Evaluation of carbapenemase screening and confirmation tests in Enterobacteriaceae and development of a practical diagnostic algorithm

RELIABLE IDENTIFICATION OF CARBAPENEMASE PRODUCING ENTEROBACTERIACEAE IS NECESSARY TO LIMIT THEIR SPREAD: This study aimed at developing a diagnostic flow-chart suitable for implementation in different types of clinical laboratories using phenotypic screening and confirmation tests. In total, 334 clinical Enterobacteriaceae isolates genetically characterized with respect to carbapenemase, extended-spectrum-beta-lactamase (ESBL), and AmpC genes were analyzed. 142/334 isolates (42.2%) were suspicious for carbapenemase production, i.e. intermediate or resistant to ertapenem AND/OR meropenem AND/OR imipenem according to EUCAST clinical breakpoints (CBPs). A group of 193/334 isolates (57.8%) showing susceptibility to ertapenem AND meropenem AND imipenem was considered as negative control group for this study. CLSI and EUCAST carbapenem CBPs and the new EUCAST MEM screening cut-off were evaluated as screening parameters. ETP, MEM and IPM +/- aminophenylboronic acid (APBA) or EDTA combined-disc tests (CDTs), and the Carba NP-II test were evaluated as confirmation assays. EUCAST temocillin cut-offs were evaluated for OXA-48 detection. The EUCAST MEM screening cut-off (< 25 mm) showed a sensitivity of 100%. The ETP APBA-CDT on Muller-Hinton agar containing cloxacillin (MH-CLX) displayed 100% sensitivity and specificity for class A carbapenemase confirmation. ETP and MEM EDTA-CDTs showed 100% sensitivity and specificity for class B carbapenemases. Temocillin diameters/MIC testing on MH-CLX was highly specific for OXA-48 producers. The overall sensitivity, specificity, PPV, and NPV of the Carba NP-II test were 78.9%, 100%, 100%, and 98.7%, respectively. Combining the EUCAST MEM carbapenemase-screening cut-off (< 25 mm), ETP (or MEM) APBA- and EDTA-CDTs, and temocillin disk diffusion on MH-CLX agar promises excellent performance for carbapenemase detection