PERFORMANCE ASSESSMENT OF THREE PHENOTYPIC TESTS FOR CARBAPENEMASE DETECTION IN ENTEROBACTERALES
Abstract
Carbapenemase-producing Enterobacterales (CPE) pose a significant threat in hospital settings, especially in intensive care units, from both therapeutic and epidemiological perspectives. Rapid identification is crucial. This study aimed to evaluate the three phenotypic methods used in routine diagnostics. The study included 56 clinical isolates of carbapenem-resistant Enterobacterales collected from patients hospitalized at the University Clinical Center (UCC) in Niš. Among these isolates, 52 were confirmed as carbapenemase producers, while four lacked carbapenemase genes. Genotypic detection was performed using multiplex polumerase chin reachon targeting the bla Klebsiella pneumoniae carbapensemae (KPC), bla Verona integron-encoded Metallo-beta-lactamase (VIM), bla New Delhi metallo-beta-lactamase (NDM), and bla-Beta lactamase (OXA)-48 genes. The evaluated phenotypic methods included the NG-Test Carba 5, the RAPIDEC Carba NP test (RCNP), and a commercial combination disk test (CDT)—KPC, MBL, and OXA-48 Confirm Kit: Carbapenemases. Multiplex PCR revealed: 2 KPC producers; 24 NDM producers; 16 OXA-48-like producers; 10 isolates producing both NDM and OXA-48 enzymes. One isolate of Enterobacter cloacae was identified as a co-producer of NDM, KPC, and OXA-48 enzymes, and one isolate of Klebsiella pneumoniae was identified as a co-producer of NDM and KPC enzymes. The sensitivity and specificity of the NG-Test Carba 5 were 98.08% and 100.00%, respectively. In the Carba NP test, after 120 minutes, sensitivity and specificity were 90.38% and 100%, respectively. For the CDT method, the sensitivity and specificity for detecting metallo-β-lactamases using dipicolinic acid were 80.56% and 100%, respectively, while for detecting class D carbapenemases using temocillin, they were 95.65% and 100%, respectively. The best results in detecting specific classes of carbapenemases were achieved with the NG-Test Carba 5 and the CDT method. These methods could be employed for rapid and reliable detection of carbapenemases in routine diagnostics.
References
Alizadeh N, Ahangarzadeh, Rezaee M, Samadi Kafil H, Hasani A, Soroush Barhaghi MH et al. Evaluation of Resistance Mechanisms in Carbapenem-Resistant Enterobacteriaceae. Infect Drug Resist 2020; 13:1377-85. [CrossRef] [PubMed]
Bartolini A, Frasson I, Cavallaro A, Richter SN, Palù G. Comparison of phenotypic methods for the detection of carbapenem non-susceptible Enterobacteriaceae. Gut Pathog 2014; 6:13. [CrossRef] [PubMed]
Bonomo RA, Burd EM, Conly J, Limbago BM, Poirel L, Segre JA. Carbapenemase-Producing Organisms: A Global Scourge. Clin Infect Dis 2018; 66(8):1290-7. [CrossRef] [PubMed]
Centers for Desease Control and Prevention. Carbapenem-resistant Enterobacterales (CRE): An urgent public health threat. Available from: URL:https://arpsp.cdc.gov/story/cre-urgent-public-health-threat
Eltahlawi RA, Jiman-Fatani A, Gad NM, Ahmed SH, Al-Rabia MW, Zakai, S et al. Detection of Carbapenem-resistance in CRE by Comparative Assessment of RAPIDEC® CARBA NP and XpertTMCarba-R Assay. Infect Drug Resist 2023; 16:1123–31. [CrossRef] [PubMed]
Galani I, Rekatsina PD, Hatzaki D, Plachouras D, Souli M, Giamarellou H. Evaluation of different laboratory tests for the detection of metallo-β-lactamase production in Enterobacterales. J Antimicrob Chemoth 2008; 61(3):548–53. [CrossRef] [PubMed]
Gallagher LC, Roundtree SS, Lancaster DP, Rudin SD, Bard JD, Roberts AL et al. Performance of the CLSI Carba NP and the Rosco Carb screen assays using North American carbapenemase-producing Enterobacteriaceae and Pseudomonas aeruginosa isolates. J Clin Microbiol 2015; 53:3370–3. [CrossRef] [PubMed]
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(12):7870–2. [CrossRef] [PubMed]
Giske CG, Gezelius L, Samuelsen Ø, Warner M, Sundsfjord A, Woodford N. A sensitive and specific phenotypic assay for detection of metallo-beta-lactamases and KPC in Klebsiella pneumoniae with the use of meropenem disks supplemented with aminophenylboronic acid, dipicolinic acid and cloxacillin. Clin Microbiol Infect 2011; 17(4):552–6. [CrossRef] [PubMed].
