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.
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Evaluation of five chromogenic agar media and the ROSCO Rapid CARB Screen kit for the
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detection and confirmation of carbapenemase production in Gram-negative bacilli
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Patricia J. Simner,a,b# Matthew W. Gilmour, a,b Pat DeGagne, a Kim Nichol, a and James A.
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Karlowsky a,b
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Diagnostic Services Manitoba, Winnipeg, Manitoba, Canadaa; University of Manitoba,
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Department of Medical Microbiology and Infectious Diseases, Winnipeg, Manitoba, Canadab
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Running Title: Detection of carbapenemase-producing Gram-negatives
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# Address correspondence to Patricia J. Simner, [email protected]
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Abstract:
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An efficient workflow to screen for and confirm carbapenemase-producing Gram-negative
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bacilli was developed by evaluating five chromogenic screening agar media and two
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confirmatory assays, the Rapid CARB Screen(Rosco Diagnostica A/S, Tasstrup, Denmark) test
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and the modified Hodge test. A panel of 150 isolates was used, including 49 carbapenemase-
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producing isolates representing a variety of β-lactamase enzyme classes. Evaluation of
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analytical performance, assay cost and turnaround time indicated that the preferred workflow
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(screening test followed by confirmatory testing) was chromID CARBA agar media
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(bioMémieux, Marcy-l’Etoile, France) followed by the Rapid CARB Screen test, yielding a
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combined sensitivity of 89.8% and a specificity of 100%. As an optional component of the
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workflow, determination of carbapenemase gene class via molecular means could be
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performed subsequent to confirmatory testing.
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Introduction: The worldwide dissemination of carbapenemase-producing Gram-negative bacilli
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(CPGNB) is a significant clinical and public health concern (10, 14). Rapid detection of these
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antibiotic resistant pathogens by the clinical microbiology laboratory is of utmost importance to
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control nosocomial spread and initiate appropriate antimicrobial therapy. Chromogenic media
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containing a carbapenem are convenient tools for screening and rapid detection of carbapenem
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resistant Gram-negative bacilli (GNB). However, growth on a chromogenic media simply
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signifies carbapenem resistance (e.g., an AmpC producer with porin loss may grow) and does
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not confirm the production of a carbapenemase. Recently, a novel rapid chromogenic test
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based on the hydrolysis of imipenem, the CarbaNP test, and a commercial version of the
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CarbaNP test, the Rapid CARB screen (RCS), have been described (3, 5, 11-13, 16). The purpose
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of the current study was to evaluate screening and confirmation methods, that when paired,
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would provide a streamlined workflow to detect CPGNB in the clinical microbiology laboratory.
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Firstly, five different chromogenic media were evaluated to select a sensitive media type to
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screen for CPGNB. Secondly, the RCS test (Rosco Diagnostica A/S, Tasstrup, Denmark) was
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evaluated to confirm carbapenemase production through a method comparison to the current
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gold standard modified Hodge (MH) test (1). A combined workflow approach was designed to
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achieve the maximum sensitivity and specificity for the detection of CPGNB, with consideration
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for cost and turnaround time of testing. Lastly, upon finding a CPGNB, it may be of value in
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some circumstances to determine the carbapenemase gene class, and therefore an established
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multiplex PCR was also incorporated as an optional endpoint to this workflow.
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Materials and Methods: Bacterial Isolates. A total of 150 isolates were tested including an assortment of 49
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isolates of GNB harboring a diverse set of carbapenemase enzyme classes and 101
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carbapenemase negative isolates. The 49 carbapenemase producers, a combination of clinical
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and ATCC strains, were previously molecularly characterized for β-lactamase genes and
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included 16 KPC producers (blaKPC-2 [n=9], -3 [n=5], -11 [n=1] ,-12 [n=1]; Citrobacter freundii, n=2;
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Citrobacter koseri, n=1; Enterobacter cloacae, n=1; Escherichia coli, n=4; Klebsiella oxytoca, n=1;
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Klebsiella pneumoniae, n=5; Pseudomonas aeruginosa, n=1; Serratia marcenscens, n=1), 12 VIM
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producers (blaVIM-1 [n=6] ,-2 [n=3] ,-4 [n=2] ,-5 [n=1] ; C. freundii, n=2; C. koseri, n=1; Enterobacter
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aerogenes, n=1; E. cloacae, n=1; K. oxytoca, n=1; Proteus mirabilis, n=1; Providencia stuartii,
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n=1; P. aeruginosa, n=5 ), 4 IMP producers (blaIMP-1,-26; E. coli, n=1; K. pneumoniae, n=2; P.
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aeruginosa, n=1), 9 NDM producers (blaNDM-1 [n=6], -4 [n=1], -5 [n=1], -7 [n=1]; Acinetobacter baumanii,
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n=1; E. coli, n=4; K. oxytoca, n=1; K. pneumoniae, n=1; P. mirabilis, n=1; Providencia rettgerii,
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n=1), 8 OXA producers (blaOXA-24 [n=1] ,-48 [n=7] ; A. baumanii, n=1; C. koseri, n=1, E. cloacae, n=1; K.
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pneumoniae, n=3; Morganella morganii, n=1, S. marcescens, n=1) and one SME producer (S.
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marcescens, n=1). Of note, one E. cloacae isolate possessed both VIM-4 and OXA-48. The 101
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carbapenemase-negative isolates included extended-spectrum β-lactamse (ESBL) and AmpC
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producers (previously molecularly characterized) with and without porin mutations, clinical
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isolates with elevated carbapenem minimum inhibitory concentrations (and negative for
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carbapenemases by PCR) and carbapenem susceptible isolates. The isolates were blinded and
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75 isolates were distributed to each of the two clinical microbiology laboratory testing sites
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(Health Sciences Centre and St. Boniface Hospital, Winnipeg, Canada). 4
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Chromogenic Media to Screen for CPGNB: The first part of this study involved
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evaluating five different chromogenic media types to screen for CPGNB. The five chromogenic
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media evaluated were: 1) Oxoid Brilliance ESBL (Besington, Hants, UK), 2) Oxoid Brilliance CRE,
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3) bioMérieux chromIDTM CARBA (Marcy-l’Etoile,France), 4) CHROMAgar Colorex C3Gr (Paris,
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France) and 5) CHROMagar Colorex KPC. The Brilliance ESBL and Colorex C3Gr media are
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designed to detect ESBLs, and in this study were evaluated both with and without an
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ertapenem disk (10 μg) to select for carbapenem resistant isolates. Inoculums for each of the
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chromogenic media types and a nonselective blood agar (BA) plate were 10 μl (~106 CFU/mL) of
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a 0.5 McFarland standard prepared in sterile saline (0.85%). Brilliance ESBL, Brilliance CRE and
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chromID CARBA were streaked using the Isoplater (Vista Technology Inc, Edmonton, Alberta,
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Canada) to mimic standard laboratory practice. The Colorex media C3Gr and Colorex KPC were
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evaluated as a biplate so the media were streaked manually. Ertapenem disks were placed
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between the 2nd and 3rd quadrant of the Brilliance ESBL and Colorex C3Gr media following
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streaking of the plate. The plates were incubated for 24 hours at 37oC and the
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presence/absence and color of growth were interpreted according to the package inserts. Any
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isolate on the ESBL chromogenic media with an ertapenem zone of inhibition of ≤27 mm were
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considered putative carbapenemase producers and would be considered for further evaluation
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using a confirmatory method. The use of the ≤27mm cut-off was established in a previous
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study using an ertapenem disk (10 μg) to screen for carbapenem resistant GNB on MacConkey
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media (6).
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Phenotypic Confirmatory Asssays: In the second part of the study, a method comparison
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between the MH test and the RCS, was performed on all study isolates, using growth from the
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BA plate inoculated concurrent with the chromogenic media. The BA plate was added to
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perform the confirmatory tests the following day since the RCS cannot be performed off the
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chromogenic media as the color of the colonies interferes with the interpretation of the test.
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The MH test using meropenem disks (10 μg) was performed and interpreted according to CLSI
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methodology (1). The RCS is a, commercially available, rapid chromogenic test for the
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detection of carbapenemases based on the hydrolysis of the imipenem in the presence of an
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inidicator [phenol red, (11)]. All the components of the assay are present within tablets called
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diatabs. Two sets of diatabs are contained in the kit, one set contains imipenem and the
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second set does not contain imipenem. The diatabs are dissolved in saline and the lysate of the
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organism is added to a tube with a dissolved diatab with imipenem and a tube with a dissolved
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diatab without imipenem (negative control tube; should remain red in color). Initially, the RCS
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was performed as previously described (5). K. pneumoniae ATCC BAA1706 and K. pneumoniae
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ATCC BAA1705 were the positive and negative controls, respectively. Any color change from
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red to yellow in the 1.5 ml test tube containing imipenem was considered a positive reaction
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for the RCS. Uninterpretable results were isolates that would give a slight positive reaction
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(peach color) in both tubes with and without imipenem.
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As there were many uninterpretable results initially with the previously described RCS
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protocol, the RCS protocol was troubleshot by determining the appropriate inoculum by testing
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one to three calibrated loops of organism and determining the optimal tube to perform the
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assay by testing 1.5 ml test tubes, cryovials, and round-bottomed 15 ml tubes. Thus, the 6
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modified RCS was performed upon repeat on all isolates to only include one 10 µl calibrated
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loopful of organism (reduced from two) and a round-bottomed 15 ml tube (instead of a conical
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1.5 ml tube). As there was variability in the degree of color change for positives with the
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modified RCS, positives were considered any color change from the negative control tubes
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(tubes without imipenem). Isolates that were orange by the modified RSC protocol in this study
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were repeated using both the RCS and the CarbaNP, as previsouly described, to assess ease of
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interpretation of the intermediate results (3, 11, 13).
