (10 μg) was performed and interpreted according to CLSI methodology (1). The RCS is a, commercially available, rapid chromogenic test for the detection of carbapenemases based on the hydrolysis of the imipenem in the presence of an inidicator [phenol red, (11)]. All the components of the assay are present within tablets called diatabs. Two sets of diatabs are contained in the kit, one set contains imipenem and the second set does not contain imipenem. The diatabs are dissolved in saline and the lysate of the organism is added to a tube with a dissolved diatab with imipenem and a tube with a dissolved diatab without imipenem (negative control tube; should remain red in color). Initially, the RCS was performed as previously described (5). K. pneumoniae ATCC BAA1706 and K. pneumoniae ATCC BAA1705 were the positive and negative controls, respectively. Any color change from red to yellow in the 1.5 ml test tube containing imipenem was considered a positive reaction for the RCS. Uninterpretable results were isolates that would give a slight positive reaction (peach color) in both tubes with and without imipenem. 108 109 110 111 As there were many uninterpretable results initially with the previously described RCS protocol, the RCS protocol was troubleshot by determining the appropriate inoculum by testing one to three calibrated loops of organism and determining the optimal tube to perform the assay by testing 1.5 ml test tubes, cryovials, and round-bottomed 15 ml tubes. Thus, the 6

112 113 114 115 116 117 118 modified RCS was performed upon repeat on all isolates to only include one 10 µl calibrated loopful of organism (reduced from two) and a round-bottomed 15 ml tube (instead of a conical 1.5 ml tube). As there was variability in the degree of color change for positives with the modified RCS, positives were considered any color change from the negative control tubes (tubes without imipenem). Isolates that were orange by the modified RSC protocol in this study were repeated using both the RCS and the CarbaNP, as previsouly described, to assess ease of interpretation of the intermediate results (3, 11, 13). 119 120 121 Conventional Multiplex PCR: Lastly, a conventional multiplex PCR for bla KPC, bla NDM, bla VIM, bla IMP and bla OXA-48/181 was performed on all isolates as previously described (8). In addition, a bla SME PCR was performed on one isolate to confirm SME production (7). 122 123 124 125 126 127 128 129 Determination of an Optimal Workflow for the Screening and Confirmation of CPGNB: The results of the screening and confirmatory assays (performed on all test isolates) was analyzed to develop the optimal workflow (i.e., test choice) for the detection and confirmation of CPGNB. By combining the individual data from the chromogenic media and the confirmatory assays we were able to determine the sensitivity and specificities of the combined assays. In addition, the cost and time to perform the different combination of assays were evaluated and considered for feasibility and full optimization of the workflow within a clinical microbiology laboratory. 130 Results: 131 132 Chromogenic media for CPGNB: The first part of this study involved evaluating five different chromogenic media types to screen for CPGNB, including two media targeting ESBLs 7

133 134 135 136 137 138 139 supplemented with and without an ertapenem disk (Table 1). Of the five different chromogenic media evaluated to detect CPGNB (Table 1), Colorex C3Gr without the ertapenem disk had the highest sensitivity, 95.9%. However, the two ESBL media types, Colorex C3Gr and Brilliance ESBL, without the use of the ertapenem disk both had a specificity of 38.6% for CPGNB. The addition of the ertapenem disk to Colorex C3Gr and Brilliance ESBL resulted in an increased specificity of 62.4% and 94.1%, however, it resulted in a loss of sensitivity of 89.8 and 42.9%, respectfully. 140 141 142 143 144 145 146 147 148 Among the three chromogenic media designed for detection of CPGNB, chromid CARBA demonstrated the highest sensitivity and specificity, followed by Colorex KPC and Brilliance CRE. The chromid CARBA media failed to identify 5 CPGNB (3 isolates with OXA-48, 1 isolate with VIM-1 and 1 isolate with VIM-5). The Colorex KPC and Brilliance CRE media failed to support the growth of these same five isolates; Colorex KPC additionally did not detect one of each of NDM-1 producer, KPC-2 producer and SME producer; Brilliance CRE additionally did not detect one of each of VIM-2, 1 IMP-26, 1 SME and one OXA-48/VIM-4 producers. All three media designed for detection of CPGNB performed poorly for the detection of OXA-48 producers, detecting at the most five of the 8 (63.5%) OXA-48 producers. 149 150 151 152 153 Phenotypic Confirmatory Assays: In the second part of the study, a method comparison between the MH test and the RCS was performed on all isolates. On the first attempt with the previously described RCS protocol, 29 (28.7%) of the carbapenemase-negative isolates gave uninterpretable results (peach color in both tubes with and without imipenem (e.g., Figure 1. C2a & C2b). In addition, 4 of the carpabenemase producers were interpreted as negative (3 8

