Parallel Validation of the NG-Test Carba 5 and the Xpert Carba-R for Detection and Characterization of Carbapenem-Resistant Enterobacterales Causing Bloodstream Infections

Abstract

The rapid detection and characterization of carbapenemases in isolates of Enterobacterales are crucial for precise antibiotic administration and infection control. This article reports the findings from a parallel evaluation of the NG-Test Carba 5 (NG Biotech, Guipry, France) and Xpert Carba-R (Cepheid, Sunnyvale, CA) assays in the detection and differentiation of five carbapenemases [imipenem-resistant phenotype (IMP), Klebsiella pneumoniae carbapenemase, New Delhi metallo-β-lactamase (NDM), oxacillin-hydrolyzing β-lactamase (OXA)-48–like, and Verona integron-encoded metallo-β-lactamase] or the genes that encode them. A total of 122 isolates recovered from blood cultures and 106 positive blood culture broth (BCB) specimens, including 134 Klebsiella pneumoniae, 54 Escherichia coli, 27 Enterobacter cloacae, 8 Klebsiella oxytoca, 2 Klebsiella aerogenes, and 3 Citrobacter freundii, were collected from two tertiary hospitals (Xi'an, China). Using PCR sequencing techniques, 89 isolates and 29 BCB specimens were determined to be Enterobacterales harboring carbapenem-resistance genes. In comparison to the PCR sequencing results, the specificities with both the NG-Test Carba 5 and Xpert Carba-R assays were 100%; the sensitivities were 92.1% and 100%, respectively, for recovered isolates and 79.3% and 100% for BCB specimens. The NG-Test Carba 5 missed eight NDM, four OXA-48–like, and one IMP β-lactamases in specimens containing two or three carbapenemase types. In summary, the NG-Test Carba 5 assay may yield false-negative results if isolates or BCB specimens contain two or three carbapenemases. The rapid detection and characterization of carbapenemases in isolates of Enterobacterales are crucial for precise antibiotic administration and infection control. This article reports the findings from a parallel evaluation of the NG-Test Carba 5 (NG Biotech, Guipry, France) and Xpert Carba-R (Cepheid, Sunnyvale, CA) assays in the detection and differentiation of five carbapenemases [imipenem-resistant phenotype (IMP), Klebsiella pneumoniae carbapenemase, New Delhi metallo-β-lactamase (NDM), oxacillin-hydrolyzing β-lactamase (OXA)-48–like, and Verona integron-encoded metallo-β-lactamase] or the genes that encode them. A total of 122 isolates recovered from blood cultures and 106 positive blood culture broth (BCB) specimens, including 134 Klebsiella pneumoniae, 54 Escherichia coli, 27 Enterobacter cloacae, 8 Klebsiella oxytoca, 2 Klebsiella aerogenes, and 3 Citrobacter freundii, were collected from two tertiary hospitals (Xi'an, China). Using PCR sequencing techniques, 89 isolates and 29 BCB specimens were determined to be Enterobacterales harboring carbapenem-resistance genes. In comparison to the PCR sequencing results, the specificities with both the NG-Test Carba 5 and Xpert Carba-R assays were 100%; the sensitivities were 92.1% and 100%, respectively, for recovered isolates and 79.3% and 100% for BCB specimens. The NG-Test Carba 5 missed eight NDM, four OXA-48–like, and one IMP β-lactamases in specimens containing two or three carbapenemase types. In summary, the NG-Test Carba 5 assay may yield false-negative results if isolates or BCB specimens contain two or three carbapenemases. The Centers for Disease Control and Prevention has reported that carbapenem-resistant Enterobacterales (CRE) are an urgent threat (https://stacks.cdc.gov/view/cdc/82532, last accessed March 15, 2021). In China, the rate of carbapenem resistance among Enterobacterales has reached up to 64.1%, which makes the treatment of serious diseases, such as bloodstream infections, difficult.1Hu F. Guo Y. Yang Y. Zheng Y. Wu S. Jiang X. Zhu D. Wang F. China Antimicrobial Surveillance Network (CHINET) Study GroupResistance reported