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 (blaKPC, blaNDM, blaVIM, blaOXA–48, blaOXA–23, blaOXA–40, and blaOXA–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.

Development of Loop-Mediated Isothermal Amplification Rapid Diagnostic Assays for the Detection of Klebsiella pneumoniae and Carbapenemase Genes in Clinical Samples

“…Our assays are also comparable to the commercialized CE-IVD LAMP Eazyplex SuperBug kits (complete A, complete B, and CRE; Amplex-Diagnostics GmbH, Germany), designed to detect carbapenemase-producing genes and selected ESBL genes in up to 30 min using the portable Genie II device ( Vergara et al, 2014 ; Findlay et al, 2015 ; Hinic et al, 2015 ; García-Fernández et al, 2016 ; Fiori et al, 2019 ; Moreno-Morales et al, 2020 ; Sekowska et al, 2020 ; Vergara et al, 2020 ; Holma et al, 2021 ). Sensitivity and specificity of the Eazyplex SuperBug kits, assessed on clinical isolates of Enterobacteriaceae ( Vergara et al, 2014 ; Findlay et al, 2015 ), were greater than 95%.…”

“…These kits were also tested on bronchoalveolar lavage fluid samples spiked with Enterobacteriaceae and carbapenem-resistant Acinetobacter spp. ( Moreno-Morales et al, 2020 ; Vergara et al, 2020 ), clinical urine samples ( Hinic et al, 2015 ), and rectal swab samples ( Holma et al, 2021 ), with 80%–100% sensitivity, a specificity greater than 95%, and a limit of detection between 10 2 and 10 3 CFUs/ml ( Hinic et al, 2015 ; Moreno-Morales et al, 2020 ; Vergara et al, 2020 ; Holma et al, 2021 ). Other CE-IVD marked portable molecular diagnostics for carbapenemases, which are cartridge-based PCR assays and require minimal sample preparation, such as Xpert Carba-R ( Tenover et al, 2013 ; Anandan et al, 2015 ; Findlay et al, 2015 ; Vergara et al, 2020 ) and Novodiag CarbaR + combining real-time PCR and microarray technologies ( Girlich et al, 2019 ; Holma et al, 2021 ), have been recently commercialized.…”

Development of a novel loop-mediated isothermal amplification assay for ß-lactamase gene identification using clinical isolates of Gram-negative bacteria

“…We developed LAMP assays for four b-lactamase genes (bla KPC , bla NDM-1 , bla IMP-1 group, and bla VIM ) and evaluated each using clinical strains isolated at diverse geographical locations (Kos et al, 2015). There are several previous reports mentioning the LAMP assay for the four b-lactamase genes identification (Moreno-Morales et al, 2020;Feng et al, 2021). While these reports used isolates from a limited number of regions, this is the first report to use clinical strains isolated at diverse geographical locations.…”