A multiplex lateral flow immunoassay for the rapid identification of NDM-, KPC-, IMP- and VIM-type and OXA-48-like carbapenemase-producing Enterobacteriaceae

Abstract

Objectives: The global spread of carbapenemase-producing Enterobacteriaceae represents a substantial challenge in clinical practice and rapid and reliable detection of these organisms is essential. The aim of this study was to develop and validate a lateral flow immunoassay (Carba5) for the detection of the five main carbapenemases (KPC-, NDM-, VIM- and IMP-type and OXA-48-like).

Methods: Carba5 was retrospectively and prospectively evaluated using 296 enterobacterial isolates from agar culture. An isolated colony was suspended in extraction buffer and then loaded on the manufactured Carba5.

Results: All 185 isolates expressing a carbapenemase related to one of the Carba5 targets were correctly and unambiguously detected in <15 min. All other isolates gave negative results except those producing OXA-163 and OXA-405, which are considered low-activity carbapenemases. No cross-reaction was observed with non-targeted carbapenemases, ESBLs, AmpCs or oxacillinases (OXA-1, -2, -9 and -10). Overall, this assay reached 100% sensitivity and 95.3% (retrospectively) to 100% (prospectively) specificity.

Conclusions: Carba5 is efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for confirmation of the five main carbapenemases encountered in Enterobacteriaceae.

Figure 1.

Carba5 principle.

Figure 2.

Data acquisition using a handheld reader. (a) Results obtained after 15 min of migration with carbapenemase-expressing strains. NDM, IMP, VIM, OXA and KPC, one colony resuspended in 150 µL of extraction buffer and 100 µL loaded on the cassette. For all, 25 µL from each previous extract pooled leading to a 1 to 5 dilution and 100 µL of this pool loaded on the cassette. (b) Previous IMP-expressing strain extract, picture of the cassette, positive test line analysis and corresponding value and carbapenemase name (table). (c) Previous pool of the five carbapenemase-expressing strain extracts, positive test line analysis and corresponding value (table). An asterisk indicates a test line not visible owing to camera sensitivity.

Figure 3.

Results obtained with different strains. One colony suspended in extraction buffer and loaded on the cassette. (a) Klebsiella pneumoniae expressing CTX-M-18. (b) K. pneumoniae expressing OXA-162. (c) K. pneumoniae expressing NDM-1 and OXA-48. N, negative result; P, positive result.