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Zimenkov D.V.,RAS Engelhardt Institute of Molecular Biology | Kulagina E.V.,RAS Engelhardt Institute of Molecular Biology | Antonova O.V.,RAS Engelhardt Institute of Molecular Biology | Krasnova M.A.,Moscow Scientific and Clinical Antituberculosis Center | And 6 more authors.
Journal of Clinical Microbiology | Year: 2015

In addition to the obligatory pathogenic species of the Mycobacterium tuberculosis complex and Mycobacterium leprae, the genus Mycobacterium also includes conditionally pathogenic species that in rare cases can lead to the development of nontuberculous mycobacterial diseases. Because tuberculosis and mycobacteriosis have similar clinical signs, the accurate identification of the causative agent in a clinical microbiology laboratory is important for diagnostic verification and appropriate treatment. This report describes a low-density hydrogel-based microarray containing oligonucleotide probes based on the species-specific sequences of the gyrB gene fragment for mycobacterial species identification. The procedure included the amplification of a 352-nucleotide fragment of the gene and its hybridization on a microarray. The triple-species-specific probe design and the algorithm for hybridization profile recognition based on the calculation of Pearson correlation coefficients, followed by the construction of a profile database, allowed for the reliable and accurate identification of mycobacterial species, including mixed-DNA samples. The assay was used to evaluate 543 clinical isolates from two regions of Russia, demonstrating its ability to detect 35 mycobacterial species, with 99.8% sensitivity and 100% specificity when using gyrB, 16S, and internal transcribed spacer (ITS) fragment sequencing as the standard. The testing of clinical samples showed that the sensitivity of the assay was 89% to 95% for smear-positive samples and 36% for smear-negative samples. The large number of identified species, the high level of sensitivity, the ability to detect mycobacteria in clinical samples, and the up-to-date profile database make the assay suitable for use in routine laboratory practice. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Balasanyants G.S.,Research Institute of Phthisiopulmonology | Sukhanov D.S.,St. Petersburg State Medical Academy
Terapevticheskii Arkhiv | Year: 2011

The review presents principles of pathogenetic therapy in tuberculosis and current trends in pharmacotherapy: immunocorrection, prophylaxis, chemotherapy side effects management. mmunocorrection is illustrated by treatment with drugs of a polyvalent action - glutoxim and cyclopheron. Mechanisms of their immune action are outlined. As to hepatoprotection, remaxol is an effective hepatoprotecting medicine improving energy supply to hepatocytes and their membranes resistance to lipid peroxidation, reactivating enzymes of antioxidant defense. Remaxol has an antioxidant/antihypoxic action, is the most promosing metabolic corrector with hepatoprotective activity.

Zimenkov D.V.,RAS Engelhardt Institute of Molecular Biology | Kulagina E.V.,RAS Engelhardt Institute of Molecular Biology | Antonova O.V.,RAS Engelhardt Institute of Molecular Biology | Zhuravlev V.Y.,Research Institute of Phthisiopulmonology | Gryadunov D.A.,RAS Engelhardt Institute of Molecular Biology
Journal of Antimicrobial Chemotherapy | Year: 2016

Background: Nucleic acid amplification tests are widely used in TB diagnostics. Priority tasks in their development consist of increasing the specificity and sensitivity of the detection of resistance to a wide spectrum of anti-TB drugs. Methods: We developed a multiplexed assay allowing the detection of 116 drug resistance-determining mutations in the rpoB, katG, inhA, ahpC, gyrA, gyrB, rrs, eis and embB genes in the Mycobacterium tuberculosis complex genome and six SNPs to identify the main lineages circulating in Russia. The assay is based on the amplification of 17 fragments of the genome using the universal primer adapter technique and heat pulses at the elongation step, followed by hybridization on a microarray. Results: The method was evaluated using 264 pairs of clinical samples and corresponding isolates. A significant proportion (25%) of smear-negative samples were correctly analysed by microarray analysis in addition to 96% of smear-positive samples. The sensitivity and specificity of the assay exceeded 90% for rifampicin, isoniazid, ofloxacin and second-line injection drugs. In agreement with previous studies, the specificity of ethambutol resistance was as low as 57%, while the sensitivity was 89.9%. Strong association of the Beijing lineage with a resistant phenotype was observed. Euro-American lineage strains, excluding Ural and LAM, were predominantly associated with the susceptible phenotype. Conclusions: The developed test has a high sensitivity and specificity and can be directly applied to clinical samples. The combination of mutation-based drug resistance profiling and basic genotyping could be useful for clinical microbiology studies and epidemiological surveillance of the M. tuberculosis complex. © The Author 2016.

