Evaluation of the Revogene Carba C Assay for Detection and Differentiation of Carbapenemase-Producing Gram-Negative Bacteria

The Revogene Carba C assay (formerly GenePOC Carba assay) is a multiplex nucleic acid-based in vitro diagnostic test intended for the detection of carbapenemase-producing Enterobacterales (CPE) from cultured colonies. This assay was evaluated directly on colonies of 118 well-characterized Enterobacterales with reduced susceptibility to carbapenems and on 49 multidrug-resistant (MDR) Pseudomonas aeruginosa and 40 MDR Acinetobacter baumannii isolates.

As CP organisms (CPOs) are a major health issue, rapid confirmation of carbapenemase production is essential not only for effective therapy but also for the prompt implementation of infection control measures able to prevent their dissemination (10). Screening protocols are based mainly on cultures of rectal swab specimens on selective media (11,12), followed by phenotypic tests to confirm a carbapenem-hydrolyzing activity, such as the Carba NP test (13), the rapid carbapenem inactivation method (rCIM) (14), lateral flow immunoassays (15,16), imipenem hydrolysis detected by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) (17), the BYG test (18), and the ␤-Carba test (19) or disk diffusion synergy test (DDST) for detection of MBLs and KPCs (e.g., meropenem disks alone and meropenem disks supplemented with aminophenylboronic acid, dipicolinic acid, or cloxacillin) (20). High-level temocillin resistance is a useful phenotypic trait for OXA-48 detection (21). Culture of rectal swab specimens followed by confirmation testing is a long process and often is not compatible with rapid implementation of reinforced hygiene measures (22).
Molecularly based techniques such as PCR and whole-genome sequencing remain the gold standard for the precise identification of carbapenemase genes (22,23). Molecular methods are now available for detecting carbapenemase genes from bacterial cultures but also directly from clinical specimens in less than an hour (22). Several assays are commercially available. Some require DNA extraction from rectal swab specimens prior to amplification, such as the PCR-based Amplidiag CARBAϩMCR assay (Mobidiag, Paris, France) (24), the Amplidiag CARBAϩVRE assay (Mobidiag, Paris, France) (25), or the Check-Direct CPE assay (Check-Points, Wageningen, The Netherlands) (26), while others do not require DNA extraction, such as the Eazyyplex SuperBug CRE (Amplex Biosystems GmbH, Gießen, Germany) (27). Others are fully automated, such as CRE ELITe MGB kits on the InGenius platform (Elitech, Les Ulis, France) (28), the BD MAX Checkpoint CPO assay (29), and the GeneXpert Carba-R assay (Cepheid, Sunnyvale, CA, USA) (30,31). Most of these assays target the "big 5" carbapenemases, i.e., KPC, OXA-48-like, NDM, VIM, and IMP, which represent more than 99% of the carbapenemases produced by Enterobacterales in countries such as France (32). Very recently, the biological performance of the Revogene Carba C assay (Meridian Bioscience, Cincinnati, OH, USA) (formerly GenePOC Carba assay [GenePOC, Québec, Canada]) has been compared to that of the Xpert Carba-R assay for the detection of the bla KPC , bla NDM , bla VIM , bla OXA-48-like , and bla IMP genes from pure colonies of Enterobacterales (33). The four most common carbapenemases (NDM, KPC, OXA-48like, and VIM) were detected with 100% sensitivity by both tests, but for IMPs, the Revogene Carba C assay showed 100% sensitivity, while that of the Xpert Carba-R assay was only 44.4% (33).

MATERIALS AND METHODS
Revogene Carba C assay. The Revogene Carba C assay was used as recommended by the manufacturer on fresh overnight bacterial colonies. Bacteria to be tested were resuspended in physiological water until a 0.5 McFarland suspension was reached. Fifteen microliters of the bacterial suspension was added to the sample buffer tube. After subsequent mixing by vortexing for 15 s, 200 l of sample buffer containing the bacteria was transferred into the sample loading chamber of the microfluidic cartridge. As recommended in the manufacturer's instructions, 8 cartridges were used for every run in the Revogene instrument, using blank cartridges when fewer than 8 samples were processed.

