JCM Accepts, published online ahead of print on 21 May 2014 J. Clin. Microbiol. doi:10.1128/JCM.00643-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Title:
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Comparative evaluation of two chromogenic tests for the rapid detection of carbapenemase in
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Enterobacteriaceae and in Pseudomonas aeruginosa isolates.
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Authors:
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Te-Din Huang*, Catherine Berhin, Pierre Bogaerts, Youri Glupczynski
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Author's affiliation:
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National Reference Laboratory for Monitoring of Antimicrobial Resistance in Gram-negative
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bacteria, CHU Dinant-Godinne | UCL Namur, Yvoir, Belgium
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*Corresponding author:
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Tel: +32-81-423212
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Fax: +32-81-423204
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Email:
[email protected] 15 16
Address of the institution at which the work was performed:
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1 Avenue Dr. G. Thérasse, 5530 Yvoir, Belgium
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Running title: Detection of carbapenemase-producing Gram-negatives
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Keywords: screening, carbapenems, resistance
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Abstract
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We compared the performance of the Carba NP test and the ROSCO Rapid CARB Screen kit
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for the detection of carbapenemase-producing Enterobacteriaceae and Pseudomonas
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aeruginosa. Both tests are rapid and highly sensitive, however Carba NP test showed superior
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specificity and several uninterpretable results were observed with Rapid CARB Screen.
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Rapid detection of carbapenemase in Enterobacteriaceae and in Pseudomonas
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aeruginosa is essential for early appropriate therapeutic management and infection control
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purposes (1). The Carba NP test has been recently proposed as a cheap and easy to perform
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imipenem hydrolysis-based test with high accuracy (sensitivity and specificity) for the
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detection of carbapenemase-producing Enterobacteriaceae (CPE) and P. aeruginosa (2, 3).
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However, the Carba NP test in its current format is an in-house technique requiring purchase
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of several reagents and home-made preparation of the test solutions (including the addition of
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imipenem). We evaluated here the ability of two imipenem hydrolysis-based rapid tests, the
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Carba NP test (CNP) and the commercially available ROSCO Rapid CARB Screen kit (RCS;
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Rosco Diagnostica A/S, Taastrup, Denmark) for the detection of CPE and of carbapenemase-
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producing P. aeruginosa (CPPA).
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A total of 135 well-characterized Enterobacteriaceae (n=100) and P. aeruginosa
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(n=35) collection strains isolated from various clinical samples (66 carbapenemase producers
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and 69 isolates expressing other representative resistance mechanisms to β-lactams) were
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tested. Additionally, all non-duplicate consecutive clinical isolates referred to the national
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reference center (NRC) from January to June 2013 for suspicion of carbapenemase production
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were included. Local laboratories using their routine testing methods and interpretative
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guidelines were requested to send all non-duplicate Enterobacteriaceae isolates showing
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decreased susceptibility to at least one carbapenem (ertapenem or meropenem) and P.
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aeruginosa isolates fulfilling all three following criteria: decreased susceptibility to at least
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one carbapenem (imipenem or meropenem), resistance to ceftazidime and/or cefepime and
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resistance to at least one aminoglycoside (amikacin, tobramycin or gentamicin).
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All tested isolates were subcultured twice and tested for imipenem hydrolysis by CNP
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previously described (2) and by RCS according to the manufacturers’ instructions using the
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same culture grown freshly on non-selective blood agar plate (4). Briefly RCS was performed
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as follows: two 10-µl-calibrated full loops of bacterial strain were incubated in a Tris-HCl 20
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mmol/L lysis buffer (B-PERII, Bacterial Protein Extraction Reagent; Thermo Scientific,
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Rockford, IL, USA) at room temperature for 30 minutes. 50 µl of the suspension was
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resuspended in 100 µl saline solution in two tubes in which one RCS test tablet and one
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negative control tablet were added. The test tubes were incubated at 37°C for up to two hours.
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Both tests were read at 30, 60 and 120 minutes of incubation. Any color change observed by
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naked eyes from red to yellow in a vial (by CNP) or in a tube (by RCS) was considered as a
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positive reaction (pink, orange or yellow). CNP result was interpreted according to the
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reference publication (2). RCS result was interpreted according to the manufacturers’
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interpretation instructions (document version DBV0040F issued 1/11/2013). Results of the
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CNP and of the RCS tests were considered negative if the test vial/tube gave a negative
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reaction; positive if the test vial/tube gave a positive reaction and the control vial/tube
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negative reaction (red); uninterpretable if the control vial/tube gave a positive reaction. All
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isolates had been verified for the presence of carbapenemase by an in-house ISO15189-
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validated multiplex PCR targeting blaVIM, blaIMP, blaNDM, blaKPC and blaOXA-48 (5). PA strains
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were additionally tested by two other in-house multiplex PCRs targeting blaGES (6) and
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blaOXA-198 (7).
