Evaluation of Five Chromogenic Agar Media and the Rosco RapidCarb Screen Kit for Detection and Confirmation of CarbapenemaseProduction in Gram-Negative Bacilli

Patricia J. Simner,a,b Matthew W. Gilmour,a,b Pat DeGagne,a Kim Nichol,a James A. Karlowskya,b

Diagnostic Services Manitoba, Winnipeg, Manitoba, Canadaa; University of Manitoba, Department of Medical Microbiology and Infectious Diseases, Winnipeg, Manitoba,Canadab

An efficient workflow to screen for and confirm the presence of carbapenemase-producing Gram-negative bacilli was developedby evaluating five chromogenic screening agar media and two confirmatory assays, the Rapid Carb screen test (Rosco Diagnos-tica A/S, Taastrup, Denmark) and the modified Hodge test. A panel of 150 isolates was used, including 49 carbapenemase-pro-ducing isolates representing a variety of �-lactamase enzyme classes. An evaluation of analytical performance, assay cost, andturnaround time indicated that the preferred workflow (screening test followed by confirmatory testing) was the chromID Carbaagar medium (bioMérieux, Marcy l’Étoile, France), followed by the Rapid Carb screen test, yielding a combined sensitivity of89.8% and a specificity of 100%. As an optional component of the workflow, a determination of carbapenemase gene class viamolecular means could be performed subsequent to confirmatory testing.

The worldwide dissemination of carbapenemase-producingGram-negative bacilli (CPGNB) is a significant clinical and

public health concern (1, 2). The rapid detection of these antibi-otic-resistant pathogens by the clinical microbiology laboratory isof utmost importance in controlling nosocomial spread and forinitiating appropriate antimicrobial therapy. Chromogenic mediacontaining a carbapenem are convenient tools for the screeningand rapid detection of carbapenem-resistant Gram-negative ba-cilli (GNB). However, growth on a chromogenic medium simplysignifies carbapenem resistance (e.g., an AmpC producer withporin loss may grow) and does not confirm the production of acarbapenemase. Recently, a novel rapid chromogenic test basedon the hydrolysis of imipenem, the Carba NP test, and a commer-cial version of the Carba NP test, the Rapid Carb screen (RCS) test,have been described (3–8). The purpose of the current study wasto evaluate screening and confirmation methods that when pairedwould provide a streamlined workflow for the detection ofCPGNB in the clinical microbiology laboratory. First, five differ-ent chromogenic media were evaluated to select a sensitive me-dium type for screening for CPGNB. Second, the RCS test (RoscoDiagnostica A/S, Taastrup, Denmark) was evaluated to confirmcarbapenemase production through a method comparison withthe current gold standard, the modified Hodge (MH) test (9). Acombined workflow approach was designed to achieve the maxi-mum sensitivity and specificity for detecting CPGNB, with a con-sideration for the cost and turnaround time of testing. Last, uponfinding a CPGNB, it may be of value in some circumstances todetermine the carbapenemase gene class, and therefore, an estab-lished multiplex PCR was also incorporated as an optional end-point to this workflow.

MATERIALS AND METHODSBacterial isolates. A total of 150 isolates were tested, including an assort-ment of 49 isolates of GNB harboring a diverse set of carbapenemaseenzyme classes and 101 carbapenemase-negative isolates. The 49 carbap-enemase producers, a combination of clinical and ATCC strains, werepreviously molecularly characterized for �-lactamase genes and included16 K. pneumoniae carbapenemase (KPC) producers (blaKPC-2 [n � 9],