Gomes L.L.,Instituto Oswaldo Cruz | Vasconcellos S.E.G.,Instituto Oswaldo Cruz | Gomes H.M.,Instituto Oswaldo Cruz | Elias A.R.,Instituto Oswaldo Cruz | And 11 more authors.
Tuberculosis | Year: 2015

SummaryIntroduction The success of the Mycobacterium tuberculosis Beijing (MtbB) lineage in different geographical regions has been attributed to high transmission, increased virulence, drug resistance and rapid adaptation to the host. In some countries of secondary MtbB dispersion like South Africa and Peru, rising prevalence of the Beijing strains is registered. However, in neighboring countries to affected regions such as Mozambique and Brazil, respectively, the prevalence of these strains is still low and this could be due to biological particularities of the circulating MtbB strains and/or differentiated host susceptibility. Objective To characterize genetically and phenotypically MtbB strains isolated in Brazil (n = 8) and Mozambique (n = 17). Methods This is a descriptive study of genotypes of the MtbB isolates, determined by spoligotyping, MIRU-VNTR typing, analysis of the IS6110 copy number in the NTF region and screening for mutations in mutT2, mutT4, rpoB, katG and pks 15/1 genes. Virulence-associated properties of the studied isolates were verified in the in vitro model of infection of human THP-1 cells. Results The genotypes defined by the 24VNTRs were distinct for all isolates included in this study and presented an HGDI of 0.997. The VNTR patterns with seven copies of MIRU26 and seven copies of QUB26, representative for the previously described MtbB genotype B0, dominant in Russia, were detected in 38.5% of the studied isolates. In addition, all isolates presented RD105 deletion and a 7 bp insertion in pks15/1 gene. Almost all tested strains belonged to the RD181 sublineage, with the exception of two strains from Mozambique of RD150 sublineage. Combined analysis of the NTF region integrity and mutations in mutT genes showed that 62.5% and 47% of isolates obtained in Brazil and Mozambique, respectively, were of the ancestral genotype. The virulence index of the ancient isolates, evaluated in the THP-1 cells, was significantly lower than that of the modern genotype group. Conclusions These data demonstrate genotype particularities of the Beijing strains isolated in Brazil and Mozambique, two countries of low prevalence of the MtbB lineage in local Mtb populations. In contrast to the neighboring countries with high prevalence of the MtbB strains of modern sublineage, significant proportions of the isolates obtained in Brazil and Mozambique were presented by the strains of the ancient sublineage. Our data suggest that lower virulence of the ancient strains, compared with the modern strains, could be involved in the slow spread of the MtbB strains in some regions. © 2015 Elsevier Ltd. All rights reserved.

Lasunskaia E.,State University of Norte Fluminense | Ribeiro S.C.M.,State University of Norte Fluminense | Manicheva O.,Research Institute of Phthisiopulmonology | Gomes L.L.,Instituto Oswaldo Cruz | And 13 more authors.
Microbes and Infection | Year: 2010

The epidemiologically important Mycobacterium tuberculosis Beijing genotype strains, highly endemic in East Asia, have become an emerging infection in certain geographic areas, including Russia, because of its increasing prevalence and association with multidrug resistance (MDR). The aim was to verify whether MDR Beijing strains circulating in the emerging regions present some biological particularities that could contribute to their success in causing disease in comparison with the sporadic strains from locations with low prevalence of the Beijing genotype. We evaluated virulence-associated characteristics of the MDR Beijing strains isolated in Russia and compared them with those of the drug-resistant and susceptible Beijing strains from Brazil and reference H37Rv strain. We found that Russian MDR strains demonstrated an increased bacterial fitness and growth in THP-1 macrophage-like cells, as well as a higher capacity to induce non-protective cytokine synthesis and necrotic macrophage death. By contrast, the biological properties of the strains isolated in Brazil largely resembled those of the H37Rv strain, with the exception of the drug-resistant isolates that presented significantly reduced fitness. The data demonstrate that the emerging MDR strains of the Beijing genotype circulating in Russia do express a pattern of properties associated with the enhanced virulence favouring its clonal dissemination in this region. © 2010 Elsevier Masson SAS. All rights reserved.

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