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CNP and RCS results for carbapenemase-producing and carbapenemase-negative
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Enterobacteriaceae and P. aeruginosa collection strains are detailed in Tables 1 and 2. The
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large majority of the characterized CPE collection strains yielded strong positive results
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(orange or yellow) by CNP (59/66) or by RCS (54/66). Two OXA-48 CPE and one IMP-13
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CPPA gave a weak positive result (pink) by CNP. CNP missed two IMP-13, one GES-18 and
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one OXA-198 CPPA strains which showed weak positive result by RCS. Two OXA-48-
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producing Enterobacteriaceae strains gave uninterpretable results with RCS. Among the 69
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characterized carbapenemase-negative strains, all gave negative results with CNP. On the
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other hand, 7 Enterobacteriaceae and 10 P. aeruginosa carbapenemase-negative strains
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yielded inconclusive (n=6) or weak false-positive results (n=11) by RCS.
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A total of 356 consecutive Enterobacteriaceae (n=135) and P. aeruginosa (n=221)
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clinical isolates were referred by 66 laboratories during the study period throughout Belgium
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to the reference laboratory for suspicion of carbapenemase production and the results of CNP
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and RCS performed on all isolates are detailed in Table 3. Seventy-two of the 135 (53%)
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Enterobacteriaceae isolates and 55 of 221 (25%) P. aeruginosa isolates were confirmed as
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carbapenemase producers. OXA-48 carbapenemase was the predominant carbapenemase
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(82%; 59/72) found in Enterobacteriaceae while VIM-type carbapenemase largely
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predominated (93%; 51/55) in P. aeruginosa. By CNP, all but 3 OXA-48-positive
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Enterobacteriaceae (two K. pneumoniae and one E. coli) and 3 OXA-198-producing P.
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aeruginosa isolates (clustered in one single hospital and most probably corresponding to one
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single clone) gave positive results. None of the false-negative (either by CNP or by RCS)
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carbapenemase-positive isolates showed mucoid colonies (8). While no uninterpretable result
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was observed with CNP, 9% of the tested isolates (16/135 Enterobacteriaceae and 17/221 P.
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aeruginosa) gave uninterpretable result showing a positive reaction with RCS negative
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control disk.
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The overall sensitivity, specificity (calculated on all tested strains), positive and negative
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predictive values (calculated on consecutive isolates referred to the reference laboratory) for
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the detection of CPE by CNP compared to the molecular detection results were of 97%,
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100%, 100% and 95% for Enterobacteriaceae; they were of 91%, 100%, 100% and 96% for
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P. aeruginosa. After excluding uninterpretable results, the sensitivity, specificity, positive and
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negative predictive values by RCS were 98%, 83%, 81% and 95% for Enterobacteriaceae;
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they were of 96%, 54%, 39% and 97% for P. aeruginosa. If considering only strong color
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changes (orange or yellow) as a positive result for RCS, the calculated specificity and positive
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predictive value increased to 99% and 96% while maintaining high sensitivity and negative
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predictive values of 90% and 96% for P. aeruginosa. In addition to the false-negative results
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observed for a small subset of OXA-48 CPE isolates and for GES-type CPPA by CNP that
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were already reported in other studies (3, 8), we also observed false negative results by CNP
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with two of the four IMP-13 and all four OXA-198 CPPA tested. Among the total 73 OXA-
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48 Enterobacteriaceae isolates tested which represent the main challenge of detection in our
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setting, the numbers of strong positive, weak positive, negative and uninterpretable results
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were 62, 8, 3 and 0 respectively by CNP, while they were 49, 14, 2 and 8 respectively by
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RCS. Based on our study experience, RCS was technically easier to perform without
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preparation of reagents required for the CNP. On the other hand, some difficulty in reading
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the color changes of RCS notably due to the turbidity of undissolved tablets together with the
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non-negligible number of false positive and uninterpretable results would be the major
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drawbacks against its routine implementation.
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Carba NP test and Rapid CARB Screen are rapid and highly sensitive screening tests for the
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exclusion of carbapenemase in Enterobacteriaceae and in P. aeruginosa. In an
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epidemiological setting with a high prevalence of VIM CPPA, Rapid CARB Screen could be
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used for the confirmation of carbapenemase production in P. aeruginosa only if a strong
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positive reaction is used as the criterion of positive result. However both screening tests
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should be used with caution in areas with higher prevalence of OXA-48 CPE and should be
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evaluated in other epidemiological settings where carbapenemases with lower hydrolytic
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activity of carbapenems may be found (e.g. IMP, GES, OXA-198). Carba NP test performed
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better than Rapid CARB Screen owing to its superior specificity and to the large number of
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uninterpretable results observed with Rapid CARB Screen.