blaKPC-3 [n � 5], blaKPC-11 [n � 1], blaKPC-12 [n � 1])—Citrobacter freun-dii, n � 2; Citrobacter koseri, n � 1; Enterobacter cloacae, n � 1; Escherichiacoli, n � 4; Klebsiella oxytoca, n � 1; Klebsiella pneumoniae, n � 5; Pseu-domonas aeruginosa, n � 1; and Serratia marcescens, n � 1; 12 VIM pro-ducers (blaVIM-1 [n � 6], blaVIM-2 [n � 3], blaVIM-4 [n � 2], blaVIM-5 [n �1])—C. freundii, n � 2; Enterobacter aerogenes, n � 1; E. cloacae, n � 1; K.oxytoca, n � 1; Proteus mirabilis, n � 1; Providencia stuartii, n � 1; and P.aeruginosa, n � 5; 4 IMP producers (blaIMP-1 [n � 3] and blaIMP-26) [n �1]—E. coli, n � 1; K. pneumoniae, n � 2; and P. aeruginosa, n � 1; 9 NDMproducers (blaNDM-1 [n � 6], blaNDM-4 [n � 1], blaNDM-5 [n � 1],blaNDM-7 [n � 1])—Acinetobacter baumannii, n � 1; E. coli, n � 4; K.oxytoca, n � 1; K. pneumoniae, n � 1; P. mirabilis, n � 1; and Providenciarettgeri, n � 1; 8 OXA producers (blaOXA-24 [n � 1], blaOXA-48 [n �7])—A. baumannii, n � 1; C. koseri, n � 1; E. cloacae, n � 1; K. pneu-moniae, n � 3; Morganella morganii, n � 1 and S. marcescens, n � 1; andone SME producer—S. marcescens, n � 1). Of note, one E. cloacae isolatepossessed both VIM-4 and OXA-48. The 101 carbapenemase-negativeisolates included extended-spectrum �-lactamase (ESBL) and AmpC pro-ducers (previously molecularly characterized) with and without porinmutations, clinical isolates with elevated carbapenem MICs (and negativefor carbapenemases by PCR), and carbapenem-susceptible isolates. Theisolates were blinded, and 75 isolates were distributed to each of the twoclinical microbiology laboratory testing sites (Health Sciences Centre andSt. Boniface Hospital, Winnipeg, Canada).

Chromogenic media to screen for CPGNB. The first part of this studyinvolved evaluating five different chromogenic medium types used to

Received 21 July 2014 Returned for modification 18 August 2014Accepted 17 October 2014

Accepted manuscript posted online 29 October 2014

Citation Simner PJ, Gilmour MW, DeGagne P, Nichol K, Karlowsky JA. 2015.Evaluation of five chromogenic agar media and the Rosco Rapid Carb screen kitfor detection and confirmation of carbapenemase production in Gram-negativebacilli. J Clin Microbiol 53:105–112. doi:10.1128/JCM.02068-14.

Editor: R. Patel

Address correspondence to Patricia J. Simner, psimner@dsmanitoba.ca.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JCM.02068-14

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screen for CPGNB. The five chromogenic media evaluated were (i) OxoidBrilliance ESBL (Besington, Hants, United Kingdom), (ii) Oxoid Bril-liance CRE, (iii) bioMérieux chromID Carba (Marcy l’Étoile, France), (iv)CHROMagar Colorex C3Gr (Paris, France), and (v) CHROMagar Col-orex KPC. The Brilliance ESBL and Colorex C3Gr media are designed todetect ESBLs, and in this study, they were evaluated both with and withoutan ertapenem disk (10 �g) to select for carbapenem-resistant isolates. Theinocula for each of the chromogenic medium types and a nonselectiveblood agar (BA) plate were 10 �l (�106 CFU/ml) of a 0.5 McFarlandstandard prepared in sterile saline (0.85%). Brilliance ESBL, BrillianceCRE, and chromID Carba were streaked using the Isoplater (Vista Tech-nology, Inc., Edmonton, Alberta, Canada) to mimic standard laboratorypractice. The Colorex C3Gr and Colorex KPC media were evaluated as abiplate, so the media were streaked manually. Ertapenem disks wereplaced between the 2nd and 3rd quadrants of the Brilliance ESBL andColorex C3Gr media following the streaking of the plate. The plates wereincubated for 24 h at 37°C, and the presence/absence and color of growthwere interpreted according to the package inserts. Any isolate on the ESBLchromogenic media with an ertapenem zone of inhibition of �27 mm wasconsidered a putative carbapenemase producer and was considered forfurther evaluation using a confirmatory method. The use of the �27-mmcutoff was established in a previous study using an ertapenem disk (10 �g)to screen for carbapenem-resistant GNB on MacConkey medium (10).