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Conventional Multiplex PCR: Lastly, a conventional multiplex PCR for blaKPC, blaNDM, blaVIM,
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blaIMP and blaOXA-48/181 was performed on all isolates as previously described (8). In addition, a
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blaSME PCR was performed on one isolate to confirm SME production (7).
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Determination of an Optimal Workflow for the Screening and Confirmation of CPGNB: The
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results of the screening and confirmatory assays (performed on all test isolates) was analyzed
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to develop the optimal workflow (i.e., test choice) for the detection and confirmation of
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CPGNB. By combining the individual data from the chromogenic media and the confirmatory
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assays we were able to determine the sensitivity and specificities of the combined assays. In
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addition, the cost and time to perform the different combination of assays were evaluated and
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considered for feasibility and full optimization of the workflow within a clinical microbiology
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laboratory.
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Results:
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Chromogenic media for CPGNB: The first part of this study involved evaluating five different
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chromogenic media types to screen for CPGNB, including two media targeting ESBLs 7
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supplemented with and without an ertapenem disk (Table 1). Of the five different chromogenic
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media evaluated to detect CPGNB (Table 1), Colorex C3Gr without the ertapenem disk had the
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highest sensitivity, 95.9%. However, the two ESBL media types, Colorex C3Gr and Brilliance
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ESBL, without the use of the ertapenem disk both had a specificity of 38.6% for CPGNB. The
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addition of the ertapenem disk to Colorex C3Gr and Brilliance ESBL resulted in an increased
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specificity of 62.4% and 94.1%, however, it resulted in a loss of sensitivity of 89.8 and 42.9%,
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respectfully.
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Among the three chromogenic media designed for detection of CPGNB, chromID CARBA
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demonstrated the highest sensitivity and specificity, followed by Colorex KPC and Brilliance
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CRE. The chromID CARBA media failed to identify 5 CPGNB (3 isolates with OXA-48, 1 isolate
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with VIM-1 and 1 isolate with VIM-5). The Colorex KPC and Brilliance CRE media failed to
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support the growth of these same five isolates; Colorex KPC additionally did not detect one of
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each of NDM-1 producer, KPC-2 producer and SME producer; Brilliance CRE additionally did not
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detect one of each of VIM-2, 1 IMP-26, 1 SME and one OXA-48/VIM-4 producers. All three
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media designed for detection of CPGNB performed poorly for the detection of OXA-48
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producers, detecting at the most five of the 8 (63.5%) OXA-48 producers.
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Phenotypic Confirmatory Assays: In the second part of the study, a method comparison
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between the MH test and the RCS was performed on all isolates. On the first attempt with the
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previously described RCS protocol, 29 (28.7%) of the carbapenemase-negative isolates gave
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uninterpretable results (peach color in both tubes with and without imipenem (e.g., Figure 1.
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C2a & C2b). In addition, 4 of the carpabenemase producers were interpreted as negative (3
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OXA-48 producers and 1 SME producer). Based off the initial results, the RCS protocol was
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troubleshot and modified upon repeat to only include one 10 µl calibrated loopful of organism
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(reduced from two) and a round-bottomed 15 ml tube (instead of a conical 1.5 ml test tube) to
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allow the diatab to dissolve completely (Figure 1).
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It was noted that different enzyme classes performed differently with the RCS. KPC
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producers would turn the bacterial suspension a characteristic bright yellow color (positive test)
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immediately or within minutes of inoculating the test, all VIM and IMP producers would be
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positive at 30 minutes, whereas NDM and OXA producers and other CPGNB with relatively low
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carbapenem MICs would turn color more slowly and would yield an intermediate orange hue
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toward the end of incubation. Three of the carbapenemase positive isolates displayed
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intermediate positive results (orange hue) which included 3 of the 4 called negative by
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unmodified RCS (2 OXA-48 producers and one SME producer). Those isolates that yielded an
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orange color towards the end of incubation in this study were repeated using both the RCS and
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the CarbaNP tests, to determine which assay was easier to interpret for intermediate results
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[Figure 2, (3, 11, 13)]. Although both the RCS and the CarbaNP tests did yield the same
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intermediate orange color, it was found that the CarbaNP was easier to interpret the
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intermediate positives. As there was variability in the degree of color change for positives with
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the modified RCS, positives were considered any color change from the negative control tubes
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(tubes without imipenem). Using this as a marker of positivity as opposed to a strict yellow
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color change, sensitivity reached 98.0% and specificity was 100%. The RCS failed to identify one
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OXA-48 producing K. pneumoniae.
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Conventional Multiplex PCR: The multiplex PCR for determination of carbapenemase gene
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class was verified with a sensitivity and specificity of 95.9% and 100%, respectively. The PCR
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failed to identify one OXA-24 producer and one SME producer. It was expected that blaOXA-24
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was not detected by the multiplex PCR as this assay targetsblaOXA-48/181 specifically. Similarly,
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blaSME is not one of the targets included in the multiplex PCR. However, a PCR specific to SME
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was performed on the isolate and was found to be positive. Considering only the genes
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targeted by the multiplex PCR, the sensitivity and specificity achieved was 100%.
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Determination of an Optimal Workflow for the Screening and Confirmation of CPGNB: The
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results of the screening and confirmatory assays (performed on all test isolates) were analyzed
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to develop the optimal workflow for the detection and confirmation of CPGNB. The best
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combination of tests in terms of analytical performance and assay cost and turnaround time
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was the use of chromID CARBA media for screening followed by the RCS test or multiplex PCR
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for confirmation as a CPGNB (89.8%, Table 1). The Colorex C3Gr media paired with the RCS did
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have the highest combined sensitivity (95.9%; partly due to the improved sensitivity to detect
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OXA producing strains, but not reaching 100% sensitivity in this enzyme class), however the low
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specificity of ESBL chromogenic screening media for CPGNB would greatly impact the overall
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cost of testing if routinely implemented. The large proportion of false positive CPGNB on
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Colorex C3Gr media that would be encountered under routine conditions (where ESBLs and
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organisms with reduced carbapenem susceptibility dominate in incidence over true CPGNB)
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would make it cost and time prohibitive.
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Discussion:
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The development of a screening and confirmatory method for the detection and
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confirmation of CPGNB is complicated by the scope of genera and enzyme classes encompassed
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in this broad group of antimicrobial resistant organisms. This study aimed to provide an
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optimal workflow for the detection of CPGNB to be implemented within the clinical
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microbiology laboratory. As such, a comprehensive evaluation of multiple chromogenic media
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and confirmatory methods were evaluated for their analytical performance and assay cost and
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turnaround time. Overall, we found that chromID CARBA media paired with the modified RCS
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protocol was the optimal approach (Figure 3), yielding a combined sensitivity of 89.8% and a
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specificity of 100%. The multiplex PCR for carbapenemase genes provided an equal analytical
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performance when paired with the chromID CARBA media, but of note, our laboratory system
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selected the RCS test as the confirmatory component for cost and time-to-reporting
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considerations. The RCS was $6.38 less per test and was anticipated to be completed at
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minimum 3 hours prior to the PCR result. However, for laboratories where high-volume
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CPGNB screening may occur, the scalability of PCR (i.e. use of 96 well plates and associated
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infrastructure) may be more cost and time effective than the manual manipulation required to
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complete the RCS test.
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The first part of this study involved evaluating five different chromogenic media types to
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screen for CPGNB. Of the five different chromogenic media evaluated to detect CPGNB,
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Colorex C3Gr without the ertapenem disk had the highest sensitivity, 95.9%. However, not 11
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surprisingly, the two ESBL media types (Colorex C3Gr and Brilliance ESBL) without the use of the
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ertapenem disk lacked specificity allowing growth of most ESBL and AmpC producers. The
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addition of the ertapenem disk to the two media types designed to detect ESBLs aided to
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substantially increase the specificity of the media for detection of CPGNB, however, it resulted
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in a loss of sensitivity. This is the first study to our knowledge that incorporated a carbapenem
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disk to select for CPGNB off of chromogenic media designed to detect ESBLs.
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Among the three chromogenic media designed for detection of CPGNB, chromID CARBA
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demonstrated the highest sensitivity and specificity, followed by Colorex KPC and Brilliance
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CRE. All three media that are designed for detection of CPGNB performed poorly for the
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detection of OXA-48 producers. A reduced ability to detect OXA-48 producers was also
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observed by Wilkinson et al in their comparison of four chromogenic media types for the
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detection of carbapenemase-producing Enterobacteriaceae (15). Some OXA-48 isolates in our
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study did co-produce other β-lactamases, but these did not appear to consistently play a role in
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OXA-48 detection on chromogenic media, as was previously described (15). The reduced
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ability of the chromogenic media evaluated in this study to detect OXA-48 producers is a known
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limitation of the media. Recently developed media such as SUPERCARBA (9) and chromID OXA-
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48 (bioMérieux) have been described to have better sensitivity to detect OXA-48 producers (4,
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9). Neither of these media were commecerially available in Canada at the time of this study.
