Performance Assessment of the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Method for Rapid Detection of Susceptibility to Ethambutol and Molecular Prediction of Extensively Drug-resistant Tuberculosis in Clinical Isolates of Mycobacterium tuberculosis.

INTRODUCTION: The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was employed for rapid detection of ethambutol (EMB) resistant clinical isolates of Mycobacterium tuberculosis. MATERIALS AND METHODS: From 182 clinical isolates of M tuberculosis collected from different regions, 103 strains were entered in the investigation. DNA was extracted by Chelex 100 method and PCR was performed using specific primers for embB gene. Polymerase chain reaction products were digested with HaeIII and NlaII restriction endonucleases and the patterns of restriction fragments were analysed. Some randomly selected samples were sequenced. RESULTS: Out of 103 studied strains, 52 were resistant to EMB. The cases of secondary tuberculosis were 53 (51.50 ± 1.77%), and primary cases 50 (48.50 ± 1.77%; p > 0.05). From 63 extensively drug-resistant (XDR), pre-XDR and multidrug-resistant (MDR) isolates, 27 (87%), 18 (81.8%) and 7 (70%) strains were resistant to EMB, respectively. Results of PCR-RFLP method showed that from 27R EMB XDR isolates, 13 (sensitivity 48% with CI: 0.307, 0.66 and specificity 100%), from 18R EMB pre-XDR strains, 4 (sensitivity 22% with CI: 0.09, 0.45 and specificity 100%) and of 7R EMB MDR, 2 (sensitivity 28% with CI: 0.082, 0.64 and specificity 100%) had mutation in ATG-Met codon 306. Results of sequencing were concordant with RFLP method. Overall, sensitivity of the molecular method was 36.5% (CI: 0.09, 0.45) and specificity 100%. None of the 40 pansusceptible strains was embB306 mutants. Extensively drug-resistant strains had a higher proportion of embB306 mutants (43%) than pre-XDR and MDR isolates (odds ratio 6.78; p < 0.001). CONCLUSION: Fast detection of susceptibility to EMB drug is possible by PCR-RFLP. The embB306 locus is a candidate marker for rapid prediction of high resistance consisting of MDR and XDR forms to anti-tuberculosis drugs using this method.

A study on demographic characteristics of drug resistant Mycobacterium tuberculosis isolates in Belarus.

OBJECTIVE: A descriptive study of drug-resistance patterns by age group and among culture-positive pulmonary tuberculosis (TB) patients referred to the Research Institute for Pulmonology and Phthisiology of Belarus between January 2007 and January 2008. METHODS: Drug susceptibility tests were performed for first- and second-line anti-tuberculosis drugs. Patients were clustered into five resistance categories: mono-resistant (Mono); multi-drug resistant (MDR); all first-line drug resistance (MDR+ES); and extensively drug resistant (XDR). The patients were divided into primary and secondary and into six groups based on age in years (<15, 15-24, 25-44, 45-54, 55-65, and >65). RESULTS: An analysis was undertaken of information gathered from 934TB patients, of whom 660 were men (70.67±1.5%) and 274 were women (29.33±1.5%) (p<0.001). In the age group 25-65years, men outnumbered women between 2.7 and 9.0 times higher. Cases of secondary TB totaled 414 (52.02±1.77%), and primary cases totaled 382 (47.98±1.77%) (p>0.05); 756 of the patients were of working age, and 170 were of non-working age, of whom 570 men of working age (18-60years) and 188 women of working age (18-55years) participated. Males were significantly more likely to have MDR-TB than females. All cases with XDR-TB were older than 14years old. CONCLUSION: As Belarus is a high-burden MDR-TB country and treatment of drug-resistant TB is long and complicated, the findings of this study provided useful information to deliver effective community-based disease control measures and a proposed plane for the effective management of drug-resistant TB at the national level.

