Brief Report
August 2014

Comparative Evaluation of Two Chromogenic Tests for Rapid Detection of Carbapenemase in Enterobacteriaceae and in Pseudomonas aeruginosa Isolates

ABSTRACT

We compared the performance of the Carba NP test and the Rosco Rapid CARB screen kit for detecting carbapenemase-producing Enterobacteriaceae and Pseudomonas aeruginosa. Both tests are rapid and highly sensitive; however, the Carba NP test showed superior specificity, and several uninterpretable results were observed with the Rapid CARB screen.

TEXT

The rapid detection of carbapenemase in Enterobacteriaceae and in Pseudomonas aeruginosa is essential for early appropriate therapeutic management and infection control purposes (1). The Carba NP test was recently proposed as a cheap and easy-to-perform imipenem hydrolysis-based test with high accuracy (i.e., sensitivity and specificity) for detecting carbapenemase-producing Enterobacteriaceae (CPE) and P. aeruginosa (2, 3). However, the Carba NP test in its current format is an in-house technique requiring the purchase of several reagents and a homemade preparation of the test solutions (including the addition of imipenem). We evaluated here the ability of two imipenem hydrolysis-based rapid tests, the Carba NP test (CNP) and the commercially available Rosco Rapid CARB screen kit (RCS) (Rosco Diagnostica A/S, Taastrup, Denmark), to detect CPE and carbapenemase-producing P. aeruginosa (CPPA).
A total of 135 well-characterized Enterobacteriaceae (n = 100) and P. aeruginosa (n = 35) collection strains isolated from various clinical samples (66 carbapenemase producers and 69 isolates expressing other representative resistance mechanisms to β-lactams) were tested. Additionally, all nonduplicate consecutive clinical isolates referred to the national reference center (NRC) from January to June 2013 for suspected carbapenemase production were included. Using their routine testing methods and interpretative guidelines, local laboratories were requested to send all nonduplicate Enterobacteriaceae isolates showing decreased susceptibility to at least one carbapenem (ertapenem or meropenem) and P. aeruginosa isolates fulfilling all three of the following criteria: decreased susceptibility to at least one carbapenem (imipenem or meropenem), resistance to ceftazidime and/or cefepime, and resistance to at least one aminoglycoside (amikacin, tobramycin, or gentamicin).
All tested isolates were subcultured twice and tested for imipenem hydrolysis by the CNP test, as previously described (2), and by the RCS, according to the manufacturer's instructions, using the same culture grown freshly on a nonselective blood agar plate (4). Briefly, the RCS was performed as follows: two 10-μl calibrated full loops of bacterial strain were incubated in a Tris-HCl-20 mmol/liter lysis buffer (Bacterial protein extraction reagent [B-PERII]; Thermo Scientific, Rockford, IL, USA) at room temperature for 30 min. Fifty microliters of the suspension was resuspended in 100 μl saline solution in two tubes, in which one RCS test tablet and one negative-control tablet were added. The test tubes were incubated at 37°C for up to 2 h. Both tests were read after 30, 60, and 120 min of incubation. Any color change observed by the naked eye from red to yellow in a vial (for the CNP test) or in a tube (for the RCS) was considered to be a positive reaction (pink, orange, or yellow). The CNP test results were