Evaluation of the BYG Carba Test, a New Electrochemical Assay for Rapid Laboratory Detection of Carbapenemase-Producing Enterobacteriaceae

Abstract

Accurate detection of carbapenemase-producing Enterobacteriaceae (CPE) constitutes a major laboratory diagnostic challenge. We evaluated an electrochemical technique (the BYG Carba test) which allows detection of CPE in less than 35 min. The BYG Carba test was first validated in triplicate against 57 collection isolates with previously characterized β-lactam resistance mechanisms (OXA-48, n = 12; KPC, n = 8; NDM, n = 8; VIM, n = 8; IMP, n = 3; GIM, n = 1; GES-6, n = 1; no carbapenemase, n = 16) and against a panel of 10 isolates obtained from the United Kingdom National External Quality Assessment Service (NEQAS). The test was then evaluated prospectively against 324 isolates referred to the national reference center for suspicion of CPE. The BYG Carba test results were compared with those obtained with the Carba NP test using multiplex PCR sequencing as the gold standard. Of the 57 collection and the 10 NEQAS isolates, all but one GES-6-producing isolate were correctly identified by the Carba BYG test. Among the 324 consecutive Enterobacteriaceae isolates tested prospectively, 146 were confirmed as noncarbapenemase producers by PCR while 178 harbored a carbapenemase gene (OXA-48, n = 117; KPC, n = 25; NDM, n = 23; and VIM, n = 13). Prospectively, in comparison with PCR results, the BYG Carba test displayed 95% sensitivity and 100% specificity versus 89% and 100%, respectively, for the Carba NP test. The BYG Carba test is a novel, rapid, and efficient assay based on an electro-active polymer biosensing technology discriminating between CPE and non-CPE. The precise electrochemical signal (electrochemical impedance variations) allows the establishment of real-time objective measurement and interpretation criteria which should facilitate the accreditation process of this technology.

FIG 1

Homemade potentiostat and eight-probe disposable electrode. Four isolates were analyzed in parallel (numbers 1 to 3 and one control [Ctrl]). Fifty-microliter drops of bacterial suspension in lysis buffer with (+) or without (−) imipenem were loaded on fingers corresponding to the isolates to be analyzed. Probe a was loaded with a drop of strain 3 suspended in buffer without imipenem; probe b was loaded with a drop of strain 3 suspended in buffer with imipenem; probe c was comprised of one working, one reference, and one counter electrode. The software subtracts the data obtained without imipenem from the data obtained with imipenem and generates a resulting real-time curve imaging the conductance of the polyaniline.

FIG 2

Real-time curve obtained with the BYG Carba test. A report of the results automatically generated by the software is shown. The gray horizontal line represents the cutoff line. The colored lines represent the real-time curve generated during the analysis. The orange (K. pneumoniae OXA-48 NEQAS 1943), blue (K. pneumoniae VIM-1 NEQAS 1945), and black (K. pneumoniae OXA-48 control strain CNR20150325) curves correspond to positive strains, and the pink flat curve corresponds to a negative Enterobacter aerogenes strain (CNR 20150311). The y axis is linked to the conductance of polyaniline, and values are expressed in arbitrary units.