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The measurement of the level of mitochondrial DNA (mtDNA) in the blood is a difficult problem due to high variability of mitochondrial genes, deletions in the mitochondrial genome in some pathological conditions, different sources of mtDNA into the bloodstream (mtDNA from tissues, from blood cells, etc.). We designed primers and TaqMan probes for highly conserved regions of the ND1 and ND2 genes outside the mitochondrial deletions "hot zones". For standardizing the technique, the true concentration of low-molecular-weight mtDNA was determined by real-time PCR for two targets: a fragment of the ND2 gene (122 bp) and the ND1 and ND2 genes (1198 bp). The sensitivity and specificity of the developed approach were verified on a DNA pool isolated from the blood plasma of healthy donors of various nationalities. The concentration of low-molecular-weight mtDNA in the blood plasma of two patients with COVID-19 was monitored over two weeks of inpatient treatment. A significant increase in the content of low-molecular-weight mtDNA was observed during the first 5 days after hospitalization, followed by a drop to the level of healthy donors. The developed technique makes it possible to assess the blood level of low-molecular-weight mtDNA regardless of the quality of sampling and makes it possible to standardize this biological marker in a wide range of infectious and non-infectious pathologies.

Ancient sublineage of the Mycobacterium tuberculosis Beijing genotype is endemic and prevalent in East Asia and rare in other world regions. While these strains are mainly drug susceptible, we recently identified a novel clonal group Beijing 1071-32 within this sublineage emerging in Siberia, Russia and present in other Russian regions. This cluster included only multi/extensive drug resistant (MDR/XDR) isolates. Based on the phylogenetic analysis of the available WGS data, we identified three synonymous SNPs in the genes Rv0144, Rv0373c, and Rv0334 that were specific for the Beijing 1071-32-cluster and developed a real-time PCR assay for their detection. Analysis of the 2375 genetically diverse M. tuberculosis isolates collected between 1996 and 2020 in different locations (European and Asian parts of Russia, former Soviet Union countries, Albania, Greece, China, Vietnam, Japan and Brazil), confirmed 100% specificity and sensitivity of this real-time PCR assay. Moreover, the epidemiological importance of this strain and the newly developed screening assay is further stressed by the fact that all identified Beijing 1071-32 isolates were found to exhibit MDR genotypic profiles with concomitant resistance to additional first-line drugs due to a characteristic signature of six mutations in rpoB450, rpoC485, katG315, katG335, rpsL43 and embB497. In conclusion, this study provides a set of three concordant SNPs for the detection and screening of Beijing 1071-32 isolates along with a validated real-time PCR assay easily deployable across multiple settings for the epidemiological tracking of this significant MDR cluster.

Background. The viral load of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the new coronavirus infection, is becoming increasingly important in clinical and epidemiological contexts. Despite this, there are significant complexities in the implementation of viral load quantitative measurement into clinical practice due to the limited approaches to its assessment.The aim of this work was to develop an approach for SARS-CoV-2 viral load analysis by the value of sample threshold cycles (Ct) relative to the Ct of the internal control sample obtained in routine PCR diagnostics of the COVID-19, and to use this approach for quantitative monitoring of viral load in patients with first positive SARS-CoV-2 test from the Irkutsk region.Materials and methods. Using regression models based on the least squares method, an approach to determine the number of copies of SARS-CoV-2 RNA in 1 ml of nasopharyngeal secretion was developed. The viral load of SARS-CoV-2 was assessed in nasopharyngeal and pharyngeal samples obtained from 1370 patients from Irkutsk and Angarsk with primary diagnosed positive PCR result in the period from July 1 to November 10, 2020.Results. A tenfold increase in the average monthly viral load among patients in September-October 2020 was revealed. We assume that the change in the epidemiological pattern of the spread of the new coronavirus infection during this period is associated with an increase in the number of contacts in the population due to the school year beginning. Higher viral loads are observed in populations at risk for COVID-19 – among healthcare workers and adults/elderly patients. Conclusion. The development of a standardized quantification of SARS-CoV-2 viral load in the nasopharyngeal samples can be a predictive clinical marker and a reliable tool for improving COVID-19 surveillance using the proposed approach to assess average viral load in a local population.