interpreted according to the guidelines by Nordmann et al. (2). The RCS results were interpreted according to the manufacturer's instructions (5). The results of the CNP and RCS tests were considered negative if the respective test vial/tube gave a negative reaction, positive if the test vial/tube gave a positive reaction and the control vial/tube gave a negative reaction (red), and uninterpretable if the control vial/tube gave a positive reaction. All isolates had been verified for the presence of carbapenemase by an in-house ISO 15189-validated multiplex PCR targeting blaVIM, blaIMP, blaNDM, blaKPC, and blaOXA-48 (6). The P. aeruginosa strains were additionally tested by two other in-house multiplex PCRs targeting blaGES (7) and blaOXA-198 (8).
The CNP and RCS test results for carbapenemase-producing and carbapenemase-negative Enterobacteriaceae and P. aeruginosa collection strains are detailed in Tables 1 and 2. The large majority of the characterized CPE collection strains yielded strong-positive results (orange or yellow) by the CNP (59/66) or RCS (54/66) test. Two OXA-48 CPE and one IMP-13 CPPA gave a weak-positive result (pink) by the CNP test. The CNP test missed two IMP-13 strains, one GES-18 strain, and one OXA-198 CPPA strain that showed a weak-positive result by the RCS. Two OXA-48-producing Enterobacteriaceae strains had uninterpretable results with the RCS. Among the 69 characterized carbapenemase-negative strains, all had negative results with the CNP test. On the other hand, 7 Enterobacteriaceae and 10 P. aeruginosa carbapenemase-negative strains yielded inconclusive (n = 6) or weak false-positive results (n = 11) by the RCS.
TABLE 1
TABLE 1 Carba NP test and Rapid CARB screen kit results for carbapenemase-producing Enterobacteriaceae and Pseudomonas aeruginosa collection strains (n = 66)
Group/species (n)Ambler classCarbapenemase enzyme(s)Total no.No. with CNP result:No. with RCS result ofa:
Positive (no. weak positive)NegativePositive (no. weak positive)Uninterpretable
Enterobacteriaceae (44)       
    K. pneumoniaeAKPC-2/KPC-31010 10 
 BNDM-133 3 (2)a 
  VIM-111 1 
  VIM-2711 1 
 DOXA-4877 (1) 6 (2)1
    Enterobacter cloacaeBVIM-122 2 
  VIM-3111 1 
  VIM-411 1 
  NDM-111 1 
 DOXA-4833 21
    E. coliBNDM-122 2 (1) 
 DOXA-4822 (1) 2 (1) 
    Klebsiella oxytocaBVIM-122 2 
 DOXA-4811 1 
    Serratia marcescensBVIM-111 1 
  VIM-411 1 
    Morganella morganiiBNDM-122 2 
    Citrobacter braakiiBVIM-111 1 
    Citrobacter freundiiDOXA-4811 1 
    Providencia vermicolaBVIM-111 1 
Pseudomonas aeruginosa (22)AGES-511 1 
  GES-181 11 (1) 
  KPC-211 1 
 BVIM-255 5 
  VIM-422 2 
  IMP-755 5 
  IMP-1331 (1)23 (2) 
  SPM-111 1 
  GIM-111 1 
  NDM-111 1 
 DOXA-1981 11 (1) 
a
No negative results were reported with RCS.
TABLE 2
TABLE 2 Carba NP test and Rapid CARB screen kit results for carbapenemase-negative Enterobacteriaceae and Pseudomonas aeruginosa collection strains (n = 69)
Group/species (no. of isolates)ESBLAmpCOther β-lactamase(s)Nonenzymatic resistance mechanism(s)Total no.No. with negative CNP result:No. with RCS result of:
PositiveaNegativeUninterpretable
Enterobacteriaceae (56)         
    E. coliCTX-M group 1 OXA-1 11 1 
 CTX-M group 1   2211 
 CTX-M group 2   66 6 
 CTX-M group 9   11 1 
 SHV-2aACC-1  11 1 
 TEM-10   11 1 
 TEM-52   11 1 
  ACC-1  11 1 
  CMY-42  11 1 
  CMY-60  11 1 
  FOX-3  11 1 
   CARB-7 11 1 
   TEM-30 11 1 
    K. pneumoniaeCTX-M group 1 OXA-9Decreased membrane permeability11 1 