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In the second part of the study, a method comparison between the MH test and the RCS
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was performed on all isolates. Initially, many uninterpretable results with the RCS occurred. At
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that point, the assay was troubleshot and improvements were made to the modified RCS
12
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protocol by using one 10 µl calibrated loopful of organism (reduced from two) and a round-
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bottomed 15 ml tube (instead of a conical 1.5 ml tube) to allow the diatab to dissolve
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completely. The diatabs did not dissolve completely in the conical bottom test tubes as there
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was not enough surface area of the diatab in contact with the small volume (150 μl) present in
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the tube. Also, it was noted after unblinding that if a high inoculum of a carbapenemase
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negative organism was present in the tubes containing imipenem, these tests would change to
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a peach color towards the end of incubation. The same uninterpreatble results were reported
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by Huang et al (5). The modification to a lowered inoculum and use of tubes with increased
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surface area is unique to this study.
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Upon repeat with the modified RCS protocol a sensitivity of 98% and specificity of 100%
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was achieved. The RCS failed to identify one OXA-48 producing K. pneumoniae. Previous
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studies have demonstrated that the RCS like the CarbaNP test does not identify all OXA-48
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producers (5, 16). Some intermediate positive results were obtained with the modified RSC and
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these isolates were tested using the CarbaNP for comparison. The CarbaNP test was easier to
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interpret than the RCS for intermediate positives; however, the advantage of the RCS is that it is
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a quality-controlled, commercially available laboratory kit, whereas the CarbaNP test requires
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preparation of reagents in-house (3, 11, 13). Overall, the vast majority of our tested CPGNB
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(91.8%) were yellow at 30 minutes, although the current clinical incidence of those enzyme
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classes that demonstrated an orange positive reaction is unknown.
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This is the first study to date that has compared the RCS to the MH. The RCS proved to
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be a more sensitive and specific test than the MH, corroborating previous findings that
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compared the CarbaNP to the MH (3, 11, 13).
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Lastly, the multiplex PCR for determination of carbapenemase gene class was verified
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with a sensitivity and specificity of 95.9% and 100%, respectfully. Although the multiplex PCR
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evaluated had a high sensitive and specificity if used for confirmation, the costs and technical
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difficulty of the assay was not ideal for routine use, and in our hands will remain an optional
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test choice for when it is of clinical value to determine the carbapenemase gene class (including
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for infection and prevention and control investigations).
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By pairing the top performing screening and confirmatory tests, the preferred workflow
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was chromID CARBA agar media followed by the Rapid CARB Screen test, yielding a combined
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sensitivity of 89.8% and a specificity of 100% (Figure 3). The combination of assays was chosen
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based off of analytical performance characteristics and cost of testing and turnaround time
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under routine conditions. Even though the combination of Colorex C3Gr with the RCS had a
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higher sensitivity than chromID CARBA and the RCS, the decreased sensitivity of the Colorex
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C3Gr media would necessitate substantially more confirmatory testing; in our panel this would
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equate to twice as many RCS tests, 51 versus 103. On the other hand, the limitation of the
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chromID CARBA media is the limited detection of OXA-48 producers which is reflected in the
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sensitivity (89.8%) of the screening algorithm chosen. Our laboratory selected the RCS test for
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the confirmatory component for cost and time-to-reporting considerations, as the RCS was
14
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$6.38 less per test and was anticipated to be completed at minimum 3 hours prior to a PCR
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result.
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Subsequent to the evaluation and verification of this optimized workflow for the
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detection and confirmation of CPGNB, the chromID CARBA and the RCS test were utilized
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clinically. A patient was admitted locally after transfer from an international hospital, and was
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placed immediately on contact precautions in a single-bed room. From a wound specimen, a
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carbapenem-resistant P. aeruginosa was isolated from the patient using routine culture. The
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isolate was positive by the RCS test and identified as a VIM producer by the multiplex PCR. A
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rectal swab was subsequently collected from the patient to screen for CPGNB using the
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chromID CARBA media and a BA plate with ertapenem disks [(2); the BA plate with ertapenem
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disks was also selected because the new, verified protocol had not yet had a verification report
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filed at the time and was not systematically used subsequent to this single collection]. A NDM-
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producing K. pneumoniae grew on both media types and an OXA-181-producing E. coli grew
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only on the BA. A ward screen was performed to investigate if these carbapenemase-producing
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organisms had spread from the isolated patient. As such, 29 rectal swabs were submitted to
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the clinical microbiology laboratory and plated to chromID CARBA media; 27 (93.1%) had no
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growth after overnight incubation. Two patient cultures had P. aeruginosa breakthrough –
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these were subbed to BA and tested negative by the RCS the following day (performed off the
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BA). In addition to the P. aeruginosa, a Gram-positive cocci and a yeast were present on the
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same chromID CARBA plates. Thus, it appeared in this single investigation that breakthrough is
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rare on the chromID CARBA media from rectal swabs, and that a Gram stain can be performed
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to quickly rule out breakthrough growth other than Gram-negative bacilli. 15
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Limitations of this study include the small number of CPGNB tested within individual
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carbapenemase classes and the use of pure cultures of stocked isolates for the evaluation as
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opposed to clinical or mocked rectal swabs. However, strengths to this study include, 1) a
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diverse subset of CPGNB, 2) the evaluation of multiple chromogenic media and the novel use of
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a ertapenem disk on ESBL chromogenic media for the selection of CPGNBs, 3) the first study to
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compare the RCS to the MH, 4) the first study to modify the RCS protocol to improve
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interpretation of results and 5) a complete workflow approach was tested for the detection to
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confirmation of CPGNB.
307
Conclusions:
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Overall, we found that chromID CARBA was the most sensitive and specific chromogenic
309
media evaluated for the detection of CPGNB and with modification to the RCS protocol we
310
were able to more easily interpret the results. By pairing the top-performing screening and
311
confirmatory tests, the preferred workflow was chromID CARBA media followed by the RCS
312
test, yielding a combined sensitivity of 89.8% and a specificity of 100%. The limitation of the
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selected algorithm is the poor detection of OXA-48 producers.
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315
Acknowledgements
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We would like to thank V. Russell and the technologists at each site for their invaluable help
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with this project and, Oxoid, ThermoFisher, bioMérieux, and Alere Canada for providing the
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chromogenic media, and Inter Medico for providing Rapid CARB Screen kits and International
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Health Management Associates, Inc (IHMA) and A. Denisuik for providing isolates for this study. 16
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of two chromogenic tests for the rapid detection of carbapenemase in
336
Enterobacteriaceae and in Pseudomonas aeruginosa isolates. J Clin Microbiol 58:3060-3.
337
6.
Lolans, K., K. Calvert, S. Won, J. Clark, and M. K. Hayden. 2010. Direct ertapenem disk
338
screening method for identification of KPC-producing Klebsiella pneumoniae and
339
Escherichia coli in surveillance swab specimens. J Clin Microbiol 48:836-41.
340 341
7.
Mataseje, L. F., D. A. Boyd, J. Delport, L. Hoang, M. Imperial, B. Lefebvre, M. Kuhn, P. Van Caeseele, B. M. Willey, and M. R. Mulvey. 2014. Serratia marcescens harbouring 17
342
SME-type class A carbapenemases in Canada and the presence of blaSME on a novel
343
genomic island, SmarGI1-1. J Antimicrob Chemother 69:1825-9.
344
8.
Mataseje, L. F., E. Bryce, D. Roscoe, D. A. Boyd, J. Embree, D. Gravel, K. Katz, P. Kibsey,
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M. Kuhn, A. Mounchili, A. Simor, G. Taylor, E. Thomas, N. Turgeon, and M. R. Mulvey.
346
2012. Carbapenem-resistant Gram-negative bacilli in Canada 2009-10: results from the
347
Canadian Nosocomial Infection Surveillance Program (CNISP). J Antimicrob Chemother
348
67:1359-67.
349
9.
Enterobacteriaceae by use of a novel screening medium. J Clin Microbiol 50:2761-6.
350 351
10.
11.
Nordmann, P., L. Poirel, and L. Dortet. 2012. Rapid detection of carbapenemaseproducing Enterobacteriaceae. Emerg Infect Dis 18:1503-7.
354 355
Nordmann, P., and L. Poirel. 2014. The difficult-to-control spread of carbapenemase producers in Enterobacteriaceae worldwide. Clin Microbiol Infect 20:821-830.
352 353
Nordmann, P., D. Girlich, and L. Poirel. 2012. Detection of carbapenemase producers in
12.
Tijet, N., D. Boyd, S. N. Patel, M. R. Mulvey, and R. G. Melano. 2013. Evaluation of the
356
Carba NP test for rapid detection of carbapenemase-producing Enterobacteriaceae and
357
Pseudomonas aeruginosa. Antimicrob Agents Chemother 57:4578-80.
358
13.
Vasoo, S., S. A. Cunningham, P. C. Kohner, P. J. Simner, J. N. Mandrekar, K. Lolans, M.
359
K. Hayden, and R. Patel. 2013. Comparison of a novel, rapid chromogenic biochemical
360
assay, the Carba NP test, with the modified Hodge test for detection of carbapenemase-
361
producing Gram-negative bacilli. J Clin Microbiol 51:3097-101.
362 363
14.
Walsh, T. R. 2010. Emerging carbapenemases: a global perspective. Int J Antimicrob Agents 36 Suppl 3:S8-14. 18
364
15.
Wilkinson, K. M., T. G. Winstanley, C. Lanyon, S. P. Cummings, M. W. Raza, and J. D.