Cross-replicating associations with rs657152 at the 9q34.2c locus and rs11385942 at the 3p21.31 locus found in patients with severe COVID-19 in the Caucasian population require the study of the discovered phenomenon in various populations, including as an independent biological marker. Primers and TaqMan probes for PCR discrimination of the A and C alleles in single nucleotide polymorphism (SNP) rs657152 have been developed. The polymorphism of the rs657152 A/C locus was determined in 129 patients with COVID-19 and in a control group of 466 healthy individuals. There were no significant differences in the frequency of distribution of the A and C alleles, 0.47/0.53 and 0.45/0.55, between patients and healthy subjects, respectively. Also, no differences were found in the distribution of alleles in patients with a high viral load in the smear (Ct in the range of 16-25) in comparison with an average and low viral load (Ct in the range of 26-40).

The Mycobacterium tuberculosis Mlva cluster 1071-32 belongs to the early ancient sublineage of the Beijing genotype. We have recently demonstrated its association with pre-XDR/XDR, increase in prevalence in parts of Russia (Northwest, Center, Siberia) and sporadic isolation in eastern Europe (Lithuania, Albania). Here, we analyzed whole genome sequencing (WGS) data in order to gain insight into genomic background of the pathogenetic properties and dissemination pattern of this cluster. The initial WGS collection (this study, GenBank, Broad Institute, GMTV) included 3089 samples, in particular, 521 from East/Southeast Asia and 1067 from Russia. Of these, 117 genomes were assigned to the Beijing early ancient sublineage. The WGS-based phylogenetic analysis subdivided samples into several clades. A large stabnd-alone branch included Russian strains of the VNTR cluster 1071-32; all were pre-XDR or XDR. Cluster-specific SNPs were determined: 15 in intergenic regions, 10 in PE_PGRS and PPE genes, 96 in other genes. Mutations leading to significant amino acid changes were found in genes of the following categories: intermediate metabolism and respiration (n=6), cell wall and cell processes (n=4), conserved hypotheticals (n=2), lipid metabolism (n=1). Three SNPs were selected for development of the real-time PCR method of detection of this resistant cluster. To conclude, the emerging highly drug-resistant Russian cluster of the M. tuberculosis Beijing genotype is characterized by a combination of potentially significant SNPs in the genes coding for intermediate metabolism and respiration and cell and cell wall processes. Supported by Russian Science Foundation (grant 19-14-00013).

Aim. To estimate the dynamics changes in the population structure of the tuberculosis (ТВ) pathogen in the Irkutsk region by comparison of genotypes of M. tuberculosis from patients of different age groups. Materials and methods. 588 epidemiologically unrelated strains of M. tuberculosis isolates from 567 ТВ patients were characterized using 24-locus MIRU-VNTR typing. 160 strains belonged to patients of different age groups. 59 strains were isolated from the «young» people with ТВ who were bom after 1990 and 101 isolates from people who were born before 1955. Results. Two-thirds of the samples (427/567) were genotype Beijing with the dominance of two subtypes belonging to the modem epidemic clonal complexes CC1 and CC2. The high level of clustering profiles of CC1 and CC2 genotype Beijing in «older» and «young» patients in the Irkutsk region indicates the presence and active transmission of epidemic CC1 and CC2 subtypes over the past fifty years.