 CTX-M group 1  Decreased membrane permeability11 1 
 TEM-10  Decreased membrane permeability11 1 
 TEM-52 OXA-1Decreased membrane permeability11  1
  DHA-1OXA-1Decreased membrane permeability111  
 CTX-M group 1 OXA-1 11  1
 CTX-M group 1   22 2 
 CTX-M group 2 SHV-11 11 1 
 CTX-M group 9 SHV-76 11 1 
  CMY-2  11 1 
  DHA-1SHV-11 332 1
   LEN 11 1 
    K. oxytocaCTX-M group 1   11 1 
 CTX-M group 2   11 1 
 CTX-M group 9, SHV-12   11 1 
 GES-7   11 1 
    E. cloacaeCTX-M group 1   11 1 
 CTX-M group 2   11 1 
 CTX-M group 9   11 1 
 CTX-M group 9, SHV-12   11 1 
    Enterobacter aerogenesCTX-M group 9cAmpCb  11 1 
 TEM-24, SHV-2a   11 1 
    Enterobacter kobeiCTX-M group 9, SHV-12   11 1 
    C. freundiiCTX-M group 1 OXA-1 11 1 
 GES-7   11 1 
    C. braakiiGES-7   11 1 
    Hafnia alvei cAmpC  22 2 
    S. marcescensCTX-M group 1   11 1 
    Proteus mirabilisCTX-M group 2   22 2 
 TEM-110   11 1 
 TEM-2   11 1 
  CMY-2  11 1 
Pseudomonas aeruginosa (13)BEL-1   222  
GES-1  OprD deficient11 1 
 PER-1 OXA-2, OXA-10OprD deficient + MexA/B-OprM11  1
 PER-1  OprD deficient111  
 PER-1   111  
 VEB-1b  OprD deficient + MexA/B-OprM11  1
 VEB-1b OXA-10OprD deficient + MexA/B-OprM11 1 
  cAmpCOXA-10OprD deficient + MexA/B-OprM111  
   OXA-2 111  
   OXA-20-like, OXA-18 11  1
  cAmpCOXA-9OprD deficient + MexA/B-OprM11 1 
   CARB 111  
a
All results were weakly positive.
b
cAmpC, overexpressed chromosomal cephalosporinase.
During the study period, a total of 356 consecutive Enterobacteriaceae (n = 135) and P. aeruginosa (n = 221) clinical isolates were referred by 66 laboratories throughout Belgium to the reference laboratory for suspected carbapenemase production, and the results of the CNP and RCS tests for all isolates are detailed in Table 3. Seventy-two of the 135 (53%) Enterobacteriaceae isolates and 55 of the 221 (25%) P. aeruginosa isolates were confirmed to be carbapenemase producers. OXA-48 carbapenemase was the predominant carbapenemase (82% [59/72]) found in Enterobacteriaceae, while VIM-type carbapenemase largely predominated (93% [51/55]) in P. aeruginosa. By the CNP test, all but 3 OXA-48-positive Enterobacteriaceae (two Klebsiella pneumoniae and one Escherichia coli) and 3 OXA-198-producing P. aeruginosa isolates (clustered in a single hospital and most probably corresponding to a single clone) had positive results. None of the false-negative (by either test) carbapenemase-positive isolates showed mucoid colonies (9). While no uninterpretable results were observed with the CNP test, 9% of the tested isolates (16/135 Enterobacteriaceae and 17/221 P. aeruginosa) gave an uninterpretable result, showing a positive reaction with the RCS negative-control disk.
TABLE 3
TABLE 3 Carba NP test and Rapid CARB screen kit results for Enterobacteriaceae and Pseudomonas aeruginosa clinical isolates referred to the Belgian national reference center (n = 356)
GroupTestaTest resultNo. of isolates with indicated carbapenemase:Total no. of isolates
OXA-48NDMKPCVIMIMPOXA-198Negative
EnterobacteriaceaeCNPPositive56 (6)b5 (3)44   69
  Negative3     6366
 RCSUninterpretable62 1  716
  Positive51 (11)3 (1)43  14 (11)75
  Negative2     4244
    Total Enterobacteriaceae  59c5d4e4f  63g135
Pseudomonas aeruginosaCNPPositive   51 (1)1 (1)  52
  Negative     3166169
 RCSUninterpretable   8  917
  Positive   43 (1)1 69 (67)113
  Negative     38891
    Total P. aeruginosa     5113166221
a