Grundmann H, Glasner C, Albiger B, Aanensen DM, Tomlinson CT, Andrasevic AT, et al; European Survey of Carbapenemase-Producing Enterobacteriaceae (EuSCAPE) Working Group. Occurrence of carbapenemase-producing Klebsiella pneumoniae and Escherichia coli in the European survey of carbapenemase-producing Enterobacteriaceae (EuSCAPE): a prospective, multinational study. Lancet Infect Dis 2017;17(2):153-63. [CrossRef] [PubMed]
Gu D, Yan Z, Cai C, Li J, Zhang Y, Wu Y et al. Comparison of the NG-Test Carba 5, Colloidal Gold Immunoassay (CGI) Test, and Xpert Carba-R for the Rapid Detection of Carbapenemases in Carbapenemase-Producing Organisms. Antibiotics 2023; 2;12(2):300. [CrossRef] [PubMed]
Hojabri Z, Arab M, Darabi N, Kia NS, Lopes BS, Pajand O. Evaluation of the commercial combined disk test and minimum inhibitory concentration (MIC) determination for detection of carbapenemase producers among Gram-negative bacilli isolated in a region with high prevalence of blaOXA-48 and blaNDM. Int Microbiol 2019; 22(1):81–9. [CrossRef] [PubMed]
Hopkins KL, Meunier D, Naas T, Volland H, Woodford N, Evaluation of the NG-Test CARBA 5 multiplex immunochromatographic assay for the detection of KPC, OXA-48-like, NDM, VIM and IMP carbapenemases. J Antimicrob Chemother 2018; 73(12):3523–6. [CrossRef] [PubMed]
Ilstrup DM. Statistical methods in microbiology. Clin Microbiol Rev 1990; 3(3):219–26. [CrossRef] [PubMed]
Josa MD, Leal R, Rojas J, Torres MI, Cortés-Muñoz F, Esparza G et al. Comparative Evaluation of Phenotypic Synergy Tests versus RESIST-4 O.K.N.V. and NG Test Carba 5 Lateral Flow Immunoassays for the Detection and Differentiation of Carbapenemases in Enterobacterales and Pseudomonas aeruginosa. Microbiol Spectr 2022;10(1):e0108021. [CrossRef] [PubMed]
Kumudunie WGM, Wijesooriya LI, Wijayasinghe YS. Comparison of four low-cost carbapenemase detection tests and a proposal of an algorithm for early detection of carbapenemase-producing Enterobacterales in resource-limited settings. PLoS One 2021;16(1):e0245290. [CrossRef] [PubMed]
Logan LK, Weinstein RA. The epidemiology of carbapenem-resistant Enterobacteriaceae: the impact and evolution of a global menace. J Infect Dis 2017; 215(1): S28–36. [CrossRef] [PubMed]
Miriagou V,Tzelepi E, Kotsakis SD, Bou Casals J,Tzouvelekis LS.Combined disc methods for the detection of KPC- and/or VIM-positive Klebsiella pneumoniae: improving reliability for the double carbapenemase producers. Clin Microbiol Infect 2013; 19(9):E412-5. [CrossRef] [PubMed]
Moloney E, Lee KW, Craig DA. Joy Allen AJ, Graziadio S, Power M et al. A PCR-based diagnostic testing strategy to identify carbapenemase-producing Enterobacterales carriers upon admission to UK hospitals: early economic modelling to assess costs and consequences. Diagn Progn 2019;3(8). [CrossRef] [PubMed]
Noël A, Huang TD, Berhin C, Hoebeke M, Bouchahrouf W, Yunus S et al. Comparative Evaluation of Four Phenotypic Tests for Detection of Carbapenemase-Producing Gram-Negative Bacteria. J Clin Microbiol 2017; 55(2):510-8. [CrossRef] [PubMed].
Pantel A, Souzy D, Sotto A, Lavigne J-P. Evaluation of two phenotypic screening tests for carbapenemase-producing Enterobacteriaceae. J Clin Microbiol 2015; 53:3359–3362. [CrossRef] [PubMed]
Queenan AM, Bush K. Carbapenemases: The versatile beta-lactamases. Clin Microbiol Rev 2007; 20(3):440–458. [CrossRef] [PubMed]
Saito K, Mizuno S, Nakano R, Tanouchi A, Mizuno T, Nakano A et al. Evaluation of NG-Test CARBA 5 for the detection of carbapenemase-producing Gram-negative bacilli. J Med Microbiol 2022; 71(6). [CrossRef] [PubMed]
Sattler J, Brunke A, Hamprecht A. Systematic Comparison of Three Commercially Available Combination Disc Tests and the Zinc-Supplemented Carbapenem Inactivation Method (zCIM) for Carbapenemase Detection in Enterobacterales Isolates. J Clin Microbiol 2021; 59(9):e0314020. [CrossRef] [PubMed]
Solgi H, Badamchi A, Shahcheraghi F, Badmasti F, Akbari M, Behzadfar M. A comparative evaluation of five phenotypic methods for identification of carbapenemase-producing Enterobacteriaceae: a modified carbapenemase detection test. Microbiol Spectr 2024; 12:e0038624. [CrossRef] [PubMed]
Tarlton NJ, Wallace MA, Potter RF, Zhang K, Dantas G, Dubberke ER, et al. Evaluation of the NG-Test CARBA 5 Lateral Flow Assay with an IMP-27-Producing Morganella morganii and Other Morganellaceae. Microbiol Spectr 2023; 11(3):11:e00793-23. [CrossRef] [PubMed]
The European Committee on Antimicrobial Susceptibility Testing - EUCAST. Resistance mechanisms. Available from: URL:https://www.eucast.org/fileadmin/src/media/ PDFs/EUCAST_files/ Resistance_mechanisms)
Tsakris A, Kristo I, Poulou A, Themeli-Digalaki K, Ikonomidis A, Petropoulou D, et al. Evaluation of boronic acid disk tests for differentiating KPC possessing Klebsiella pneumoniae isolates in the clinical laboratory. J Clin Microbiol 2009; 47(2): 362–7. [CrossRef] [PubMed]
van Dijk K, Voets GM, Scharringa J, Voskuil S, Fluit AC, Rottier WC et al. A disc diffusion assay for detection of class A, B and OXA-48 carbapenemases in Enterobacterales using phenyl boronic acid, dipicolinic acid and temocillin. Clin Microbiol Infect 2014; 20(4):345-9. [CrossRef] [PubMed]