Introduction. Globally, an estimated 10.0 million (range, 9.0–11.1 million) people fell ill with tuberculosis (TB) in 2018, a number that has been relatively stable in recent years [1]. Despite the obvious differences in the virulence and transmissibility of different genotypes [2], the pathogenesis of tuberculosis, especially in the later stages, is still poorly understood [3]. At the same time, some researchers suggest that the ability of Mycobacterium tuberculosis to form biofilms in vivo [3] is an important aspect of pathogenesis TB that should be used in the development of new generation vaccines [4]. Previously, we suggested that in the long treatment of TB, a specific lung microbiota can develop in the caseous contents of tuberculosis foci, forming a polymicrobial biofilm, resistant to anti-tuberculosis therapy and contributing to the survival of M. tuberculosis [5]. Here, we present results of the metagenomic analysis of 14 tuberculomas and discuss the results obtained. Methods. The study recruited 13 patients undergoing planned surgical treatment at the Irkutsk Regional TB Hospital, Russia. DNA was isolated from caseous contents of 14 tuberculomas obtained during surgery. Two samples were obtained from one patient at two time points separated by six months. Next-generation sequencing of DNA samples was performed at the NextSeq 550 platform (Illumina, USA). The genomic DNA library was prepared by Nextera XT kit (Illumina). High Performance Sequence Analysis was completed on the NextSeq 550 platform (Illumina, USA) with NextSeq 500/550 v2.5 sequencing reagent kits (Illumina, USA). The NGS short sequencing reads (fastq files) were deposited to the NCBI (PRJNA659860). The assembly of short reads pertaining to mycobacterial DNA from the total pool of metagenomic sequences was carried out with the Magic-BLAST tools [6]. Mapping of short reads to genome of the reference strain M. tuberculosis H37Rv (NC_000962) was performed using Burrows-Wheeler Aligner [7]. The extraction of sequences from the mapped genome with the calculation of the coverage of each position was performed with SamTools [8]. Taxonomic classification of high-throughput sequencing reads from metagenomic whole genome sequencing was performed by using Kraken 2 [9]. Results. Among 5167 bacterial reads, a total of 105 families were identified. Of them, 68 taxa belonged to Gram-negative bacteria, and 35 to Gram-positive. At the same time, the number of reads of Gram-negative and Gram-positive taxa was very similar - 2430 and 2746, respectively. This was probably due to the presence of 1801 reads from the family Mycobacteriaceae (M. tuberculosis). The microbial content of the tuberculomas was estimated as 104-108 bacterial genomes per 1 gram of sample. The vast majority of reads (over 95%) were attributed to the human DNA. We have subdivided the samples into two types of bacterial communities: (i) paucibacillary bacterial community with a predominance of TB (63%-96%) and (ii) low-bacterial community with a predominance of non-mycobacterial taxa (13%-38%). Staphylococcaceae, Pseudomonadaceae, Eggerthellaceae, Pasteurellaceae, and Acetobacteraceae prevailed among non-mycobacterial communities. Conclusions. It can be unambiguously concluded that the microbiota of about half of the tuberculomas did not have a dominant taxon or it differed from M. tuberculosis. Acknowledgments. This work was supported by Russian Foundation for Basic Research (Grant No. 19-515-55009).

Currently, Mycobacterium tuberculosis isolates of Latin-American Mediterranean (LAM) family may be detected far beyond the geographic areas that coined its name 15years ago. Here, we established the framework phylogeny of this geographically intriguing and pathobiologically important mycobacterial lineage and hypothesized how human demographics and migration influenced its phylogeography. Phylogenetic analysis of LAM isolates from all continents based on 24 variable number of tandem repeats (VNTR) loci and other markers identified three global sublineages with certain geographic affinities and defined by large deletions RD115, RD174, and by spoligotype SIT33. One minor sublineage (spoligotype SIT388) appears endemic in Japan. One-locus VNTR signatures were established for sublineages and served for their search in published literature and geographic mapping. We suggest that the LAM family originated in the Western Mediterranean region. The most widespread RD115 sublineage seems the most ancient and encompasses genetically and geographically distant branches, including extremely drug resistant KZN in South Africa and LAM-RUS recently widespread across Northern Eurasia. The RD174 sublineage likely started its active spread in Brazil; its earlier branch is relatively dominated by isolates from South America and the derived one is dominated by Portuguese and South/Southeastern African isolates. The relatively most recent SIT33-sublineage is marked with enigmatic gaps and peaks across the Americas and includes South African clade F11/RD761, which likely emerged within the SIT33 subpopulation after its arrival to Africa. In addition to SIT388-sublineage, other deeply rooted, endemic LAM sublineages may exist that remain to be discovered. As a general conclusion, human mass migration appears to be the major factor that shaped the M. tuberculosis phylogeography over large time-spans.

