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. 2023 Apr 18;11(4):1052.
doi: 10.3390/microorganisms11041052.

Capture ELISA for KPC Detection in Gram-Negative Bacilli: Development and Standardisation

André Valencio  1 , Miriam Aparecida da Silva  2 , Fernanda Fernandes Santos  1 , Juliana Moutinho Polatto  2 , Marcelo Marcondes Ferreira Machado  1 , Roxane Maria Fontes Piazza  2 , Ana Cristina Gales  1
Affiliations

Capture ELISA for KPC Detection in Gram-Negative Bacilli: Development and Standardisation

André Valencio et al. Microorganisms. .

Abstract

The detection of KPC-type carbapenemases is necessary for guiding appropriate antibiotic therapy and the implementation of antimicrobial stewardship and infection control measures. Currently, few tests are capable of differentiating carbapenemase types, restricting the lab reports to their presence or not. The aim of this work was to raise antibodies and develop an ELISA test to detect KPC-2 and its D179 mutants. The ELISA-KPC test was designed using rabbit and mouse polyclonal antibodies. Four different protocols were tested to select the bacterial inoculum with the highest sensitivity and specificity rates. The standardisation procedure was performed using 109 previously characterised clinical isolates, showing 100% of sensitivity and 89% of specificity. The ELISA-KPC detected all isolates producing carbapenemases, including KPC variants displaying the ESBL phenotype such as KPC-33 and -66.

Keywords: Klebsiella pneumoniae; antibodies; carbapenemases; immunodiagnostic.

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Conflict of interest statement

A.C.G. has received honoraria and/or consultation fees from Entasis Therapeutics, BioMerieux, Eurofarma, MSD, Pfizer, Roche, Sandoz, and United Medical, and research funding from Eurofarma.

Figures

Figure 1
Figure 1
Graphical summary of protocols for the preparation of bacterial isolates before the ELISA test.
Figure 2
Figure 2
Presence of purified rKPC-2 on SDS–PAGE gel. The top two fractions were collected from AKTA equipment. Legend, 1: Tube 17, 2: Tube 18, MM: molecular marker.
Figure 3
Figure 3
Titration of animal serum containing antibodies against rKPC-2. Legend: (A) titre of anti-rKPC-2 antibodies in rabbit serum; (B) titre of anti-rKPC-2 antibodies in mouse serum.
Figure 4
Figure 4
Purification of polyclonal antibodies from (MM) molecular marker; (A) rabbit and (B) mice on SDS–PAGE gel. Antibodies have two subunits, one with 50 KDa and the other with 25 KDa.
Figure 5
Figure 5
Statistical parameters of the four protocols of bacterial inoculum preparation: (A) ROC DC curve with cut-off 0.269, sensitivity: 77.8%, and specificity: 75%; (a) t-test p-value < 0.05; (B) ROC PD-OD1 curve, cut-off of 0.486, sensitivity of 100%, and specificity of 100%; (b) t-test **** p-value < 0.0001. (C) ROC PD-G curve, cut-off of 0.437; 100% sensitivity, and 100% specificity; (c) t-test with **** p-value < 0.0001; (D) ROC GMHB curve, cut-off 0.159, sensitivity 88.9%, and specificity 100%, (d) t-test * p-value < 0.05; legend: P: positive isolates; N: negative isolates.
Figure 6
Figure 6
T-Student analysis of the KPC-ELISA-GMHB test verification. Caption: KPC+: KPC-like-producing samples. KPC-: isolates not producing KPC-like (p-value: <0.0001).
Figure 7
Figure 7
ROC curve of the performance of the ELISA-KPC-GMHB. This methodology obtained a sensitivity of 100% and a specificity of 89%, with 95% IC and a cut-off of 0.401.
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