Resistencia cruzada entre isoniacida y etionamida y su alta correlación con la mutación C-15T en aislamientos de Mycobacterium tuberculosis de Perú / Cross-resistance between isoniazid and ethionamide and its strong association with mutation C-15T in Mycobacterium tuberculosis isolates from Peru

Resumen Diversos estudios han evidenciado una resistencia cruzada entre isoniacida y etionamida, 2 de los fármacos utilizados en el tratamiento de la tuberculosis multirresistente.El objetivo del presente estudio fue determinar la resistencia cruzada entre ambos fármacos en aislados de Mycobacterium tuberculosis obtenidos en un hospital de Lima (Perú), conalta proporción de pacientes con tuberculosis. Se calculó la frecuencia de mutaciones asociadas con la resistencia a la isoniacida (INH) evaluando el gen katG y la región promotorainhA mediante la prueba molecular Genotype MTBDRplus v2.0. El método gold standard conocido como agar proporciones en placa (APP) permitió la identificación de resistencia a INH yetionamida. De 107 aislamientos resistentes a INH, 54 fueron multirresistentes (identificadosmediante la prueba Genotype MTBDRplus) y 49 (es decir, el 45,8% del total) también fueronresistentes a etionamida por el método APP. En los aislamientos resistentes a INH, se encontraron mutaciones en el gen katG en el 50,5% (54/107); en la región promotora inhA en el23,3% (25/107), y un 14,0% (15/107) presentaron mutaciones en ambos. Un 12,1% (13/107)fueron resistentes a INH por ausencia de banda wild type y banda de mutación. La mutaciónC-15T en la región promotora inhA presentó una fuerte asociación con la resistencia a etionamida y alcanzó el 73,4% (36/49) de los aislamientos resistentes a dicho fármaco. Los resultadosdel presente estudio sugieren que la identificación de mutaciones relacionadas con resistenciaa INH, sobre todo en la región promotora inhA, podría ser de gran utilidad para identificarla resistencia cruzada a etionamida y mejorar el tratamiento de las personas afectadas portuberculosis.© 2019 Asociacion Argentina de Microbiolog´ía. Publicado por Elsevier Espana, S.L.U. Este es unart´ículo Open Access bajo la licencia CC BY-NC-ND (https://creativecommons.org/licenses/by-nc-nd/4.0/).

Abstract Several studies have shown cross-resistance between isoniazid and ethionamide, 2of the drugs used in the treatment of multidrug-resistant tuberculosis. The objective of this study was to determine the cross-resistance between both drugs in Mycobacterium tuberculosis isolates from a hospital with high incidence of tuberculosis in Lima, Peru. The frequency of mutations to isoniazid in the katG gene and the inhA promoter region was identified by the Genotype MTBDRplus v2.0 molecular test. The gold standard Agar Proportion method (APM) allowed todetect resistance to isoniazid and ethionamide. Of 107 isoniazid-resistant isolates (54 multidrug-resistant isolates identified by the Genotype MTBDRplus test, 45.8% (49/107) were also resistant to ethionamide by the APM. Mutations were found in the katG gene in 50.5% (54/107), in the promoter region inhA in 23.3% (25/107) and 14.0% (15/107) that share both mutations in the resistant isolates to INH. The absence of the wild type and mutation bands indicated that 12.1% (13/107) of the isolates were resistant to INH. The mutation C-15T in the inhA promoter region showed a strong association with resistance to ethionamide in 73.4% (36/49) of the isolates analyzed. The results of the present study suggest that the identification of mutations related to resistance to isoniazid, especially in the inhA promoter region, could be very useful to identify cross-resistance to ethionamide and improve the treatment of individuals suffering from this disease.

[Cross-resistance between isoniazid and ethionamide and its strong association with mutation C-15T in Mycobacterium tuberculosis isolates from Peru]. / Resistencia cruzada entre isoniacida y etionamida y su alta correlación con la mutación C-15T en aislamientos de Mycobacterium tuberculosis de Perú.