The population structure of Mycobacterium tuberculosis from Republic of Sakha (Yakutia) was studied using 24-loci MIRU-VNTR genotyping. Most of the studied 199 strains isolated from 199 pulmonary tuberculosis patients (34.2% or 68 of 199 strains) belonged to the Beijing genotype. Multiple drug resistant and extensively drug resistant (MDR/XDR) isolates were predominant (χ2 = 15.5; p < 0.001) among representatives of the CC2/W148 subtype of the Beijing genotype (9.5% or 19 of 199 isolates). Strains of the genotype S (15.6% or 31 of 199 isolates) were the second most common genotype after Beijing. Most of the strains of S genotype had an identical profile, 233325153325141344222372. Among non-Beijing isolates, representatives of the S genotype included the most significant proportion of MDR or XDR ((χ2 = 59.8; p < 0.001). Isolates of Ural genotype were the third most common (10% or 20 of 199 isolates). The strains belonging to LAM genotype (8.5% or 17 of 199) were genetically heterogeneous. Minor genotypes T and Haarlem were also highly heterogeneous. The epidemic spread of MBT isolates belonging to S genotype and CC2/W148 of the Beijing genotype in Yakutia was reconstructed based on a phylogenetic model. The probable time of origin was estimated using the scale proposed by M. Merker et al. (2015). It was shown that isolates of the CC2/W148 subtype divided into four phylogenetic sublineages and were introduced in the recent historical period (the 20th century). Phylogenetic relationships between 30 MIRU-VNTR profiles of S genotype representatives from Yakutia and 31 reference S-profiles profiles from Europe and Canada were studied. Profiles of the isolates of S-genotype from Yakutia formed compact phylogenetic group. It suggested that these strains have been evolving in Yakutia. It was revealed that the ancestral genotype S was imported onto the territory of Yakutia 300–600 years ago.

The existence of "transrenal" DNA (tr-DNA), i.e. cell-free DNA that has distributed through the renal barrier to the urine, was first shown from a pathogen in 2000 (Botezatu et al., 2000). However, a targeted search for tr-DNA from Mycobacterium tuberculosis (MBT) started relatively recently (Cannas et al., 2008; Green et al., 2009). While other MBT cellular components found in the urine, e.g. lipoarabinomannan, have been used as an enhanced diagnostic tool, tr-DNA has the potential for strain specific identification or a more persistent biomarker during treatment of active disease. We therefore sought to identify by high-throughput next generation sequencing (NGS) MBT genome fragments in the urine of people with human immunodeficiency virus and tuberculosis (HIV-TB) co-infection living in a co-epidemic setting, and to evaluate whether these DNA targets are suitable for the development a quantitative TaqMan polymerase chain reaction with real-time detection (rt-PCR). Selection and mapping to the reference MBT genome of strain H37Rv (NC_000962) revealed 158 fragments of mycobacterial DNA with length from 19 to 44 base pairs (bp) repeated in different DNA samples. Five targets were chosen for design of rt-PCR primers and probes. Comparative analysis of the newly developed tests that were based on the results of NGS did not reveal a significant increase in sensitivity and specificity relative to the previous empirically designed targets. Howver, highly reproducible NGS reads of mycobacterial tr-DNA were obtained. rt-PCR test development suitable for more practical clinical use was likely limited by the small size of the secreted DNA fragments. It is necessary to develop further molecular approaches for the detection of mycobacterial tr-DNA or rely on NGS techniques with inherent bioinformatics requirements.