Phenotypic confirmatory assays. In the second part of the study, amethod comparison between the MH test and the RCS test was performedon all study isolates using growth from the BA plate inoculated concur-rently with the chromogenic media. The BA plate was added to performthe confirmatory tests the following day, since the RCS test cannot beperformed from the chromogenic media, as the color of the colonies in-terferes with the interpretation of the test. The MH test using meropenemdisks (10 �g) was performed and interpreted according to CLSI method-ology (9). The RCS is a commercially available rapid chromogenic test forthe detection of carbapenemases based on the hydrolysis of imipenem inthe presence of an indicator (phenol red [5]). All the components of theassay are present within tablets called diatabs. Two sets of diatabs arecontained in the kit, one containing imipenem and one without. Thediatabs are dissolved in saline, and the lysate of the organism is added to atube with a dissolved diatab with imipenem and a tube with a dissolveddiatab without imipenem (negative-control tube; it should remain red incolor). Initially, the RCS test was performed as previously described (4). K.pneumoniae strain ATCC BAA1706 and K. pneumoniae strain ATCCBAA1705 were the positive and negative controls, respectively. Any colorchange from red to yellow in the 1.5-ml test tube containing imipenemwas considered a positive reaction for the RCS. Uninterpretable resultswere isolates that gave a slight positive reaction (peach color) in both thetubes with and without imipenem.

As there were many uninterpretable results initially using the previ-ously described RCS protocol, troubleshooting was performed in the RCSprotocol; the appropriate inoculum was determined by testing one tothree calibrated loops of organism and finding the optimal tube for per-forming the assay by testing 1.5-ml test tubes, cryovials, and round-bot-tom 5-ml tubes. Thus, the modified RCS test was performed upon repeaton all isolates to include only one 10-�l calibrated loopful of organism(reduced from two) and a round-bottom 5-ml tube (instead of a conical1.5-ml tube). As there was variability in the degree of color change for thepositives with the modified RCS protocol, positives were considered thosedisplaying any color change from that of the negative-control tubes (tubeswithout imipenem). The isolates that were orange by the modified RCSprotocol in this study were repeated using both the RCS test and the CarbaNP test, as previously described, to assess the ease of interpretation of theintermediate results (3, 5, 7).

Conventional multiplex PCR. Last, a conventional multiplex PCR forblaKPC, blaNDM, blaVIM, blaIMP, and blaOXA-48 and blaOXA-181 was per-formed on all isolates, as previously described (11). In addition, a blaSME

PCR was performed on one isolate to confirm SME production (12).

Determination of an optimal workflow for the screening and confir-mation of CPGNB. The results of the screening and confirmatory assays(performed on all test isolates) were analyzed to develop the optimalworkflow (i.e., test choice) for the detection and confirmation of CPGNB.By combining the individual data from the chromogenic media and theconfirmatory assays, we were able to determine the sensitivities and spec-ificities of the combined assays. In addition, the cost and time to performthe different combination of assays were evaluated and considered forfeasibility and full optimization of the workflow within a clinical micro-biology laboratory setting.

RESULTSChromogenic media for CPGNB. The first part of this study in-volved evaluating five different chromogenic medium types usedto screen for CPGNB, including two media targeting ESBLs sup-plemented with and without an ertapenem disk (Table 1). Of thefive different chromogenic media evaluated to detect CPGNB (Ta-ble 1), Colorex C3Gr without the ertapenem disk had the highestsensitivity, at 95.9%. However, the two ESBL medium types, Col-orex C3Gr and Brilliance ESBL, without the use of the ertapenemdisk both had a specificity of 38.6% for CPGNB. The addition ofthe ertapenem disk to Colorex C3Gr and Brilliance ESBL resultedin increased specificities of 62.4% and 94.1%, respectively; how-ever, it resulted in a loss of sensitivity, at 89.8 and 42.9%, respec-tively.

