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ABSTRACTAs carbapenemase-producing Gram-negative bacilli (CP-GNB) (Enterobacteriaceae, Pseudomonadaceae, and Acinetobacterspp.) are becoming a major public health issue, there is an urgent need for accurate and fast diagnostic tests. The Amplidiag CarbaR+VRE assay is a multiplex nucleic acid-based in vitrodiagnostic test intended for the detection of CP-GNB and vancomycin-resistant enterococci (VRE) from cultured colonies. We have evaluated its ability to detect carbapenemase genes in 100 well-characterized GNB and in 200 consecutive enterobacterial isolates with reduced susceptibility to carbapenems that were referred to the French National Reference Center for carbapenem resistance. The assay has been validated on purified DNA but also directly on colonies. The Amplidiag CarbaR+VRE assay could detect all KPC, NDM, VIM, IMP, and OXA-48-like variants tested and all acquired carbapenem-hydrolyzing oxacillinases from Acinetobacter baumannii(OXA-23, OXA-24/-40, and OXA-58) as well as the overexpressed chromosomally encoded OXA-51-like ß-lactamase associated with an upstream inserted ISAba1. However, as claimed by the manufacturer, other carbapenemases such as GES-like carbapenemases (GES-2, GES-5, and GES-14), GIM-1, AIM-1, SPM-1, DIM-1, OXA-198 in Pseudomonas aeruginosa, or OXA-143-like in A. baumanniiwere not detected. Amplidiag CarbaR+VRE's performance values were high (100% sensitivity and 99% specificity) as it could detect the five major carbapenemases—NDM, VIM, IMP, KPC, and OXA-48—as well as OXA-type carbapenemases from Acinetobacterspp. that are currently emerging also among Proteus mirabilisand other enterobacterial isolates. It can provide a result directly from colonies growing on Mueller-Hinton (MH) agar or on selective screening medium in less than 2 h. Further evaluations are now necessary to determine the performance values directly on rectal swabs.