154 155 156 157 OXA-48 producers and 1 SME producer). Based off the initial results, the RCS protocol was troubleshot and modified upon repeat to only include one 10 µl calibrated loopful of organism (reduced from two) and a round-bottomed 15 ml tube (instead of a conical 1.5 ml test tube) to allow the diatab to dissolve completely (Figure 1). 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 It was noted that different enzyme classes performed differently with the RCS. KPC producers would turn the bacterial suspension a characteristic bright yellow color (positive test) immediately or within minutes of inoculating the test, all VIM and IMP producers would be positive at 30 minutes, whereas NDM and OXA producers and other CPGNB with relatively low carbapenem MICs would turn color more slowly and would yield an intermediate orange hue toward the end of incubation. Three of the carbapenemase positive isolates displayed intermediate positive results (orange hue) which included 3 of the 4 called negative by unmodified RCS (2 OXA-48 producers and one SME producer). Those isolates that yielded an orange color towards the end of incubation in this study were repeated using both the RCS and the CarbaNP tests, to determine which assay was easier to interpret for intermediate results [Figure 2, (3, 11, 13)]. Although both the RCS and the CarbaNP tests did yield the same intermediate orange color, it was found that the CarbaNP was easier to interpret the intermediate positives. As there was variability in the degree of color change for positives with the modified RCS, positives were considered any color change from the negative control tubes (tubes without imipenem). Using this as a marker of positivity as opposed to a strict yellow color change, sensitivity reached 98.0% and specificity was 100%. The RCS failed to identify one OXA-48 producing K. pneumoniae. 9

175 176 177 178 179 180 181 182 Conventional Multiplex PCR: The multiplex PCR for determination of carbapenemase gene class was verified with a sensitivity and specificity of 95.9% and 100%, respectively. The PCR failed to identify one OXA-24 producer and one SME producer. It was expected that bla OXA-24 was not detected by the multiplex PCR as this assay targetsbla OXA-48/181 specifically. Similarly, bla SME is not one of the targets included in the multiplex PCR. However, a PCR specific to SME was performed on the isolate and was found to be positive. Considering only the genes targeted by the multiplex PCR, the sensitivity and specificity achieved was 100%. 183 184 185 186 187 188 189 190 191 192 193 194 195 Determination of an Optimal Workflow for the Screening and Confirmation of CPGNB: The results of the screening and confirmatory assays (performed on all test isolates) were analyzed to develop the optimal workflow for the detection and confirmation of CPGNB. The best combination of tests in terms of analytical performance and assay cost and turnaround time was the use of chromid CARBA media for screening followed by the RCS test or multiplex PCR for confirmation as a CPGNB (89.8%, Table 1). The Colorex C3Gr media paired with the RCS did have the highest combined sensitivity (95.9%; partly due to the improved sensitivity to detect OXA producing strains, but not reaching 100% sensitivity in this enzyme class), however the low specificity of ESBL chromogenic screening media for CPGNB would greatly impact the overall cost of testing if routinely implemented. The large proportion of false positive CPGNB on Colorex C3Gr media that would be encountered under routine conditions (where ESBLs and organisms with reduced carbapenem susceptibility dominate in incidence over true CPGNB) would make it cost and time prohibitive. 10

196 197 Discussion: 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 The development of a screening and confirmatory method for the detection and confirmation of CPGNB is complicated by the scope of genera and enzyme classes encompassed in this broad group of antimicrobial resistant organisms. This study aimed to provide an optimal workflow for the detection of CPGNB to be implemented within the clinical microbiology laboratory. As such, a comprehensive evaluation of multiple chromogenic media and confirmatory methods were evaluated for their analytical performance and assay cost and turnaround time. Overall, we found that chromid CARBA media paired with the modified RCS protocol was the optimal approach (Figure 3), yielding a combined sensitivity of 89.8% and a specificity of 100%. The multiplex PCR for carbapenemase genes provided an equal analytical performance when paired with the chromid CARBA media, but of note, our laboratory system selected the RCS test as the confirmatory component for cost and time-to-reporting considerations. The RCS was $6.38 less per test and was anticipated to be completed at minimum 3 hours prior to the PCR result. However, for laboratories where high-volume CPGNB screening may occur, the scalability of PCR (i.e. use of 96 well plates and associated infrastructure) may be more cost and time effective than the manual manipulation required to complete the RCS test. 214 215 216 The first part of this study involved evaluating five different chromogenic media types to screen for CPGNB. Of the five different chromogenic media evaluated to detect CPGNB, Colorex C3Gr without the ertapenem disk had the highest sensitivity, 95.9%. However, not 11

