[The genome polymorphism of the Mycobacterium avium subsp. hominissuis strains].

The non-tuberculosis mycobacteria Mycobacterium avium subsp. hominissuis (MAH) are able to cause human mycobacteriosis. In this work, the results of the first comprehensive study of the genome polymorphism of the clinical strains of MAH were reported using the typing scheme by 13 loci MATR-VNTR (TR292, TRX3, TR25, TR47, MATR-1, MATR-4, MATR-5, MATR-6, MATR-8, MATR-11, MATR-14, MATR-15, MATR-16) containing tandem nucleotide sites and IS1245-RFLP-typing sites. A total of 90 MAH strains isolated from patients with lung mycobacteriosis without epidemiological connection (including HIV infected) were tested in 2008-2011. The inhomogeneity of the MAH strains by 36 profiles of 13 loci MATR-VNTR was observed. The majority of the strains (68.8%) were included in the 8 MATR-VNTR clusters; most large cluster contained 37 strains with 13-bitnumerical profile 2222223145443'. The nucleotide sequence of the MATR-16 (3') locus contains the long deletion (GenBank accession no. KF479191). The MAH strains of the MATR-VNTR clusters were found to be inhomogeneous by the IS1245 marker. The MATR-VNTR-typing method by 13 loci is recommended for preliminary differentiation of domestic MAH strains with further analysis of the MATR-VNTR clusters using the IS1245-RFLP-typing method.

[Genotypic characteristics of Mycobacterium avium subsp. hominissuis strains].

Mycobacterium avium are typical environmental, non-tuberculosis microorganisms that occasionally cause mycotuberculosis, an infectious disease in wild and domestic animals, birds, and humans. Here, we report the results of the first study on the genetic diversity of the Russian population of M. avium. A total of 85 M. avium subsp. hominissuis (MAH) clinical strains were isolated from patients (including 30 HIV-positive individuals) with mycobacteriosis in St. Petersburg, 2008-2011. The biochemical identification of the microorganisms was carried out using the PCR detection of the mobile elements IS901 and IS900, as well as of the polymorphism of restriction fragments of the hsp65 gene. The genetic diversity of the isolates was evaluated by VNTR typing based on eight variable-number tandem repeats (VNTRs) (292, X3, 25, 47, 3, 7, 10, and 32 [Thibault et al., 2007]). The MAH population studied was characterized by 15 VNTR types, including nine unique patterns and six clusters of isolates with identical eight-digit profiles. The largest clusters (22221128 and 24221128) included 45 (59.2%) and 15 (19.7%) isolates, respectively; the others contained from 2-7 strains. The strains of the cluster 2533112'8 possessed a truncated TR10 locus (allele 2'). Taking into account the absence of the epidemiological links between the patients and the fact that the infection was delivered from the environment, the high rate of clustering of MAH isolates can be explained by the low discriminatory power of the eight-locus VNTR-typing scheme (HGDI 0-0.61).

[Molecular-genetic characteristics of Mycobacterium tuberculosis strains isolated from patients with tuberculous spondylitis].

AIM: Molecular-genetic characteristic of M. tuberculosis strains isolated from operation material of patients with tuberculous spondylitis. MATERIALS AND METHODS: 107 strains of M. tuberculosis isolated in 2007 - 2011 from patients with spine tuberculosis were studied by methods of spoligotyping and MIRU-VNTR by 12 and 24 loci. RESULTS: Strains of genetic family Beijing dominated (n = 80), 78% of those had multiple drug resistance (MDR). Strains of genetic families T, H3 (Ural), LAM, Manu, H4 and S were also detected. Differentiating of 80 strains of Beijing genotype by MIRU-VNTR method by 24 loci revealed 24 variants (HGI = 0.83) including 7 clusters, the largest of those (100-32) included 23 strains (87% MDR). CONCLUSION: The leading role of Beijing genotype M. tuberculosis strains in development of tuberculous spondylitis with multiple drug resistance of the causative agent is shown.

[Characteristics of Mycobacterium tuberculosis strains circulating in Pskov region].

