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

[This corrects the article DOI: 10.1371/journal.pone.0241600.].

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 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.

Mycobacterium tuberculosis ribosomal protein S1 (RpsA) and variants with truncated C-terminal end show absence of interaction with pyrazinoic acid.

Pyrazinamide (PZA) is an antibiotic used in first- and second-line tuberculosis treatment regimens. Approximately 50% of multidrug-resistant tuberculosis and over 90% of extensively drug-resistant tuberculosis strains are also PZA resistant. Despite the key role played by PZA, its mechanisms of action are not yet fully understood. It has been postulated that pyrazinoic acid (POA), the hydrolyzed product of PZA, could inhibit trans-translation by binding to Ribosomal protein S1 (RpsA) and competing with tmRNA, the natural cofactor of RpsA. Subsequent data, however, indicate that these early findings resulted from experimental artifact. Hence, in this study we assess the capacity of POA to compete with tmRNA for RpsA. We evaluated RpsA wild type (WT), RpsA ∆A438, and RpsA ∆A438 variants with truncations towards the carboxy terminal end. Interactions were measured using Nuclear Magnetic Resonance spectroscopy (NMR), Isothermal Titration Calorimetry (ITC), Microscale Thermophoresis (MST), and Electrophoretic Mobility Shift Assay (EMSA). We found no measurable binding between POA and RpsA (WT or variants). This suggests that RpsA may not be involved in the mechanism of action of PZA in Mycobacterium tuberculosis, as previously thought. Interactions observed between tmRNA and RpsA WT, RpsA ∆A438, and each of the truncated variants of RpsA ∆A438, are reported.

Estudio genotóxico de una bebida experimental de quinua, kiwicha y kañiwa / Genotoxic study of an experimental drink of quinoa, kiwicha and kañiwa

La evaluación genotóxica de un producto es un paso importante para determinar su viabilidad para consumo humano. Se ha elaborado una bebida experimental a base de pseudocereales de alto valor nutricional como son quinua (Chenopodium quinoa Willd.), kiwicha (Amaranthus caudatus L.) y kañiwa (Chenopodium pallidicaule Aellen), preparada para inducir un posible efecto hipolipemiante en un grupo de personas. El objetivo de este estudio fue evaluar el potencial genotóxico de esta bebida experimental mediante dos pruebas in vitro validadas por agencias internacionales. En la prueba de Ames se utilizaron las cepas TA98 y TA10 de Salmonella typhimurium, con y sin fracción microsomal (S9). Se evaluaron 4 dosis de bebida y además un posible efecto antimutagénico (mismas 4 dosis más mutágeno). Para la prueba de micronúcleos se usó cultivos de linfocitos con células binucleadas, en presencia de cinco dosis de la bebida. Ambas pruebas indican que la bebida estudiada en sus distintas dosis, no presenta efecto genotóxico. Sin embargo, en la evaluación del posible efecto protector de la bebida, se evidenciaría que por el contrario, se potencia el efecto mutagénico de los mutágenos empleados para cada cepa. Por lo tanto, es importante que esta bebida experimental sea sometida a pruebas adicionales in vitro e in vivo para evaluar el potencial genotóxico antes de su consumo.