[Molecular and genetic characterization of rifampicin- and/or izoniazid-resistent strains of M. tuberculosis, isolated in Novosibirsk and Tomsk regions].

The purpose of the work was to study rifampicin- and izoniazid-resistent strains of M. tuberculosis, circulating in Western Siberia, by VNTR and IS6110 typing. The authors also studied genetic causes of resistance to these antibiotics and undertook a search of new VNTR loci, displaying polymorphism in genomes of closely related clonally-disseminated variants of Mycobacterium tuberculosis in W-Beijing family model analysis.

[The genetic diversity of Mycobacterium tuberculosis and an assessment of risk factors of tuberculosis spread in Russia's Siberian region by molecular epidemiological methods]. / Geneticheskoe raznoobrazie Mycobacterium tuberculosis i otsenka faktorov riska rasprostraneniia zabolevaniia tuberkulezom v Sibirskom regione Rossii metodami molekuliarnoi épidemiologii.

Molecular epidemiology approaches provided for a new interpretation of the TB infection transmission dynamics, contributed to changing the focuses of attention and updated the monitoring practice. On the basis of 101 cases of isolates of Mycobacterium tuberculosis (MBT) complex sampled from 84 patients with pulmonary tuberculosis in the Siberian region, we proved that the independent methods of IS6110 RFLP genetic typing and VNTR-typing by five accurate repeat tandems of ETR A, B, C, D, and E bring about similar results and can be used in studying the MTB clonal structure population in the Siberian region for the purpose of defining the TB infection transmission dynamics. The most widespread genetic types were detected, i.e. Beeijing family strains, the S42 spoligotype, and the 31323 VNTR type, which account for 52.3% of all samples. The general parameters describing the epidemic process intensity were evaluated, i.e. those characterizing the strains (91.6%) and the transmission activity factor (72%). Consequently, each three of the four analyzed TB cases resulted from a recent transmission. However, there is a trend, within the analyzed samples, towards a higher percentage of clusterization in the age group ranging from 40 to 60. Such trend is typical of a prevalence of TB reactivation cases caused by MBT complex strains spread intensively in the discussed territory. As for the clusterized isolates, which are endemic for the territory, such data should be interpreted as a recent transmission only cautiously. 28.5% of the studied isolates are resistant to anti-TB drugs used in medical practice; and 35.7% of them are resistant to izoniazide and rifampicin, therefore, according to the WHO classification they are considered to be poly-antibiotics-resistant (PAR). No strict associations were found between the spectrum of antibiotics-resistance and any of genotypes, however, 30% of PAR strains are 32525 and 42525 types VNTR (spoligotype S1 or Beejing type).

[KatG Ser3 15Thr mutation as the main reason of isoniazid resistance in Mycobacterium tuberculosis isolated in the Novosibirsk and Kemerovo Regions]. / Mutatsiia Ser315Thr gena katG--osnovnaia prichina ustoichivosti k izoniazidu u izoliatov Mycobacterium tuberculosis, rasprostranennykh v Novosibirskoi i Kemerovskoi oblastiakh.

KatG Ser3 15Thr mutation is one of the main reasons of resistance to isoniazid in Mycobacterium tuberculosis isolates. The frequency rate of the above mutation among isoniazid-resistant isolates made 94% in Novosibirsk Region and 93% in Kemerovo Region. The use of PCR-restriction fragment length polymorphism (PCR-RFLP) can be regarded as an adequate method for rapid screening of isoniazid resistance in Mycobacterium tuberculosis isolated in the West-Siberian Region.

[Improvement of a method for detecting of strains of the plague microbe using polymerase chain reaction]. / Optimizatsiia sposoba detektsii shtammov chumnogo mikroba pri pomoshchi polimeraznoi tsepnoi reaktsii.

Three pairs of oligonucleotide primers, complementary to nucleotide sequences of Yersinia pestis plasmids (pPst, 9.5 kb; pCad, 70 kb; pFra, 95 kb), were used in polymerase chain reaction for high-sensitive specific detection of plague pathogen. Primer pairs P1,P2,C1,C2, and F1,F2 were used to amplify fragments of pla gene (plasmid pPst), yop1 gene (plasmid pCad of plague microbe), and caf1 gene (plasmid pFra), respectively. The method developed enables specific detection of strains from all natural loci in Russia and contiguous states as well as strains of oceanic origin. Sensitivity of method is 50-100 CFU/ml. Primer sequences enable to amplify gene fragments, located in three own plasmides of plague microbe, in the same reaction mixture. The method offers identification of plague microbe and determination of its virulence and epidemic significance.