365
Perry. 2012. Comparison of four chromogenic culture media for carbapenemase-
366
producing Enterobacteriaceae. J Clin Microbiol 50:3102-4.
367
16.
Yusuf, E., S. Van Der Meeren, A. Schallier, and D. Pierard. 2014. Comparison of the
368
Carba NP test with the Rapid CARB Screen Kit for the detection of carbapenemase-
369
producing Enterobacteriaceae and Pseudomonas aeruginosa. Eur J Clin Microbiol Infect
370
Dis. [Epub ahead of print]
371 372
19
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 (%) VIM IMP NDM KPC (12) (4) (9) (16) blaIMP- blaNDMblaKPC- blaVIM-
OXA (8) blaOXA-
2, 3,11,12
24,48
a 1,2,4 ,5
1,26
1,2,4,5
a
SME (1) blaSME
Total (49)
Carbapenemase negative GNB : N (%) ESBL AmpC Carb Total (12) (45) S (101) (43)
Performance for CPGNB Detection (%) Sensitivity Specificity
Detection on Chromogenic Media
# of tests
Brilliance 14 ESBL (87.5) Brilliance 7 ESBL with (46.7) ERT Brilliance 15 CRE (93.8) ChromID 16 CARBA (100) Colorex 16 C3Gr (100) Colorex 16 C3Gr with (100) ERT Colorex 15 KPC (93.8) Confirmation Assays
11 (91.6) 4 (33.3)
4 (100) 1 (25.0)
9 (100) 6 (66.7)
4 (50.0) 4 (50.0)
0
8 (66.7) 10 (83.3) 12 (100) 10 (83.3)
3 (75.0) 4 (100) 4 (100) 4 (100)
9 (100) 9 (100) 9 (100) 8 (88.9)
4 (50.0) 5 (62.5) 7 (87.5) 6 (75.0)
0
10 (83.3)
4 (100)
8 (88.9)
5 (62.5)
Modified Hodge Rapid CARB Screen Multiplex
7 (58.3) 10 (83.3) 12
14 (87.5) 16 (100) 16
# of confirma -tory tests
0
1 (100) 0 1 (100) 0
41 (83.7) 21 (42.9)
12 (100) 2 (16.7)
32 (71.1) 2 (4.4)
18 (41.9) 2 (4.7)
62 (61.4) 6 (5.9)
38 (77.6) 44 (89.8) 47 (95.9) 44 (89.8)
3 (25.0) 2 (16.7) 12 (100) 2 (16.7)
9 (20.0) 3 (6.7) 41 (91.1) 34 (75.6)
1 (2.3) 0 9 (20.9) 2 (4.7)
13 (12.9) 5 (5.0) 62 (61.4) 38 (37.6)
41 (83.7)
2 (16.7)
5 (11.1)
1 (2.3)
8 (7.9)
83.7
38.6
NA
6 (66.7) 9 (100) 9
8 (100) 7 (87.5) 7b
0 1 (100) 0c
37 (75.5) 48 (98.0) 47
0
0
0
7 (15.6) 0
0
7 (6.9) 0
0
0
0
0
Time
*List price CAD per plate
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
NA 42.9
94.1
77.6
87.1
89.8 95.9
95.0 38.6
NA NA NA NA
89.8
62.4
83.7
92.1
NA # of tests
3 (75.0) 4 (100) 4
Cost
75.5
93.1
98.0
100
95.9
100
NA NA NA
$2.79
18-24 hrs
Cost CAD per assay
TAT
$0.27
24 hours
$4.57
2 hours
$10.95
4 hours
20
Convention (100) (100) (100) (100) (87.5) (95.9) al PCR Algorithms for the detection and confirmation of carbapenemase producers Brilliance ESBL & RCS Brilliance CRE & RCS Chrom ID & RCS Colorex C3Gr & RCS Colorex KPC & RCS Colorex C3Gr & Multiplex PCR chromID CARBA & Multiplex PCR
1 2 3 4 5 6 7 8
14 (87.5) 15 (93.8) 16 (100) 16 (100) 15 (93.8) 16 (100)
11 (91.6) 8 (66.7) 10 (83.3) 12 (100) 10 (83.3) 12 (100)
4 (100) 3 (75.0) 4 (100) 4 (100) 4 (100) 4 (100)
9 (100) 9 (100) 9 (100) 9 (100) 8 (88.9) 9 (100)
4 (50.0) 4 (50.0) 4 (50.0) 7 (87.5) 5 (62.5) 6 (75.0)
16 (100)
10 (83.3)
4 (100)
9 (100)
4 (50.0)
0 0 1 (100) 0 0 0
1 (100)
41 (83.7) 38 (77.6) 44 (89.8) 47 (95.9) 41 (83.7) 45 (91.8)
44 (89.8)
# of tests 0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
83.7
100
103
TAT d
Cost per isolate $9.99
50 hours
$12.13
77.6
100
51
50 hours
89.8
100
49
$7.82
50 hours
95.9
100
109
$7.36
50 hours
83.7
100
49
$7.36
50 hours
91.7
100
103
$13.74
53 hours
89.8
100
49
$14.20
53 hours
0
a
One Enterobacter cloacae isolate possessed both VIM-4 and OXA-48 One OXA-24 producer was not identified by the multiplex conventional PCR as it is specific for blaOXA-48 (6). c blaSME is not a target in the multiplex PCR. However, a simplex PCR for blaSME was performed and was positive for the isolate (70). RCS: Rapid CARB Screen, GNB: Gram-negative bacilli, CPGNB: Carbapenemase-producing Gram-negative bacilli, mins: minutes, hrs: hours. NA: Not applicable. d Factoring in the likelihood of the need for a pure culture to initiate any confirmatory testing b
21
9 10
Figure 1: Troubleshooting the Rapid Carb Screen test with the use of different tubes and increasing inoculum.
A
11 12
1a
1b
2a
1a
1b
2a
2b
3a
3b
13 14 15 16
B
17
2b
3a
3b
18 19 20 21
C 22 23
1a
1b
2a
2b
3a
3b
24 25 26
Rapid CARB Screen comparing the test in A) round bottom 15 ml tubes, B) Cryovials and C) test
27
tubes using a AmpC-producing Enterobacter cloacae (should yield a negative result by RCS). 1
28
a) Tube with imipenem 1b) Tube without imipenem; using one calibrated 10 μl loop of
29
organism. 2a) Tube with imipenem, 2b) Tube without imipenem; using two calibrated 10 μl
30
loops of organism. 3a) Tube with imipenem, 3b) Tube without imipenm; using three calibrated 22
31
10 μl loops of organism. Of note, as the inoculum increases tube with imipenem (Tube A) in
32
the Cryovials and Test tubes demonstrate that it turns a peach color in comparison to the tube
33
without Imipenem (Tube B). In addition, the color change in the test tubes (C) is not apparent
34
as the diatabs were not able to properly dissolve and result in uninterpretatble results
35
especially with increased inoculum. The round bottom 15 ml tubes A) demonstrated the most
36
easy to interpret results.
37
23
38 39
Figure 2: Comparison of the Rapid CARB Screen to the CarbaNP for a SME producing Serratia
40
marcsens at two hours of incubation (intermediate positive; orange hue).
A 1a
41
1b
3b
3a
2b
2a
B 1a
1b
2a
2b
3a
3b
42 43
A. Rapid CARB Screen Results using round bottom 15 ml tubes and one full 10 μl calibrated loop
44
of organism. B. CarbaNP Assay using 1.5ml conical test tubes and one full 10 μl calibrated loop
45
of organism. 1. Positive Control: K. pneumoniae ATCC BAA1705; 2. Negative Control: K.
46
pneumoniae ATCC BAA1706 and 3. SME-producing Serratia marcenscens. Tubes a) without
47
imipenem and tubes b) with imipenem. In section A, note the dissolved diatabs settling at the
48
bottom of the tubes.
49
24
50 51 52 53
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
54 55 56
Growth – perform Gram stain to confirm Gram-negative bacilli
No Growth
57 58 59 60
GNB
No GNB
Issue final report with NOCP
No further workup
61
Note: color of growth is indicative of:
62
• Pink-Burgundy: E. coli • Bluish green-gray: Klebsiella – Enterobacter – Serratia – Citrobacter group
63 64 65
• 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
66 67 68 69
Any isolate is RCS
All isolates are RCS
• Report ID of RCS positive isolates, and add CARBP comment • Notify IP&C and IDS
No ID are reported, and add NOCP comment
70 71
NOCP
No carbapenemase-producing Gram-negative bacteria isolated.
This isolate contains a carbapenemase and demonstrates reduced susceptibility to carbapenems. Consultation with the Infectious Disease Service is recommended. CARBP 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
1
Evaluation of five chromogenic agar media and the ROSCO Rapid CARB Screen kit for the
2
detection and confirmation of carbapenemase production in Gram-negative bacilli
3
4
Patricia J. Simner,a,b# Matthew W. Gilmour, a,b Pat DeGagne, a Kim Nichol, a and James A.