CNP, Carba NP test; RCS, Rapid CARB screen kit.
b
The number of weak-positive results is in parentheses for all positive results.
c
Includes 42 K. pneumoniae, 10 E. coli, 3 K. oxytoca, 2 E. cloacae, 1 Enterobacter asburiae, and 1 E. kobei isolates.
d
Includes 2 E. cloacae, 1 K. pneumoniae, 1 E. coli, and 1 C. freundii isolates.
e
Includes 3 K. pneumoniae and 1 E. cloacae isolates.
f
Includes 2 E. cloacae, 1 K. pneumoniae, and 1 C. freundii isolates.
g
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 1 P. mirabilis isolates.
The overall sensitivity, specificity (calculated on all tested strains), and positive and negative predictive values (calculated on consecutive isolates referred to the reference laboratory) for detecting CPE using the CNP test compared to the molecular detection results were 97%, 100%, 100%, and 95%, respectively, for Enterobacteriaceae, and 91%, 100%, 100%, and 96%, respectively, for P. aeruginosa. After excluding the uninterpretable results, the sensitivity, specificity, and positive and negative predictive values using the RCS were 98%, 83%, 81%, and 95%, respectively, for Enterobacteriaceae, and 96%, 54%, 39%, and 97%, respectively, for P. aeruginosa. If only strong color changes (orange or yellow) were considered a positive result using the RCS, the calculated specificity and positive predictive values increased to 99% and 96%, respectively, while maintaining high sensitivity and negative predictive values of 90% and 96%, respectively, for P. aeruginosa. In addition to the false-negative results observed for a small subset of OXA-48 CPE isolates and for GES-type CPPA isolates using the CNP test that were already reported in other studies (3, 9), we also observed false-negative results using the CNP test with two of the four IMP-13 and all four OXA-198 CPPA isolates tested. Among the total 73 OXA-48 Enterobacteriaceae isolates tested, which represent the main detection challenge in our setting, the numbers of strong-positive, weak-positive, negative, and uninterpretable results were 62, 8, 3, and 0, respectively, by the CNP test, while they were 49, 14, 2, and 8, respectively, by the RCS. Based on our study experience, the RCS was technically easier to perform, as preparation of the reagents was not required as it is for the CNP test. On the other hand, some difficulty in reading the color changes in the RCS, notably due to the turbidity of the undissolved tablets, together with the nonnegligible number of false-positive and uninterpretable results, are the major drawbacks against its routine implementation.
The Carba NP test and Rapid CARB screen are rapid and highly sensitive screening tests used to exclude carbapenemase in Enterobacteriaceae and P. aeruginosa. In an epidemiological setting with a high prevalence of VIM CPPA isolates, the Rapid CARB screen can be used for confirmation of carbapenemase production in P. aeruginosa only if a strong-positive reaction is used as the criterion for a positive result. However, both screening tests should be used with caution in areas with higher prevalence of OXA-48 CPE and should be evaluated in other epidemiological settings in which carbapenemases with lower hydrolytic activity of carbapenems may be found (e.g., IMP, GES, and OXA-198). The Carba NP test performed better than the Rapid CARB screen, owing to its superior specificity and the large number of uninterpretable results observed with the Rapid CARB screen.

