|Year : 2017 | Volume
| Issue : 1 | Page : 116-119
Strengths and limitations of various screening methods for carbapenem-resistant Enterobacteriaceae including new method recommended by clinical and laboratory standards institute, 2017: A tertiary care experience
Agila Kumari Pragasam, Balaji Veeraraghavan, Yamuna Devi Bakthavatchalam, Radha Gopi, Raziya Fathima Aslam
Department of Clinical Microbiology, Christian Medical College, Vellore, Tamil Nadu, India
|Date of Web Publication||16-Mar-2017|
Department of Clinical Microbiology, Christian Medical College, Vellore - 632 004, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Carbapenemase-mediated carbapenem resistance is a major concern across the world. Rapid detection of carbapenemase-producing organisms is of great importance in clinical settings. However, it is essential to have a test with good sensitivity and specificity. The aim of the study was to compare the performance of RAPIDEC® CARBA NP and modified carbapenem inactivation method (mCIM) recommended by Clinical and Laboratory Standards Institute guideline 2017. A total of ninety carbapenem resistant Escherichia coli and Klebsiella pneumoniae have been tested. The presence of various carbapenemases was screened by conventional multiplex polymerase chain reaction. RAPIDEC® CARBA NP detected 90%, whereas mCIM detected 99% of the study isolates tested. Although RAPIDEC® CARBA NP is a rapid test, the sensitivity is reduced for blaOxa-48Likedetection; while mCIM could pick up blaOxa-48Likeenzymes with excellent sensitivity. Further, organisms producing low carbapenemase activity enzymes, thickness of the inoculum and the disc potency are likely to influence the test results of mCIM with an overnight delay.
Keywords: Carbapenem, carbapenem inactivation method, carbapenemase, RAPIDEC® CARBA NP, resistance
|How to cite this article:|
Pragasam AK, Veeraraghavan B, Bakthavatchalam YD, Gopi R, Aslam RF. Strengths and limitations of various screening methods for carbapenem-resistant Enterobacteriaceae including new method recommended by clinical and laboratory standards institute, 2017: A tertiary care experience. Indian J Med Microbiol 2017;35:116-9
|How to cite this URL:|
Pragasam AK, Veeraraghavan B, Bakthavatchalam YD, Gopi R, Aslam RF. Strengths and limitations of various screening methods for carbapenem-resistant Enterobacteriaceae including new method recommended by clinical and laboratory standards institute, 2017: A tertiary care experience. Indian J Med Microbiol [serial online] 2017 [cited 2017 Jun 25];35:116-9. Available from: http://www.ijmm.org/text.asp?2017/35/1/116/202349
| ~ Introduction|| |
Infections due to carbapenem-resistant organism have become a great concern for the clinicians due to the limited therapeutic options. Carbapenem resistance is mostly mediated by the production of carbapenemase enzymes that are present on the mobile genetic elements, followed by chromosomal-mediated porin loss and efflux pumps overexpression. In these circumstances, detection of carbapenemase producers is of significant importance for guiding effective antimicrobial therapy and for infection control.
Several tests are described for the screening and detection of carbapenemases, but it requires more time, expertise and a well-established laboratory to perform these assays.,,,, For these reasons, to have a point of care test for the detection of carbapenemase producers; Nordmann et al. have developed a biochemical-based assay Carba NP, which can detect the presence of carbapenemase in <2 h of time. Carba NP has been extensively evaluated and proved to be an excellent rapid test. In Carba NP, metallo-beta-lactamases (MBLs) detection rate was excellent with good sensitivity and specificity. However, it lacks the sensitivity for detecting blaOxa-48Like producers and some enzymes possessing weak carbapenemase activity such as blaSME and blaGES., Following this, a series of modifications has been done to improve the sensitivity and specificity of these assays. In addition, various phenotypic screening tests and their strength and limitations are mentioned in [Table 1]. Such modifications include the use of bromothymol blue (0.04%) as a pH indicator and use of Triton X-100 in place of lysis buffer. RAPIDEC ® CARBA NP kit (BioMerieux, France), a point of care test, based on the Carba NP procedure is commercially available for the detection of carbapenemase in Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp.