from China antimicrobial surveillance network (CHINET) in 2018.Eur J Clin Microbiol Infect Dis. 2019; 38: 2275-2281Crossref PubMed Scopus (79) Google Scholar The World Health Organization has called for the development of new diagnostic tests for the accurate diagnosis of CRE infection to aid in antimicrobial stewardship (https://www.who.int/publications/i/item/WHO-MVP-EMP-2019.05, last accessed February 14, 2021). Carbapenemases, the major cause of resistance among CRE, are classified as Ambler classes A, B, and D, according to the amino acid sequences, each with different hydrolytic activity against carbapenems. The accurate identification of serine versus metallo-based (MBL) carbapenemases provides valuable information for guiding antibacterial drug therapy. For example, the β-lactam/β-lactamase inhibitor combination ceftazidime/avibactam is active against class A carbapenemases but does not inhibit class B metallo-enzymes.2Pogue J.M. Bonomo R.A. Kaye K.S. Ceftazidime_avibactam, meropenem_vaborbactam, or both_ clinical and formulary considerations.Clin Infect Dis. 2019; 68: 519-524Crossref PubMed Scopus (80) Google Scholar,3van Duin D. Lok J.J. Earley M. Cober E. Richter S.S. Perez F. Salata R.A. Kalayjian R.C. Watkins R.R. Doi Y. Kaye K.S. Fowler Jr., V.G. Paterson D.L. Bonomo R.A. Evans S. Antibacterial Resistance Leadership GroupColistin versus ceftazidime-avibactam in the treatment of infections due to carbapenem-resistant Enterobacteriaceae.Clin Infect Dis. 2018; 66: 163-171Crossref PubMed Scopus (284) Google Scholar Bloodstream infections are difficult to treat, and prompt and effective treatment is crucial for shortening hospital stay and decreasing mortality.4Stewardson A.J. Marimuthu K. Sengupta S. Allignol A. El-Bouseary M. Carvalho M.J. et al.Effect of carbapenem resistance on outcomes of bloodstream infection caused by Enterobacteriaceae in low-income and middle-income countries (PANORAMA): a multinational prospective cohort study.Lancet Infect Dis. 2019; 19: 601-610Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar Therefore, the rapid identification of suspected resistance mechanisms in CRE should be a priority in treatment monitoring, given that this identification can be useful for clinicians in optimizing antimicrobial therapy. The NG-Test Carba 5 (NG Biotech, Guipry, France) and Xpert Carba-R (Cepheid, Sunnyvale, CA) assays are two newly developed methods of detecting the five major carbapenemases [imipenem-resistant phenotype (IMP), Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-β-lactamase (NDM), oxacillin-hydrolyzing β-lactamase (OXA)-48–like, and Verona integron-encoded metallo-β-lactamase (VIM)] or carbapenemase genes (blaIMP, blaKPC, blaNDM, blaOXA-48–like, and blaVIM). NG-Test Carba 5 uses monoclonal antibodies for detecting the five carbapenemases in bacterial colonies, with a turnaround time of 30 minutes, simple operations, and easy reading of colloid immunochromatographic test strips.5Giordano L. Fiori B. D'Inzeo T. Parisi G. Liotti F.M. Menchinelli G. De Angelis G. De Maio F. Luzzaro F. Sanguinetti M. Posteraro B. Spanu T. Simplified testing method for direct detection of carbapenemase-producing organisms from positive blood cultures using the NG-test Carba 5 assay.Antimicrob Agents Chemother. 2019; 63: e00550-19Crossref PubMed Scopus (16) Google Scholar, 6Bogaerts P. Berger A.S. Evrard S. Huang T.D. Comparison of two multiplex immunochromatographic assays for the rapid detection of major carbapenemases in Enterobacterales.J Antimicrob Chemother. 2020; 75: 1491-1494Crossref PubMed Google Scholar, 7Boutal H. Vogel A. Bernabeu S. Devilliers K. Creton E. Cotellon G. Plaisance M. Oueslati S. Dortet L. Jousset A. Simon S. Naas T. Volland H. A multiplex lateral flow immunoassay for the rapid identification of NDM-, KPC-, IMP- and VIM-type and OXA-48-like carbapenemase-producing Enterobacteriaceae.J Antimicrob Chemother. 