5
Karlowsky a,b
6
7
Diagnostic Services Manitoba, Winnipeg, Manitoba, Canadaa; University of Manitoba,
8
Department of Medical Microbiology and Infectious Diseases, Winnipeg, Manitoba, Canadab
9
10
Running Title: Detection of carbapenemase-producing Gram-negatives
11
12
# Address correspondence to Patricia J. Simner, [email protected]
1
13
Abstract:
14
An efficient workflow to screen for and confirm carbapenemase-producing Gram-negative
15
bacilli was developed by evaluating five chromogenic screening agar media and two
16
confirmatory assays, the Rapid CARB Screen(Rosco Diagnostica A/S, Tasstrup, Denmark) test
17
and the modified Hodge test. A panel of 150 isolates was used, including 49 carbapenemase-
18
producing isolates representing a variety of β-lactamase enzyme classes. Evaluation of
19
analytical performance, assay cost and turnaround time indicated that the preferred workflow
20
(screening test followed by confirmatory testing) was chromID CARBA agar media
21
(bioMémieux, Marcy-l’Etoile, France) followed by the Rapid CARB Screen test, yielding a
22
combined sensitivity of 89.8% and a specificity of 100%. As an optional component of the
23
workflow, determination of carbapenemase gene class via molecular means could be
24
performed subsequent to confirmatory testing.
25
2
26
27
Introduction: The worldwide dissemination of carbapenemase-producing Gram-negative bacilli
28
(CPGNB) is a significant clinical and public health concern (10, 14). Rapid detection of these
29
antibiotic resistant pathogens by the clinical microbiology laboratory is of utmost importance to
30
control nosocomial spread and initiate appropriate antimicrobial therapy. Chromogenic media
31
containing a carbapenem are convenient tools for screening and rapid detection of carbapenem
32
resistant Gram-negative bacilli (GNB). However, growth on a chromogenic media simply
33
signifies carbapenem resistance (e.g., an AmpC producer with porin loss may grow) and does
34
not confirm the production of a carbapenemase. Recently, a novel rapid chromogenic test
35
based on the hydrolysis of imipenem, the CarbaNP test, and a commercial version of the
36
CarbaNP test, the Rapid CARB screen (RCS), have been described (3, 5, 11-13, 16). The purpose
37
of the current study was to evaluate screening and confirmation methods, that when paired,
38
would provide a streamlined workflow to detect CPGNB in the clinical microbiology laboratory.
39
Firstly, five different chromogenic media were evaluated to select a sensitive media type to
40
screen for CPGNB. Secondly, the RCS test (Rosco Diagnostica A/S, Tasstrup, Denmark) was
41
evaluated to confirm carbapenemase production through a method comparison to the current
42
gold standard modified Hodge (MH) test (1). A combined workflow approach was designed to
43
achieve the maximum sensitivity and specificity for the detection of CPGNB, with consideration
44
for cost and turnaround time of testing. Lastly, upon finding a CPGNB, it may be of value in
45
some circumstances to determine the carbapenemase gene class, and therefore an established
46
multiplex PCR was also incorporated as an optional endpoint to this workflow.
3
47
48
Materials and Methods: Bacterial Isolates. A total of 150 isolates were tested including an assortment of 49
49
isolates of GNB harboring a diverse set of carbapenemase enzyme classes and 101
50
carbapenemase negative isolates. The 49 carbapenemase producers, a combination of clinical
51
and ATCC strains, were previously molecularly characterized for β-lactamase genes and
52
included 16 KPC producers (blaKPC-2 [n=9], -3 [n=5], -11 [n=1] ,-12 [n=1]; Citrobacter freundii, n=2;
53
Citrobacter koseri, n=1; Enterobacter cloacae, n=1; Escherichia coli, n=4; Klebsiella oxytoca, n=1;
54
Klebsiella pneumoniae, n=5; Pseudomonas aeruginosa, n=1; Serratia marcenscens, n=1), 12 VIM
55
producers (blaVIM-1 [n=6] ,-2 [n=3] ,-4 [n=2] ,-5 [n=1] ; C. freundii, n=2; C. koseri, n=1; Enterobacter
56
aerogenes, n=1; E. cloacae, n=1; K. oxytoca, n=1; Proteus mirabilis, n=1; Providencia stuartii,
57
n=1; P. aeruginosa, n=5 ), 4 IMP producers (blaIMP-1,-26; E. coli, n=1; K. pneumoniae, n=2; P.
58
aeruginosa, n=1), 9 NDM producers (blaNDM-1 [n=6], -4 [n=1], -5 [n=1], -7 [n=1]; Acinetobacter baumanii,
59
n=1; E. coli, n=4; K. oxytoca, n=1; K. pneumoniae, n=1; P. mirabilis, n=1; Providencia rettgerii,
60
n=1), 8 OXA producers (blaOXA-24 [n=1] ,-48 [n=7] ; A. baumanii, n=1; C. koseri, n=1, E. cloacae, n=1; K.
61
pneumoniae, n=3; Morganella morganii, n=1, S. marcescens, n=1) and one SME producer (S.
62
marcescens, n=1). Of note, one E. cloacae isolate possessed both VIM-4 and OXA-48. The 101
63
carbapenemase-negative isolates included extended-spectrum β-lactamse (ESBL) and AmpC
64
producers (previously molecularly characterized) with and without porin mutations, clinical
65
isolates with elevated carbapenem minimum inhibitory concentrations (and negative for
66
carbapenemases by PCR) and carbapenem susceptible isolates. The isolates were blinded and
67
75 isolates were distributed to each of the two clinical microbiology laboratory testing sites
68
(Health Sciences Centre and St. Boniface Hospital, Winnipeg, Canada). 4
69
70
Chromogenic Media to Screen for CPGNB: The first part of this study involved
71
evaluating five different chromogenic media types to screen for CPGNB. The five chromogenic
72
media evaluated were: 1) Oxoid Brilliance ESBL (Besington, Hants, UK), 2) Oxoid Brilliance CRE,
73
3) bioMérieux chromIDTM CARBA (Marcy-l’Etoile,France), 4) CHROMAgar Colorex C3Gr (Paris,
74
France) and 5) CHROMagar Colorex KPC. The Brilliance ESBL and Colorex C3Gr media are
75
designed to detect ESBLs, and in this study were evaluated both with and without an
76
ertapenem disk (10 μg) to select for carbapenem resistant isolates. Inoculums for each of the
77
chromogenic media types and a nonselective blood agar (BA) plate were 10 μl (~106 CFU/mL) of
78
a 0.5 McFarland standard prepared in sterile saline (0.85%). Brilliance ESBL, Brilliance CRE and
79
chromID CARBA were streaked using the Isoplater (Vista Technology Inc, Edmonton, Alberta,
80
Canada) to mimic standard laboratory practice. The Colorex media C3Gr and Colorex KPC were
81
evaluated as a biplate so the media were streaked manually. Ertapenem disks were placed
82
between the 2nd and 3rd quadrant of the Brilliance ESBL and Colorex C3Gr media following
83
streaking of the plate. The plates were incubated for 24 hours at 37oC and the
84
presence/absence and color of growth were interpreted according to the package inserts. Any
85
isolate on the ESBL chromogenic media with an ertapenem zone of inhibition of ≤27 mm were
86
considered putative carbapenemase producers and would be considered for further evaluation
87
using a confirmatory method. The use of the ≤27mm cut-off was established in a previous
88
study using an ertapenem disk (10 μg) to screen for carbapenem resistant GNB on MacConkey
89
media (6).
5
90
Phenotypic Confirmatory Asssays: In the second part of the study, a method comparison
91
between the MH test and the RCS, was performed on all study isolates, using growth from the
92
BA plate inoculated concurrent with the chromogenic media. The BA plate was added to
93
perform the confirmatory tests the following day since the RCS cannot be performed off the
94
chromogenic media as the color of the colonies interferes with the interpretation of the test.
95
The MH test using meropenem disks (10 μg) was performed and interpreted according to CLSI
96
methodology (1). The RCS is a, commercially available, rapid chromogenic test for the
97
detection of carbapenemases based on the hydrolysis of the imipenem in the presence of an
98
inidicator [phenol red, (11)]. All the components of the assay are present within tablets called
99
diatabs. Two sets of diatabs are contained in the kit, one set contains imipenem and the
100
second set does not contain imipenem. The diatabs are dissolved in saline and the lysate of the
101
organism is added to a tube with a dissolved diatab with imipenem and a tube with a dissolved
102
diatab without imipenem (negative control tube; should remain red in color). Initially, the RCS
103
was performed as previously described (5). K. pneumoniae ATCC BAA1706 and K. pneumoniae
104
ATCC BAA1705 were the positive and negative controls, respectively. Any color change from
105
red to yellow in the 1.5 ml test tube containing imipenem was considered a positive reaction
106
for the RCS. Uninterpretable results were isolates that would give a slight positive reaction
107
(peach color) in both tubes with and without imipenem.
108
As there were many uninterpretable results initially with the previously described RCS
109
protocol, the RCS protocol was troubleshot by determining the appropriate inoculum by testing
110
one to three calibrated loops of organism and determining the optimal tube to perform the
111
assay by testing 1.5 ml test tubes, cryovials, and round-bottomed 15 ml tubes. Thus, the 6
112
modified RCS was performed upon repeat on all isolates to only include one 10 µl calibrated
113
loopful of organism (reduced from two) and a round-bottomed 15 ml tube (instead of a conical
114
1.5 ml tube). As there was variability in the degree of color change for positives with the
115
modified RCS, positives were considered any color change from the negative control tubes
116
(tubes without imipenem). Isolates that were orange by the modified RSC protocol in this study
117
were repeated using both the RCS and the CarbaNP, as previsouly described, to assess ease of
118
interpretation of the intermediate results (3, 11, 13).
