Development of simplex and multiplex immunochromatographic assays for rapid detection of GES-, PER-, and VEB-producing gram-negative bacteria.

Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae are increasingly reported worldwide, emphasizing the need for rapid diagnostic tools to limit their spread and prevent outbreaks. Although CTX-M enzymes remain predominant, less common ESBLs such as GES, PER, and VEB are emerging and gaining clinical relevance. We developed simplex and multiplex lateral flow immunoassays (LFIAs) for the rapid detection of these ESBLs. Monoclonal antibodies were produced and incorporated into dedicated LFIA strips. The assays were evaluated using 116 WGS-characterized gram-negative bacteria (GNB) isolates expressing diverse β-lactamases and grown on various media. For testing, three colonies were suspended in 150 µL of extraction buffer, applied to the strips, and results were visually interpreted after 15 min. Both simplex and multiplex LFIAs achieved 100% sensitivity and specificity for detecting GES-, PER-, and VEB-producing isolates, irrespective of bacterial species, β-lactamase content, or growth conditions. The panel included prevalent variants of PER (n = 5, including PER-2), VEB (n = 9), and GES (n = 9, including ESBLs and carbapenemases). Detection limits for recombinant PER-1, VEB-1, and GES-1 were 5, 50, and 100 pg/mL, respectively, and 105, 106, and 106 CFU/mL for corresponding Acinetobacter baumannii producers grown in tryptic soy broth. Overall, these simplex and multiplex GES-, PER-, and VEB-targeting LFIAs provide a rapid, accurate, and user-friendly approach suitable for routine use in clinical microbiology laboratories. When combined with the NG-Test CTX-M MULTI assay, which detects the five major CTX-M groups, they may further enhance the overall detection of the most common ESBLs, particularly among non-fermenting GNB.IMPORTANCEAlthough less prevalent globally than CTX-M-type ESBLs, GES-, PER-, and VEB-type β-lactamases represent an important and often underrecognized mechanism of resistance, particularly in non-fermenting gram-negative pathogens. These enzymes are frequently associated with multidrug-resistant organisms and may significantly compromise available therapeutic options. The detection of the genes encoding these enzymes is often based on homemade PCR. Rapid diagnostic tests based on lateral flow immunoassays (LFIAs) have been successfully used to detect CTX-M-like ESBLs and the five main carbapenemases. Here, we demonstrated that GES, PER, and VEB enzymes can be readily detected by simplex or multiplex LFIAs from Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii colonies. These assays are rapid (≤15 min), highly sensitive (105-106 CFU/mL), requiring no specialized equipment, thus offering a user-friendly alternative to molecular assays in clinical laboratories. These features align well with the World Health Organization ASSURED criteria (Affordable, Sensitive, Specific, User-friendly, Rapid, Equipment-free, and Deliverable).