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]

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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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2. 3. 4.

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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

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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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Comparative evaluation of two chromogenic tests for rapid detection of carbapenemase in Enterobacteriaceae and in Pseudomonas aeruginosa isolates.

We compared the performance of the Carba NP test and the Rosco Rapid CARB screen kit for detecting carbapenemase-producing Enterobacteriaceae and Pseu...
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