Comparative Evaluation of Phenotypic Synergy Tests versus RESIST-4 O.K.N.V. and NG Test Carba 5 Lateral Flow Immunoassays for the Detection and Differentiation of Carbapenemases in Enterobacterales and Pseudomonas aeruginosa

Abstract

The spread of carbapenem-resistant Pseudomonas aeruginosa and carbapenemase-producing Enterobacterales (CPE) has dramatically impacted morbidity and mortality. COVID-19 pandemic has favored the selection of these microorganisms because of the excessive and prolonged use of broad-spectrum antibiotics and the outbreaks related to patient transfer between hospitals and inadequate personal protective equipment. Therefore, early CPE detection is considered essential for their control. We aimed to compare conventional phenotypic synergy tests and two lateral flow immunoassays for detecting carbapenemases in Enterobacterales and P. aeruginosa. We analyzed 100 carbapenem-resistant Gram-negative bacilli isolates, 80 Enterobacterales, and 20 P. aeruginosa (86 isolates producing KPC, NDM, OXA-48, IMP, and VIM carbapenemases and 14 non-carbapenemase-producing isolates). We performed a modified Hodge test, boronic acid and ethylenediaminetetraacetic acid (EDTA) synergy tests, and two lateral flow immunoassays: RESIST-4 O.K.N.V. (Coris Bioconcept) and NG Test Carba 5 (NG Biotech). In total, 76 KPC, seven VIM, one NDM, one OXA-48, and one isolate coproducing KPC + NDM enzymes were included. The concordance of different methods estimated by the Kappa index was 0.432 (standard error: 0.117), thus showing a high variability with the synergy tests with boronic acid and EDTA and reporting 16 false negatives that were detected by the two immunochromatographic methods. Co-production was only detected using immunoassays. Conventional phenotypic synergy tests with boronic acid and EDTA for detecting carbapenemases are suboptimal, and their routine use should be reconsidered. These tests depend on the degree of enzyme expression and the distance between disks. Lateral flow immunoassay tests are a rapid and cost-effective tool to detect and differentiate carbapenemases, improving clinical outcomes through targeted therapy and promoting infection prevention measures. IMPORTANCE Infections due to multidrug-resistant pathogens are a growing problem worldwide. The production of carbapenemases in Pseudomonas aeruginosa and Enterobacterales cause a high impact on the mortality of infected patients. Therefore, it is of great importance to have methods that allow the early detection of these multi-resistant microorganisms, achieving the confirmation of the type of carbapenemase present, with high sensitivity and specificity, with the aim of improving epidemiological control, dissemination, the clinical course to through targeted antibiotic therapy and promoting infection control in hospitals.

Keywords: EDTA; boronic acid; carbapenemases; immunoassay; multidrug resistance; synergy test.

FIG 1

Errors in synergy tests. (A) KPC-producing K. pneumoniae not detected with boronic acid and EDTA (1a). Positive result adjusting distance to 5 mm (1b) and positive result by NG Test Carba 5 and RESIST-4 O.K.N.V immunoassays (1c). (B) VIM-producing P. aeruginosa with inconclusive EDTA result (2a), positive result adjusting distance to 5 mm (2b), confirmation of VIM by immunoassay (2c). (C) K. pneumoniae with KPC and NDM co-production without synergistic effect with a boronic acid or EDTA (3a), positivity for both enzymes by immunoassay (3b). (D) Impact of the distance between disks: False-negative for serine-carbapenemases with 15 mm (4a) positive result at 10 mm (4b). Differences in boronic acid tests for serin-carbapenemases at 10 mm, 5 mm, and 0 mm distance (4c, 4d). There are differences in synergy between imipenem, meropenem, and ertapenem discs at 0 mm distance (4e).