AIM: Characteristics of drug resistance (DR) and population structure of Mycobacterium tuberculosis in Pskov region. MATERIALS AND METHODS: In 90 strains of M. tuberculosis drug resistance was studied by culture method and by using "TB-BIOCHIP"; genotyping was determined by spoligotyping method. RESULTS: 55 (61.1%) of 90 M.tuberculosis strains had drug resistance, with 40 (44.4%) being multi-resistant. M. tuberculosis population was presented by SIT1 spoligotype strains of genetic families Beijing--44.4%, LAM--21.1%, T--14.4%, Haarlem--11.1% and Ural--5.6%, according to SpolDB4. Among M. tuberculosis strains circulating in Pskov region the most widespread (44.4%) was SIT1 spoligotype (p < 0.0001). DR and multi-resistant DR (MDR) in Beijing strains occurred more frequently than in "non-Beijing" strains (p < 0.001 and p = 0.03 respectively) and were determined by rpoB mutations Ser531-->Ley and katG Ser315-->Thr. All the SIT252 spoligotype strains were multi-resistant, and their resistance to rifampicin was determined by rpoB Asp516-->Ser substitution, to isoniazid --katG Ser315-->Thr and inhA_T15 substitutions. CONCLUSION: The data obtained gives evidence on tuberculosis epidemiological unfavorability and wide circulation of MDR M. tuberculosis strains in Pskov region.

[Molecular genetic tools for etiologic diagnostics of disseminated lung tuberculosis].

AIM: Improvement of etiologic diagnostics of disseminated lung tuberculosis (DLT) and determination of Mycobacterium tuberculosis (MBT) drug susceptibility on the basis of molecular genetic methods. MATERIALS AND METHODS: Samples from respiratory tract of patients with DLT were studied using real time polymerase chain reaction and the "TB-BIOCHIP" assay developed by Institute of Molecular Biology. Methods of spoligotyping and reverse hybridization were used for identification, genotyping and express-detection of drug resistance of MBT to rifampicin in sputum samples stained for bacterioscopy. RESULTS: In 76 (41.5%) of 183 patients with radiological signs of DLT, DNA of tuberculosis complex mycobacteria was detected in respiratory tract samples (specificity 87.7%); mutations in genes rpoB, katG, inhA as well as region ahpC-oxyR associated with resistance to rifampicin and isoniazide were revealed in 67% and 79.5% of patients with DLT respectively. In 48.8% of sputum samples, DNA of MBT of epidemically significant genotype Beijing associated with multidrug resistance of MBT in Russia was identified. CONCLUSION: Molecular genetic methods allow to use both fresh and archived respiratory tract specimens for rapid verification of DLT diagnosis during oligobacillar forms of tuberculosis as well as timely prescribe and correct the treatment regimen of the patient according to individual drug susceptibility spectrum of the agent.

[Monitoring of chemotherapy in patients with pulmonary tuberculosis by the blood bacteriostatic activity-determining test].

The paper gives the results of studies to determine blood bacteriostatic activity (BBA) in the use of a patient's autostrain and semiliquid medium versus the clinical and laboratory parameters of the course of a process in 101 patients with pulmonary tuberculosis. There is evidence for the relationship of the BBA to the sensitivity to isoniazid and the structure of drug resistance. The zero values of BBA correspond to the severest course of the disease. The efficiency of treatment is much higher in patients with high and moderate BBA. The latter's determination using the semiliquid medium permits an objective evaluation of the efficiency of chemotherapy, identification of patients with a poor prognosis, and then choice of an individual treatment regimen on day 7 after the test just before obtaining the data on drug sensitivity.

[The rate and pattern of drug resistance in Mycobacterium tuberculosis at various sites of the disease].

Drug resistance of Mycobacterium tuberculosis (MBT) to essential and reserve antituberculous drugs (ATD) was monitored in patients with tuberculosis of respiratory organs (ROT) and at extrapulmonary sites (EPT) in 2005-2007. The standard indirect absolute concentration technique was used to study the drug resistance of 578 MBT strains from pretreated patients with ROT and that of 159 MT strains from those with EPT. With all sites of tuberculosis, a poor trend continues to show an increase in the total frequency of MBT resistance (from 82.3 to 85.2% in ROT and from 43.7 to 59.3% in EPT) and its severer pattern due to MRD (from 69.5 to 78.0% in patients with ROT and from 20.8 to 35.5% in those with EPT). The rate of MTB resistance to 4 ATDs or more is 73.7 and 34.6% in patients with ROT and in those with ERT, respectively. The total resistance of MBT to 7-8 ATDs was revealed in every three patients with ROT (33.0%) and in 12.6% of those with EPT.