The population structure of the M. tuberculosis in Yakutia was estimated by the MIRU-VNTR method of 24 loci genotyping. 199 strains from 199 patients with pulmonary tuberculosis were tested. The greatest number of the strains (34.2%, 68/199) belonged to the genotype Beijing. The significant predominance (X² = 15.5; p0.001) of the multidrug and extensively drug-resistance (MDR/XDR) among the isolates of Beijing genotype was revealed in subtype CC2/ W148 - 9.5% (19/199). Strains of the genotype S (15.6%, 31/199) were the second most common genotype after Beijing. The majority of S-strains had an identical profile 233325153325141344222372. S genotype strains also significantly more frequently carried the MDR/ XDR (X² = 59.8;p0.001) among non-Beijing isolates. The genotype strain Ural ranks the third in the prevalence - 10.0% (20/199). The strains belonging to the family LAM (8.5%, 17/199) had considerable genetic heterogeneity. A great genetic diversity was also found in minor genotypes T and Haarlem. A phylogenetic reconstruction of the epidemic spread of the S-genotype and subtype CC2/W148 of the Beijing genotype in Yakutia was performed with estimation of the probable time of origin in the scale proposed by Merker M. et al. (2015). It was shown that the strains of the subtype CC2/W148 had been formed from four distinct phylogenetic sublines in recent historical period (XX century). It was estimated that phylogenetic relationships accounted for 30 MIRU-VNTR profiles of S-strains from Yakutia and 31 reference S-profiles from Europe and Canada. The profiles of the S-genotype from Yakutia form a phylogenetically compact group, indicating that all evolutionary history of these strains happened in the Sakha Republic. The time of the ancestral S-genotype spreading in Yakutia was estimated to be in the range from 300 to 600 years.

Background Ural genetic family is a part of the Euro-American lineage of Mycobacterium tuberculosis and is endemic in Northern Eurasia (former Soviet Union [FSU]). These strains were long described as drug susceptible and of low virulence, but recent studies reported an increasing circulation of the multidrug-resistant (MDR) and extensively drug-resistant (XDR) Ural strains. Here, we analyzed all publicly available whole genome sequence data of Ural genotype isolates, in order to elucidate their phylogenomic diversity with a special focus on MDR and potentially epidemic clones. Results A total of 149 M. tuberculosis genomes of Ural isolates from FSU countries were mined from the GMTV database and TB-ARC project. We identified 6002 variable amino acid positions that were assessed for functional significance and used to build ML, NJ trees and for Bayesian TMRCA estimation. Three robust monophyletic clades were identified: Clade A (31 isolates from Russia, Belarus, Moldova), Clade B (52 isolates from Russia), and Clade C (37 isolates from Moldova, 2 from Belarus). Clade C was significantly associated with XDR or pre-XDR status compared to the pooled Clades A and B (33/39 versus 5/83, P T, RpoB Ser450Leu, RpsL Lys88Arg, eis -12G > A and EmbB Ser297Ala/T > G. Its further dissemination may occur towards both Russia and European Union and should be taken into consideration by health authorities. The identified spoligotyping signatures can serve for rapid preliminary detection and surveillance of the more hazardous pre-XDR associated strains of the Ural family, both in populations from countries of their endemic circulation and migrant communities. Electronic supplementary material The online version of this article (10.1186/s12864-018-5162-3) contains supplementary material, which is available to authorized users.