ACKNOWLEDGMENTS

This study was supported in part by a research grant from the Fondation Mont-Godinne. The national reference center is partially supported by the Belgian Ministry of Social Affairs through a fund within the health insurance system.
We thank our microbiologist colleagues for referring the isolates to the national reference center and to Jose Bou Casals from Rosco Diagnostica for providing the Rosco Rapid CARB screen kits.

REFERENCES

1.
Cantón R, Akóva M, Carmeli Y, Giske CG, Glupczynski Y, Gniadkowski M, Livermore DM, Miriagou V, Naas T, Rossolini GM, Samuelsen Ø, Seifert H, Woodford N, Nordmann P, and European Network on Carbapenemases. 2012. Rapid evolution and spread of carbapenemases among Enterobacteriaceae in Europe. Clin. Microbiol. Infect. 18:413–431.
2.
Nordmann P, Poirel L, and Dortet L. 2012. Rapid detection of carbapenemase-producing Enterobacteriaceae. Emerg. Infect. Dis. 18:1503–1507.
3.
Dortet L, Poirel L, and Nordmann P. 2012. Rapid detection of carbapenemase-producing Pseudomonas spp. J. Clin. Microbiol. 50:3773–3776.
4.
Dortet L, Bréchard L, Poirel L, and Nordmann P. 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.
5.
Rosco Diagnostica. 2013. Rapid CARB screen kit 98021, version DBV0040F, package insert. Rosco Diagnostica A/S, Taastrup Denmark. http://www.rosco.dk/gfx/insert%2098021.pdf.
6.
Bogaerts P, Rezende de Castro R, de Mendonca R, Huang TD, Denis O, and Glupczynski Y. 2013. Validation of carbapenemase and extended-spectrum β-lactamase multiplex endpoint PCR assays according to ISO 15189. J. Antimicrob. Chemother. 68:1576–1582.
7.
Bogaerts P, Naas T, El Garch F, Cuzon G, Deplano A, Delaire T, Huang TD, Lissoir B, Nordmann P, and Glupczynski Y. 2010. GES extended-spectrum β-lactamases in Acinetobacter baumannii isolates in Belgium. Antimicrob. Agents Chemother. 54:4872–4878.
8.
El Garch F, Bogaerts P, Bebrone C, Galleni M, and Glupczynski Y. 2011. OXA-198, an acquired carbapenem-hydrolyzing class D beta-lactamase from Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 55:4828–4833.
9.
Tijet N, Boyd D, Patel SN, Mulvey MR, and Melano RG. 2013. Evaluation of the Carba NP test for rapid detection of carbapenemase-producing Enterobacteriaceae and Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 57:4578–4580.

Information & Contributors

Information

Published In

cover image Journal of Clinical Microbiology
Journal of Clinical Microbiology
Volume 52Number 8August 2014
Pages: 3060 - 3063
Editor: P. Bourbeau
PubMed: 24850357

History

Received: 4 March 2014
Returned for modification: 14 April 2014
Accepted: 17 May 2014
Published online: 21 December 2020

Permissions

Request permissions for this article.

Contributors

Authors

Te-Din Huang
National Reference Laboratory for Monitoring of Antimicrobial Resistance in Gram-Negative Bacteria, CHU Dinant-Godinne, UCL Namur, Yvoir, Belgium
Catherine Berhin
National Reference Laboratory for Monitoring of Antimicrobial Resistance in Gram-Negative Bacteria, CHU Dinant-Godinne, UCL Namur, Yvoir, Belgium
Pierre Bogaerts
National Reference Laboratory for Monitoring of Antimicrobial Resistance in Gram-Negative Bacteria, CHU Dinant-Godinne, UCL Namur, Yvoir, Belgium
Youri Glupczynski
National Reference Laboratory for Monitoring of Antimicrobial Resistance in Gram-Negative Bacteria, CHU Dinant-Godinne, UCL Namur, Yvoir, Belgium

Editor

P. Bourbeau
Editor

Notes

Address correspondence to Te-Din Huang, [email protected].

Metrics & Citations

Metrics

Note:

  • For recently published articles, the TOTAL download count will appear as zero until a new month starts.
  • There is a 3- to 4-day delay in article usage, so article usage will not appear immediately after publication.
  • Citation counts come from the Crossref Cited by service.

Citations

If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. For an editable text file, please select Medlars format which will download as a .txt file. Simply select your manager software from the list below and click Download.

View Options

Figures and Media

Figures

Media

Tables

Share

Share

Share the article link

Share with email

Email a colleague

Share on social media

American Society for Microbiology ("ASM") is committed to maintaining your confidence and trust with respect to the information we collect from you on websites owned and operated by ASM ("ASM Web Sites") and other sources. This Privacy Policy sets forth the information we collect about you, how we use this information and the choices you have about how we use such information.
FIND OUT MORE about the privacy policy