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Immunological detection of pyrazine-2-carboxylic acid for the detection of pyrazinamide resistance in Mycobacterium tuberculosis.

Florentini, EA; Angulo, N; Gilman, RH; Alcántara, R; Roncal, E; Antiparra, R; Toscano, E; Vallejos, K; Kirwan, D; Zimic, M; et al. Florentini, EA; Angulo, N; Gilman, RH; Alcántara, R; Roncal, E; Antiparra, R; Toscano, E; Vallejos, K; Kirwan, D; Zimic, M; Sheen, P (2020) Immunological detection of pyrazine-2-carboxylic acid for the detection of pyrazinamide resistance in Mycobacterium tuberculosis. PLoS One, 15 (11). e0241600. ISSN 1932-6203 https://doi.org/10.1371/journal.pone.0241600
SGUL Authors: Kirwan, Daniela Elisa

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Abstract

Pyrazinamide (PZA) susceptibility testing in Mycobacterium tuberculosis (Mtb) is a current area of development and PZA-resistant strains are increasingly prevalent. Previous studies have demonstrated that the detection of pyrazinoic acid (POA), the metabolite produced by the deamidation of PZA, is a good predictor for PZA resistance since a resistant strain would not convert PZA into POA at a critical required rate, whereas a susceptible strain will do, expelling POA to the extracellular environment at a certain rate, and allowing for quantification of this accumulated analyte. In order to quantify POA, an indirect competitive ELISA (icELISA) test using hyperimmune polyclonal rabbit serum against POA was developed: for this purpose, pure POA was first covalently linked to the highly immunogenic Keyhole Limpet Hemocyanine, and inoculated in rabbits. A construct made of bovine serum albumin (BSA) linked to pure POA and fixed at the bottom of wells was used as a competitor against spiked samples and liquid Mtb culture supernatants. When spiked samples (commercial POA alone) were analyzed, the half maximal inhibitory concentration (IC50) was 1.16 mg/mL, the limit of detection 200 μg/mL and the assay was specific (it did not detect PZA, IC50 > 20 mg/mL). However, culture supernatants (7H9-OADC-PANTA medium) disrupted the competition and a proper icELISA curve was not obtainable. We consider that, although we have shown that it is feasible to induce antibodies against POA, matrix effects could damage its analytical usefulness; multiple, upcoming ways to solve this obstacle are suggested.

Item Type: Article
Additional Information: Correction available at https://doi.org/10.1371/journal.pone.0259439 Copyright: © 2020 Florentini et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Keywords: Animals, Antibodies, Antitubercular Agents, Drug Resistance, Bacterial, Enzyme-Linked Immunosorbent Assay, Immunoconjugates, Inhibitory Concentration 50, Mycobacterium tuberculosis, Pyrazinamide, Rabbits, Serum Albumin, Bovine, Toxicity Tests, MD Multidisciplinary, General Science & Technology
SGUL Research Institute / Research Centre: Academic Structure > Infection and Immunity Research Institute (INII)
Journal or Publication Title: PLoS One
ISSN: 1932-6203
Language: eng
Dates:
DateEvent
5 November 2020Published
17 October 2020Accepted
Publisher License: Creative Commons: Attribution 4.0
Projects:
Project IDFunderFunder ID
MR/P019978/1Medical Research Councilhttp://dx.doi.org/10.13039/501100000265
099805/Z/112/ZWellcome Trusthttp://dx.doi.org/10.13039/100004440
MR/K007467/1Medical Research Councilhttp://dx.doi.org/10.13039/501100000265
MR/P019978/2Medical Research Councilhttp://dx.doi.org/10.13039/501100000265
PubMed ID: 33151985
Web of Science ID: WOS:000591376200062
Go to PubMed abstract
URI: https://openaccess.sgul.ac.uk/id/eprint/114142
Publisher's version: https://doi.org/10.1371/journal.pone.0241600

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