Evaluation of a Loop-Mediated Isothermal Amplification Assay to Detect Carbapenemases Directly From Bronchoalveolar Lavage Fluid Spiked With Acinetobacter spp

Abstract

Carbapenem-resistant Acinetobacter spp. mainly Acinetobacter baumannii are frequently causing nosocomial infections with high mortality. In this study, the efficacy of the Eazyplex^® SuperBug Complete A system, based on loop-mediated isothermal amplification (LAMP), to detect the presence of carbapenemases in Acinetobacter spp. directly from bronchoalveolar lavage (BAL) samples was assessed. A total of 22 Acinetobacter spp. strains producing OXA-23, OXA-40, OXA-58, NDM, and IMP were selected. Eazyplex SuperBug Complete A kit, used with the Genie II device, is a molecular diagnostics kit that detects a selection of genes that express carbapenemases (bla _KPC , bla _NDM , bla _VIM , bla _OXA-48 , bla _OXA-23 , bla _OXA-40 , and bla _OXA-58 ). Negative BAL samples were identified, McFarland solutions were prepared from each of the 22 Acinetobacter strains and serial dilutions in saline solution were made to finally spike BAL samples to a concentration of 10² and 10³ CFU/ml. Fifteen concentrations out of the 44 tested out did not provide detection of the carbapenemase-producing gene, all but one being at the lowest concentration tested at 10² CFU/ml; therefore, the limit of sensitivity is 10³ CFU/ml. This assay represents the kind of advantages that investing in molecular diagnostics brings to the clinical practice, allowing the identification of carbapenemases in less than 30 min with a sensitivity of 10³ CFU/ml.

Keywords: Acinetobacter spp.; bronchoalveolar lavage; carbapenemases; detection; oxacillinases.