To the Editor: Next-generation sequencing (NGS) technology is becoming more affordable and is increasingly being widely used for high-resolution molecular epidemiology of tuberculosis. Using an example of the emerging multidrug-resistant strain of Mycobacterium tuberculosis, we showed the value of informed understanding when in silico prediction from NGS data achieved with available bioinformatics tools is placed within the context of the existing genotyping framework. Spoligotyping is a classical method of M. tuberculosis genotyping, and the SITVIT_WEB database contains data on 7,105 spoligotype patterns of 58,180 isolates from 153 countries (http://www.pasteur-guadeloupe.fr:8081/SITVIT_ONLINE). Spoligotyping targets a variation of the DR/CRISPR locus, whose evolution in M. tuberculosis occurs through deletion of single or multiple spacers. By virtue of the orientation of the associated cas genes, the locus is situated on the minus strand, whereas its spacers are numbered within the locus, not the genome. Spoligotype international type (SIT) 266 (Figure, panel A) is an epidemiologically significant genotype. It constitutes a substantial proportion of the population structure of M. tuberculosis in Belarus, a post-Soviet state in Eastern Europe (1,2), and has been described sporadically in the neighboring provinces in northwestern and central Russia (2–5) and in Latvia (6). More important, it is multidrug resistant (MDR) (and most likely extensively drug resistant). In a recent Belarus study, SIT266 was found in 25 of 163 strains; all 25 were MDR (1). This situation contrasts clearly with its apparently parental type SIT264, which differs from SIT266 in a single spacer 8 (Figure, panel A). SIT264 is more widespread across Eastern Europe but at very low prevalence and is not associated with multidrug resistance (3,6,7). On the basis of 24 mycobacterial interspersed repetitive unit variable number tandem repeats clustering and robust phylogenetic single-nucleotide polymorphisms, SIT264 and SIT266 isolates are assigned to the Latin American–Mediterranean lineage of M. tuberculosis (2). Figure A) Spoligotyping hybridization profiles of H37Rv and BCG reference strains, Mycobacterium tuberculosis SIT266 (2 strains in this study) and SIT264 (previously published strain in [3]). SIT, spoligotype international type, designated according to SITVIT_WEB ... We recovered 2 MDR M. tuberculosis isolates of SIT266 from pulmonary tuberculosis patients from northwestern Russia in 2014. Bacterial DNA was subjected to macroarray-based spoligotyping (8) and whole-genome sequencing on the MiSeq platform (Illumina, San Diego, CA, USA). M. tuberculosis NGS data were deposited in the National Center for Biotechnology Information Sequence Read Archive (project no. PRJNA305488). The short sequencing reads were subjected to analysis by using the SpoTyping program (https://github.com/xiaeryu/SpoTyping) (9) and the TGS-TB online tool (https://gph.niid.go.jp/tgs-tb/) (10) to deduce their spoligoprofile. We used the TGS-TB tool to map the IS6110 insertion sites and detect drug resistance mutations. The reads also were mapped to the genome of reference strain H37Rv (GenBank accession no. NC_00962.3) by using the Geneious 9.0 package (Biomatters Ltd, Auckland, New Zealand). We obtained 1,294,895 and 816,693 paired reads for strains 4542 and 8279, respectively, and mapped them to the reference. Mean read length was 300 bp, and the average genome coverage was 72. Strains 4542 and 8279 were phenotypically MDR and harbored mutations associated with resistance to all 5 first line-drugs. The macroarray hybridization spoligotyping assigned both strains to spoligotype SIT266. However, by in silico typing, their spoligotype was predicted to be SIT264 (Figure, panel A). To reconcile these findings, we hypothesized that this discrepancy resulted from an IS6110 asymmetrically inserted in the direct repeat unit adjacent to the spacer 8 in a SIT266 isolate. This insertion would disrupt a target sequence for biotin-labeled DRa primer, thus preventing spacer 8 from amplification. Indeed, in both SIT266 isolates, the in silico analysis identified a forward IS6110 insertion that was mapped to position 3122916 in H37Rv genome (Technical Appendix Figure). This location correlates with the location of spacer 8 in this same genome from positions 3122954 to 3122918. Thus, IS6110 precedes spacer 8 in the genome of isolate with spoligotype SIT266, or follows it, within the DR locus (Figure, panel B; Technical Appendix Figure). An immediate excellent contribution of NGS with regard to tuberculosis treatment and control is its capacity to rapidly screen for multiple gene targets linked to the development of drug resistance. However, knowledge of strain genotype is no less clinically and epidemiologically relevant. A superspreading strain might be marked with other pathobiologically important features. In the case presented here (indeed emerging and MDR), the NGS-based in silico spoligotyping would confuse the MDR/extensively drug resistant SIT266 with “less dangerous” SIT264. To be precise, the revealed discrepancy is not inherent to the NGS technology itself. Although the general limitation of the use of short sequencing reads to infer repetitive genome regions is known, it did not pose a problem in our study. However, both bioinformatics tools predicted the spoligoprofile solely from the presence or absence of spacer sequences and did not take into account a “hiding” effect exerted by a putative IS6110 insertion on adjacent spacer under classical spoligotyping. In conclusion, we suggest that an accurate NGS-based prediction requires an integrative approach to all relevant information obtained by in silico analysis of a given genome locus. In particular, not only presence of CRISPR spacers but also presence and location of potentially interfering IS6110 insertion(s) should be considered for correct NGS-based assignment to internationally recognized spoligotypes. Technical Appendix: Whole-genome, short-sequencing reads of Mycobacterium tuberculosis strain 4542 mapped to complete genome of reference strain H37Rv (spacers 7–9 in CRISPR locus). Click here to view.(3.1M, pdf)

