[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.

[Methods of extraction of Mycobacterium tuberculosis DNA from clinical samples for PCR: comparison and evaluation].

Several commonly used methods of extraction of Mycobacterium tuberculosis DNA from clinical samples for further use in PCR were compared. We used the commercial sets "DNA-express" (Litekh, Russia) and "DNA-sorb-B" (Amplisens, Russia) as well as phenol extraction, fast alkaline lysis and DNA sorption on silicone gel for DNA extraction. All methods were compared by the quantity of isolated DNA and stability of DNA preparation as well as by efficiency and cost of extraction. "DNA-express" proved to be most effective in the extraction of Mycobacterium tuberculosis DNA with its cost being the smallest.

[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.

[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.

Highest prevalence of the Mycobacterium tuberculosis Beijing genotype isolates in patients newly diagnosed with tuberculosis in the Novosibirsk oblast, Russian Federation.

In order to assess the genetic diversity of Mycobacterium tuberculosis in the Novosibirsk Region and determine profiles of resistance, 106 M. tuberculosis isolates were analysed. Fifty (47 %) isolates were identified using variable number tandem repeat typing as being in the Beijing family, of which eight (16 %) were type M2 isolates with the genetic profile 233325153533424 and eight (16 %) were type M11 isolates with the genetic profile 233325173533424, both of which are widespread in Russia. Mutations associated with resistance to isoniazid and rifampicin were identified. Of 48 isolates with resistance to isoniazid, 42 (87.5 %) contained a Ser(315)→Thr substitution in the katG gene and one contained a T→A substitution at position -34 of the promoter region of the ahpC gene. Of 31 isolates with resistance to rifampicin, 19 (61 %) each contained a mutation (TCG→TTG) at codon 531 of the rpoB gene. Two isolates each contained a mutation (GAC→GTC) at codon 516 of the rpoB gene and two others each contained a substitution at codon 526 of the rpoB gene, leading to a His→Asn substitution in one case and a His→Asp substitution in another case. One isolate contained a mutation (CTG→CCG) at codon 533 of the rpoB gene. An association between the Beijing genotype and multidrug resistance was demonstrated (R = 0.2, P = 0.032). However, it was interesting to note that a significant proportion (46 %) of isolates were sensitive to all drugs tested.