Study of promoter and structural gene sequence of whiB7 in MDR and XDR forms of Mycobacterium tuberculosis / Estudio del promotor del ADN y la secuencia de genes estructurales de whiB7 en las formas MDR y XDR de Mycobacterium tuberculosis

Resistance phenomenon in M tuberculosis is mainly based on decreased permeability of the bacterial envelope and function of effluent pumps. The regulatory gene of the whiB7 transcription determines drug resistance in these bacteria. Increases in WhiB7 protein activity induce transcription of resistance genes leading to intrinsic multidrug resistance. The aim of this work was to evaluate the whiB7 gene sequence in susceptible, MDR and XDR clinical isolates of M tuberculosis in order to further design an inhibitor. Thirty-three clinical isolates of MTB identified as susceptible, MDR and XDR-TB were investigated by PCRfor sequencing of the entire promoter (429 bp), structural gene (279 bp) and the end of the upstream gene uvrD (265 bp). No differences were detected in the sequences of the structural gene in susceptible and MDR with XDR isolates and all of them terminated at TGA as stop codon. Examination of sequence profiles of the promoter part of whiB7 by several sets ofprimers proved that there were no differences between sequence ofsusceptible, MDR and XDR isolates by type strain (H37Rr). Furthermore, the structure of WhiB7 protein was studied in achieved sequences from clinical isolates. We found that the promoter and structural gene of whiB7 are highly conservative in clinical susceptible and resistant isolates. It is a key finding that would assist in the design ofan inhibitor for the WhiB7 protein in all clinical forms in further studies.

El fenómeno de resistencia en M tuberculosis se basa principalmente en la disminución de la permeabilidad de la envoltura bacterial y la función de las bombas efluentes. El gene regulador de la trascripción de whiB7 determina la resistencia al medicamento en estas bacterias. Los aumentos en la actividad de proteína de WhiB7 inducen la trascripción de genes de resistencia que llevan a la resistencia intrínseca de multimedicamentos. El objetivo de este trabajo fue evaluar la secuencia de genes de whiB7 en aislados clínicos susceptibles MDR y XDR de M tuberculosis para mejorar el diseño de un inhibidor. Treinta y tres aislados clínicos de MTB identificados como MDR y XDR-TB susceptibles, fueron investigados por PCR para la secuenciación del promotor entero (429 bp), el gene estructural (279 bp) y el extremo del uvrD gen arriba (265 bp). No se detectó diferencia alguna en las secuencias del gene estructural en aislados susceptibles, MDR y XDR, terminando todos ellos en TGA como codón de terminación. El examen de perfiles de la secuencia de la parte de promotor de whiB7 por varios conjuntos de iniciadores (primers), demostró que no había ninguna diferencia entre la secuencia de aislados susceptibles MDR y XDR por tipo de cepa (H37Rv). Además, la estructura de la proteína de WhiB7 se estudió en secuencias logradas de aislados clínicos. Se encontró que el promotor y el gene estructural whiB7 son muy conservadores en aislados clínicos susceptibles y resistentes. Se trata de un hallazgo clave que ayudaría a designar un inhibidor para la proteína WhiB7 en todas las formas de este patógeno en estudios ulteriores.

Study of promoter and structural gene sequence of whiB7 in MDR and XDR forms of Mycobacterium tuberculosis.

Resistance phenomenon in M tuberculosis is mainly based on decreased permeability of the bacterial envelope and function of effluent pumps. The regulatory gene of the whiB7 transcription determines drug resistance in these bacteria. Increases in WhiB7 protein activity induce transcription of resistance genes leading to intrinsic multidrug resistance. The aim of this work was to evaluate the whiB7 gene sequence in susceptible, MDR and XDR clinical isolates of M tuberculosis in order to further design an inhibitor. Thirty-three clinical isolates of MTB identified as susceptible, MDR and XDR-TB were investigated by PCR for sequencing of the entire promoter (429 bp), structural gene (279 bp) and the end of the upstream gene uvrD (265 bp). No differences were detected in the sequences of the structural gene in susceptible and MDR with XDR isolates and all of them terminated at TGA as stop codon. Examination of sequence profiles of the promoter part of whiB7 by several sets of primers proved that there were no differences between sequence of susceptible, MDR and XDR isolates by type strain (H37Rr). Furthermore, the structure of WhiB7 protein was studied in achieved sequences from clinical isolates. We found that the promoter and structural gene of whiB7 are highly conservative in clinical susceptible and resistant isolates. It is a key finding that would assist in the design of an inhibitor for the WhiB7 protein in all clinical forms in further studies.