2018; 73: 909-915Crossref PubMed Scopus (110) Google Scholar, 8Bianco G. Boattini M. van Asten S.A.V. Iannaccone M. Zanotto E. Zaccaria T. Bernards A.T. Cavallo R. Costa C. RESIST-5 O.O.K.N.V. and NG-test Carba 5 assays for the rapid detection of carbapenemase-producing Enterobacterales from positive blood cultures: a comparative study.J Hosp Infect. 2020; 105: 162-166Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar The Xpert assay is run on an automated, random-access PCR system that integrates nucleic acid extraction, amplification, and detection together to identify the five major carbapenem-resistance genes qualitatively on samples from rectal swabs and pure colonies, with a turnaround time of approximately 60 minutes.9Traczewski M.M. Carretto E. Canton R. Moore N.M. Carba-R Study TeamMulticenter evaluation of the Xpert Carba-R assay for detection of carbapenemase genes ingram-negative isolates.J Clin Microbiol. 2018; 56: e00272-18Crossref PubMed Scopus (47) Google Scholar, 10Tato M. Ruiz-Garbajosa P. Traczewski M. Dodgson A. McEwan A. Humphries R. Hindler J. Veltman J. Wang H. Canton R. Multisite evaluation of Cepheid Xpert Carba-R assay for detection of carbapenemase-producing organisms in rectal swabs.J Clin Microbiol. 2016; 54: 1814-1819Crossref PubMed Scopus (84) Google Scholar, 11Jin X. Zhang H. Wu S. Qin X. Jia P. Tenover F.C. Tang Y.W. Li M. Hu F. Yang Q. Yu Y. Multicenter evaluation of Xpert Carba-R assay for detection and identification of the carbapenemase genes in rectal swabs and clinical isolates.J Mol Diagn. 2021; 23: 111-119Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar In this study, the performance of the NG-Test Carba 5 and Xpert Carba-R tests was assessed as the capacity for the detection and differentiation of the five carbapenemases or carbapenemase genes in Enterobacterales isolates recovered from blood cultures and positive blood culture broth (BCB), with PCR sequence analysis used as a reference standard. Nonduplicate Enterobacterales isolates recovered from blood cultures and positive BCB specimens were collected from the Second Affiliated Hospital of Xi'an Jiaotong University and the Xijing Hospital of the Air Force Medical University (both, Xi'an, China) between January 1, 2019, and December 22, 2020. The study protocol was approved by the Medical Ethics Committee of the Second Affiliated Hospital of Xi'an Jiaotong University (protocol 2021017). BCB specimens were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Marcy l'Etoile, bioMérieux, France). In brief, 5 μL of the BCB was injected into a separator gel tube, followed by centrifugation at 845 × g for 10 minutes. The enrichment was transferred into 500 μL of sterile water with a 1-μL disposable sterile inoculation loop and centrifuged at 3660 × g for 2 minutes. The supernatant was discarded and then 500 μL of sterile water was added and vortexed vigorously prior to centrifugation as described above. After repeating with 500 μL of sterile water once, 700 μL of ethanol was added and centrifuged as described previously. After the ethanol was aspirated, 30 μL of 70% formic acid was added to the tube and vortexed vigorously, and then the tube was centrifuged as described in the previous paragraph after the addition of 30 μL of acetonitrile. One microliter of supernatant was placed on the target plate and air-dried at room temperature, followed by the loading of 1 μL of matrix liquid. To identify the isolates, spectrum information was uploaded, and the mass spectrometry data were collected for the comparison and analysis with the profiles in the database using Myla software version 4.71 (bioMérieux, Marcy l'Etoile, France). A score above 99.9 was considered acceptable.12Chen Y. Porter V. Mubareka S. Kotowich L. Simor A.E. Rapid identification of bacteria directly from positive blood cultures by use of a serum separator tube, smudge plate preparation, and matrix-assisted laser desorption ionization-time of flight mass spectrometry.J Clin Microbiol. 