This study was performed in the clinical microbiology laboratory at the Baruch Padeh Medical Center, Israel. Bacterial clinical or screening samples previously found to be resistant to carbapenems using our routine methods, were retested. Out of the 48 isolates, 10 were non-CP CRE, and 38 were CP-CRE: 1 IMP, 10 NDM, 8 OXA-48, 6 VIM, and 13 KPC. Briefly, testing involved Enterobacterales identification by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) technology (Bruker Daltonics, Bremen, Germany), and then with the β CARBA kit (Bio-Rad Laboratories Ltd., Rishon Lezion, Israel), which detects strains with a decreased susceptibility to carbapenems. Colonies that gave a positive result were further analysed using the Xpert® Carba-R (Cepheid, Sunnyvale, CA, USA) PCR assay, that detects the five most prevalent carbapenemases (KPC, NDM, VIM, OXA-48, and IMP). In order to demonstrate that bacteria type plays no role in kit performance, different types of CRE bacteria were tested, such as Escherichia coli and Klebsiella pneumoniae. Moreover, we examined bacteria with various types of carbapenemases (NDM, KPC, OXA, VIM, IMP).
Additionally, 10 non-CP CRE isolates were collected. All isolates were stored at -80°C until analysis. Prior to analysis by the NG-Test kit, the isolates were grown on MacConkey agar (BD Diagnostics, Sparks, MD), at 36±1oC, in 5% CO2, for 24 h.
Blood culture preparation
Suspensions simulating patient blood samples were prepared by diluting human blood with saline, at a ratio of 45% blood unit and 55% saline. Then, a bacterial suspension of each isolate, was prepared in saline, to a turbidity of 0.5 McFarland, which is equivalent to 1-2*108 CFU/ml. The bacterial suspension was then further diluted, to create variable concentrations of bacteria that were later mixed with the blood suspensions. Blood-bacteria suspensions (9 ml) were introduced to an anaerobic blood culture bottle [Plus Anaerobic/F Culture Vials (BD Diagnostics, Sparks, MD)], producing "blood cultures" with a final concentration of 6.75* 107 CFU/ml. Samples from each bottle were then tested with the NG-Test CARBA 5 (NG Biotech) kit. The lowest concentration recognized by the NG Test CARBA 5 kit was determined for each pathogen tested.
Detection of carbapenemases by the NG-Test CARBA 5 kit
The NG-Test CARBA 5 kit (NG Biotech, Guipry, France) is a qualitative rapid immunoassay, in which mouse monoclonal antibodies directed against KPC (K), OXA (O), VIM (V), IMP (I), and NDM (N), are immobilized on the nitrocellulose membrane test zones K, O, V, I and N, respectively. The tested sample is mixed with 150 μL extraction buffer; 100 μL of this mixture is then dispensed into the cassette well and allowed to migrate towards the conjugate pad. If a carbapenemase is present in the suspension, it reacts with labelled monoclonal anti-carbapenemase antibodies. The carbapenemase-antibodies complex migrates through the nitrocellulose membrane and is captured by corresponding monoclonal anti-carbapenemase antibodies immobilized on the membrane, resulting in a red line (or lines) on the test zone (s) and on the control zone.
Samples from each “blood culture” (1 ml) were transferred into an Eppendorf tube, after which, 1ml lysis buffer (Accuprep Genomic DNA extraction kit, Bioneer, Korea) was added. The Eppendorf tube was vortexed for 20 sec and then centrifuged for 1 min at 13,000 rpm/min. Pellets were then washed with 1 ml wash buffer (Accuprep Genomic DNA extraction kit, Bioneer, Korea) vortexed for 20 sec, and centrifuged for 1 min at 13,000 rpm/min. Pellets were then resuspended in 150 µl extraction buffer (supplied with the kit) and placed on the test zone of the cassette and later analyzed for results.
As mentioned above, for each isolate, we tested several concentrations of isolate colonies per ml "blood culture" in order to define what is the lowest concentration that the kit can identify. It should be mentioned that each of the isolates was correctly identified by the kit, when we tested directly the isolate's colonies (without preparing a blood-bacteria suspension).
The Xpert® Carba-R (Cepheid, Sunnyvale, CA, USA) PCR assay served as the reference method for calculating sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV). Therefore, specimens that were found positive or negative by the Xpert® Carba-R were defined as "True Positive" or "True Negative", respectively.
Agreement between Xpert® Carba-R and the NG-Test CARBA 5 kit was calculated as the percentage of samples that had the same results for both assays, out of the total number of tested samples. Data were analyzed using SAS® version 9.3 (SAS Institute, Cary, NC, US).