The bio-information search was carried out and the design of primers and TaqMan probes was developed to detect DNA of agent of tuberculosis subtypes CC1 and CC2-W148 of Beijing genotype and also Ural genotype in various clinical material (phlegm, spinal fluid, pleural fluid, etc.) using real-time polymerase chain reaction technique. The 180 clinical samples from 143 patients with tuberculosis of lungs were used to carry out an approval of sensitivity and specificity of the developed tests concerning studies at the genetic analyzer GeneXpert. The sensitivity of tests on CC1, CC2-W148 и Ural relating to polymerase chain reaction of analyzer Gene Expert made up to 91%, 106% and 81% correspondingly. In all cases, the specificity made up to 100%. In parallel studies the samples with DNA of СС2-W148 genotype were more often positive on mutation of resistance to Rifampicin-Rif (+) according the results of GeneXpert (χ² = 27,1; p0,01) related to other genotypes. At the same time, detection of СС2-W148 strain in patient was more often accompanied by discrepancy of results: GeneXpert - Rif (+) and resistance to Rifampicin in bacteriological study under ultimate validation of multiple medicinal resistance of tuberculosis (χ² = 5,1; p0,05). The analysis was applied to negative effect of combination of allele-336G of CD209 gene of patient with genotype of tuberculosis mycobacterium Beijing detected previously (Ogarkov et al., 2012). The significant prevalence was observed related to widespread medicinal resistance (χ² =4,3; p0,05) in patients with allele-336G of CD209 gene in combination with CC2-W148 clone in comparison with other combinations in patients. The obtained results testify a possibility of application of genetic typing of tuberculosis agent and patient for early diagnosis of development of various complications of tuberculosis at the stages of primary examination of primarily detected patients with tuberculosis.

All known genotypes (3925 spoligoprofiles of M. tuberculosis) were selected using the SITVIT database in nine countries, including countries of the former Soviet Union: Russia, Latvia, Estonia, Poland, Finland, Italy, Portugal, Japan, and Vietnam. The programs SpolTools and DESTUS were used to construct consensus networks to identify the epidemic genotypes of M. tuberculosis in the studied countries. In Russia, Latvia, and Estonia, the Beijing strain was determined as the main epidemic genotype. In Finland, Poland, Portugal, and Italy, all epidemic genotypes belonged to other families. The hypothesis on the explosive nature of the spreading of the Beijing genotype of M. tuberculosis was put forward for Russia and other former Soviet countries in the 20th century. The basic idea of the hypothesis was that the pattern of dissemination of the Beijing genotype occurred at the CER (Chinese Eastern Railway), in the Gulag, and in the civilian society of Soviet Union. The Beijing genotype of M. tuberculosis affected Russian builders of the CER during the end the 19th and early 20th centuries. With the repression of CER builders in the Soviet Union, the Beijing genotype was spread among Gulag prisoners; after 1953, it had spread throughout the civilian society of the entire country. The distribution of epidemic genotypes of M. tuberculosis in the studied countries was interpreted as evidence of the suggested hypothesis.