[Drug sensitivity in Mycobacterium tuberculosis versus its viability, cytotoxicity, genotype, and the course of the process in patients with pulmonary tuberculosis].

The authors studied drug sensitivity, mutations in the katG, in-hA, alpC, rpoB genes, virulence via the cytotoxicity test on THP-1 cells, and the viability and genetic affiliation of 53 clinical M. tuberculosis isolates versus data on the form and dynamics of a process. Sensitive and resistant strains did not significantly differ in viability and cytotoxicity. The highest death of infected macrophages was observed was seen with infection of M. tuberculosis of the Beijing B0 genotype, the least one seen with that of LAM with the similar rate of multiple drug resistance. There was a correlation of the changes in the count of lymphocytes in patients with the genetic affiliation of a causative agent. The severest course of the tuberculous process was observed in baseline lymphopenia (before treatment) in combination with multidrug resistance of mycobacteria, high and moderate cytotoxicity and high viability. Ser-Leu 531 mutation resulted in cross resistance to rifampicin and mycobutin in most cases.

[Some regularities of an immune response in patients with pulmonary tuberculosis and drug-resistant Mycobacterium strains].

Clinical and immunological studies were conducted in 62 patients with infiltrative pulmonary tuberculosis and 53 with disseminated one. As drug resistance and viability of mycobacteria increased, an immune response was found to develop as the humoral type and cellular immunity was suppressed. A more marked reduction in the activity of T helper cells type 1 of an immune response and the neutrophilic granulocytic system was revealed in patients with disseminated pulmonary tuberculosis. The maximum suppression of cell immunity was found in patients with multidrug-resistant mycobacterial strains. The observed changes in an immune response and in the production of cytokines (IL-2, IL-8) are informative signs correlating with deterioration of a specific process. Mycobacterial drug resistance and its suppressed cell immunity make chemotherapy policy difficult. The findings identify patients with mycobacterial drug resistance and high viability as a group of priority in the context of immunomodulator therapy.

[The specific features of bacterial isolation and drug resistance of Mycobacteria in extrapulmonary tuberculosis].

The reduced viability and enzymatic activity of the pathogen from the foci of extrapulmonary tuberculosis are twice more frequently than those in pulmonary tuberculosis. The isolation of mycobacterial L-forms and the use of polymerase chain reaction significantly enhance the efficiency of the etiological diagnosis of extrapulmonary tuberculosis. There are extremely poor trends for drug resistance to develop in mycobacteria, in 2005 the rate of which was 92.5% in pulmonary tuberculosis and 83.4% in extrapulmonary tuberculosis. However, multidrug resistance is much less common in extrapulmonary tuberculosis than in pulmonary tuberculosis; the rates were 33.9 and 73.9%, respectively. Levofloxacin resistance was also completely preserved so the reserves for the treatment of extrapulmonary tuberculosis are higher than those in pulmonary tuberculosis.

[Levofloxacin susceptibility of Mycobacterium tuberculosis].

Drug susceptibility of 195 M. tuberculosis clinical isolates was tested with the indirect method of absolute concentrations on the Löwenstein-Jensen medium. 151 and 44 M. tuberculosis strains were isolated from patients with respiratory tract tuberculosis and tuberculosis of extrapulmonary localization respectivily. The resistance of the isolates to the main and reserve antituberculosis drugs amounted to 79.5%, including 62.1% of the isolates with multiple drug resistance. Levofloxacin was used in a concentration of 10 mcg/ml. The resistance to levofloxacin was detected only in 4 isolates from the patients with respiratory tract tuberculosis (2.05%), whereas the resistance to ofloxacin amounted to 11.8%.

[Immunological parameters and mycobacterial biological properties in infiltrative pulmonary tuberculosis].

Thirty-one patients with infiltrative pulmonary tuberculosis were clinically and immunologically examined. A relationship was found between the definite immunological parameters and the biological properties of mycobacteria. The higher viability of mycobacteria and the increase in the drug resistance have been shown to be associated with the decreased activity of lymphocytes and the suppressed production of interleukin-2, which suggests a decrease in the activity of type 1 T helper cells and a significant increase in the synthesis of tuberculosis antibodies with the lower serum concentrations of circulating immune complexes. This is an informative indicator of a severe specific process, as previously established. Moreover, the authors show certain differences in the changes of immunological parameters in patients with infiltrative pulmonary tuberculosis with varying mycobacterial properties. Thus, the results of the performed study make investigators pay a particular attention to the immunopathogenetic value of the biological properties of Mycobacterium tuberculosis.

