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

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...

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Autores principales:	Edgar A Florentini, Noelia Angulo, Robert H Gilman, Roberto Alcántara, Elisa Roncal, Ricardo Antiparra, Emily Toscano, Katherine Vallejos, Danni Kirwan, Mirko Zimic, Patricia Sheen
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2020
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/323a61b43f9241a1b4b39d221ba6862c
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spelling	oai:doaj.org-article:323a61b43f9241a1b4b39d221ba6862c2021-12-02T20:11:28ZImmunological detection of pyrazine-2-carboxylic acid for the detection of pyrazinamide resistance in Mycobacterium tuberculosis.1932-620310.1371/journal.pone.0241600https://doaj.org/article/323a61b43f9241a1b4b39d221ba6862c2020-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0241600https://doaj.org/toc/1932-6203Pyrazinamide (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.Edgar A FlorentiniNoelia AnguloRobert H GilmanRoberto AlcántaraElisa RoncalRicardo AntiparraEmily ToscanoKatherine VallejosDanni KirwanMirko ZimicPatricia SheenPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 15, Iss 11, p e0241600 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Edgar A Florentini Noelia Angulo Robert H Gilman Roberto Alcántara Elisa Roncal Ricardo Antiparra Emily Toscano Katherine Vallejos Danni Kirwan Mirko Zimic Patricia Sheen Immunological detection of pyrazine-2-carboxylic acid for the detection of pyrazinamide resistance in Mycobacterium tuberculosis.
description	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.
format	article
author	Edgar A Florentini Noelia Angulo Robert H Gilman Roberto Alcántara Elisa Roncal Ricardo Antiparra Emily Toscano Katherine Vallejos Danni Kirwan Mirko Zimic Patricia Sheen
author_facet	Edgar A Florentini Noelia Angulo Robert H Gilman Roberto Alcántara Elisa Roncal Ricardo Antiparra Emily Toscano Katherine Vallejos Danni Kirwan Mirko Zimic Patricia Sheen
author_sort	Edgar A Florentini
title	Immunological detection of pyrazine-2-carboxylic acid for the detection of pyrazinamide resistance in Mycobacterium tuberculosis.
title_short	Immunological detection of pyrazine-2-carboxylic acid for the detection of pyrazinamide resistance in Mycobacterium tuberculosis.
title_full	Immunological detection of pyrazine-2-carboxylic acid for the detection of pyrazinamide resistance in Mycobacterium tuberculosis.
title_fullStr	Immunological detection of pyrazine-2-carboxylic acid for the detection of pyrazinamide resistance in Mycobacterium tuberculosis.
title_full_unstemmed	Immunological detection of pyrazine-2-carboxylic acid for the detection of pyrazinamide resistance in Mycobacterium tuberculosis.
title_sort	immunological detection of pyrazine-2-carboxylic acid for the detection of pyrazinamide resistance in mycobacterium tuberculosis.
publisher	Public Library of Science (PLoS)
publishDate	2020
url	https://doaj.org/article/323a61b43f9241a1b4b39d221ba6862c
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Immunological detection of pyrazine-2-carboxylic acid for the detection of pyrazinamide resistance in Mycobacterium tuberculosis.

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