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Acknowledgments
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The study was supported in part by the research grant of Fondation Mont-Godinne. The
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national reference center is partially supported by the Belgian Ministry of Social Affairs
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through a fund within the Health Insurance System. We are thankful to our microbiologists
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colleagues for referring the isolates to the national reference center and to Jose Bou Casals
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from Rosco Diagnostica for providing the ROSCO Rapid CARB Screen kits.
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References
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Canton, R., M. Akova, Y. Carmeli, C. G. Giske, Y. Glupczynski, M. Gniadkowski, D. M. Livermore, V. Miriagou, T. Naas, G. M. Rossolini, O. Samuelsen, H. Seifert, N. Woodford, and P. Nordmann. 2012. Rapid evolution and spread of carbapenemases among Enterobacteriaceae in Europe. Clin Microbiol Infect 18:413-431. Nordmann, P., L. Poirel, and L. Dortet. 2012. Rapid detection of carbapenemase-producing Enterobacteriaceae. Emerg Infect Dis 18:1503-1507. Dortet, L., L. Poirel, and P. Nordmann. 2012. Rapid detection of carbapenemase-producing Pseudomonas spp. J Clin Microbiol 50:3773-3776. Dortet, L., L. Brechard, L. Poirel, and P. Nordmann. 2014. Impact of the isolation medium for detection of carbapenemase-producing Enterobacteriaceae using an updated version of the Carba NP test. J Med Microbiol 63:772-776. Bogaerts, P., R. Rezende de Castro, R. de Mendonca, T. D. Huang, O. Denis, and Y. Glupczynski. 2013. Validation of carbapenemase and extended-spectrum beta-lactamase multiplex endpoint PCR assays according to ISO 15189. J Antimicrob Chemother 68:15761582. Bogaerts, P., T. Naas, F. El Garch, G. Cuzon, A. Deplano, T. Delaire, T. D. Huang, B. Lissoir, P. Nordmann, and Y. Glupczynski. 2010. GES extended-spectrum beta-lactamases in Acinetobacter baumannii isolates in Belgium. Antimicrob Agents Chemother 54:4872-4878. El Garch, F., P. Bogaerts, C. Bebrone, M. Galleni, and Y. Glupczynski. 2011. OXA-198, an acquired carbapenem-hydrolyzing class D beta-lactamase from Pseudomonas aeruginosa. Antimicrob Agents Chemother 55:4828-4833. Tijet, N., D. Boyd, S. N. Patel, M. R. Mulvey, and R. G. Melano. 2013. Evaluation of the Carba NP test for rapid detection of carbapenemase-producing Enterobacteriaceae and Pseudomonas aeruginosa. Antimicrob Agents Chemother 57:4578-4580.
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Table 1. Carba NP test and Rapid CARB Screen kit results for carbapenemase-producing Enterobacteriaceae and Pseudomonas aeruginosa
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collection strains (n=66)
CNP result Group Enterobacteriaceae (n=44)
Species K. pneumoniae
E. cloacae
Ambler class A B
D B
S. marcescens
D B D B D B
M. morganii C. braakii C. freundii P. vermicola Pseudomonas aeruginosa (n=22)
B B D B A
E. coli K. oxytoca
Carbapenemase enzymes KPC-2/-3 NDM-1 VIM-1 VIM-27 OXA-48 VIM-1 VIM-31 VIM-4 NDM-1 OXA-48 NDM-1 OXA-48 VIM-1 OXA-48 VIM-1 VIM-4 NDM-1 VIM-1 OXA-48 VIM GES-5 GES-18 KPC-2
RCS result
a
Total n Positive Negative Positivea Negative Uninterpretable 10 3 1 1 7 2 1 1 1 3 2 2 2 1 1 1 2 1 1 1 1 1 1
10 3 1 1 7 (1) 2 1 1 1 3 2 2 (1) 2 1 1 1 2 1 1 1 1 1 1
10 3 (2) 1 1 6 (2) 2 1 1 1 2 2 (1) 2 (1) 2 1 1 1 2 1 1 1 1 1 (1) 1
1
1
B
166 167 168
a
D in brackets the number of weak positive results
VIM-2 VIM-4 IMP-7 IMP-13 SPM-1 GIM-1 NDM-1 OXA-198
5 2 5 3 1 1 1 1
5 2 5 1 (1) 1 1 1
2
1
5 2 5 3 (2) 1 1 1 1 (1)
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Table 2. Carba NP test and Rapid CARB Screen kit results for carbapenemase-negative Enterobacteriaceae and Pseudomonas aerugionsa