[Bacteriological diagnosis of mycobacteriosis: possibilities and prospects]. / Vozmozhnosti i perspektivy bakteriologicheskoi diagnostiki mikobakterioza.

The results of identification of Mycobacterium non-tuberculosis isolated in the North-West Region of the Russian Federation, which have been obtained in the past 20 years, are presented. The identification was made by bacteriological and biochemical studies. M. avium-intracellulare complex is the man causative agent of mycobacteriosis. The currently available methods of chromatography and molecular genetics, which are designed for accelerated detection and species-specific identification of Mycobacterium, are briefly reviewed.

[Pulmonary tuberculosis caused by Mycobacterium tuberculosis of different genotypes]. / Tuberkulez legkikh, vyzvannyi Mycobacterium tuberculosis razlichnykh genotipov.

A relationship of the clinical picture of destructive pulmonary tuberculosis to the genotype, drug resistance, and virulence of Mycobacterium tuberculosis (MBT) strains was studied. As compared with the processes induced by individual genotypes, pulmonary tuberculosis caused by MBT from the family Beijing was found to be characterized by more marked clinical symptoms, multisegmental lung tissue lesions with multiple decay cavities, by low bacteriostatic blood activity, abundant bacterial isolation, and progression. MBT of the Beijing genotype were characterized by higher rates of resistance to antituberculous drugs and their combinations than were the strains of individual genotypes; they have a high virulence, which is likely to enhance their transmissibility and to determine the poor course of a specific process.

[Microbiological and molecular genetic characterization of Mycobacterium tuberculosis strains isolated from patients with multiorgan and generalized tuberculosis]. / Mikrobiologicheskaia i molekuliarno-geneticheskaia kharakteristika shtammov Mycobacterium tuberculosis, vydelennykh ot bol'nykh poliorgannym i generalizovannym tuberkulezom.

The paper presents the microbiological and molecular genetic characteristics of Mycobacterium tuberculosis (MBT) isolated from multiple lesion foci in 26 patients with multiorgan and generalized tuberculosis. Cultures of MBT of the family Beijing were isolated from the pathological specimens taken from 17 (65.4%) patients; those with individual genotypes were in 9 (34.6%) patients. The study indicated that cultured MBT from different types of pathological material from 25 of the 26 patients had identical spoligotypes irrespective of their clinical diagnosis. At the same time minor profile IS6110 changes might occur in the Mycobacteria of a patient during persistence. It may be concluded that multiple foci of tuberculous affliction in the patients are of unique origin and have the mechanism of development, associated with endogenous reactivation of infection.

[Accelerated determination of the Mycobacterium tuberculosis drug resistance with a semiliquid culture medium]. / Uskorennoe opredelenie lekarstvennoi ustoichivosti Mycobacterium tuberculosis s pomoshch'iu poluzhidkoi pitatel'noi sredy.

A medium was worked out whose consistence is close to liquid ones (the terminal concentration of agar-agar is 0.08%) and which enable a visual registration of growth of Mycobacterium tuberculosis (MBT) on days 3-4 after culture inoculation. The medium is based on the liquid synthetic medium of Soton, which is added 0.35% of nutrition agar and 10% (medium 1) or 25% (medium 2) of horse serum. The elaborated semi-liquid medium was used simultaneously with the standard method of absolute concentrations base on Levenstein-Jensen's medium to determine the drug resistance of 294 MBT clinical strains. The highest coincidence rate of results, in percent, was found for medium 2: for streptomycin--90.1%, for isoniazid--96.7%, and for rifampycin--96.3%, therefore, the mentioned medium can be referred to as an optimal one in a fast determination of the MBT resistance to three main anti-TB drugs. The deadline of registering the results for sensitive strains is 7 days, and for resistant strains it is 3-4 days, i.e. after the onset of primary growth signs.

[Drug resistance of Mycobacterium tuberculosis in the North-West of Russia]. / Lekarstvennaia ustoichivost' mikobakterii tuberkuleza na severo-zapade Rossii.