2015; 53: 3349-3352Crossref PubMed Scopus (26) Google Scholar,13Carretero O. Rivas G. Loras C. Orellana M.A. Rapid identification of bacteria directly from positive blood cultures by a modified method using a serum separator tube and matrix-assisted laser desorption ionization - time of flight MS.J Med Microbiol. 2020; 69: 1373-1380Crossref PubMed Scopus (1) Google Scholar To determine carbapenem susceptibilities phenotypically, bacterial isolates and BCB specimens were inoculated on blood agar plates in three-phase streaking, followed by incubation at 35°C ± 2°C for 18 to 24 hours. Identification and antimicrobial susceptibility testing were performed using the Vitek 2 Compact automated microbial identification system (bioMérieux). Carbapenem susceptibility testing data were interpreted according to the Clinical and Laboratory Standards Institute minimum inhibitory concentration breakpoints.14Clinical and Laboratory Standards InstitutePerformance Standards for Antimicrobial Susceptibility Testing. ed 30. CLSI supplement. M100. ed: CLSI.2020Google Scholar All nonduplicate isolates were tested with the NG-Test Carba 5 kit for the detection of the five major carbapenemases (IMP-1, KPC, NDM, OXA-48–like, and VIM) according to the manufacturer's instructions. Briefly, five drops (approximately 150 μL) of extraction buffer were added into an Eppendorf tube, and the bacterial isolate was seeded into the tube with a 1-μL inoculation loop. After brief vortexing, 100 μL of mixture was loaded into the sample well marked with "S" in the test cassette with a disposable transfer pipette. The reading was completed within 20 minutes after the additional incubation for 15 minutes at room temperature.15Potron A. Fournier D. Emeraud C. Triponney P. Plesiat P. Naas T. Dortet L. Evaluation of the immunochromatographic NG-test Carba 5 for rapid identification of carbapenemase in nonfermenters.Antimicrob Agents Chemother. 2019; 63: e00968Crossref PubMed Scopus (13) Google Scholar For the identification of positive BCB specimens, the mixture was prepared by adding 1 mL of specimen into 1 mL of lysis buffer, and the supernatant was discarded after centrifugation. Further washing was completed by filling with 1 mL of washing buffer and centrifugation. Then 5 drops of buffer were added and mixed well. The mixture of 100 μL was loaded for reading as described above.16Takissian J. Bonnin R.A. Naas T. Dortet L. NG-test Carba 5 for rapid detection of carbapenemase-producing Enterobacterales from positive blood cultures.Antimicrob Agents Chemother. 2019; 63: e00011Crossref PubMed Scopus (37) Google Scholar For the detection of carbapenemase genes in the blood culture isolates with Xpert Carba-R, a 0.5 MacFarland suspension (about 1 to 2 × 108 colony-forming units/mL) was prepared by picking several colonies from an 18- to 24-hour bacterial culture plate with a swab. Ten microliters of the bacterial suspension was added into 5 mL of sample reagent and vortexed vigorously for 10 seconds, then 1.7 mL of the sample reagent was loaded into an Xpert Carba-R cartridge and placed into the GeneXpert instrument.17Ko Y.J. Kim J. Kim H.N. Yoon S.Y. Lim C.S. Lee C.K. Diagnostic performance of the Xpert Carba-R assay for active surveillance of rectal carbapenemase-producing organisms in intensive care unit patients.Antimicrob Resist Infect Control. 2019; 8: 127Crossref PubMed Scopus (6) Google Scholar For the detection of the carbapenemase genes in positive BCB specimens, 10 μL of BCB specimen was added into 5 mL of sample reagent. The mixture was vortexed vigorously for 10 seconds and 1.7 mL of sample was loaded for detection as described in the previous paragraph.18Cointe A. Walewski V. Hobson C.A. Doit C. Bidet P. Dortet L. Bonacorsi S. Birgy A. Rapid carbapenemase detection with Xpert Carba-R V2 directly on positive blood vials.Infect Drug Resist. 