Evaluation of an Immunochromatographic Lateral Flow Assay (OXA-48 K -SeT) for Rapid Detection of OXA-48-Like Carbapenemases in Enterobacteriaceae

Abstract

We evaluated an immunochromatographic lateral flow assay to detect OXA-48-like carbapenemases (OXA-48 K-SeT) in Enterobacteriaceae (n ‫؍‬ 82). One hundred percent sensitivity and specificity were observed using bacteria recovered from both solid medium and spiked blood culture bottles, and the results were obtained in <10 min.T he identification, treatment, and control of multidrug-resistant (MDR) bacterial infections are global health priorities. Enterobacteriaceae with plasmids carrying genes encoding class A (Klebsiella pneumoniae carbapenemases [KPC]), B (IMP, VIM, and NDM), and D (OXA) carbapenemases (carbapenemase-producing Enterobacteriaceae [CPE]) are one of the most important groups of pathogens due to the burden of disease, lack of any new treatments, and potential for dissemination (1). Rapid and effective diagnostics therefore underpin any strategy aimed at tackling the problem of CPE. Considerable effort has been made to develop novel assays using both genotypic and phenotypic approaches. These approaches include the genetic detection of resistance gene profiles (PCR, loop-mediated isothermal amplification [LAMP], microarrays, and genome sequencing) (2), selective culture medium (chromogenic or supplemented) (3), combination disk testing (4), and direct or indirect detection of carbapenem-hydrolyzing enzymes (matrix-assisted laser desorption ionization-time of flight mass spectrometry [MALDI-TOF MS], acidometric Carba NP, and Blue-Carba) (5-7). These methods require varied levels of technical skill, investment into equipment, and quality assurance optimization. Strains producing OXA-48-like carbapenemhydrolyzing class D enzymes (CHDL) have proven to be particularly difficult to detect in clinical laboratories. This is due in part to relatively low MICs, conflicting interpretive rules associated with automated systems (8), and a lack of suitable inhibitor compounds for use in confirmatory tests. A novel means of detecting OXA-48-like enzymes using an antibody-mediated approach was recently developed (9). The OXA-48 K-SeT assay relies on the immunological capture of two epitopes specific to the OXA-48 enzyme using colloidal gold nanoparticles bound to a nitrocellulose membrane within a lateral flow device. Capture and detection antibodies were designed to bind all current CHDL OXA-48-like variants .In this study, we assessed the performance of the Coris OXA-48 K-SeT assay for detecting OXA-48-like-mediated carbapenem resistance in a large collection of carbapenem-resistant Enterobacteriaceae and also determined whether it could provide robust results when working directly with organisms recovered from blood culture bottles.Eighty-two enterobacterial isolates were used in the evaluation. Seventy-eight were clinical isolates (K. pneumoniae, n ϭ 60; Escherichia coli, n ϭ 11; Enterobacter cloacae, n ϭ 6; Enterobacter aerogenes, n ϭ 1) with resistance to one or more carbapenems (ertapenem, imipenem, and/or meropenem) along with a susceptible type strain as a representative control for each bacterial specie...

Rapid Identification of OXA-48 and OXA-163 Subfamilies in Carbapenem-Resistant Gram-Negative Bacilli with a Novel Immunochromatographic Lateral Flow Assay

“…Independent tests were performed in triplicate, and the LoD was calculated as recommended (24). Using viable colony counts, the LoDs of the assay were 9.2 ϫ 10 6 (OXA-163), 4.6 ϫ 10 7 (OXA-247), and 2.1 ϫ 10 7 CFU/ml (OXA-438), while for OXA-48, the LoD was 1.9 ϫ 10 6 CFU/ml, which was similar to that of a previous report (2.41 ϫ 10 6 CFU/ml [7]). Purified proteins were detected at up to 0.4 (OXA-48), 0.49 (OXA-163), and 0.625 ng/ml (OXA-247 and OXA-438), similar to the LoD indicated by the manufacturer for another K-SeT version (OXA-48 K-SeT; 0.125 ng/ml).…”

“…The OXA-163/48 Duo K-SeT test differentiates between subfamilies with distinct substrate profiles, such as OXA-48 and OXA-163, obviating the need for more costly and lengthy characterization with molecular amplification methods. The assay was highly sensitive and specific and detected the presence of OXA-48-producing strains within seconds to minutes, similar to results previously reported for the OXA-48 subfamily and KPC carbapenemases (7)(8)(9)(10)25). No significant difference in band intensities was observed between the different subfamilies and/or variants tested or between the bacterial species, in carbapenem MICs, or in associations of the OXA protein to other ␤-lactamases (data not shown).…”

“…Bottles were incubated until the blood culture was flagged positive by the system (from 6 to 11 h). Aliquots of a 0.1-ml volume were harvested in 10 drops of LY-A buffer, and subsequently, 3 drops of the mix were applied to the sample well (7). We confirmed that the final inoculum of positive cultures was higher than the LoD by performing serial-diluted subcultures.…”

“…The OXA-48 K-SeT test (Coris BioConcept, Belgium) relies on immunological capture of two epitopes specific to the OXA-48 variants OXA-48, OXA-181, OXA-204, OXA-232, and OXA-244 using colloidal gold nanoparticles bound to a nitrocellulose membrane within a lateral flow device (6). The reported sensitivity and specificity were both 100%, with the result obtained in less than 10 min (6)(7)(8)(9)(10). Noteworthy is that new allelic variants of OXA-48 have emerged, namely, OXA-163 and the related variants OXA-247, OXA-405, and OXA-438 (11)(12)(13)(14), which are not recognized by the OXA-48 K-SeT test (6,8,10).…”

A highly multiplexed melt-curve assay for detecting the most prevalent carbapenemase, ESBL and AmpC genes

“…These steps usually take 48-96 hours with antibiotic prescription initiated empirically, resulting in the unnecessary use of last resort antibiotics and an increased risk of drug resistance 20 . Recently rapid diagnostic tests have been developed for testing bacterial colonies, utilising antibody mediated capture of carbapenem or ESBL enzymes and providing a visual read out within 15 minutes 21 . Rapid phenotypic tests based on the colormetric detection of carbapenem hydrolysis are also commercially available, notably the Rapidec Carba NP 22 , which is sensitive for the detection of most carbapenemases, but insensitive for detecting OXA-type enzymes 23 .…”