RESUMO
Multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis cases in the Ukraine are increasing. Pyrazinamide (PZA) is critically important for first- and second-line tuberculosis (TB) treatment regimes. However, PZA drug susceptibility testing is time consuming and technically challenging. The present study utilized Next-generation sequencing (NGS) to identify mutations in the pncA gene from clinical isolates and to assess the prevalence of pncA gene mutations in MDR/XDR-TB patients. Clinical isolates were inactivated in molecular transport media and shipped from Kharkiv, Ukraine, to San Antonio, TX. Whole-genome and targeted pncA gene sequencing was carried out using Illumina MiSeq instrumentation. Mutations were noted in 67 of 91 (74%) clinical isolates comprising substitutions, insertions, and deletions in the pncA coding and upstream promoter region. Of 45 mutation types, there were 11 novel, i.e., to date unknown, pncA mutations identified of which 3 were confirmed PZA resistant. Seven isolates contained mixed base mutations, whereas 4 harbored doubled mutations. Data reported here further support use of NGS for pncA gene characterization and may contribute in significant fashion to PZA therapy, especially in MDR- and XDR-TB patients.
Assuntos
Amidoidrolases/genética , Proteínas Mutantes/genética , Mutação , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mycobacterium tuberculosis/isolamento & purificação , Prevalência , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Ucrânia/epidemiologiaRESUMO
The Ukraine ranks among the top 20 countries with the highest number of multidrug-resistant (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis cases in the world. However, little is known of the genetic diversity, i.e., resistance signatures, in clinical isolates from this region. We analyzed seven of most prevalent MDR/XDR antibiotic resistance-conferring genes from clinical isolates (n = 75) collected from geographically diverse Ukrainian oblasts and the southern Crimean peninsula. Genomic analysis revealed that 6 (8%) were sensitive, 3 (4%) were resistant to at least one antibiotic but were not MDR, 40 (53%) were MDR, and 26 (35%) were XDR. The majority of isolates (81%) were of the Beijing-like lineage. This is the first study to use next-generation sequencing (NGS) of clinical isolates from the Ukraine to characterize mutations in genes conferring M. tuberculosis drug resistance. Several isolates harbored drug resistance signatures that have not been observed in other countries with high-burden tuberculosis. Most notably, the absence of inhA gene promoter mutations, a diversity of mutation types in the rpoB resistance-determining region, and detection of heteroresistance provide a broader understanding of MDR/XDR from this area of the world.
Assuntos
Tuberculose Extensivamente Resistente a Medicamentos/epidemiologia , Genes Bacterianos , Variação Genética , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Adulto , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/genética , Farmacorresistência Bacteriana Múltipla/genética , Tuberculose Extensivamente Resistente a Medicamentos/microbiologia , Feminino , Genoma Bacteriano , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Oxirredutases/genética , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Escarro/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Ucrânia/epidemiologiaRESUMO
In the conditions of high relative density of multidrug resistant tuberculosis in Ukraine, rapid diagnostics of M. tuberculosis susceptibility to antituberculous drugs is the important tool in treatment of patients with lung tuberculosis. 78 patients with lung TB that were treated in TB dispensary in Kharkiv during 2004-2005 years were analyzed. Mutations in 531 codon of rpoB gene of isolates M. tuberculosis were studied. Among 48 (61.5%) resistant isolates of M tuberculosis according to phenotypic method, mutations were found only at 29 (60.4%) isolates. Among 30 (38.5%) phenotypic sensitive M. tuberculosis isolates only 1 (3.3%) isolate had mutation in rpoB gene M. tuberculosis.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Mutação , Mycobacterium/genética , Rifampina/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Tuberculose Pulmonar/microbiologia , Antituberculosos/uso terapêutico , Códon , RNA Polimerases Dirigidas por DNA , Farmacorresistência Bacteriana Múltipla/genética , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium/efeitos dos fármacos , Mycobacterium/isolamento & purificação , Rifampina/uso terapêutico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Pulmonar/tratamento farmacológicoRESUMO
Endogenic intoxication (EI) tests were conducted in 191 patients with pulmonary tuberculosis. 40 healthy subjects served control. In tuberculosis patients EI depended on the disease clinical form, dissemination, destruction. Administration of detoxication therapy at the start of the combined treatment promoted rapid EI reduction. Due to detoxication, side effects of the drugs occurred 3 times less frequently, the efficacy of the treatment rose. Minor hemosorption resulted in detoxication effects superior to those of transfusions. EI classification is suggested, the prognostic value of EI tests is ascertained.