RESULTS AND DISCUSSION
The Revogene Carba C assay was tested on a collection of 118 well-characterized Enterobacterales with reduced susceptibility to carbapenems and on 49 P. aeruginosa and 40 Acinetobacter sp. isolates resistant to carbapenems and expressing various ␤-lactamases (Tables 1 to 3).
During this evaluation, the Revogene Carba C assay was used as recommended by the manufacturer, on fresh overnight bacterial colonies. The procedure for sample preparation was easy to perform, taking ca. 15 Table 1) (38). The chromosomally encoded OXA-535 identified in a Shewanella species (39), which has only 91.3% amino acid identity with OXA-48, has not been detected (data not shown). OXA-535, although not present in Enterobacterales, was used because it is the progenitor of OXA-436, a distantly related OXA-48 variant responsible for an outbreak associated with several enterobacterial species in Denmark (39,40). It is thus likely that OXA-436 would not be detected by the Revogene Carba C assay, and further studies on the Revogene Carba C assay should be carried out to test OXA-436 producers. Moreover, OXA-163 and OXA-405, two OXA-48 variants that lack significant imipenemase activity (34,41) but that show strong expanded-spectrum hydrolytic activity, were consistently not detected by the Revogene Carba C assay. It is still debated whether these enzymes are or are not carbapenemases (35,42). For example, OXA-163 is considered a carbapenemase by the CLSI, although from an enzymatic point of view with respect to imipenem, these variants are not carbapenemases (43); however, their nucleotide sequences are too similar to that of the bla OXA-48 gene to be distinguished by most molecular detection assays (24,28,30,33). The Revogene Carba C assay has this particular advantage of distinguishing these bla OXA-48 variants. Indeed, OXA-163 producers hydrolyze extended-spectrum cephalosporins and are most prevalent in places such as South America, but they need to be individualized as such and not be considered genuine CPE. All the tested IMP variants were correctly identified with Revogene Carba C assay, even those that are not detected by Xpert Carba (Cepheid), such as IMP-2, IMP-8, IMP-11, and IMP-14, or by NG-test Carba5, an immunochromatographic assay, which misses IMP-14 (30,33,44). In multiple-carbapenemase producers, all resistance determinants were correctly identified. None of the non-carbapenemase producers or nontargeted-carbapenemase producers yielded positive PCR results.
Conclusion. The Revogene Carba C assay showed excellent sensitivity and specificity for the five most common carbapenemases regardless of the host bacteria, including IMP variants that constitute a very heterogeneous family of enzymes and that are not well detected by most molecular or immunochromatographic assays (46). Our study demonstrated that the Revogene Carba C assay is well adapted to the French epidemiology of CPE and CP-Pa, which reflects the epidemiology in many European countries. Its simplicity and short turnaround time make it suitable for use in the routine microbiology laboratory. It can provide results from colonies that grow on Mueller-Hinton (MH) agar but also on those from selective screening media.
The assay detects the main carbapenemases encountered in Enterobacterales (KPC, NDM, VIM, IMP, and OXA-48-like) and in P. aeruginosa (VIM, IMP, NDM, and KPC) but only the minor ones in A. baumannii (VIM, IMP, and NDM), as the most widespread OXA carbapenemases usually identified in A. baumannii (OXA-23-like, OXA-24/-40-like, and OXA-58-like) are not targeted by this assay. When extrapolated to the global French epidemiology of CPOs, we would expect (at best) 99.28% sensitivity for CPE detection and 93.33% for CP-Pa but only 12.5% for CP-Ab. Thus, Younden's index for the French situation would be 99.27, 93.3, and 10 for CPE, CP-Pa, and CP-Ab, respectively.
As for most molecular assays, mutation and/or polymorphisms in the primer/probe binding region of the targeted gene may lead to false-negative results, and thus some variants may not be detected. It is of outmost importance that a molecular test be evaluated on the local epidemiology in order to know which are the alleles that might be missed. Regular evaluation of novel variants is required to assess the sensitivity and specificity of this assay in a constantly evolving carbapenemase field. The main drawback of this assay is that it so far has been validated only on pure cultures. Evaluation directly on rectal swabs is a mandatory next step for the assay to be fully useful for carbapenemase gene detection. a GES-carbas is a GES ␤-lactamase with carbapenemase activity.