217 218 219 220 221 222 surprisingly, the two ESBL media types (Colorex C3Gr and Brilliance ESBL) without the use of the ertapenem disk lacked specificity allowing growth of most ESBL and AmpC producers. The addition of the ertapenem disk to the two media types designed to detect ESBLs aided to substantially increase the specificity of the media for detection of CPGNB, however, it resulted in a loss of sensitivity. This is the first study to our knowledge that incorporated a carbapenem disk to select for CPGNB off of chromogenic media designed to detect ESBLs. 223 224 225 226 227 228 229 230 231 232 233 234 Among the three chromogenic media designed for detection of CPGNB, chromid CARBA demonstrated the highest sensitivity and specificity, followed by Colorex KPC and Brilliance CRE. All three media that are designed for detection of CPGNB performed poorly for the detection of OXA-48 producers. A reduced ability to detect OXA-48 producers was also observed by Wilkinson et al in their comparison of four chromogenic media types for the detection of carbapenemase-producing Enterobacteriaceae (15). Some OXA-48 isolates in our study did co-produce other β-lactamases, but these did not appear to consistently play a role in OXA-48 detection on chromogenic media, as was previously described (15). The reduced ability of the chromogenic media evaluated in this study to detect OXA-48 producers is a known limitation of the media. Recently developed media such as SUPERCARBA (9) and chromid OXA- 48 (biomérieux) have been described to have better sensitivity to detect OXA-48 producers (4, 9). Neither of these media were commecerially available in Canada at the time of this study. 235 236 237 In the second part of the study, a method comparison between the MH test and the RCS was performed on all isolates. Initially, many uninterpretable results with the RCS occurred. At that point, the assay was troubleshot and improvements were made to the modified RCS 12

238 239 240 241 242 243 244 245 246 protocol by using one 10 µl calibrated loopful of organism (reduced from two) and a roundbottomed 15 ml tube (instead of a conical 1.5 ml tube) to allow the diatab to dissolve completely. The diatabs did not dissolve completely in the conical bottom test tubes as there was not enough surface area of the diatab in contact with the small volume (150 μl) present in the tube. Also, it was noted after unblinding that if a high inoculum of a carbapenemase negative organism was present in the tubes containing imipenem, these tests would change to a peach color towards the end of incubation. The same uninterpreatble results were reported by Huang et al (5). The modification to a lowered inoculum and use of tubes with increased surface area is unique to this study. 247 248 249 250 251 252 253 Upon repeat with the modified RCS protocol a sensitivity of 98% and specificity of 100% was achieved. The RCS failed to identify one OXA-48 producing K. pneumoniae. Previous studies have demonstrated that the RCS like the CarbaNP test does not identify all OXA-48 producers (5, 16). Some intermediate positive results were obtained with the modified RSC and these isolates were tested using the CarbaNP for comparison. The CarbaNP test was easier to interpret than the RCS for intermediate positives; however, the advantage of the RCS is that it is a quality-controlled, commercially available laboratory kit, whereas the CarbaNP test requires 254 preparation of reagents in-house (3, 11, 13). Overall, the vast majority of our tested CPGNB 255 256 (91.8%) were yellow at 30 minutes, although the current clinical incidence of those enzyme classes that demonstrated an orange positive reaction is unknown. 13