Several studies have shown cross-resistance between isoniazid and ethionamide, 2of the drugs used in the treatment of multidrug-resistant tuberculosis. The objective of this study was to determine the cross-resistance between both drugs in Mycobacterium tuberculosis isolates from a hospital with high incidence of tuberculosis in Lima, Peru. The frequency of mutations to isoniazid in the katG gene and the inhA promoter region was identified by the Genotype MTBDRplus v2.0 molecular test. The gold standard Agar Proportion method (APM) allowed todetect resistance to isoniazid and ethionamide. Of 107 isoniazid-resistant isolates (54 multidrug-resistant isolates identified by the Genotype MTBDRplus test, 45.8% (49/107) were also resistant to ethionamide by the APM. Mutations were found in the katG gene in 50.5% (54/107), in the promoter region inhA in 23.3% (25/107) and 14.0% (15/107) that share both mutations in the resistant isolates to INH. The absence of the wild type and mutation bands indicated that 12.1% (13/107) of the isolates were resistant to INH. The mutation C-15T in the inhA promoter region showed a strong association with resistance to ethionamide in 73.4% (36/49) of the isolates analyzed. The results of the present study suggest that the identification of mutations related to resistance to isoniazid, especially in the inhA promoter region, could be very useful to identify cross-resistance to ethionamide and improve the treatment of individuals suffering from this disease.

Mechanochemistry applied to reformulation and scale-up production of Ethionamide: Salt selection and solubility enhancement.

Ethionamide (ETH), a Biopharmaceutics Classification System class II drug, is a second-line drug manufactured as an oral dosage form by Pfizer to treat tuberculosis. Since its discovery in 1956, only one reformulation was proposed in 2005 as part of the efforts to improve its solubility. Due to the limited scientific research on active pharmaceutical ingredients (APIs) for the treatment of neglected diseases, we focused on the development of an approachable and green supramolecular synthesis protocol for the production of novel solid forms of ETH. Initially, three salts were crystal engineered and supramolecular synthesized via slow evaporation of the solvent: a saccharinate, a maleate and an oxalate. The crystal structures of all salts were determined by single crystal X-ray diffraction. In sequence, mechanochemical protocols for them were developed, being the scale-up production of the maleate salt successfully reproducible and confirmed by powder X-ray diffraction. Finally, a more complete solid-state characterization was carried out for the ETH maleate salt, including thermal analysis, infrared spectroscopy, scanning electron microscopy and equilibrium solubility at different dissolution media. Although ETH maleate is thermodynamically less stable than ETH, the equilibrium solubility results revealed that this novel salt is much more soluble in purified water than ETH, thus being a suitable new candidate for future formulations.

GenoType MTBDR plus 1.0 ® para la detección de resistencia cruzada entre isoniacida y etionamida en aislamientos de Mycobacterium tuberculosis multirresistentes / GenoType MTBDR plus 1.0 ® for the detection of cross-resistance between isoniazide and ethionamide in isolates of multidrug-resistant Mycobacterium tuberculosis