Among the three chromogenic media designed to detectCPGNB, chromID Carba demonstrated the highest sensitivity andspecificity, followed by Colorex KPC and Brilliance CRE. ThechromID Carba medium failed to identify 5 CPGNB (3 isolateswith OXA-48, 1 isolate with VIM-1, and 1 isolate with VIM-5).The Colorex KPC and Brilliance CRE media failed to support thegrowth of these same five isolates; the Colorex KPC medium ad-ditionally did not detect one of each of the NDM-1, KPC-2, andSME producers, and Brilliance CRE additionally did not detectone of each of the VIM-2, IMP-26, SME, and OXA-48 and VIM-4producers. All three media designed for the detection of CPGNBperformed poorly for the detection of OXA-48 producers, detect-ing at the most 5 of the 8 (63.5%) OXA-48 producers.

Phenotypic confirmatory assays. In the second part of thestudy, a method comparison between the MH test and the RCStest was performed on all isolates. On the first attempt with thepreviously described RCS protocol, 29 (28.7%) of the carbapen-emase-negative isolates gave uninterpretable results (i.e., a peachcolor in both tubes with and without imipenem [e.g., Fig. 1C,tubes 2a and 2b]). In addition, 4 of the carbapenemase producerswere interpreted as negative (3 OXA-48 producers and 1 SMEproducer). Based on the initial results, troubleshooting was per-formed for the RCS protocol, and it was modified upon repeat toonly include one 10-�l calibrated loopful of organism (reducedfrom two) and a round-bottom 5-ml tube (instead of a conical1.5-ml test tube) to allow the diatab to dissolve completely (Fig. 1).

It was noted that different enzyme classes performed differ-ently with the RCS test. KPC producers turned the bacterial sus-pension a characteristic bright yellow color (positive test) imme-diately or within minutes of inoculating the test, all VIM and IMPproducers were positive at 30 min, and NDM and OXA producersand other CPGNB with relatively low carbapenem MICs turnedcolor more slowly and yielded an intermediate orange hue towardthe end of incubation. Three of the carbapenemase-positive iso-lates displayed intermediate positive results (orange hue), whichincluded 3 of the 4 that were called negative by the unmodified

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Detection of Carbapenemase-Producing GNB

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RCS (2 OXA-48 producers and 1 SME producer). Those isolatesthat yielded an orange color toward the end of incubation in thisstudy were repeated using both the RCS and the Carba NP tests todetermine which assay was easier to interpret for the intermediateresults (Fig. 2) (3, 5, 7). Although both the RCS and the Carba NPtests yielded the same intermediate orange color, it was found thatthe Carba NP test was easier for interpreting the intermediatepositives. As there was variability in the degree of color change forthe positives with the modified RCS test, the positives were con-sidered those displaying any color change from that of the nega-tive-control tubes (tubes without imipenem). Using this as a

marker of positivity, as opposed to a strict yellow color change, thesensitivity reached 98.0% and the specificity was 100%. The RCSfailed to identify one OXA-48-producing K. pneumoniae isolate.

Conventional multiplex PCR. The multiplex PCR for the de-termination of carbapenemase gene class was verified, with a sen-sitivity and specificity of 95.9% and 100%, respectively. The PCRfailed to identify one OXA-24 producer and one SME producer. Itwas expected that blaOXA-24 would not be detected by the multi-plex PCR, as this assay specifically targets blaOXA-48 and blaOXA-181.Similarly, blaSME is not one of the targets included in the multiplexPCR. However, a PCR specific to SME was performed on the iso-

FIG 1 Troubleshooting the Rapid Carb screen test with the use of different tubes and increasing inocula. Shown is the Rapid Carb screen comparing the test inround-bottom 5-ml tubes (A), cryovials (B), and test tubes (C) using an AmpC-producing E. cloacae isolate (which should yield a negative result by RCS). Tube1a has imipenem, and tube 1b is without imipenem; these used one calibrated 10-�l loop of the organism. Tube 2a has imipenem, and tube 2b is withoutimipenem; these used two calibrated 10-�l loops of the organism. Tube 3a has imipenem, and tube 3b is without imipenem; these used three calibrated 10-�lloops of the organism. In addition, the color change in the test tubes (C) is not apparent, as the diatabs were not able to properly dissolve and resulted inuninterpretable results, especially with increased inoculum. The round-bottom 5-ml tubes (A) demonstrated the easiest-to-interpret results.