|Table 1: Strength and limitations of various phenotypic screening tests for carbapenemase-producing organisms|
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Meanwhile, van der Zwaluw et al. described a new method for detecting carbapenemase producers. This test was based on the inactivation of carbapenem (meropenem disc - 10 µg) by the carbapenemase produced by the test organisms. The inactivated disc is then checked for the meropenem activity against the carbapenem susceptible Escherichia coli ATCC 25922. The appearance of zone of inhibition indicates the meropenem activity, whereas the absence of zone indicates the inactivated meropenem by the carbapenemases produced by the test organism. This method was shown to have 100% and 98% concordance rate when compared with polymerase chain reaction (PCR) for Enterobacteriaceae and nonfermenters, respectively. Overall, this test is simple, does not require special reagents, but it is time consuming (24 h). Carbapenem inactivation method (CIM) test is modified to improve the sensitivity and specificity and are summarised in [Table 1].
Clinical and Laboratory Standards Institute (CLSI), 2015 included Carba NP as a screening test for the carbapenemase detection (CLSI M100-S25). Recently, CLSI 2017 (CLSI M100-S27) included “modified CIM (mCIM)” as a carbapenemase screening test for Enterobacteriaceae (CSLI M100-S27). Compared to Carba NP, which was a rapid method with a turnaround time of 2 h, mCIM requires an overnight incubation for the detection of carbapenemases. Although mCIM is time consuming, it is relatively simple with the sensitivity and specificity of >99%.,
In developing countries like India, the rates of carbapenemase producers are high. A reliable, rapid and robust test for detecting carbapenemase producers is the need of the time. This study was carried out to evaluate and compare the ready to use RAPIDEC ® CARBA NP test versus recently recommended mCIM method by CLSI, in a tertiary care centre.
| ~ Materials and Methods|| |
In this study, a total of ninety carbapenem-resistant isolates were included for the evaluation of RAPIDEC ® CARBA NP test and for mCIM. Of which, 34 and 56 were E. coli and Klebsiella pneumoniae, respectively. These were isolated from bloodstream infections from patients at Christian Medical College (CMC), Vellore, South India. All the isolates were subjected to RAPIDEC ® Carba NP and mCIM test to screen for the presence of carbapenemase activity as described previously.,K. pneumoniae ATCC BAA 1705 and K. pneumoniae ATCC BAA 1706 were used as positive and negative controls for all the assays, respectively. Further, the isolates were screened for carbapenemase such as blaIMP, blaVIM, blaNDM, blaOxa-48Like and blaKPC by conventional multiplex PCR, as described previously. Known positive controls for the respective genes were used in every run.
| ~ Results|| |
Results of RAPIDEC ® CARBA NP and mCIM test are summarised in [Table 2]. RAPIDEC ® CARBA NP detected 97% (n = 33) and 89% (n = 50) of carbapenemase-producing E. coli and K. pneumoniae, respectively, whereas mCIM detected 100% (n = 34) and 98% (n = 55) of carbapenemase-producing E. coli and K. pneumoniae, respectively. The overall sensitivity and specificity of the RAPIDEC ® CARBA NP and mCIM were found to be >90%. In RAPIDEC ® CARBA NP, for class B carbapenemases (MBLs), colour change from red to yellow was observed in <10 min, whereas class D blaOxa type carbapenemases showed colour change of orange to up to an hour. Isolates that showed weak colour change, when tested with in-house Carba NP, showed clear positive of yellow colour, following which they were considered positive.
|Table 2: Results of RAPIDEC® CARBA NP and modified carbapenem inactivation method test|
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| ~ Discussion|| |
These study findings are in concurrence with the previously published reports on the evaluation of RAPIDEC ® CARBA NP, where the sensitivity and specificity are >90%. Although many investigators have evaluated Carba NP test, the sensitivity and specificity were found to be >90% for class A and class B MBLs. While sensitivity and specificity were less for detecting blaOxa-48Like enzymes, due to its weak carbapenemase activity., This was recently addressed by Pasteran et al., wherein use of Triton X-100 reagent in place of lysis buffer improves the sensitivity of detecting blaOxa-48Like enzymes. This modification cannot be performed with point of care test RAPIDEC ® CARBA NP.