The morbidity of primary and secondary drug resistance (DR) of Mycobacterium tuberculosis (MBT) was studied in the North-West of Russia during 1991-2001. The frequency rate and structure of, mainly, secondary DR MBT was investigated in tuberculosis of different localizations on the basis of data provided by clinics of Saint-Petersburg Research Institute for Phthisiopulmonology (PRIP) during 1990-2000. A sharp increase of primary DR MBT (in the North-West of Russia) up to 35-45.5% was detected in the Murmansk, Arkhangelsk and Saint-Petersburg Regions as well as in the Republic of Karelia, an increase of up to 61% was registered in the Kaliningrad Region. The frequency rate of secondary DR MBT went up by 1.5-2 times reaching 80-86% in the Murmansk, Arkhangelsk and Kaliningrad Regions. According to the PRIP clinics, there is also an essential growth of DR MBT in all tuberculosis localizations with a trend of the stability structure shifting towards poly-resistance, which accounts for 65.2% in pulmonary tuberculosis and for 33.6% in extra-pulmonary tuberculosis.

[Sensitivity to levofloxacin of Mycobacterium tuberculosis strains with various phenotype isolated from patients with newly diagnosed and chronic tuberculosis]. / Chuvstvitel'nost' k levofloksatsinu shtammov Mycobacterium tuberculosis s razlichnym genotipom, vydelennykh ot bol'nykh s vpervye vyiavlennym i khronicheskim tuberkuleznym protsessom.

Susceptibility to levofloxacin of 56 strains of Mycobacterium tuberculosis, isolated from 22 patients with first estimated and 84 patients with chronic tuberculosis was investigated. Ratio of multi-drug resistance achieved 54.5 per cent in first group and 94.1 per cent in the second group. 23 (41.1 per cent) isolated strains belonged to individual genotypes, 33 (58.9 per cent)--to Beijing group. All mycobacteria isolates were sensitive to critical concentration of levofloxacin--10 mcg/mL. The results of the investigation proves the ability to recommend levofloxacin application at the patients with first estimated tuberculosis and at the patients with most hazardous tuberculosis pathogen of Beijing group.

[Sensitivity of Mycobacterium tuberculosis to levofloxacin]. / Issledovanie chuvstvitel'nosti mikobakterii tuberkuleza k levofloksatsinu.

Susceptibility of 92 strains of mycobacteria to levofloxacin (5, 10 and 50 mcg/mL) was investigated by indirect method of absolute concentrations on Levenstain-Jensen media. The investigation was performed on 85 strains of Mycobacterium tuberculosis isolated from 83 patients with different types of tuberculosis and also on drug-sensitive laboratory strains of M. tuberculosis H37Rv-M, H37Rv-GISK, Academia, M. bovis-bovinus 8, M. bovis BCG and on two strains of M. fortuitum with total resistance to antimycobacterial agents. 87.8 per cent of clinical isolates were multi-drug resistant. From one patient treated with ciprofloxacin two strains of M. tuberculosis were isolated--one resistant to 5 mcg/mL of levofloxacin and second strain-resistant to 10 mcg/mL of levofloxacin. All other clinical and laboratory strains of mycobacteria (97.8 per cent) were susceptible to all three concentrations of levofloxacin.

[Sensitivity to levofloxacin of various types of non-tuberculosis Mycobacterium]. / Chuvstvitel'nost' k levofloksatsinu razlichnykh vidov netuberkuleznykh bakterii.

Activity of levofloxacin (Tavanic) against 10 species of nontuberculosis of mycobacteria was investigated by indirect method of absolute concentrations on Levenstain-Jensen media (levofloxacin concentration 5 and mcg/mL). The investigation was performed on 71 strains of nontubercolosis mycobacteria: Mycobacterium avium-intracellulare--24 strains, M. fortuitum--17 strains, M. chelonae--10 strains, M. malmoense--13 strains and 6 other species of mycobacteria. Susceptible to critical levofloxacin concentration were 8 species of 10. Resistance to levofloxacin (10 mcg/mL) was estimated for 16.7 per cent of M. avium-intracellulare and 30 per cent of M. chelonei strains. It is concluded that levofloxacin may be a drug of choice for management of mycobacteriosis caused by M. fortuitum, M. kansasii, M. xenopi, M. malmoense, and in the most of cases due to M. avium-intracellulare and M. chelonae.