[Design of a chromosomal DNA probe species specific for Yersinia pestis]. / Konstruirovanie vidospetsifichnogo dlia Yersinia pestis khromosomnogo DNA-zonda.

A 14.8 kb DNA fragment from the chromosome of Yersinia pestis TWJ was cloned and the restriction map constructed. The fragment designated as T16 and its subfragments were tested in dot-hybridization with strains of Yersinia genus and other members of Enterobacteriaceae. A species-specific DNA probe (designated MK) was constructed on the basis of the T16 fragment. As judged from restriction analysis, blot-hybridization experiments and, partially, sequencing, significant homology exists between the MK DNA probe and this one developed by Bardarov et. al. (1990). A repeated sequence in two copies was discovered in the MK fragment.

[Molecular genetic characteristics of rifampicin-resistant Mycobacterium tuberculosis isolates in Novosibirsk]. / Molekuliarno-geneticheskaia kharakteristika ustoichivykh k rifampitsinu izoliatov Mycobacterium tuberculosis, vydelennykh v Novosibirske.

Forty rifampicin-resistant clinical isolates from patients living in Novosibirsk were studied. Six alleles earlier described in the literature were identified by the sequencing technique. The frequency of mutations in the studied samples slightly differs from that earlier reported for other geographic regions: 21 (52.5%) strains carried the mutated codon TTG in position 531 (Ser-->Leu), 7 (17.5%) had GTC in position 516 (Asp-->Val) and 2 (5%) had the GAC substitution in position 526 (His-->Asp), which is prevalent elsewhere. Sequence analysis revealed no mitations in 5 (12.5%) of the 40 isolates although this isolate was repeatedly resistant to rifampicin. VNTR-typing targeted to tandem repeats (ETR A, B, C, D, and E) was carried out to establish a genetic relationship for rifampicin-resistant isolates. Nine genetic types with VNTR-profiles termed as 12322, 32122, 32123, 32124, 32125, 32522, 23524, 12223, 22222, 33433 were revealed. There was no strict correlation between the type of mutation in the rpoB gene and the VNTR-type, which reflects different rates of evolution and the level of selective pressure on these genetic targets. The isolates of VNTR-types 32123 and 32125 with mutations in codon 531, and type 32122 in codons 531, 526, 516 showed a high clustering. This is likely to reflect the recent transmission and clonal dissemination of the epidemic strains of Mycobacterium tuberculosis. Thus, mutations in the rpoB gene did not reduce the virulence and transmissivity of these clones. Twenty-six of 27 clinical isolates selected by rifampicin-resistance were also resistant to isoniazid, which confirms the known fact that rifampicin-resistance may be used as a marker of isoniazid-resistance.

Development of a diagnostic test for Yersinia pestis by the polymerase chain reaction.

A 501 bp caf1 gene fragment and a 443 bp of pla gene fragment carried by 100 kb (pFra) and 10 kb (pPst) species-specific extrachromosomal replicons, respectively, were used as targets to study the conditions under which DNA amplification by polymerase chain reaction (PCR) may be applied to detect and identify Yersinia pestis DNA in cell lysates of pure cultures and biological samples. The sensitivity limit of PCR with the crude cell lysates of Y. pestis EV was estimated as 10-50 cfu in reaction mixture. When target Y. pestis EV cells were mixed with fresh blood of white mice, which contained 0.4% potassium citrate, the PCR detection level varied from 400 to 100 cfu ml-1 of blood depending on the method used for preparing the sample. In our tests PCR was effective for the detection of yersinia in the blood of white laboratory mice experimentally infected with virulent Y. pestis KM638 strain. This method can be considered convenient for routine detection and identification of Y. pestis.

[Involvement of nitric oxide in the development of tuberculin anergy in patients with pulmonary tuberculosis]. / Uchastie oksida azota v razvitii tuberkulinovoi anergii u bol'nykh tuberkulezom legkikh.

The production of nitric oxide (NO) and the magnitude of an antigen-specific proliferative response of the human lymphocytes stimulated by M. tuberculosis antigen [a purified protein derivative (PPD)] were investigated. PPD-reactive T lymphocytes were found in the peripheral blood of healthy donors. Normal values (mean values, the range of the minimum and maximum values) of PPD-induced proliferation and NO production were determined. Patients with pulmonary tuberculosis were found to have different levels of PPD-stimulated proliferation and NO production. The lymphocytes are shown to preserve their PPD reactivity in patients with normal NO production whereas the PPD-induced proliferative response was significantly decreased in those with high NO production. Patients with reduced tuberculin reactivities and high NO production were less responsive to treatment. The findings suggest that nitric oxide is involved in the development of tuberculin anergy with pulmonary tuberculosis.