2019; 12: 3311-3316Crossref PubMed Scopus (10) Google Scholar Note, the use of the Xpert Carba-R test directly from positive BCBs has not been reviewed by any regulatory authority. A phenotypic method using four disks, one containing imipenem alone, a second with imipenem and 3-aminophenylboronic acid (APB), a third with imipenem and EDTA, and a fourth with imipenem and both APB and EDTA, was used for differentiating MBL and KPC carbapenemases, as previously described.19Tsakris A. Poulou A. Pournaras S. Voulgari E. Vrioni G. Themeli-Digalaki K. Petropoulou D. Sofianou D. A simple phenotypic method for the differentiation of metallo-beta-lactamases and class A KPC carbapenemases in Enterobacteriaceae clinical isolates.J Antimicrob Chemother. 2010; 65: 1664-1671Crossref PubMed Scopus (165) Google Scholar The test was performed by inoculation of Mueller-Hinton agar with the 0.5 McFarland suspension of the test organism, and placement of the four disks onto the agar plate. The agar plates were incubated at 37°C overnight. The zones of inhibition around the imipenem disks with and without the inhibitors were measured, and increases in zone diameters of ≥5 mm were considered significant. A serine carbapenemase (ie, KPC) was indicated by zone diameters around APB but not to the imipenem of a was indicated by zone around but not APB to the and the of both of carbapenemases was indicated by zone diameters around both APB and to the of the three zone the isolate was considered to be for and serine carbapenemase The of APB and in the study not any on bacterial Y. G. Evans K. G. A method with agar plate assay for phenotypic of KPC, MBL and carbapenemases among Enterobacteriaceae.J Microbiol. 2016; PubMed Scopus (6) Google Scholar For five carbapenemase genes (blaIMP, blaKPC, blaOXA-48–like, blaNDM, and were by followed by sequencing with as described D. S. R. A multiplex PCR for the and detection of the carbapenemase genes blaKPC, blaNDM, and Microbiol PubMed Scopus (10) Google Scholar The of the positive PCR were to the in The and of the NG-Test Carba 5 and the Xpert Carba-R tests were in comparison with the PCR sequencing For this study, the five carbapenemase genes were to be by A total of 122 nonduplicate isolates recovered from blood cultures and 106 positive BCB specimens were collected the study The blood culture isolates Klebsiella pneumoniae, Escherichia coli, 10 Enterobacter cloacae, 3 Klebsiella oxytoca, 2 Citrobacter freundii, and 1 Klebsiella BCB specimens pneumoniae, 30 coli, cloacae, and 3 A total of 89 isolates and 29 BCB specimens were to at one carbapenem as determined by Vitek 2 The carbapenemase in the study KPC, NDM, and were and clinical information on the isolates and BCB specimens in the study are in The specimen and carbapenemases were between the two of the and of the Clinical in the by Second Affiliated Hospital of Xi'an Jiaotong University Hospital of the Air Force Medical University ± ± ± ± ± ± culture Klebsiella Escherichia Enterobacter Klebsiella Citrobacter Klebsiella Klebsiella Escherichia Enterobacter Klebsiella Citrobacter Klebsiella in a new The performance of NG-Test Carba 5 and Xpert Carba-R tests was using the PCR sequencing results as the reference standard. NG-Test Carba the sensitivities were 92.1% for isolates and 79.3% for BCB specimens, the specificities were 100% for both specimen sensitivities were the NG-Test Carba 5 was used for detecting NDM, or carbapenemases The sensitivities and specificities of the Xpert test for the detection and differentiation of the five carbapenemase genes were 100% for both isolates and BCB specimens and by NG-Test Carba 5 recovered from blood culture the be positive on on positive on on in a new and by Xpert Carba-R recovered from blood culture the be positive on on positive on on in a new the be positive on on positive on on the be positive on on positive on on the CRE