Development and evaluation of a novel rapid immunochromatographic assay for the detection of DHA-producing Enterobacterales.

Background: The increasing rates of infections caused by broad-spectrum β-lactamase-producing Enterobacterales is a major public health concern. DHA-type cephalosporinases are a rising cause of expanded-spectrum cephalosporin (ESC) resistance in Enterobacterales. Here, we have developed and validated lateral flow immunoassays (LFIAs) for the detection of the DHA-type cephalosporinases.

Materials And Methods: LFIA compatible monoclonal antibodies (mAbs) specific for the DHA-2 were produced. The DHA-LFIA strip, consisting of a test line (TL) that detects DHA-type cephalosporinases and a control line capturing all excess mAbs, was retrospectively evaluated on 111 well-characterized Gram-negative isolates grown on different culture media and expressing various β-lactamases (BL). One colony was resuspended in 150 µL of extraction buffer and subsequently loaded on the DHA-LFIA strip. Results were read by eye after 15 minutes of migration.

Results And Discussions: Irrespective of the bacterial species or the β-lactamase gene content, all isolates expressing a DHA-type β-lactamase were detected with a sensitivity and specificity of 100%. In addition, intensities of DHA-TL were stronger with plasmid-encoded or hyper-producing DHA Morganella spp. isolates compared to WT Morganella spp. isolates. DHA-TL intensities were correlated with the MIC of cefotaxime and specific activities using cephalothin as substrate.

Conclusions: The DHA-LFIA allows reliable and rapid detection of all tested DHA variants in <15 minutes irrespective of the chromosomal or plasmid location. It is affordable and easy to implement in the routine laboratory workflow for the confirmation of DHA-type-BL. Multiplexing it with CMY-type and cefotaxime-M ESBLs would improve the overall sensitivity for the detection of ESC resistance.