119
Conventional Multiplex PCR: Lastly, a conventional multiplex PCR for blaKPC, blaNDM, blaVIM,
120
blaIMP and blaOXA-48/181 was performed on all isolates as previously described (8). In addition, a
121
blaSME PCR was performed on one isolate to confirm SME production (7).
122
Determination of an Optimal Workflow for the Screening and Confirmation of CPGNB: The
123
results of the screening and confirmatory assays (performed on all test isolates) was analyzed
124
to develop the optimal workflow (i.e., test choice) for the detection and confirmation of
125
CPGNB. By combining the individual data from the chromogenic media and the confirmatory
126
assays we were able to determine the sensitivity and specificities of the combined assays. In
127
addition, the cost and time to perform the different combination of assays were evaluated and
128
considered for feasibility and full optimization of the workflow within a clinical microbiology
129
laboratory.
130
Results:
131
Chromogenic media for CPGNB: The first part of this study involved evaluating five different
132
chromogenic media types to screen for CPGNB, including two media targeting ESBLs 7
133
supplemented with and without an ertapenem disk (Table 1). Of the five different chromogenic
134
media evaluated to detect CPGNB (Table 1), Colorex C3Gr without the ertapenem disk had the
135
highest sensitivity, 95.9%. However, the two ESBL media types, Colorex C3Gr and Brilliance
136
ESBL, without the use of the ertapenem disk both had a specificity of 38.6% for CPGNB. The
137
addition of the ertapenem disk to Colorex C3Gr and Brilliance ESBL resulted in an increased
138
specificity of 62.4% and 94.1%, however, it resulted in a loss of sensitivity of 89.8 and 42.9%,
139
respectfully.
140
Among the three chromogenic media designed for detection of CPGNB, chromID CARBA
141
demonstrated the highest sensitivity and specificity, followed by Colorex KPC and Brilliance
142
CRE. The chromID CARBA media failed to identify 5 CPGNB (3 isolates with OXA-48, 1 isolate
143
with VIM-1 and 1 isolate with VIM-5). The Colorex KPC and Brilliance CRE media failed to
144
support the growth of these same five isolates; Colorex KPC additionally did not detect one of
145
each of NDM-1 producer, KPC-2 producer and SME producer; Brilliance CRE additionally did not
146
detect one of each of VIM-2, 1 IMP-26, 1 SME and one OXA-48/VIM-4 producers. All three
147
media designed for detection of CPGNB performed poorly for the detection of OXA-48
148
producers, detecting at the most five of the 8 (63.5%) OXA-48 producers.
149
Phenotypic Confirmatory Assays: In the second part of the study, a method comparison
150
between the MH test and the RCS was performed on all isolates. On the first attempt with the
151
previously described RCS protocol, 29 (28.7%) of the carbapenemase-negative isolates gave
152
uninterpretable results (peach color in both tubes with and without imipenem (e.g., Figure 1.
153
C2a & C2b). In addition, 4 of the carpabenemase producers were interpreted as negative (3
8
154
OXA-48 producers and 1 SME producer). Based off the initial results, the RCS protocol was
155
troubleshot and modified upon repeat to only include one 10 µl calibrated loopful of organism
156
(reduced from two) and a round-bottomed 15 ml tube (instead of a conical 1.5 ml test tube) to
157
allow the diatab to dissolve completely (Figure 1).
158
It was noted that different enzyme classes performed differently with the RCS. KPC
159
producers would turn the bacterial suspension a characteristic bright yellow color (positive test)
160
immediately or within minutes of inoculating the test, all VIM and IMP producers would be
161
positive at 30 minutes, whereas NDM and OXA producers and other CPGNB with relatively low
162
carbapenem MICs would turn color more slowly and would yield an intermediate orange hue
163
toward the end of incubation. Three of the carbapenemase positive isolates displayed
164
intermediate positive results (orange hue) which included 3 of the 4 called negative by
165
unmodified RCS (2 OXA-48 producers and one SME producer). Those isolates that yielded an
166
orange color towards the end of incubation in this study were repeated using both the RCS and
167
the CarbaNP tests, to determine which assay was easier to interpret for intermediate results
168
[Figure 2, (3, 11, 13)]. Although both the RCS and the CarbaNP tests did yield the same
169
intermediate orange color, it was found that the CarbaNP was easier to interpret the
170
intermediate positives. As there was variability in the degree of color change for positives with
171
the modified RCS, positives were considered any color change from the negative control tubes
172
(tubes without imipenem). Using this as a marker of positivity as opposed to a strict yellow
173
color change, sensitivity reached 98.0% and specificity was 100%. The RCS failed to identify one
174
OXA-48 producing K. pneumoniae.
9
175
176
Conventional Multiplex PCR: The multiplex PCR for determination of carbapenemase gene
177
class was verified with a sensitivity and specificity of 95.9% and 100%, respectively. The PCR
178
failed to identify one OXA-24 producer and one SME producer. It was expected that blaOXA-24
179
was not detected by the multiplex PCR as this assay targetsblaOXA-48/181 specifically. Similarly,
180
blaSME is not one of the targets included in the multiplex PCR. However, a PCR specific to SME
181
was performed on the isolate and was found to be positive. Considering only the genes
182
targeted by the multiplex PCR, the sensitivity and specificity achieved was 100%.
183
Determination of an Optimal Workflow for the Screening and Confirmation of CPGNB: The
184
results of the screening and confirmatory assays (performed on all test isolates) were analyzed
185
to develop the optimal workflow for the detection and confirmation of CPGNB. The best
186
combination of tests in terms of analytical performance and assay cost and turnaround time
187
was the use of chromID CARBA media for screening followed by the RCS test or multiplex PCR
188
for confirmation as a CPGNB (89.8%, Table 1). The Colorex C3Gr media paired with the RCS did
189
have the highest combined sensitivity (95.9%; partly due to the improved sensitivity to detect
190
OXA producing strains, but not reaching 100% sensitivity in this enzyme class), however the low
191
specificity of ESBL chromogenic screening media for CPGNB would greatly impact the overall
192
cost of testing if routinely implemented. The large proportion of false positive CPGNB on
193
Colorex C3Gr media that would be encountered under routine conditions (where ESBLs and
194
organisms with reduced carbapenem susceptibility dominate in incidence over true CPGNB)
195
would make it cost and time prohibitive.
10
196
197
Discussion:
198
The development of a screening and confirmatory method for the detection and
199
confirmation of CPGNB is complicated by the scope of genera and enzyme classes encompassed
200
in this broad group of antimicrobial resistant organisms. This study aimed to provide an
201
optimal workflow for the detection of CPGNB to be implemented within the clinical
202
microbiology laboratory. As such, a comprehensive evaluation of multiple chromogenic media
203
and confirmatory methods were evaluated for their analytical performance and assay cost and
204
turnaround time. Overall, we found that chromID CARBA media paired with the modified RCS
205
protocol was the optimal approach (Figure 3), yielding a combined sensitivity of 89.8% and a
206
specificity of 100%. The multiplex PCR for carbapenemase genes provided an equal analytical
207
performance when paired with the chromID CARBA media, but of note, our laboratory system
208
selected the RCS test as the confirmatory component for cost and time-to-reporting
209
considerations. The RCS was $6.38 less per test and was anticipated to be completed at
210
minimum 3 hours prior to the PCR result. However, for laboratories where high-volume
211
CPGNB screening may occur, the scalability of PCR (i.e. use of 96 well plates and associated
212
infrastructure) may be more cost and time effective than the manual manipulation required to
213
complete the RCS test.
214
The first part of this study involved evaluating five different chromogenic media types to
215
screen for CPGNB. Of the five different chromogenic media evaluated to detect CPGNB,
216
Colorex C3Gr without the ertapenem disk had the highest sensitivity, 95.9%. However, not 11
217
surprisingly, the two ESBL media types (Colorex C3Gr and Brilliance ESBL) without the use of the
218
ertapenem disk lacked specificity allowing growth of most ESBL and AmpC producers. The
219
addition of the ertapenem disk to the two media types designed to detect ESBLs aided to
220
substantially increase the specificity of the media for detection of CPGNB, however, it resulted
221
in a loss of sensitivity. This is the first study to our knowledge that incorporated a carbapenem
222
disk to select for CPGNB off of chromogenic media designed to detect ESBLs.
223
Among the three chromogenic media designed for detection of CPGNB, chromID CARBA
224
demonstrated the highest sensitivity and specificity, followed by Colorex KPC and Brilliance
225
CRE. All three media that are designed for detection of CPGNB performed poorly for the
226
detection of OXA-48 producers. A reduced ability to detect OXA-48 producers was also
227
observed by Wilkinson et al in their comparison of four chromogenic media types for the
228
detection of carbapenemase-producing Enterobacteriaceae (15). Some OXA-48 isolates in our
229
study did co-produce other β-lactamases, but these did not appear to consistently play a role in
230
OXA-48 detection on chromogenic media, as was previously described (15). The reduced
231
ability of the chromogenic media evaluated in this study to detect OXA-48 producers is a known
232
limitation of the media. Recently developed media such as SUPERCARBA (9) and chromID OXA-
233
48 (bioMérieux) have been described to have better sensitivity to detect OXA-48 producers (4,
234
9). Neither of these media were commecerially available in Canada at the time of this study.