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collection strains (n=69) CNP result
Group Species Enterobacteriaceae (n=56) E. coli
ESBL
AmpCb
CTX-M group 1 CTX-M group 1 CTX-M group 2 CTX-M group 9 SHV-2a TEM-10 TEM-52 -
ACC-1 ACC-1 CMY42 CMY60 FOX-3 -
K. pneumoniae
RCS result
Other βlactamases
Non-enzymatic resistance mechanisms
OXA-1 -
-
1 2 6 1 1 1 1 1
1 2 6 1 1 1 1 1
-
-
1
1
1
CARB-7 TEM-30
decreased membrane permeability decreased membrane permeability decreased membrane permeability
1 1 1 1
1 1 1 1
1 1 1 1
1
1
1
1
1
1
1
1
1
CTX-M group 1
-
OXA-9
CTX-M group 1
-
-
TEM-10
-
-
Total n Negative Positivea Negative Uninterpretable
1
1 1 6 1 1 1 1 1
TEM-52
K. oxytoca
E. cloacae
E. aerogenes
E. kobei C. freundii C. braakii H. alvei S. marcescens P. mirabilis
-
DHA-1 CTX-M group 1 CTX-M group 1 CTX-M group 2 CTX-M group 9 CMY-2 DHA-1 CTX-M group 1 CTX-M group 2 CTX-M group 9, SHV-12 GES-7 CTX-M group 1 CTX-M group 2 CTX-M group 9 CTX-M group 9, SHV-12 CTX-M group 9 cAmpC TEM-24, SHV2a CTX-M group 9, SHV-12 CTX-M group 1 GES-7 GES-7 cAmpC CTX-M group 1 CTX-M group 2 -
OXA-1 OXA-1 SHV-11 SHV-76 SHV-11 LEN -
decreased membrane permeability decreased membrane permeability -
-
-
1 1 1 1 1
1 1 1 1 1
1 1 1 1 1
-
-
1 1
1 1
1 1
-
-
1
1
1
OXA-1 -
-
1 1 1 1 2 1 2
1 1 1 1 2 1 2
1 1 1 1 2 1 2
OXA-1
1
1
1 1 2 1 1 1 3 1 1 1
1 1 2 1 1 1 3 1 1 1
1 1 1 2 1 1 1 2
1 1 1 1
Pseudomonas aeruginosa (n=13)
TEM-110 TEM-2 -
CMY-2
-
-
1 1 1
1 1 1
BEL-1 GES-1
-
2 1
-
1 1 1
1 1 1
VEB-1b
-
-
1
1
VEB-1b
-
OXA-10
1
1
-
cAmpC -
1 1
1 1
-
-
OXA-10 OXA-2 OXA-20 like, OXA-18
OprD deficient OprD deficient + MexA/B-OprM OprD deficient OprD deficient + MexA/B-OprM OprD deficient + MexA/B-OprM OprD deficient + MexA/B-OprM -
2 1
PER-1 PER-1 PER-1
OXA-2, OXA-10 -
1
1
-
cAmpC -
OXA-9 CARB
OprD deficient + MexA/B-OprM -
1 1
1 1
171
a
all were weak positive results
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b
cAmpC, overexpressed chromosomal cephalosporinase
173 174
1 1 1 2 1 1 1 1 1 1 1 1 1 1 1
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Table 3. Carba NP test and Rapid CARB Screen kit results for Enterobacteriaceae and Pseudomonas aerugionsa clinical isolates referred to the
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Belgian national reference center (n=356) Carbapenemase Test result OXA-48 NDM Positivea 56 (6) 5 (3) Negative 3 RCS Uninterpretable 6 2 Positivea 51 (11) 3 (1) Negative 2 Total Enterobacteriaceae 59 b 5c
Group Enterobacteriaceae CNP
Pseudomonas aeruginosa
CNP RCS
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Test
Positivea Negative Uninterpretable Positivea Negative
Total P. aeruginosa CNP, Carba NP test; RCS, Rapid CARB Screen kit
KPC 4
VIM 4
4
1 3
4d
4e 51 (1)
IMP
OXA-198 Negative Total n 69 63 66 7 16 14 (11) 75 42 44 63 f 135
1 (1) 3
8 43 (1)
1
51
1
178
a
in brackets the number of weak positive result
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b
includes 42 K. pneumoniae, 10 E. coli, 3 K. oxytoca, 2 E. cloacae, 1 E. asburiae and 1 E. kobei isolates
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c
includes 2 E. cloacae, 1 K. pneumoniae, 1 E. coli and 1 C. freundii isolates
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d
includes 3 K. pneumoniae and 1 E. cloacae isolates
3 3
166 9 69 (67) 88 166
52 169 17 113 91 221
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e
includes 2 E. cloacae, 1 K. pneumoniae and 1 C. freundii isolates
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f
includes 24 K. pneumoniae, 10 E. coli, 10 E. cloacae, 8 E. aerogenes, 4 K. oxytoca, 2 E. asburiae, 2 E. kobei, 1 C. freundii, 1 S. marcescens and
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1 P. mirabilis isolates
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