The main challenge with CIM lies in the interpretation of the assay. van der Zwaluw et al. interpreted the test as positive if no zone of inhibition is observed, whereas negative if zone of inhibition appeared., Subsequently, Tijet et al. improvised the interpretation as positive when zone of inhibition is 6 mm and negative when the zone of inhibition is ≥20 mm. More recently, Yamada et al. reported <15 mm zone as positive and >15 mm as negative. The same protocol of mCIM method was adopted by CLSI, with a slight change in the interpretative criteria as <19 mm zone as positive and negative if it is ≥19 mm. Notably, amongst the positives in this study, 79% of the tested isolates did not produce zone of inhibition (6 mm), whereas 21% had a zone of inhibition ranging 9–18 mm. Although the majority of the carbapenemase producers does not produce zone of inhibition, for which interpretation of the test results is straightforward. While isolates with an enzyme with low carbapenemase enzyme activity form zone of inhibition up to 19 mm.
The limitation of this study is Gram-negative organisms other than E. coli and K. pneumoniae were not evaluated. In addition, isolates with carbapenem resistance mechanisms other than carbapenemase productions such as AmpC hyperproduction, overexpression of efflux pumps and loss of porins are not included.
The overall sensitivity and specificity were found to be >90% for the carbapenemase detection by RAPIDEC ® CARBA NP. However, detection of blaOxa-48Like producers is going to be challenging. While mCIM is relatively simple and reliable, it requires good technical skill compared to RAPIDEC ® CARBA NP. Wherein, organisms producing low carbapenemase activity enzymes, thickness of the inoculum and the disc potency are likely to influence the test results of mCIM with an overnight delay.
The authors thank Fluid Research Grant, CMC, for providing financial support.
Financial support and sponsorship
The authors thank Fluid Research Grant, CMC, for providing financial support.
Conflicts of interest
There are no conflicts of interest.
| ~ References|| |
Poirel L, Walsh TR, Cuvillier V, Nordmann P. Multiplex PCR for detection of acquired carbapenemase genes. Diagn Microbiol Infect Dis 2011;70:119-23.
Bernabeu S, Poirel L, Nordmann P. Spectrophotometry-based detection of carbapenemase producers among Enterobacteriaceae. Diagn Microbiol Infect Dis 2012;74:88-90.
Cuzon G, Naas T, Bogaerts P, Glupczynski Y, Nordmann P. Probe ligation and real-time detection of KPC, OXA-48, VIM, IMP, and NDM carbapenemase genes. Diagn Microbiol Infect Dis 2013;76:502-5.
Jeremiah SS, Balaji V, Anandan S, Sahni RD. A possible alternative to the error prone modified Hodge test to correctly identify the carbapenemase producing Gram-negative bacteria. Indian J Med Microbiol 2014;32:414-8.
] [Full text]
Anandan S, Damodaran S, Gopi R, Bakthavatchalam YD, Veeraraghavan B. Rapid screening for carbapenem resistant organisms: Current results and future approaches. J Clin Diagn Res 2015;9:DM01-3.
Nordmann P, Poirel L, Dortet L. Rapid detection of carbapenemase-producing Enterobacteriaceae. Emerg Infect Dis 2012;18:1503-7.
Tijet N, Boyd D, Patel SN, Mulvey MR, Melano RG. Evaluation of the Carba NP test for rapid detection of carbapenemase-producing Enterobacteriaceae and Pseudomonas aeruginosa
. Antimicrob Agents Chemother 2013;57:4578-80.