Introducción. Una parte de los aislamientos de Mycobacterium tuberculosis multirresistente también presenta resistencia a la etionamida. Es importante determinar si la resistencia a la isoniacida es independiente o se cruza con la resistencia a la etionamida, ya que si sucede lo segundo habría que reevaluar el tratamiento antituberculoso de segunda línea. La prueba molecular GenoType MTBDR plus ® detecta las mutaciones asociadas con la resistencia a isoniacida y podría detectar la resistencia cruzada a la etionamida. Objetivo. Evaluar la prueba GenoType MTBDR plus ® y comparar su desempeño con el de la secuenciación, en la detección de mutaciones en el gen katG y en el promotor inhA en aislamientos clínicos de M. tuberculosis multirresistente. Materiales y métodos. Se utilizaron el estuche comercial GenoType MTBDR plus 1.0 ® y la secuenciación para evaluar mutaciones en el gen katG y en el promotor inhA en 30 aislamientos de M. tuberculosis multirresistente con resistencia a la etionamida. La cepa de laboratorio H37Rv y tres aislamientos sensibles a los medicamentos de primera línea, sirvieron de control. Resultados. Al comparar los resultados de la secuenciación y de la prueba GenoType MTBDR plus ® , el índice kappa fue de 1. Todos los aislamientos resistentes a la isoniacida y la etionamida tenían las mutaciones detectadas con la prueba GenoTypeMTBDR plus ® en el gen katG, y 40 % de ellos, las detectadas en el promotor inhA. Mediante secuenciación se encontraron, además, mutaciones en katG en posiciones diferentes a las detectadas por la prueba GenoType MTBDR plus ® . Conclusión. La prueba GenoTypeMTBDR plus ® tiene la capacidad de detectar rápidamente la resistencia a isoniacida. Además, los resultados del estudio sugieren que también podría utilizarse como prueba de tamización para detectar la resistencia cruzada a etionamida.

Introduction: A variable proportion of isolates of multidrug-resistant Mycobacterium tuberculosis also presents resistance to ethionamide. It is important to determine whether resistance to isoniazid is independent or crossed with resistance to ethionamide, given that this could lead to the re-evaluation of second-line anti-tuberculosis treatment. The GenoType MTBDR plus ® molecular test is used for the detection of MDR-MTB, as it identifies mutations associated with resistance to isoniazide and could detect cross-resistance with ethionamide. Objective: To evaluate the performance of GenoType MTBDR plus ® in comparison with sequencing in the detection of mutations in gene katG and promotor inhA in clinical isolates of multidrug-resistant M. tuberculosis . Materials and methods: The GenoType MTBDR plus 1.0 ® commercial kit and sequencing were used to evaluate mutations in gene katG and promotor inhA in 30 multidrug-resistant M. tuberculosis isolates that were resistant to ethionamide. The laboratory strain H37Rv and three pan-sensitive isolates acted as controls. Results: The kappa index for the comparison between the results of sequencing and GenoType MTBDR plus ® was 1. All the isolates resistant to isoniazid and ethionamide had the mutations detected by GenoTypeMTBDR plus ® in the katG gene and 40% of them in promotor inhA. Sequencing also revealed katG mutations in positions different to those detected by GenoType MTBDR plus ® . Conclusion: GenoType MTBDR plus ® is able to detect resistance to isoniazid rapidly. Our results suggest that it could also be used to screen for cross-resistance with ethionamide.

Inhibitory activity of isoniazid and ethionamide against Cryptococcus biofilms.

In recent years, the search for drugs to treat systemic and opportunistic mycoses has attracted great interest from the scientific community. This study evaluated the in vitro inhibitory effect of the antituberculosis drugs isoniazid and ethionamide alone and combined with itraconazole and fluconazole against biofilms of Cryptococcus neoformans and Cryptococcus gattii. Antimicrobials were tested at defined concentrations after susceptibility assays with Cryptococcus planktonic cells. In addition, we investigated the synergistic interaction of antituberculosis drugs and azole derivatives against Cryptococcus planktonic cells, as well as the influence of isoniazid and ethionamide on ergosterol content and cell membrane permeability. Isoniazid and ethionamide inhibited both biofilm formation and viability of mature biofilms. Combinations formed by antituberculosis drugs and azoles proved synergic against both planktonic and sessile cells, showing an ability to reduce Cryptococcus biofilms by approximately 50%. Furthermore, isoniazid and ethionamide reduced the content of ergosterol in Cryptococcus spp. planktonic cells and destabilized or permeabilized the fungal cell membrane, leading to leakage of macromolecules. Owing to the paucity of drugs able to inhibit Cryptococcus biofilms, we believe that the results presented here might be of interest in the designing of new antifungal compounds.