The investigation was aimed at testing the hypothesis on the possible role of some genes of the renin-angiotensin system in the formation of innate immunity to tuberculosis. It is known that insertion-deletion (I/D) polymorphism of the gene encoding the angiotensin-converting enzyme (ACE) influences the concentration of angiotensin II in the human organism and, indirectly, many stages of metabolism. On one hand, homozygotes of deletion in the ACE gene (DD genotype) are characterized with obesity, arterial hypertension, hypocholesterolemia, and some other pathological conditions. On the other hand, it was demonstrated that hypercholesterolemia is common pathology for patients with pulmonary tuberculosis (C. Perez-Guzman et al., 2005).

An interaction of different human alleles and endemic bacterial strains may be clinically manifested as different outcome of the disease in different hosts infected with the same genotype. The primary objective of this study was to investigate this issue in the model of Mycobacterium tuberculosis and human DC-SIGN encoding CD209 promoter SNP (rs4804803) in Russian Siberian population. We sought to find a possible combination of M. tuberculosis lineage and human host allele/genotype correlating with unfavorable outcome of the disease. The 101 paired DNA samples from patients with pulmonary TB (human and M. tuberculosis DNA) were studied by 12-loci MIRU-VNTR typing (M. tuberculosis strains) and CD209 −336 A/G typing (human DNA). Ninety autopsy DNA samples as a source of human and mycobacterial nucleic acids from persons who died from TB were also subjected to the same genotyping procedures. A human control group consisted of 177 healthy individuals. The Beijing genotype was more frequently identified in autopsy versus patient samples, in 70.0% and 51.5%, respectively (χ2 = 6.06, P = 0.01). Regarding other M. tuberculosis genetic families, no significant difference in LAM family distribution among patient strains and autopsy samples has been found. In contrast, Ural genotype was significantly less frequently detected in the autopsy samples (χ2 = 6.12, P = 0.01). Allelic and genotypic frequencies of the CD209 −336A/G did not differ significantly under global comparison when contrasting controls versus patients versus autopsy samples. However intriguing and contrasting significant associations were found in the male subgroup under M. tuberculosis genotype-stratified comparisons. Firstly, male carriers of −336AA genotype were more frequently infected with Beijing genotype (χ2 = 5.2, P = 0.02). Secondly and remarkably, this association was inverted in the autopsy sample: male carriers of −336AA genotype died less frequently due to TB caused by a Beijing rather than a non-Beijing strain (χ2 = 5.37, P = 0.02). In conclusion, we hypothesize that although carriers of CD209 −336A allele are more sensitive to infection with a Beijing strain, a combination of human CD209 −336G allele and M. tuberculosis Beijing genotype leads more frequently to the lethal outcome in pulmonary TB male patients in Russian (Caucasian) population.

This paper studies a full nucleotide sequence of cryptic plasmid pTP33, which was isolated from the typical plague strain of the Tuvinian natural focus, Yersinia pestis I-2638. Sequencing was carried out using the 454 GS Junior platform (Roche). In analysis using the software package GS De Novo Assembler v. 2.7 (Roche) and the algorithm Newbler v. 2.7, 1855 nucleotide reads, which contained 1101246 nucleotides, were assembled to a contig of 33 978 bp. The GC content of the obtained nucleotide sequence was 50.25%. During annotation, we found 56 open reading frames. Homologs of the predicted reading frames were sought in the BLAST databases. We detected 22 reading frames coding hypothetical proteins, 23 frames coding phagerelated proteins, and 11 frames coding proteins with known functions, including toxin–antitoxin system YefM-YoeB, nucleic acids and polysaccharides metabolism proteins (exopolysaccharide production protein ExoZ, exodeoxyribonuclease VIII), and replication proteins (ParA). Some predicted pTP33 proteins were found to be homologs (from 45 to 75%) with sequences of phage-related proteins of certain microorganisms—endosymbionts of insects (Sodalis glossinidius) and endosymbionts of entomopathogenic nematodes (Photorhabdus luminescens, P. asymbiotica, Xenorhabdus bovienii).