257 258 259 This is the first study to date that has compared the RCS to the MH. The RCS proved to be a more sensitive and specific test than the MH, corroborating previous findings that compared the CarbaNP to the MH (3, 11, 13). 260 261 262 263 264 265 Lastly, the multiplex PCR for determination of carbapenemase gene class was verified with a sensitivity and specificity of 95.9% and 100%, respectfully. Although the multiplex PCR evaluated had a high sensitive and specificity if used for confirmation, the costs and technical difficulty of the assay was not ideal for routine use, and in our hands will remain an optional test choice for when it is of clinical value to determine the carbapenemase gene class (including for infection and prevention and control investigations). 266 267 268 269 270 271 272 273 274 275 276 By pairing the top performing screening and confirmatory tests, the preferred workflow was chromid CARBA agar media followed by the Rapid CARB Screen test, yielding a combined sensitivity of 89.8% and a specificity of 100% (Figure 3). The combination of assays was chosen based off of analytical performance characteristics and cost of testing and turnaround time under routine conditions. Even though the combination of Colorex C3Gr with the RCS had a higher sensitivity than chromid CARBA and the RCS, the decreased sensitivity of the Colorex C3Gr media would necessitate substantially more confirmatory testing; in our panel this would equate to twice as many RCS tests, 51 versus 103. On the other hand, the limitation of the chromid CARBA media is the limited detection of OXA-48 producers which is reflected in the sensitivity (89.8%) of the screening algorithm chosen. Our laboratory selected the RCS test for the confirmatory component for cost and time-to-reporting considerations, as the RCS was 14

277 278 $6.38 less per test and was anticipated to be completed at minimum 3 hours prior to a PCR result. 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 Subsequent to the evaluation and verification of this optimized workflow for the detection and confirmation of CPGNB, the chromid CARBA and the RCS test were utilized clinically. A patient was admitted locally after transfer from an international hospital, and was placed immediately on contact precautions in a single-bed room. From a wound specimen, a carbapenem-resistant P. aeruginosa was isolated from the patient using routine culture. The isolate was positive by the RCS test and identified as a VIM producer by the multiplex PCR. A rectal swab was subsequently collected from the patient to screen for CPGNB using the chromid CARBA media and a BA plate with ertapenem disks [(2); the BA plate with ertapenem disks was also selected because the new, verified protocol had not yet had a verification report filed at the time and was not systematically used subsequent to this single collection]. A NDMproducing K. pneumoniae grew on both media types and an OXA-181-producing E. coli grew only on the BA. A ward screen was performed to investigate if these carbapenemase-producing organisms had spread from the isolated patient. As such, 29 rectal swabs were submitted to the clinical microbiology laboratory and plated to chromid CARBA media; 27 (93.1%) had no growth after overnight incubation. Two patient cultures had P. aeruginosa breakthrough these were subbed to BA and tested negative by the RCS the following day (performed off the BA). In addition to the P. aeruginosa, a Gram-positive cocci and a yeast were present on the same chromid CARBA plates. Thus, it appeared in this single investigation that breakthrough is rare on the chromid CARBA media from rectal swabs, and that a Gram stain can be performed to quickly rule out breakthrough growth other than Gram-negative bacilli. 15

299 300 301 302 303 304 305 306 Limitations of this study include the small number of CPGNB tested within individual carbapenemase classes and the use of pure cultures of stocked isolates for the evaluation as opposed to clinical or mocked rectal swabs. However, strengths to this study include, 1) a diverse subset of CPGNB, 2) the evaluation of multiple chromogenic media and the novel use of a ertapenem disk on ESBL chromogenic media for the selection of CPGNBs, 3) the first study to compare the RCS to the MH, 4) the first study to modify the RCS protocol to improve interpretation of results and 5) a complete workflow approach was tested for the detection to confirmation of CPGNB. 307 Conclusions: 308 309 310 311 312 313 Overall, we found that chromid CARBA was the most sensitive and specific chromogenic media evaluated for the detection of CPGNB and with modification to the RCS protocol we were able to more easily interpret the results. By pairing the top-performing screening and confirmatory tests, the preferred workflow was chromid CARBA media followed by the RCS test, yielding a combined sensitivity of 89.8% and a specificity of 100%. The limitation of the selected algorithm is the poor detection of OXA-48 producers. 314 315 316 317 318 319 Acknowledgements We would like to thank V. Russell and the technologists at each site for their invaluable help with this project and, Oxoid, ThermoFisher, biomérieux, and Alere Canada for providing the chromogenic media, and Inter Medico for providing Rapid CARB Screen kits and International Health Management Associates, Inc (IHMA) and A. Denisuik for providing isolates for this study. 16