Pragasam AK, Sahni RD, Anandan S, Sharma A, Gopi R, Hadibasha N, et al.
A pilot study on carbapenemase detection: Do we see the same level of agreement as with the CLSI observations. J Clin Diagn Res 2016;10:DC09-13.
Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fifth Informational Supplement. CLSI Document M100-S25. Wayne, PA: Clinical and Laboratory Standards Institute; January, 2015.
Nordmann P, Girlich D, Poirel L. Detection of carbapenemase producers in Enterobacteriaceae by use of a novel screening medium. J Clin Microbiol 2012;50:2761-6.
Bogaerts P, Yunus S, Massart M, Huang TD, Glupczynski Y. Evaluation of the BYG Carba test, a new electrochemical assay for rapid laboratory detection of carbapenemase-producing Enterobacteriaceae. J Clin Microbiol 2016;54:349-58.
Dortet L, Poirel L, Nordmann P. Further proofs of concept for the Carba NP test. Antimicrob Agents Chemother 2014;58:1269.
Dortet L, Poirel L, Errera C, Nordmann P. CarbAcineto NP test for rapid detection of carbapenemase-producing Acinetobacter
spp. J Clin Microbiol 2014;52:2359-64.
Pires J, Novais A, Peixe L. Blue-carba, an easy biochemical test for detection of diverse carbapenemase producers directly from bacterial cultures. J Clin Microbiol 2013;51:4281-3.
Pasteran F, Veliz O, Ceriana P, Lucero C, Rapoport M, Albornoz E, et al.
Evaluation of the Blue-Carba test for rapid detection of carbapenemases in gram-negative bacilli. J Clin Microbiol 2015;53:1996-8.
Poirel L, Nordmann P. Rapidec ®
Carba NP test for rapid detection of carbapenemase producers. J Clin Microbiol 2015;53:3003-8.
van der Zwaluw K, de Haan A, Pluister GN, Bootsma HJ, de Neeling AJ, Schouls LM. The carbapenem inactivation method (CIM), a simple and low-cost alternative for the Carba NP test to assess phenotypic carbapenemase activity in gram-negative rods. PLoS One 2015;10:e0123690.
Tijet N, Patel SN, Melano RG. Detection of carbapenemase activity in Enterobacteriaceae: Comparison of the carbapenem inactivation method versus the Carba NP test. J Antimicrob Chemother 2016;71:274-6.
Aktas E, Malkoçoglu G, Otlu B, Çopur Çiçek A, Külah C, Cömert F, et al.
Evaluation of the carbapenem inactivation method for detection of carbapenemase-producing Gram-negative bacteria in comparison with the Rapidec Carba NP. Microb Drug Resist 2016. [Epub ahead of print].
Yamada K, Kashiwa M, Arai K, Nagano N, Saito R. Comparison of the Modified-Hodge test, Carba NP test, and carbapenem inactivation method as screening methods for carbapenemase-producing Enterobacteriaceae. J Microbiol Methods 2016;128:48-51.
Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Seventh Informational Supplement. CLSI Document M100-S27. Wayne, PA: Clinical and Laboratory Standards Institute; January, 2017.
Pasteran F, Tijet N, Melano RG, Corso A. Simplified protocol for Carba NP Test for enhanced detection of carbapenemase producers directly from bacterial cultures. J Clin Microbiol 2015;53:3908-11.
Hombach M, von Gunten B, Castelberg C, Bloemberg GV. Evaluation of the Rapidec ®
Carba NP test for detection of carbapenemases in Enterobacteriaceae. J Clin Microbiol 2015;53:3828-33.
Garg A, Garg J, Upadhyay GC, Agarwal A, Bhattacharjee A. Evaluation of the Rapidec Carba NP test kit for detection of carbapenemase-producing Gram-negative bacteria. Antimicrob Agents Chemother 2015;59:7870-2.
[Table 1], [Table 2]