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late and was found to be positive. Considering only the genestargeted by the multiplex PCR, the sensitivity and specificityachieved were both 100%.

Determination of an optimal workflow for the screening andconfirmation of CPGNB. The results of the screening and confir-matory assays (performed on all test isolates) were analyzed todevelop the optimal workflow for the detection and confirmationof CPGNB. The best combination of tests in terms of analyticalperformance, assay cost, and turnaround time was the use of chro-mID Carba medium for screening, followed by the RCS test ormultiplex PCR for confirmation of detected CPGNB (89.8%; Ta-ble 1). The Colorex C3Gr medium paired with the RCS test hadthe highest combined sensitivity (95.9%, which was partly due tothe improved sensitivity for detecting OXA-producing strains, butit did not reach 100% sensitivity in this enzyme class); however,the low specificity of the ESBL chromogenic screening media forCPGNB would greatly impact the overall cost of testing if rou-tinely implemented. The large proportion of false-positiveCPGNB on Colorex C3Gr medium that would be encounteredunder routine conditions (in which ESBLs and organisms withreduced carbapenem susceptibility dominate in incidence overtrue CPGNB) would make it cost- and time-prohibitive.

DISCUSSION

The development of a screening and confirmatory method for thedetection and confirmation of CPGNB is complicated by thescope of the genera and enzyme classes encompassed in this broadgroup of antimicrobial-resistant organisms. This study aimed toprovide an optimal workflow for the detection of CPGNB to beimplemented within the clinical microbiology laboratory. Assuch, a comprehensive evaluation of multiple chromogenic mediaand confirmatory methods was conducted to evaluate their ana-lytical performance, assay cost, and turnaround time. Overall, wefound that chromID Carba medium paired with the modified RCS

protocol was the optimal approach (Fig. 3), yielding a combinedsensitivity of 89.8% and a specificity of 100%. The multiplex PCRfor carbapenemase genes provided an equal analytical perfor-mance when paired with the chromID Carba medium, but, ofnote, our laboratory system selected the RCS test as the confirma-tory component for cost and time-to-reporting considerations.The RCS cost Can$6.38 less per test and was anticipated to becompleted at a minimum of 3 h prior to the PCR result. However,for laboratories in which high-volume CPGNB screening may oc-cur, the scalability of PCR (i.e., use of 96-well plates and associatedinfrastructure) may be more cost- and time-effective than themanual manipulation required to complete the RCS test.

The first part of this study involved evaluating five differentchromogenic medium types used to screen for CPGNB. Of the fivedifferent chromogenic media evaluated to detect CPGNB, Col-orex C3Gr without the ertapenem disk had the highest sensitivity,at 95.9%. However, not surprisingly, the two ESBL medium types(Colorex C3Gr and Brilliance ESBL) without the use of the ertap-enem disk lacked specificity, allowing the growth of most ESBLand AmpC producers. The addition of the ertapenem disk to thetwo medium types designed to detect ESBLs helped to substan-tially increase the specificity of the media for detecting CPGNB;however, it resulted in a loss of sensitivity. This is the first study, toour knowledge, that incorporated a carbapenem disk to select forCPGNB from chromogenic media designed to detect ESBLs.

Among the three chromogenic media designed to detectCPGNB, chromID Carba demonstrated the highest sensitivity andspecificity, followed by Colorex KPC and Brilliance CRE. All threemedia that were designed to detect CPGNB performed poorly forthe detection of OXA-48 producers. A reduced ability to detectOXA-48 producers was also observed by Wilkinson et al. (13) intheir comparison of four chromogenic medium types used for thedetection of carbapenemase-producing Enterobacteriaceae. Some

FIG 2 Comparison of the Rapid Carb screen with the Carba NP test for an SME-producing Serratia marcescens isolate at 2 h of incubation (intermediate positive;orange hue). (A) Rapid Carb screen results using round-bottom 5-ml tubes and one full 10-�l calibrated loop of the organism. (B) Carba NP assay using 1.5-mlconical test tubes and one full 10-�l calibrated loop of the organism. Tubes labeled with a 1 are positive controls of K. pneumoniae ATCC BAA1705, tubes labeledwith a 2 are K. pneumoniae ATCC BAA1706, and tubes labeled with a 3 are SME-producing S. marcescens. Tubes with numbers followed by “a” lack imipenem,and tubes with numbers followed by “b” contain imipenem. (A) Note the dissolved diatabs settling at the bottom of the tubes.

