[MOLECULAR CHARACTERISTICS OF THE MULTIDRUG-RESISTANT MYCOBACTERIUM TUBERCULOSIS STRAINS IN THE NORTHWEST RUSSIA].

The goal of this work was to study the genotypic characteristics of the multidrug-resistant (MDR, i.e., resistant to at least rifampicine and isoniazid) Mycobacterium tuberculosis strains isolated in 2011-2012 from tuberculosis (TB) patients in the Northwest Russia. Spoligotyping of 195 M. tuberculosis isolates identified 14 different spoligotypes and assigned isolates to the genetic families Beijing (n = 162, 83%), LAM (n = 15), H3/URAL (n = 14), as well as T, Haarlem and X. Spoligotypes SIT1 (Beijing), SIT42 (LAM) and SIT262 (H3/URAL) were the most prevalent. Irrespective to the genotype, all the isolates were resistant to streptomycin. The multidrug resistance was accompanied by the resistance to ethionamide (56%), amikacin (31%), kanamycin (40%), and capreomycin (33%). The ethambutol resistance was found in 71% (n = 115) and 42% (n = 14) of the Beijing and non-Beijing strains, respectively (p < 0.05). In conclusion, the multidrug resistant M. tuberculosis population circulating in the Northwest Russia continues to be dominated by the Beijing family strains.

[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).

[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.

[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.

[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.

[The problem of mixed mycobacterial infections--mycobacteriosis and tuberculosis].

Mycobacteriosis occurs in our country as sporadic cases; nevertheless the clinical significance of this disease is great since its course is severe and its treatment presents great difficulties. Eighteen patients with mycobacteriosis caused by M. avium (n = 14), M. malmoense (n = 2), M. kansasii (n = 1), and M. xenopi (n = 1) were followed up. Subsequently, all the examinees fell ill with tuberculosis due to nosocomial infection. The change of a pathogen was bacteriologically documented by the isolation of M. tuberculosis. Ten tuberculosis-infected persons died. In this connection, it is necessary to legislatively determine the status of mycobacteriosis as an independent entity, to develop a proposition on mycobacteriosis, including its diagnosis, treatment, and follow-up, as well as to enhance the knowledge of phthisiologists on how to manage patients with mycobacteriosis, and to improve the bacteriological diagnosis of the disease.

[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.

[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.

[Lung tissue destructive lesions caused by nontuberculous mycobacteria]. / Destruktivnye porazheniia legochnoi tkani, vyzvannye netuberkuileznymi mikobakteriiami.

Forty-two patients with pulmonary mycobacteriosis were clinically characterized. In most patients, mycobacteriosis developed in the presence of residual changes in the lungs after cured tuberculosis. The torpid onset of mycobacteriosis with a gradual augmentation of symptoms of intoxication was observed. All the examinees were found to have destructive lung tissue changes that were characterized by the presence of caverns with a significantly transformed lung pattern.

[BCG osteitis in children (algorithms of diagnosis and treatment)]. / BTsZh-ostity u detei (algoritmy diagnostiki i lechenia).

In 156 children with osseous complications due BCG vaccination, the clinical features of disease, radiation and laboratory changes were studied. An algorithm of diagnosis of BCG ostitis and a system of organizational measures whose implementation make it possible to objective the statistical registration of BGC ostitis in Russia are proposed.

[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.

[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.

[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.

[Characteristics of non-tuberculous Mycobacterium -- pathogens of diseases in man]. / Kharakteristika netuberkuleznykh mikobakterii -- potentsialnykh vozbuditelei zabolevanii cheloveka.

The paper characterizes nontuberculosis mycobacteria (NTMB) by their species, shows the structure of mycobacteriasis pathogens, drug sensibility in NTMB and the degree of their virulence for laboratory animals. The specific proportion of NTMB is Mycobacterium avium (50.8%). A total of 136 cases of mycobacterium infection were examined. As high as 20.7% of isolates from mycobacteriasis patients have been found to have a high natural resistance to most antibacterial drugs. A combination of kanamycin, ethionamide, and ethambutol is the most effective in the treatment, additionally aminoglucosides--amikacin, gentamicin, sulfamides, such as biseptol, sulfadimethoxin, as well as the quinolones ciptobay and tarivid.

[Determination of the activity of broad-spectrum antibiotics against Mycobacterium fortuitum by the agar diffusion method]. / Opredelenie aktivnosti antibiotikov shirokogo spektra deistviia protiv Mycobacterium fortuitum metodom diffuzii v agare.

Rapidly growing M. fortuitum bring about such conditions in man, like mycobacterioses. Management of patients presents a problem since potentially pathogenic mycobacteria are highly resistant to most antituberculous drugs. To find effective antibacterial drugs for the treatment of human patients, the definition of sensitivity of M. fortuitum to broad--spectrum antibiotics by agar disk-diffusion method on Soton's medium was carried out. Sensitivity of 53 strains of mycobacteria to 13 drugs was investigated. It was demonstrated that the aminoglycoside group (gentamicin, kanamycin and monomycin inhibiting the growth of 96, 98 and 76% % of the strains, respectively) had been the most active. Tetracycline, ristomycin and erythromycin actively affected 35-15% of the tested strains. The agar disk-diffusion method is easy to handle and use in laboratory conditions.

[Clinical course of mycobacteriosis in young persons]. / Techenie mikobakterioza u lits molodogo vozrasta.

Clinical manifestations of mycobacteriosis in 22 patients aged 16-35 are described. In males and females the disease was caused by M. avium and M. fortuitum, predisposed by exposure to occupational hazards and previous bronchopulmonary and general somatic diseases, respectively. In x-ray pictures of the lungs there appeared unilateral lesion in the upper lobe of the right lung.

[The tactics of management of patients with mycobacteriosis of the lungs]. / K voprosu o taktike vedeniia bol'nykh mikobakteriozom legkikh.

Recently, pulmonary mycobacteriosis patients more often attract researchers' attention. On the basis of observations at the institute clinic and the literature data an opinion was voiced concerning diagnosis, registration and taking on record, therapeutic tactics and dispensary follow-up. Discussion of the work by phthisiatrists who follow up these patients will contribute to the elaboration of a unified effective approach to pulmonary mycobacteriosis patients.

[Plasmid profile of mycobacteria spread on the territory of the Russian Federation]. / Plasmidnyi profil' mikobakterii, rasprostranennykh na territorii Rossiiskoi Federatsii.

The plasmid profile of different species of mycobacteria vegetating in the Russian Federation was first studied. All the drug-resistant mycobacterial strains of tuberculosis were found to be truly polyresistant. There was a certain association between the plasmid carriage and the high viability of the mycobacterial population in nontuberculous mycobacteria.