specimens, 30 one carbapenemase including blaNDM, blaNDM, 2 blaOXA-48–like, 2 and 1 1 and The NG-Test Carba 5 missed eight genes in specimens in which both and genes were In the Xpert test and carbapenemase genes the specimens one or resistance genes 1 and the eight specimens in which was missed by the NG-Test Carba were positive for carbapenemases on the Xpert test and of MBL and KPC metallo-based carbapenemases. in a new metallo-based carbapenemases. The of from the transfer of and resistance genes between or G. The rapid of carbapenem-resistant Resist 2016; PubMed Scopus Google Scholar The rapid and accurate and differentiation of carbapenemase valuable information for guiding effective antibiotic and methods of detection and characterization been described including the modified Carba agar modified carbapenem modified carbapenem method EDTA, and M. L. J. D. C. Evaluation of three and KPC, for the detection of Klebsiella pneumoniae Clin 66: PubMed Scopus Google K. A. A.J. The carbapenem method a simple and for the Carba test to phenotypic carbapenemase activity in 2015; PubMed Scopus Google Scholar of methods a second a turnaround time 24 to carbapenemase and and and of For example, the modified test may yield results used for testing bacteria with spectrum and and false-negative results used for testing bacteria with F. E. G. A.J. A. protocol for modified test for detection of and carbapenemase Clin Microbiol. 2016; 54: PubMed Scopus Google R.A. J. L. a Infect Dis. 2018; 66: PubMed Scopus Google Scholar the NG-Test Carba 5 and Xpert Carba-R assays were developed for the rapid detection of five carbapenemases KPC, NDM, and VIM) or the genes that encode them. NG-Test Carba 5 is on a immunochromatographic and the kit has the capacity to identify 5 within 15 minutes. Xpert Carba-R is a PCR that approximately minutes to the five of resistance genes from colonies in pure culture or from rectal detection the of antimicrobial therapy. assays can serine β-lactamases from which is useful in not optimizing antibiotic stewardship and but the of drug resistance antibiotic In susceptibility to ceftazidime-avibactam of Enterobacteriaceae isolates collected the Surveillance study to Agents Chemother. 2016; PubMed Scopus Google C. O. L. F. C. C. C. M. P. J. A. Clinical and of infections caused by carbapenemase-producing Enterobacteriaceae in with J Antimicrob 2019; 53: PubMed Scopus Google Scholar In the study, carbapenemases of isolates were by Xpert Carba-R, NG-Test Carba 5 to identify of the carbapenemases in organisms with of KPC KPC and KPC the 106 positive BCB specimens, the carbapenemase genes in the isolates were on Xpert in organisms with the combination of KPC or KPC not of the carbapenemases were identified on NG-Test Carba the NG-Test Carba 5 and the Xpert assays sensitivities of 92.1% and 100%, respectively, for isolates, and sensitivities of 79.3% and 100% for BCB specimens, to the reference PCR sequencing For the detection of a carbapenemase or carbapenemase the of the assays with the sequencing was 100% with both Xpert Carba-R was accurate resistance genes were R. Q. Wu S. D. M. D. Zheng Y. Guo Y. Zhang R. Hu F. China Antimicrobial Surveillance Network (CHINET) Study of carbapenemases NDM, and VIM) among carbapenem-resistant Enterobacteriaceae from and in Infect Microbiol. 2020; PubMed Scopus Google Scholar For specimens containing both MBL and KPC carbapenemases, the MBL detection can major in administration of β-lactam/β-lactamase inhibitor such as ceftazidime-avibactam or In both the NG-Test Carba 5 and the Xpert Carba-R assays rapid detection and identification of five carbapenemases in both isolates recovered from blood cultures and BCB specimens. The NG-Test Carba 5 assay missed the detection of of carbapenemases, NDM, in specimens with one carbapenemase the of the clinical at the two hospitals for

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