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OXA-48 isolates in our study coproduced other �-lactamases, butthese did not appear to consistently play a role in OXA-48 detec-tion on chromogenic media, as was previously described (13). Thereduced ability of the chromogenic media evaluated in this studyto detect OXA-48 producers is a known limitation of the media.Recently developed media, such as Supercarba (14) and chromIDOXA-48 (bioMérieux), have been described to have better sensi-tivity for detecting OXA-48 producers (14, 15). Neither of thesemedia were commercially available in Canada at the time of thisstudy.

In the second part of the study, a method comparison betweenthe MH test and the RCS test was performed on all isolates. Ini-tially, many uninterpretable results occurred with the RCS test. Atthat point, troubleshooting was performed for the assay, and im-provements were made for the modified RCS protocol by usingone 10-�l calibrated loopful of organism (reduced from two) anda round-bottom 5-ml tube (instead of a conical 1.5-ml tube) toallow the diatab to dissolve completely. The diatabs did not dis-solve completely in the conical-bottom test tubes, as there was notenough surface area of the diatab in contact with the small volume(150 �l) present in the tube. Also, it was noted after unblindingthat if a high inoculum of a carbapenemase-negative organism

was present in the tubes containing imipenem, these tests wouldchange to a peach color toward the end of incubation. The sameuninterpretable results were reported by Huang et al. (4). Themodifications of a lowered inoculum and the use of tubes withincreased surface area are unique to this study.

Upon repeat with the modified RCS protocol, a sensitivity of98% and a specificity of 100% were achieved. The RCS failed toidentify one OXA-48-producing K. pneumoniae isolate. Previousstudies have demonstrated that the RCS test, like the Carba NPtest, does not identify all OXA-48 producers (4, 8). Some interme-diate positive results were obtained with the modified RCS test,and these isolates were tested using the Carba NP test for compar-ison. The Carba NP test was easier to interpret than the RCS testfor the intermediate positives; however, the advantage of the RCStest is that it is a quality-controlled commercially available labo-ratory kit, whereas the Carba NP test requires the preparation ofreagents in-house (3, 5, 7). Overall, the vast majority (91.8%) ofour tested CPGNB were yellow at 30 min, although the currentclinical incidences of those enzyme classes that demonstrated anorange positive reaction are unknown.

This is the first study to date that has compared the RCS test tothe MH test. The RCS test proved to be a more sensitive and

NOCP

CARBP ious Disease Service is recommended.

After 18-24 hours of incubation, examine the chromID CARBA

Issue final report with NOCP

No Growth Growth – perform Gram stain to confirm Gram-negative bacilli

• 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

• Report ID of RCS positive isolates, and add CARBP comment

• Notify IP&C and IDS

Note: color of growth is indicative of:

• Pink-Burgundy: E. coli • Bluish green-gray: Klebsiella –

Enterobacter – Serratia – Citrobacter group

No further workup

GNB No GNB

Any isolate is RCS positive All isolates are RCS negative

No ID are reported, and add NOCP comment

FIG 3 The chosen workflow for the detection and confirmation of carbapenemase-producing Gram-negative bacilli using chromID Carba medium for detectionand the Rapid Carb screen kit for confirmation. GNB, Gram-negative bacilli; IP&C, Infection Prevention and Control; IDS, Infectious Disease Service.

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specific test than the MH test, corroborating previous findingsthat compared the Carba NP test with the MH test (3, 5, 7).

Last, the multiplex PCR for the determination of carbapen-emase gene class was verified, with a sensitivity and specificity of95.9% and 100%, respectively. Although the multiplex PCR eval-uated had a high sensitivity and specificity if used for confirma-tion, the costs and technical difficulty of the assay are not ideal forroutine use, and in our hands, it will remain an optional testchoice for when it is of clinical value to determine the carbapen-emase gene class (including for infection, prevention, and controlinvestigations).

