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f We compared the diagnostic accuracy of the Carba NP test with that of a straightforward matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) method for detecting carbapenemase-producing Enterobacteriaceae (CPE). Using PCR as the reference method, both tests demonstrated a sensitivity of 87% and a specificity of 100%. MALDI-TOF MS offers a potential alternative for the rapid detection of CPE in the clinical laboratory setting. The global spread of carbapenemase-producing Enterobacteriaceae (CPE) is a major threat to public health (1). The efficient laboratory detection of these agents requires a rapid phenotypic test that provides accurate results while awaiting confirmation by PCR. In this study, a method was developed for detecting CPE using an imipenem hydrolysis assay and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), using the Vitek MS research use only (RUO) system (bioMérieux, Marcy l'Étoile, France). Using PCR as a reference method, the diagnostic accuracy of MALDI-TOF MS was compared with that of the Carba NP test (CNP).The investigation was conducted at Melbourne Pathology and Swinburne University of Technology, Melbourne, Australia, between October 2013 and April 2014. A panel of 105 genetically characterized Enterobacteriaceae was assembled from nine Australian laboratories. All strains were identified by MALDI-TOF MS (Vitek MS [bioMérieux]) using the Vitek MS Database version 2.0 (bioMérieux) and had MICs for meropenem determined by Etest (bioMérieux). Molecular characterization had been performed by various methods (2-11); at a minimum, each strain had been subjected to PCR, including targets for bla KPC , bla NDM , bla IMP , bla VIM , and bla OXA-48 (2-11). The entire collection was assembled prior to the study, and all of the strains obtained were included.The CNP was performed according to a previously published method (12) on strains cultured on Columbia blood agar (CBA) for 18 to 24 h. The results were read by a microbiologist who was blinded to all other results in the study.The MALDI-TOF MS method was adapted from a previous