A Lateral Flow Immunoassay for the Rapid Identification of CTX-M-Producing Enterobacterales from Culture Plates and Positive Blood Cultures.

Sandrine Bernabeu,Kayaththiry Caroline Ratnam,Hervé Boutal,Hervé Volland,Nicolas Fortineau,Anaïs Vogel,Surbhi Malhotra-Kumar,Saoussen Oueslati,Stéphanie Simon,Camille Gonzalez,Marc Plaisance,Laurent Dortet,Karine Devilliers,Thierry Naas

doi:10.3390/diagnostics10100764

Abstract

We have developed a lateral flow immunoassay (LFIA), named NG-Test CTX-M MULTI (NG-Test), to detect group 1, 2, 8, 9, 25 CTX-M producers from agar plates and from positive blood cultures in less than 15 min. The NG-Test was validated retrospectively on 113 well-characterized enterobacterial isolates, prospectively on 102 consecutively isolated ESBL-producers from the Bicêtre hospital and on 100 consecutive blood cultures positive with a gram-negative bacilli (GNB). The NG-Test was able to detect all CTX-M producers grown on the different agar plates used in clinical microbiology laboratories. No false positive nor negative results were observed. Among the 102 consecutive ESBL isolates, three hyper mucous isolates showed an incorrect migration leading to invalid results (no control band). Using an adapted protocol, the results could be validated. The NG-Test detected 99/102 ESBLs as being CTX-Ms. Three SHV producers were not detected. Among the 100 positive blood cultures with GNB tested 10/11 ESBL-producers were detected (8 CTX-M-15, 2 CTX-M-27). One SHV-2-producing-E. cloacae was missed. The NG-Test CTX-M MULTI showed 100% sensitivity and specificity with isolates cultured on agar plates and was able to detect 98% of the ESBL-producers identified in our clinical setting either from colonies or from positive blood cultures.

Highlights

The production of β-lactamases is a commonly encountered mechanism of resistance in Gram-negative bacteria, especially Enterobacterales [1]
The aim of the study was to develop a rapid lateral flow immunoassay (LFIA), named NG-Test CTX-M MULTI, for the detection of the CTX-Ms belonging to the five groups directly from colonies isolated on different culture media widely used in routine laboratory and from positive blood cultures
20 monoclonal antibodies were tested, and the pairs of antibodies showing the best limit of detection (LOD) for CTX-M-expressing bacteria were selected for the NG-Test CTX-M MULTI assay as previously described [24,25]

Summary

Introduction

The production of β-lactamases is a commonly encountered mechanism of resistance in Gram-negative bacteria, especially Enterobacterales [1]. In healthcare settings, extended-spectrum-β-lactamase (ESBL)-producing Enterobacterales (ESBL-E) are of particular concern. The CDC has estimated that ESBL-Es account for 19% of healthcare-related infections annually and that infections involving by these bacteria are associated with increased mortality and cost of care [2]. Within the ESBLs, the CTX-M family appeared in the early 80 s and represent the most prevalent ESBLs worldwide. They are divided into five groups based on amino acid sequence identity: the CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25 groups. In each group are included minor allelic variants differing one from the other by one or few amino-acid substitutions [3,4,5]

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