[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.

[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.

[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.

[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.

[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.

[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 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.

[Characterization of Mycobacterium tuberculosis strains prevalent in North-West of Russia by spoligotyping]. / Kharakteristika tsirkuliruiushchikh na Severo-Zapade Rossii shtammov Mycobacterium tuberculosis s ispol'zovaniem spoligotipirovaniia.

Spoligotyping was used for genotyping of 238 M. tuberculosis cultures isolated from 302 patients with pulmonary tuberculosis living in the north-west of Russia, including those in Saint Petersburg, in 1998-2001, and the M. tuberculosis strains H37Rv, M. bovis, and M. bovic BCG and M. tuberculosis in the specimens painted for microscopy. The quality of spoligotyping was high and similar to that when DNA from the cultured mycobacteria and slide scrapes. The findings were compared with the data available in the International database. There was heterogenicity in the microbial population: the clinical strains showed 58 (RO-R57) types of profiles, of them 56% were revealed in single cases. The spoligotype RO(1; S1) was found in 153 (51%) patients. This spoligotype is predominant in the world and characteristic of polyresistant high-transmissive strains of the genetic family Beijing. The spoligotypes R24 (251), R14 (252), and R8 (253) first described by the authors are now encountered only in Russia. To extend the Russian database that contains M. tuberculosis spologotyping profiles from different regions will promote the improvement of an epidemiological surveillance system and tuberculosis control programmes not only within administrative territorial entities, but also within the whole country.

[Molecular genetic characteristics of Mycobacterium tuberculosis isolated from patients operated on for pulmonary tuberculosis]. / Molekuliarno-geneticheskaia kharakteristika shtammov Mycobacterium tuberculosis, vydelennykh ot bol'nykh, operirovannykh po povodu tuberkuleza legkikh.

The RFLP-IS6110 assay was used to genotype 67 Mycobacterium tuberculosis isolated from different specimens (including intraoperative ones) of 24 patients operated on for chronic progressive pulmonary tuberculosis who lived in north-western Russia. More than half (53%) of 17 types of RFLP profiles of isolates are identical and typical of M. tuberculosis of the Beijing family. Comparing the results of isolate genotyping with clinical, microbiological, and pathomorphological findings allows cases of endogenous reactivation to be differentiated from exogenous (nosocomial, in particular) reinfection in recurrence. Beijing genotype strains are shown to cause more severe unarrested course of drug-resistant fibrocavernous tuberculosis. At the same time such strains are more frequently detected in relatively young persons with a shorter duration of the disease, which reflects current clonal Beijing genotype dissemination that present a severe epidemiological hazard.

[Virulence of Mycobacteria tuberculosis]. / Virulentnost' mikobakterii tuberkuleza.

The virulence of 43 Mycobacteria tuberculosis strains isolated from 21 patients with new cases of pulmonary tuberculosis and from 20 patients with chronic destructive pulmonary tuberculosis (CDPT) was studied in non-inbred albino mice. Twenty four (56%) and 19 (44%) M. tuberculosis strains belonged to the Beijing and individual genotypes, respectively. The virulence of isolates from the new cases of pulmonary tuberculosis was higher than that from CDPT patients. The M. tuberculosis strains studied showed no correlation between the virulence, viability, and affiliation to a certain genotype. However, there was a lower rate of decreased virulence in multidrug-resistant strains of the Beijing genotype than in the isolates of individual genotypes. This is likely to be a factor of the high transmissible capacity of M. tuberculosis strains of the Beijing family.

[Significance of mycobacterial drug resistance in pulmonary tuberculosis surgery]. / Znachenie lekarstvennoi ustoichivosti mikobakterii v khirurgii tuberkuleza legkikh.

The outcomes of surgical treatment of 546 patients with pulmonary tuberculosis were analyzed in relation to the bacteriological characteristics. Seeding detected sputum bacterial isolation in 52.4% of cases. Mycobacterial drug resistance was detected in 83.6% of the tested cultures. It has been concluded that the resistance is a first-order infectious agent whose action is shown in lowering the efficiency of preoperative courses of chemotherapy by 2.5 times, in increasing the incidence and severity of postoperative pleural and pulmonary complications by 6-7 times, in deteriorating the immediate outcomes of surgical treatment to 82.4% with a 7.4% mortality rate. Intensive bacterial isolation unarrested by surgery is a second-order infectious agent which more clearly shows a relationship of the efficiency of surgical treatment to a reduction in the cure rate to 74.2% with a total mortality of 15.2%. There is evidence for that it is advisable to apply an active surgical policy in a group of patients having an infectious risk factor.

Line probe assay in the rapid detection of rifampin-resistant Mycobacterium tuberculosis directly from clinical specimens.

The sensitivity of a PCR-based line probe assay (Inno-LiPA Rif. TB Assay; Innogenetics NV Zwijndrecht, Belgium) was studied by using nested-PCR technique. A total of 75 specimens, representing various body locations from 70 suspected tuberculosis patients were obtained. LiPA yielded 30 Mycobacterium tuberculosis complex positive results (sensitivity 58.8%, compared with final diagnoses) whereas culture for M. tuberculosis was positive in 18 specimens (sensitivity 35.3%). Genotypic rifampin resistance testing by LiPA showed that 7 specimens contained rpoB mutations associated with RMP resistance, and sequencing data of the rpoB gene and LiPA patterns agreed in 29 of 30 M. tuberculosis positive specimens (96.7%). This indicates reliable performance, which makes the test suitable for the rapid determination of resistance to rifampin directly in clinical samples. However, the best results are obtained if LiPA is combined with conventional staining and culture methods.