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Table 1: Performance of various chromogenic media, multiple confirmation assays and algorithms for the detection and confirmation of carbapenemase-producing Gram-negative bacilli (GNB). Genotype or Phenotype (150) Carbapenemase-producing GNB: N (%) KPC (16) bla KPC- 2, 3,11,12 VIM (12) bla VIM- 1,2,4 a,5 IMP (4) bla IMP- 1,26 NDM (9) bla NDM- 1,2,4,5 OXA (8) bla OXA- 24,48 a SME (1) bla SME Total (49) Carbapenemase negative GNB : N (%) ESBL (12) AmpC (45) Carb S (43) Total (101) Performance for CPGNB Detection (%) Sensitivity Specificity # of confirma -tory tests Cost Time Detection on Chromogenic Media # of tests *List price CAD per plate Brilliance 14 11 4 9 4 0 41 12 32 18 62 NA 83.7 38.6 ESBL (87.5) (91.6) (100) (100) (50.0) (83.7) (100) (71.1) (41.9) (61.4) Brilliance ESBL with ERT Brilliance CRE ChromID CARBA Colorex C3Gr Colorex C3Gr with ERT Colorex KPC 7 (46.7) 15 (93.8) 16 (100) 16 (100) 16 (100) 4 (33.3) 8 (66.7) 10 (83.3) 12 (100) 10 (83.3) 1 (25.0) 3 (75.0) 4 (100) 4 (100) 4 (100) 6 (66.7) 9 (100) 9 (100) 9 (100) 8 (88.9) 4 (50.0) 4 (50.0) 5 (62.5) 7 (87.5) 6 (75.0) 0 21 (42.9) 0 38 (77.6) 1 44 (100) (89.8) 0 47 (95.9) 1 44 (100) (89.8) 2 (16.7) 3 (25.0) 2 (16.7) 12 (100) 2 (16.7) 2 (4.4) 9 (20.0) 3 (6.7) 41 (91.1) 34 (75.6) 2 (4.7) 1 13 (2.3) (12.9) 0 5 (5.0) 9 62 (20.9) (61.4) 2 (4.7) 6 (5.9) 42.9 94.1 77.6 87.1 89.8 95.0 95.9 38.6 38 (37.6) 89.8 62.4 NA NA NA NA NA 20 Turn Around Time (TAT) $5.42 18-24 hrs $5.62 18-24 hrs $7.56 18-24 hrs $3.25 18-24 hrs $2.79 18-24 hrs $2.99 18-24 hrs 15 10 4 8 5 0 41 2 5 1 8 NA 83.7 92.1 (93.8) (83.3) (100) (88.9) (62.5) (83.7) (16.7) (11.1) (2.3) (7.9) $2.79 18-24 hrs Confirmation Assays # of tests Cost CAD per assay TAT Modified 14 7 3 6 8 0 37 0 7 0 7 NA 75.5 93.1 Hodge (87.5) (58.3) (75.0) (66.7) (100) (75.5) (15.6) (6.9) $0.27 24 hours Rapid CARB 16 10 4 9 7 1 48 0 0 0 0 NA 98.0 100 Screen (100) (83.3) (100) (100) (87.5) (100) (98.0) $4.57 2 hours Multiplex 16 12 4 9 7 b 0 c 47 0 0 0 0 95.9 100 NA $10.95 4 hours

Convention al PCR (100) (100) (100) (100) (87.5) (95.9) Algorithms for the detection and confirmation of carbapenemase producers # of tests Cost per isolate TAT d Brilliance 14 11 4 9 4 0 41 0 0 0 0 $9.99 83.7 100 103 ESBL & RCS (87.5) (91.6) (100) (100) (50.0) (83.7) 50 hours Brilliance 15 8 3 9 4 0 38 0 0 0 0 $12.13 77.6 100 51 CRE & RCS (93.8) (66.7) (75.0) (100) (50.0) (77.6) 50 hours Chrom ID & 16 10 4 9 4 1 44 0 0 0 0 RCS (100) (83.3) (100) (100) (50.0) (100) (89.8) 89.8 100 49 $7.82 50 hours Colorex 16 12 4 9 7 0 47 0 0 0 0 C3Gr & RCS (100) (100) (100) (100) (87.5) (95.9) 95.9 100 109 $7.36 50 hours Colorex KPC 15 10 4 8 5 0 41 0 0 0 0 & RCS (93.8) (83.3) (100) (88.9) (62.5) (83.7) 83.7 100 49 $7.36 50 hours Colorex 16 12 4 9 6 0 45 0 0 0 0 PCR C3Gr & (100) (100) (100) (100) (75.0) (91.8) Multiplex 91.7 100 103 $13.74 53 hours chromid 16 10 4 9 4 1 44 0 0 0 0 PCR CARBA & (100) (83.3) (100) (100) (50.0) (100) (89.8) Multiplex 89.8 100 49 $14.20 53 hours 1 2 a One Enterobacter cloacae isolate possessed both VIM-4 and OXA-48 3 b One OXA-24 producer was not identified by the multiplex conventional PCR as it is specific for bla OXA-48 (6). 4 c bla SME is not a target in the multiplex PCR. However, a simplex PCR for bla SME was performed and was positive for the isolate (70). 5 RCS: Rapid CARB Screen, GNB: Gram-negative bacilli, CPGNB: Carbapenemase-producing Gram-negative bacilli, mins: minutes, hrs: 6 hours. NA: Not applicable. 7 d Factoring in the likelihood of the need for a pure culture to initiate any confirmatory testing 8 21