By pairing the top-performing screening and confirmatorytests, the preferred workflow was chromID Carba agar medium,followed by the Rapid Carb screen test, yielding a combined sen-sitivity of 89.8% and a specificity of 100% (Fig. 3). The combina-tion of assays was chosen based on the analytical performancecharacteristics, cost of testing, and turnaround time under routineconditions. Even though the combination of Colorex C3Gr withthe RCS test had a higher sensitivity than that of the chromIDCarba and the RCS test, the decreased sensitivity of the ColorexC3Gr medium would necessitate substantially more confirmatorytesting; in our panel, this would equate to twice as many RCS tests(51 versus 103, respectively). On the other hand, the limitation of thechromID Carba medium is the limited detection of OXA-48 produc-ers, which is reflected in the sensitivity (89.8%) of the screening algo-rithm chosen. Our laboratory selected the RCS test for the confirma-tory component for cost and time-to-reporting considerations, as theRCS test was Can$6.38 less per test and was anticipated to be com-pleted at a minimum of 3 h prior to a PCR result.

Subsequent to the evaluation and verification of this optimizedworkflow for the detection and confirmation of CPGNB, thechromID Carba and the RCS test were utilized clinically. A patientwas admitted locally after transfer from an international hospitaland was placed immediately on contact precautions in a single-bed room. From a wound specimen, carbapenem-resistant P.aeruginosa was isolated from the patient using routine culture.The isolate was positive by the RCS test and identified as a VIMproducer by the multiplex PCR. A rectal swab was subsequentlycollected from the patient to screen for CPGNB using thechromID Carba medium and a BA plate with ertapenem disks(16) (the BA plate with ertapenem disks was also selected becausethe new verified protocol had not yet had a verification report filedand was not systematically used subsequent to this single collec-tion). An NDM-producing K. pneumoniae isolate grew on bothmedium types, and an OXA-181-producing E. coli isolate grew onthe BA only. A ward screen was performed to investigate if thesecarbapenemase-producing organisms had spread from the iso-lated patient. As such, 29 rectal swabs were submitted to the clin-ical microbiology laboratory and plated on chromID Carba me-dium; 27 (93.1%) had no growth after overnight incubation. Twopatient cultures had P. aeruginosa breakthrough; these were sub-cultured onto BA and tested negative by the RCS test the followingday (performed from the BA). In addition to the P. aeruginosa, aGram-positive coccus and a yeast were present on the samechromID Carba plates. Thus, it appeared in this single investiga-tion that breakthrough from rectal swabs on the chromID Carbamedium is rare, and a Gram stain can be performed to quickly ruleout breakthrough growth other than that from Gram-negativebacilli.

The limitations of this study include the small number of

CPGNB tested within individual carbapenemase classes and theuse of pure cultures of stocked isolates for the evaluation, as op-posed to clinical or mocked rectal swabs. However, the strengthsof this study include (i) a diverse subset of CPGNB, (ii) the eval-uation of multiple chromogenic media and the novel use of aertapenem disk on ESBL chromogenic media for the selection ofCPGNBs, (iii) being the first study to compare the RCS test withthe MH test, (iv) being the first study to modify the RCS protocolto improve the interpretation of the results, and (v) a completeworkflow approach was tested for confirmation of CPGNB detec-tion.

Overall, we found that chromID Carba was the most sensitiveand specific chromogenic medium evaluated for the detection ofCPGNB, and with modification to the RCS protocol, we were ableto more easily interpret the results. By pairing the top-performingscreening and confirmatory tests, we found that the preferredworkflow was chromID Carba medium, 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 ofOXA-48 producers.

ACKNOWLEDGMENTS

We thank V. Russell and the technologists at each site for their invaluablehelp with this project, Oxoid, Thermo Fisher, bioMérieux, and Alere Can-ada for providing the chromogenic media, Inter Medico for providing theRapid Carb screen kits, and International Health Management Associates,Inc. (IHMA) and A. Denisuik for providing the isolates for this study.

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