Study on genes for cross-resistance to isoniazid and ethionamidein Mycobacterium tuberculosis / 预防医学

Objective@#To examine the association between the cross-resistance to ethionamide (Eto) and isoniazid (INH) and mutations of drug resistant genes in Mycobacterium tuberculosis (MTB), so as to provide the evidence for clinical diagnosis and treatment for multidrug-resistant (MDR) tuberculosis.@*Methods@#Totally 126 MTB clinical isolates were selected, including 88 MDR-MTB clinical isolates and 38 INH- and rifampicin (RFP)-sensitive isolates. The resistance to INH and Eto was tested in MTB clinical isolates using the drug susceptibility test, and the mutations in the spacer region of INH and Eto resistance-related katG, inhA, ethA, mshA, ndh, spacer region of oxyR-ahpC and inhA promoter were detected using PCR assay. The phenotypic resistance served as a gold standard, and the sensitivity, specificity and accuracy of gene mutation tests were calculated for detection of MTB clinical isolates cross-resistant to INH and Eto.@*Results@#Of the 126 MTB clinical isolates, there were 37 isolates cross-resistant to INH and Eto (29.37%), 51 isolates with resistance to INH and susceptibility to Eto (40.48%), 4 isolates with susceptibility to INH and resistance to Eto (3.17%) and 34 isolates with susceptibility to INH and Eto (26.98%). Among the 41 Eto-resistant MTB clinical isolates, there were 37 isolates with resistance to INH (90.24%). There were 64 MTB clinical isolates detected with katG mutations (50.79%), 4 isolates with mutation in the spacer region of oxyR-ahpC (3.17%), 2 isolates with inhA mutations (1.59%), and these isolates were all resistant to INH. There were 11 MTB clinical isolates detected with mutation in the inhA promoter (8.73%) and one isolate with ndh mutation, and all these isolates were cross-resistant to INH and Eto. There were 23 MTB clinical isolates detected with ethA mutations (18.25%) and 40 isolates with mshA mutations (31.75%), in which Eto-susceptible and -resistant isolates were detected. The diagnostic sensitivity, specificity and accuracy of inhA promoter tests for detection of cross-resistance to INH and Eto were 29.73% (95%CI: 16.44%-47.17%), 100.00% (95%CI: 87.36%-100.00%) and 63.38% (95%CI: 51.76%-73.63%) in MTB clinical isolates.@*Conclusions@#The prevalence of INH resistance is high in Eto-resistant MTB clinical isolates. Mutation in the inhA promoter region correlates with the cross-resistance to INH and Eto in MTB clinical isolates, and detection of mutation in the inhA promoter may be feasible to detect the cross-resistance to INH and Eto in MTB clinical isolates.

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.

Multiparticulate Drug Delivery System For Gastrointestinal Tuberculosis

Drug solubility poses numerous challenges in design of formulations for drugs with poor aqueous solubility. Ethionamide is an antitubercular drug belonging to biopharmaceutical classification system class II drug having less aqueous solubility. Nanosuspensions were prepared by using various solvents such as methanol, ethanol, acetone and chloroform and it was prepared using anti-solvent precipitation technique by using probe sonication. Various stabilizers such as tocopherolpolythytlene glycol succinate, polyvinylpyrrolidone and tween 80 singly or in combination were studied. A 32 factorial design was employed to study the effect of independent variables, concentration of stabilizers and stirring speed on particle size and cumulative percent drug release. The particle size of the optimized batch was 97.54 ± 8.47 nm with polydispersity index of 0.36 and zeta potential -10.1 ± 2.3 mV. The cumulative percent drug release of optimized batch was found to be 95.01 ± 1.16% in 60 min. Optimized batch was ultracentrifuged and evaluated for saturation solubility studies, stability and powder X-ray Diffraction studies. Optimized nanosuspension was loaded on Espheres by spraying in a coating pan and then coating of Eudragit controlled release polymers. The coated Espheres were evaluated for drug content, friability, scanning electron microscopy, ex-vivo permeation studies and drug release kinetics studies. The friability value for primary coated sphere was found to be 0.8 ± 0.12% and for secondary was 1% and the best fit model was found to be Korsmeyer-Peppas model which is indicative of diffusion controlled release. Ex vivo diffusion studies revealed a moderate increase in permeability.

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.

Revisiting the susceptibility testing of Mycobacterium tuberculosis to ethionamide in solid culture medium.

Background & objectives: Increase in the isolation of drug resistant phenotypes of Mycobacterium tuberculosis necessitates accuracy in the testing methodology. Critical concentration defining resistance for ethionamide (ETO), needs re-evaluation in accordance with the current scenario. Thus, re-evaluation of conventional minimum inhibitory concentration (MIC) and proportion sensitivity testing (PST) methods for ETO was done to identify the ideal breakpoint concentration defining resistance. Methods: Isolates of M. tuberculosis (n=235) from new and treated patients were subjected to conventional MIC and PST methods for ETO following standard operating procedures. Results: With breakpoint concentration set at 114 and 156 μg/ml, an increase in specificity was observed whereas sensitivity was high with 80 μg/ml as breakpoint concentration. Errors due to false resistant and susceptible isolates were least at 80 μg/ml concentration. Interpretation & conclusions: Performance parameters at 80 μg/ml breakpoint concentration indicated significant association between PST and MIC methods.

Consistency of standard laboratory strain Mycobacterium tuberculosis H37Rv with ethionamide susceptibility testing.

Drug susceptibility pattern of standard Mycobacterium tuberculosis strain H37Rv showed discrepancy in minimum inhibitory concentration method for ethionamide and consistent results were obtained for the other second line drugs namely, kanamycin and ofloxacin. It is, therefore, necessary to revisit the susceptibility testing method for ethionamide for effective clinical management of patients with drug resistant tuberculosis.

Ethionamide-induced pellagroid dermatitis resembling lichen simplex chronicus: A report of two cases.

Pellagra is a niacin deficiency disorder characterized clinically by diarrhea, dermatitis, and dementia. However, few drugs also cause pellagroid dermatitis. Recently, we encountered two cases of pellagroid dermatitis; both were on second line of antituberculosis drugs. Case 1 was of multidrug-resistant pulmonary tuberculosis. Patient was on ethionamide since one year before developing pellagroid dermatitis. Case 2 was of central nervous system tuberculoma and was on second line of antitubercular drugs. This patient was on ethionamide and isoniazid (INH) since six months before developing pellagroid dermatitis. This patient had previously taken first line of antituberculous therapy, inclusive of INH, for 1 year without any dermatitis. The skin lesions in both patients were symmetric hyperpigmented thickened plaques with prominent skin markings resembling lichen simplex chronicus. Nicotinamide 300 mg in three divided doses healed the lesions completely within 4 weeks and 3 weeks in first and second patient, respectively.