235
In the second part of the study, a method comparison between the MH test and the RCS
236
was performed on all isolates. Initially, many uninterpretable results with the RCS occurred. At
237
that point, the assay was troubleshot and improvements were made to the modified RCS
12
238
protocol by using one 10 µl calibrated loopful of organism (reduced from two) and a round-
239
bottomed 15 ml tube (instead of a conical 1.5 ml tube) to allow the diatab to dissolve
240
completely. The diatabs did not dissolve completely in the conical bottom test tubes as there
241
was not enough surface area of the diatab in contact with the small volume (150 μl) present in
242
the tube. Also, it was noted after unblinding that if a high inoculum of a carbapenemase
243
negative organism was present in the tubes containing imipenem, these tests would change to
244
a peach color towards the end of incubation. The same uninterpreatble results were reported
245
by Huang et al (5). The modification to a lowered inoculum and use of tubes with increased
246
surface area is unique to this study.
247
Upon repeat with the modified RCS protocol a sensitivity of 98% and specificity of 100%
248
was achieved. The RCS failed to identify one OXA-48 producing K. pneumoniae. Previous
249
studies have demonstrated that the RCS like the CarbaNP test does not identify all OXA-48
250
producers (5, 16). Some intermediate positive results were obtained with the modified RSC and
251
these isolates were tested using the CarbaNP for comparison. The CarbaNP test was easier to
252
interpret than the RCS for intermediate positives; however, the advantage of the RCS is that it is
253
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
(91.8%) were yellow at 30 minutes, although the current clinical incidence of those enzyme
256
classes that demonstrated an orange positive reaction is unknown.
13
257
This is the first study to date that has compared the RCS to the MH. The RCS proved to
258
be a more sensitive and specific test than the MH, corroborating previous findings that
259
compared the CarbaNP to the MH (3, 11, 13).
260
Lastly, the multiplex PCR for determination of carbapenemase gene class was verified
261
with a sensitivity and specificity of 95.9% and 100%, respectfully. Although the multiplex PCR
262
evaluated had a high sensitive and specificity if used for confirmation, the costs and technical
263
difficulty of the assay was not ideal for routine use, and in our hands will remain an optional
264
test choice for when it is of clinical value to determine the carbapenemase gene class (including
265
for infection and prevention and control investigations).
266
By pairing the top performing screening and confirmatory tests, the preferred workflow
267
was chromID CARBA agar media followed by the Rapid CARB Screen test, yielding a combined
268
sensitivity of 89.8% and a specificity of 100% (Figure 3). The combination of assays was chosen
269
based off of analytical performance characteristics and cost of testing and turnaround time
270
under routine conditions. Even though the combination of Colorex C3Gr with the RCS had a
271
higher sensitivity than chromID CARBA and the RCS, the decreased sensitivity of the Colorex
272
C3Gr media would necessitate substantially more confirmatory testing; in our panel this would
273
equate to twice as many RCS tests, 51 versus 103. On the other hand, the limitation of the
274
chromID CARBA media is the limited detection of OXA-48 producers which is reflected in the
275
sensitivity (89.8%) of the screening algorithm chosen. Our laboratory selected the RCS test for
276
the confirmatory component for cost and time-to-reporting considerations, as the RCS was
14
277
$6.38 less per test and was anticipated to be completed at minimum 3 hours prior to a PCR
278
result.
279
Subsequent to the evaluation and verification of this optimized workflow for the
280
detection and confirmation of CPGNB, the chromID CARBA and the RCS test were utilized
281
clinically. A patient was admitted locally after transfer from an international hospital, and was
282
placed immediately on contact precautions in a single-bed room. From a wound specimen, a
283
carbapenem-resistant P. aeruginosa was isolated from the patient using routine culture. The
284
isolate was positive by the RCS test and identified as a VIM producer by the multiplex PCR. A
285
rectal swab was subsequently collected from the patient to screen for CPGNB using the
286
chromID CARBA media and a BA plate with ertapenem disks [(2); the BA plate with ertapenem
287
disks was also selected because the new, verified protocol had not yet had a verification report
288
filed at the time and was not systematically used subsequent to this single collection]. A NDM-
289
producing K. pneumoniae grew on both media types and an OXA-181-producing E. coli grew
290
only on the BA. A ward screen was performed to investigate if these carbapenemase-producing
291
organisms had spread from the isolated patient. As such, 29 rectal swabs were submitted to
292
the clinical microbiology laboratory and plated to chromID CARBA media; 27 (93.1%) had no
293
growth after overnight incubation. Two patient cultures had P. aeruginosa breakthrough –
294
these were subbed to BA and tested negative by the RCS the following day (performed off the
295
BA). In addition to the P. aeruginosa, a Gram-positive cocci and a yeast were present on the
296
same chromID CARBA plates. Thus, it appeared in this single investigation that breakthrough is
297
rare on the chromID CARBA media from rectal swabs, and that a Gram stain can be performed
298
to quickly rule out breakthrough growth other than Gram-negative bacilli. 15
299
Limitations of this study include the small number of CPGNB tested within individual
300
carbapenemase classes and the use of pure cultures of stocked isolates for the evaluation as
301
opposed to clinical or mocked rectal swabs. However, strengths to this study include, 1) a
302
diverse subset of CPGNB, 2) the evaluation of multiple chromogenic media and the novel use of
303
a ertapenem disk on ESBL chromogenic media for the selection of CPGNBs, 3) the first study to
304
compare the RCS to the MH, 4) the first study to modify the RCS protocol to improve
305
interpretation of results and 5) a complete workflow approach was tested for the detection to
306
confirmation of CPGNB.
307
Conclusions:
308
Overall, we found that chromID CARBA was the most sensitive and specific chromogenic
309
media evaluated for the detection of CPGNB and with modification to the RCS protocol we
310
were able to more easily interpret the results. By pairing the top-performing screening and
311
confirmatory tests, the preferred workflow was chromID CARBA media followed by the RCS
312
test, yielding a combined sensitivity of 89.8% and a specificity of 100%. The limitation of the
313
selected algorithm is the poor detection of OXA-48 producers.
314
315
Acknowledgements
316
We would like to thank V. Russell and the technologists at each site for their invaluable help
317
with this project and, Oxoid, ThermoFisher, bioMérieux, and Alere Canada for providing the
318
chromogenic media, and Inter Medico for providing Rapid CARB Screen kits and International
319
Health Management Associates, Inc (IHMA) and A. Denisuik for providing isolates for this study. 16
320 321
References
322
1.
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Antimicrobial Susceptibility Testing; 24th Informational Supplement Document M100-
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S24. Clinical and Laboratory Standards Institute, Wayne, PA.
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Blackburn, J., C. Tsimiklis, V. Lavergne, J. Pilotte, S. Grenier, A. Gilbert, B. Lefebvre, M.
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C. Domingo, C. Tremblay, and A. M. Bourgault. 2013. Carbapenem disks on MacConkey
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agar in screening methods for detection of carbapenem-resistant Gram-negative rods in
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producing Pseudomonas spp. J Clin Microbiol 50:3773-6.
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Dortet, L., L. Poirel, and P. Nordmann. 2012. Rapid detection of carbapenemase-
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Girlich, D., C. Anglade, G. Zambardi, and P. Nordmann. 2013. Comparative evaluation
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of a novel chromogenic medium (chromID OXA-48) for detection of OXA-48 producing
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Enterobacteriaceae. Diagn Microbiol Infect Dis 77:296-300.
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5.
Huang, T. D., C. Berhin, P. Bogaerts, and Y. Glupczynski. 2014. Comparative evaluation
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of two chromogenic tests for the rapid detection of carbapenemase in
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Enterobacteriaceae and in Pseudomonas aeruginosa isolates. J Clin Microbiol 58:3060-3.
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6.
Lolans, K., K. Calvert, S. Won, J. Clark, and M. K. Hayden. 2010. Direct ertapenem disk
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screening method for identification of KPC-producing Klebsiella pneumoniae and
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Escherichia coli in surveillance swab specimens. J Clin Microbiol 48:836-41.
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Mataseje, L. F., D. A. Boyd, J. Delport, L. Hoang, M. Imperial, B. Lefebvre, M. Kuhn, P. Van Caeseele, B. M. Willey, and M. R. Mulvey. 2014. Serratia marcescens harbouring 17
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SME-type class A carbapenemases in Canada and the presence of blaSME on a novel
343
genomic island, SmarGI1-1. J Antimicrob Chemother 69:1825-9.
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Mataseje, L. F., E. Bryce, D. Roscoe, D. A. Boyd, J. Embree, D. Gravel, K. Katz, P. Kibsey,
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M. Kuhn, A. Mounchili, A. Simor, G. Taylor, E. Thomas, N. Turgeon, and M. R. Mulvey.
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2012. Carbapenem-resistant Gram-negative bacilli in Canada 2009-10: results from the
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Canadian Nosocomial Infection Surveillance Program (CNISP). J Antimicrob Chemother
348
67:1359-67.
349
9.
Enterobacteriaceae by use of a novel screening medium. J Clin Microbiol 50:2761-6.
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Nordmann, P., L. Poirel, and L. Dortet. 2012. Rapid detection of carbapenemaseproducing Enterobacteriaceae. Emerg Infect Dis 18:1503-7.
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Nordmann, P., and L. Poirel. 2014. The difficult-to-control spread of carbapenemase producers in Enterobacteriaceae worldwide. Clin Microbiol Infect 20:821-830.
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Nordmann, P., D. Girlich, and L. Poirel. 2012. Detection of carbapenemase producers in
12.
Tijet, N., D. Boyd, S. N. Patel, M. R. Mulvey, and R. G. Melano. 2013. Evaluation of the
356
Carba NP test for rapid detection of carbapenemase-producing Enterobacteriaceae and
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Pseudomonas aeruginosa. Antimicrob Agents Chemother 57:4578-80.
