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

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.

Occurrence and Diversity of CTX-M-Producing Escherichia coli From the Seine River

“…WGS identified bla CTX–M– 1 ( n = 5), bla CTX–M– 15 ( n = 7), bla CTX–M– 14 ( n = 1), bla CTX–M– 27 ( n = 2), bla TEM– 52 ( n = 1) ESBL genes, bla DHA– 1 ( n = 1) cephalosporinase gene, and bla TEM– 1 ( n = 3) and bla OXA– 1 ( n = 2) penicillinase genes ( Table 1 ). The results of the WGS were in accordance with those of the NG-Test CTX-M- MULTI LFIA, validating this latest test for the rapid detection of the five groups of CTX-M-producing Enterobacterales, as previously reported ( Bernabeu et al, 2020 ). As observed in other studies, group 1 CTX-M-producing E. coli isolates were dominant in our study ( Zarfel et al, 2017 ; Hooban et al, 2020 ).…”

“…NG-Test CTXM-Multi, a rapid Lateral Flow Immuno Assay (LFIA, NG-Biotech, Guipry, France) was used to detect all five CTX-M-groups, as previously described ( Bernabeu et al, 2020 ). Briefly, one colony was resuspended in the extraction buffer, vortexed, and 100 μl was dropped on the LFIA strip.…”

Rapid Detection of VanA/B-Producing Vancomycin-Resistant enterococci using Lateral Flow Immunoassay from colonies and blood culture

“…Currently, there are eight different acquired vancomycin resistance operons described: vanA, vanB, vanD, vanE, vanG, vanL, vanM and vanN (4-6). In Europe, the two Merk, Darmstadt, Germany) (12)(13)(14). Recombinant plasmids pET22b+ vanB and pET22b-plyV12 (12,15), which encodes a broadly active phage lytic enzyme with lethal activity against E. faecalis and E. faecium were transformed into Escherichia coli BL 21 (DE3).…”

“…Recombinant plasmids pET22b+ vanB and pET22b-plyV12 (12,15), which encodes a broadly active phage lytic enzyme with lethal activity against E. faecalis and E. faecium were transformed into Escherichia coli BL 21 (DE3). Upon induction, the recombinant VanB and plyV12 proteins were purified using Ni-NTA agarose affinity resin as previously described (12)(13)(14). The VanB recombinant protein was then used to immunize mice and as a standard for the selection of monoclonal antibody (mAb) pairs as previously described (12)(13)(14).…”

“…Upon induction, the recombinant VanB and plyV12 proteins were purified using Ni-NTA agarose affinity resin as previously described (12)(13)(14). The VanB recombinant protein was then used to immunize mice and as a standard for the selection of monoclonal antibody (mAb) pairs as previously described (12)(13)(14). All animal experiments were performed in compliance with…”