Comparison of five methods for detection of carbapenemases in Enterobacterales with proposal of a new algorithm

Abstract

The aim of this study was to evaluate the performance of five different carbapenemase tests and to develop an algorithm which will permit the detection of most common and rare carbapenemases in routine microbiology laboratories. Methods: The immunochromatographic tests CARBA-5 (NG), RESIST-4 O.K.N.V. (Coris), the colorimetric b-CARBA (BioRad), a newly developed carbapenem-inactivation method (CIM) supplemented with zinc (zCIM), and the Xpert Carba-R (Cepheid) were challenged with a collection of 189 molecularly characterized Enterobacterales isolates, including 146 carbapenemase producers (CPE): VIM (n ¼ 48), OXA-48like (n ¼ 40), NDM (n ¼ 29), KPC (n ¼ 13), IMI (n ¼ 9), IMP (n ¼ 9), OXA-58 (n ¼ 2), and GES (n ¼ 2). Results: The overall sensitivity/specificity values for the five carbapenemase detection tests were 84.2% (CI 77.6e89.2%)/100% (CI 91.8e100%) for RESIST-4, 88.2% (CI 82.1e92.4%)/100% (CI 91.8e100%) for CARBA-5, 88.2% (CI 82.1e92.4%)/100% (CI 91.8e100%) for Xpert Carba-R, 73.7% (CI 66.2e80.0%)/100% (CI 93.4e99.0%) for b-CARBA, and 97.4% (CI 87.9e99.6%)/97.7% (CI 87.9e99.6%) for zCIM. The four common carbapenemases (KPC, OXA-48-like, NDM, and VIM) were detected with 97.6% sensitivity by all tests except for b-CARBA (76.6% (CI 68.4e83.2%)). IMI and GES were only detected by zCIM (sensitivity 90.9%(CI 62.3e98.4%)). Based on these results a new algorithm was developed, consisting of an immunochromatographic assay as the first test followed by zCIM, which allows detection of 99.3% of all carbapenemases assessed.Conclusions: Except for b-CARBA, all methods showed excellent sensitivity/specificity for the detection of the four most frequent carbapenemases. With the new algorithm, rare variants can also be detected. It is rapid, simple, and inexpensive and can be performed in any microbiology laboratory, as no PCR equipment is required.

Direct β-Lactam Inactivation Method: a New Low-Cost Assay for Rapid Detection of Carbapenemase- or Extended-Spectrum-β-Lactamase-Producing Enterobacterales Directly from Positive Blood Culture Bottles

“…Our method originates from the CIM test, a simple and accurate assay recently developed to detect carbapenemase production in pure cultures of GN bacterial isolates. Various studies demonstrated a higher sensitivity of the CIM test and its improved variants over those of traditional colorimetric methods, especially for the detection of low-activity carbapenemases (26,31,32). As recently highlighted by Humphries (33), the interest of the clinical microbiology community regarding CIM-based tests is shown by the many articles posted on this topic since Breakpoints and interpretation criteria are as follows: for ES/ACBL/CARB-nonproducing EB, the MEM diameter was Ͼ15 mm and the CTX diameter was Ͼ14 mm; for ESBL/ACBL-producing EB, the MEM diameter was Ͼ15 mm and the CTX diameter was Յ12 mm; for carbapenemase-producing EB, the MEM diameter was Յ12 mm; and for disks with ATUs, the MEM diameter was 13 to 14 mm and the CTX diameter was 13 mm.…”

Rapid and Easy Detection of Carbapenemases in Enterobacterales in the Routine Laboratory Using the New GenePOC Carba/Revogene Carba C Assay

“…Rare carbapenemases (e.g., GES, IMI, GIM, and OXA-58) are currently not targeted by the two assays evaluated or most other commercially available PCR assays. Therefore, other diagnostic tests, such as the Carba NP test (20) or the zCIM test (4), which have previously proved to be sensitive for rare carbapenemase detection, are necessary to detect all CPE strains.…”

“…The presence of the ␤-lactamase (bla) genes encoding OXA-48-like, VIM, IMP, NDM, and KPC enzymes was analyzed for all strains by PCR and subsequent DNA Sanger sequencing, as described previously (4). For phenotypic identification of carbapenemases, a combination disk test (KPC and metallo-␤-lactamase [MBL] disk kit; Liofilchem, Roseto degli Abruzzi, Italy) was used as recommended by EUCAST (14), with meropenem and one of the following ␤-lactamase inhibitors: EDTA (MBL), cloxacillin (AmpC), or phenylboronic acid (class A).…”

“…A number of phenotypic methods for carbapenemase detection (e.g., modified Hodge test or various combination disk tests) show long turnaround times and variable sensitivity according to carbapenemase type and microorganism. Colorimetric tests, such as the Rapidec Carba NP assay (bioMérieux, Nürtingen, Germany) or the ␤-Carba assay (Bio-Rad, Marnes-la-Coquette, France), have demonstrated variable sensitivity depending on ␤-lactamase activity and culture medium used (3,4). Hydrolysis of carbapenems can also be detected by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (5), but this method is only rarely used in routine microbiology.…”

“…Novel immunochromatographic tests (ICTs), such as the Resist-4 O.K.N.V. assay (Coris BioConcept, Gembloux, Belgium) and the Carba-5 assay (NG Biotech, Guipry, France), have been shown to be rapid and cost-effective methods with high sensitivity and specificity (4,(6)(7)(8). However, PCR-based assays, although able to detect only targeted genes, currently remain the gold standard for carbapenemase detection.…”