9 10 11 12 Figure 1: Troubleshooting the Rapid Carb Screen test with the use of different tubes and increasing inoculum. A 1a 1b 2a 2b 3b 3a 13 14 15 16 B 17 1a 1b 2a 2b 3a 3b 18 19 20 21 22 23 24 C 1a 1b 2a 2b 3b 3a 25 26 27 28 29 30 Rapid CARB Screen comparing the test in A) round bottom 15 ml tubes, B) Cryovials and C) test tubes using a AmpC-producing Enterobacter cloacae (should yield a negative result by RCS). 1 a) Tube with imipenem 1b) Tube without imipenem; using one calibrated 10 μl loop of organism. 2a) Tube with imipenem, 2b) Tube without imipenem; using two calibrated 10 μl loops of organism. 3a) Tube with imipenem, 3b) Tube without imipenm; using three calibrated 22

31 32 33 34 35 36 10 μl loops of organism. Of note, as the inoculum increases tube with imipenem (Tube A) in the Cryovials and Test tubes demonstrate that it turns a peach color in comparison to the tube without Imipenem (Tube B). In addition, the color change in the test tubes (C) is not apparent as the diatabs were not able to properly dissolve and result in uninterpretatble results especially with increased inoculum. The round bottom 15 ml tubes A) demonstrated the most easy to interpret results. 37 23

38 39 40 Figure 2: Comparison of the Rapid CARB Screen to the CarbaNP for a SME producing Serratia marcsens at two hours of incubation (intermediate positive; orange hue). A 1a 1b 2a 2b 3a 3b 41 B 1a 1b 2a 2b 3a 3b 42 43 44 45 46 47 48 A. Rapid CARB Screen Results using round bottom 15 ml tubes and one full 10 μl calibrated loop of organism. B. CarbaNP Assay using 1.5ml conical test tubes and one full 10 μl calibrated loop of organism. 1. Positive Control: K. pneumoniae ATCC BAA1705; 2. Negative Control: K. pneumoniae ATCC BAA1706 and 3. SME-producing Serratia marcenscens. Tubes a) without imipenem and tubes b) with imipenem. In section A, note the dissolved diatabs settling at the bottom of the tubes. 49 24

50 51 52 53 54 Figure 3: The chosen workflow for the detection and confirmation of carbapenemase-producing Gram-negative bacilli using chromid CARBA media for the detection and the Rapid CARB Screen for confirmation. After 18-24 hours of incubation, examine the chromid CARBA 55 56 57 58 59 60 61 62 63 64 65 66 67 68 No Growth Issue final report with NOCP No GNB No further workup Growth perform Gram stain to confirm Gram-negative bacilli GNB Note: color of growth is indicative of: Pink-Burgundy: E. coli Bluish green-gray: Klebsiella Enterobacter Serratia Citrobacter group Subculture to a BA to perform the Rapid Carb Screen the following day. Initiate ID of GNB if sufficient growth is present on chromid CARBA, or ID from BA the following day 69 70 Any isolate is RCS All isolates are RCS 71 NOCP CARBP Report ID of RCS positive isolates, and add CARBP comment Notify IP&C and IDS No carbapenemase-producing Gram-negative bacteria isolated. No ID are reported, and add NOCP comment This isolate contains a carbapenemase and demonstrates reduced susceptibility to carbapenems. Consultation with the Infectious Disease Service is recommended. Follow the Infection Prevention & Control guidelines for Antibiotic Resistant Organisms (ARO) and/or contact Infection Prevention and Control practitioner. 25

72 73 74 GNB: Gram-negative bacilli, IP&C: Infection Prevention and Control, ID: Infectious Disease Service 75 26