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Vasoo, S., S. A. Cunningham, P. C. Kohner, P. J. Simner, J. N. Mandrekar, K. Lolans, M.
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K. Hayden, and R. Patel. 2013. Comparison of a novel, rapid chromogenic biochemical
360
assay, the Carba NP test, with the modified Hodge test for detection of carbapenemase-
361
producing Gram-negative bacilli. J Clin Microbiol 51:3097-101.
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Walsh, T. R. 2010. Emerging carbapenemases: a global perspective. Int J Antimicrob Agents 36 Suppl 3:S8-14. 18
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Wilkinson, K. M., T. G. Winstanley, C. Lanyon, S. P. Cummings, M. W. Raza, and J. D.
365
Perry. 2012. Comparison of four chromogenic culture media for carbapenemase-
366
producing Enterobacteriaceae. J Clin Microbiol 50:3102-4.
367
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Yusuf, E., S. Van Der Meeren, A. Schallier, and D. Pierard. 2014. Comparison of the
368
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369
producing Enterobacteriaceae and Pseudomonas aeruginosa. Eur J Clin Microbiol Infect
370
Dis. [Epub ahead of print]
371 372
19
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 (%) VIM IMP NDM KPC (12) (4) (9) (16) blaIMP- blaNDMblaKPC- blaVIM-
OXA (8) blaOXA-
2, 3,11,12
24,48
a 1,2,4 ,5
1,26
1,2,4,5
a
SME (1) blaSME
Total (49)
Carbapenemase negative GNB : N (%) ESBL AmpC Carb Total (12) (45) S (101) (43)
Performance for CPGNB Detection (%) Sensitivity Specificity
Detection on Chromogenic Media
# of tests
Brilliance 14 ESBL (87.5) Brilliance 7 ESBL with (46.7) ERT Brilliance 15 CRE (93.8) ChromID 16 CARBA (100) Colorex 16 C3Gr (100) Colorex 16 C3Gr with (100) ERT Colorex 15 KPC (93.8) Confirmation Assays
11 (91.6) 4 (33.3)
4 (100) 1 (25.0)
9 (100) 6 (66.7)
4 (50.0) 4 (50.0)
0
8 (66.7) 10 (83.3) 12 (100) 10 (83.3)
3 (75.0) 4 (100) 4 (100) 4 (100)
9 (100) 9 (100) 9 (100) 8 (88.9)
4 (50.0) 5 (62.5) 7 (87.5) 6 (75.0)
0
10 (83.3)
4 (100)
8 (88.9)
5 (62.5)
Modified Hodge Rapid CARB Screen Multiplex
7 (58.3) 10 (83.3) 12
14 (87.5) 16 (100) 16
# of confirma -tory tests
0
1 (100) 0 1 (100) 0
41 (83.7) 21 (42.9)
12 (100) 2 (16.7)
32 (71.1) 2 (4.4)
18 (41.9) 2 (4.7)
62 (61.4) 6 (5.9)
38 (77.6) 44 (89.8) 47 (95.9) 44 (89.8)
3 (25.0) 2 (16.7) 12 (100) 2 (16.7)
9 (20.0) 3 (6.7) 41 (91.1) 34 (75.6)
1 (2.3) 0 9 (20.9) 2 (4.7)
13 (12.9) 5 (5.0) 62 (61.4) 38 (37.6)
41 (83.7)
2 (16.7)
5 (11.1)
1 (2.3)
8 (7.9)
83.7
38.6
NA
6 (66.7) 9 (100) 9
8 (100) 7 (87.5) 7b
0 1 (100) 0c
37 (75.5) 48 (98.0) 47
0
0
0
7 (15.6) 0
0
7 (6.9) 0
0
0
0
0
Time
*List price CAD per plate
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
NA 42.9
94.1
77.6
87.1
89.8 95.9
95.0 38.6
NA NA NA NA
89.8
62.4
83.7
92.1
NA # of tests
3 (75.0) 4 (100) 4
Cost
75.5
93.1
98.0
100
95.9
100
NA NA NA
$2.79
18-24 hrs
Cost CAD per assay
TAT
$0.27
24 hours
$4.57
2 hours
$10.95
4 hours
20
Convention (100) (100) (100) (100) (87.5) (95.9) al PCR Algorithms for the detection and confirmation of carbapenemase producers Brilliance ESBL & RCS Brilliance CRE & RCS Chrom ID & RCS Colorex C3Gr & RCS Colorex KPC & RCS Colorex C3Gr & Multiplex PCR chromID CARBA & Multiplex PCR
1 2 3 4 5 6 7 8
14 (87.5) 15 (93.8) 16 (100) 16 (100) 15 (93.8) 16 (100)
11 (91.6) 8 (66.7) 10 (83.3) 12 (100) 10 (83.3) 12 (100)
4 (100) 3 (75.0) 4 (100) 4 (100) 4 (100) 4 (100)
9 (100) 9 (100) 9 (100) 9 (100) 8 (88.9) 9 (100)
4 (50.0) 4 (50.0) 4 (50.0) 7 (87.5) 5 (62.5) 6 (75.0)
16 (100)
10 (83.3)
4 (100)
9 (100)
4 (50.0)
0 0 1 (100) 0 0 0
1 (100)
41 (83.7) 38 (77.6) 44 (89.8) 47 (95.9) 41 (83.7) 45 (91.8)
44 (89.8)
# of tests 0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
83.7
100
103
TAT d
Cost per isolate $9.99
50 hours
$12.13
77.6
100
51
50 hours
89.8
100
49
$7.82
50 hours
95.9
100
109
$7.36
50 hours
83.7
100
49
$7.36
50 hours
91.7
100
103
$13.74
53 hours
89.8
100
49
$14.20
53 hours
0
a
One Enterobacter cloacae isolate possessed both VIM-4 and OXA-48 One OXA-24 producer was not identified by the multiplex conventional PCR as it is specific for blaOXA-48 (6). c blaSME is not a target in the multiplex PCR. However, a simplex PCR for blaSME was performed and was positive for the isolate (70). RCS: Rapid CARB Screen, GNB: Gram-negative bacilli, CPGNB: Carbapenemase-producing Gram-negative bacilli, mins: minutes, hrs: hours. NA: Not applicable. d Factoring in the likelihood of the need for a pure culture to initiate any confirmatory testing b
21
9 10
Figure 1: Troubleshooting the Rapid Carb Screen test with the use of different tubes and increasing inoculum.
A
11 12
1a
1b
2a
1a
1b
2a
2b
3a
3b
13 14 15 16
B
17
2b
3a
3b
18 19 20 21
C 22 23
1a
1b
2a
2b
3a
3b
24 25 26
Rapid CARB Screen comparing the test in A) round bottom 15 ml tubes, B) Cryovials and C) test
27
tubes using a AmpC-producing Enterobacter cloacae (should yield a negative result by RCS). 1
28
a) Tube with imipenem 1b) Tube without imipenem; using one calibrated 10 μl loop of
29
organism. 2a) Tube with imipenem, 2b) Tube without imipenem; using two calibrated 10 μl
30
loops of organism. 3a) Tube with imipenem, 3b) Tube without imipenm; using three calibrated 22
31
10 μl loops of organism. Of note, as the inoculum increases tube with imipenem (Tube A) in
32
the Cryovials and Test tubes demonstrate that it turns a peach color in comparison to the tube
33
without Imipenem (Tube B). In addition, the color change in the test tubes (C) is not apparent
34
as the diatabs were not able to properly dissolve and result in uninterpretatble results
35
especially with increased inoculum. The round bottom 15 ml tubes A) demonstrated the most
36
easy to interpret results.
37
23
38 39
Figure 2: Comparison of the Rapid CARB Screen to the CarbaNP for a SME producing Serratia
40
marcsens at two hours of incubation (intermediate positive; orange hue).
A 1a
41
1b
3b
3a
2b
2a
B 1a
1b
2a
2b
3a
3b
42 43
A. Rapid CARB Screen Results using round bottom 15 ml tubes and one full 10 μl calibrated loop
44
of organism. B. CarbaNP Assay using 1.5ml conical test tubes and one full 10 μl calibrated loop
45
of organism. 1. Positive Control: K. pneumoniae ATCC BAA1705; 2. Negative Control: K.
46
pneumoniae ATCC BAA1706 and 3. SME-producing Serratia marcenscens. Tubes a) without
47
imipenem and tubes b) with imipenem. In section A, note the dissolved diatabs settling at the
48
bottom of the tubes.
49
24
50 51 52 53
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
54 55 56
Growth – perform Gram stain to confirm Gram-negative bacilli
No Growth
57 58 59 60
GNB
No GNB
Issue final report with NOCP
No further workup
61
Note: color of growth is indicative of:
62
• Pink-Burgundy: E. coli • Bluish green-gray: Klebsiella – Enterobacter – Serratia – Citrobacter group
63 64 65
• 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
66 67 68 69
Any isolate is RCS
All isolates are RCS
• Report ID of RCS positive isolates, and add CARBP comment • Notify IP&C and IDS
No ID are reported, and add NOCP comment
70 71
NOCP
No carbapenemase-producing Gram-negative bacteria isolated.
This isolate contains a carbapenemase and demonstrates reduced susceptibility to carbapenems. Consultation with the Infectious Disease Service is recommended. CARBP 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
