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

Edgar A Florentini,Roberto Alcántara,Elisa Roncal,Ricardo Antiparra,Robert H Gilman,Noelia Angulo,Katherine Vallejos,Patricia Sheen,Emily Toscano,Mirko Zimic,Danni Kirwan

doi:10.1371/journal.pone.0241600

Edgar A Florentini, Roberto Alcántara + Show 9 more

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https://doi.org/10.1371/journal.pone.0241600

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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.

Highlights

Tuberculosis (TB) is the leading infectious cause of morbidity and mortality worldwide
PZA resistance is associated with resistance to other TB medications: current estimates indicate that 50% of MDR-TB cases are PZA resistant [6,7,8] and, alarmingly, the emergence of Mycobacterium tuberculosis (Mtb) strains resistant to all known anti-TB drugs has been reported [9]
These constructs were diluted in PBS and optimum dilutions that elicited the maximum absorbance in the absence of the analyte (Amax) using the minimum amount of analyte capable of inhibiting 50% of the signal (IC50) were found to be 1:400 for LB, 1:5,000 for POA.OH linker BSA (PLB), 1:4,500 for HRP and an antiserum dilution of 1:50,000

Summary

Introduction

Tuberculosis (TB) is the leading infectious cause of morbidity and mortality worldwide. In 2017, approximately 1.3 million deaths and 10 million new cases were reported [1]. PZA resistance is associated with resistance to other TB medications: current estimates indicate that 50% of MDR-TB cases are PZA resistant [6,7,8] and, alarmingly, the emergence of Mycobacterium tuberculosis (Mtb) strains resistant to all known anti-TB drugs has been reported [9]. This is likely to be caused by the mismanagement of pharmacological protocols. This scenario invokes the notion that testing for PZA resistance would be key to effective prescribing in TB, yet this is not routinely performed

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