SARS-CoV-2 Recombination and Coinfection Events Identified in Clinical Samples in Russia.

Recombination is one of the mechanisms of SARS-CoV-2 evolution along with the occurrence of point mutations, insertions, and deletions. Recently, recombinant variants of SARS-CoV-2 have been registered in different countries, and some of them have become circulating forms. In this work, we performed screening of SARS-CoV-2 genomic sequences to identify recombination events and co-infections with various strains of the SARS-CoV-2 virus detected in Russia from February 2020 to March 2022. The study included 9336 genomes of the COVID-19 pathogen obtained as a result of high-throughput sequencing on the Illumina platform. For data analysis, we used an algorithm developed by our group that can identify viral recombination variants and cases of co-infections by estimating the frequencies of characteristic substitutions in raw read alignment files and VCF files. The detected cases of recombination were confirmed by alternative sequencing methods, principal component analysis, and phylogenetic analysis. The suggested approach allowed for the identification of recombinant variants of strains BA.1 and BA.2, among which a new recombinant variant was identified, as well as a previously discovered one. The results obtained are the first evidence of the spread of recombinant variants of SARS-CoV-2 in Russia. In addition to cases of recombination we identified cases of coinfection: eight of them contained the genome of the Omicron line as one of the variants, six of them the genome of the Delta line, and two with the genome of the Alpha line.

Genomic Variability of SARS-CoV-2 Omicron Variant Circulating in the Russian Federation during Early December 2021 and Late January 2022.

Analysis of genomic variability of pathogens associated with heightened public health concerns is an opportunity to track transmission routes of the disease and helps to develop more effective vaccines and specific diagnostic tests. We present the findings of a detailed genomic analysis of the genomic variability of the SARS-CoV-2 Omicron variant that spread in Russia between 8 December 2021 and 30 January 2022. We performed phylogenetic analysis of Omicron viral isolates collected in Moscow (n = 589) and downloaded from GISAID (n = 397), and identified that the BA.1 lineage was predominant in Russia during this period. The BA.2 lineage was also identified early in December 2021. We identified three cases of BA.1/BA.2 coinfections and one case of Delta/Omicron coinfection. A comparative genomic analysis of SARS-CoV-2 viral variants that spread in other countries allowed us to identify possible cases of transmission. We also found that some mutations that are quite rare in the Global Omicron dataset have a higher incidence rate, and identified genetic markers that could be associated with ways of Omicron transmission in Russia. We give the genomic variability of single nucleotide variations across the genome and give a characteristic of haplotype variability of Omicron strains in both Russia and around the world, and we also identify them.

First insight into the whole-genome sequence variations in Mycobacterium bovis BCG-1 (Russia) vaccine seed lots and their progeny clinical isolates from children with BCG-induced adverse events.

BACKGROUND: The only licensed live Bacille Calmette-Guérin (BCG) vaccine used to prevent severe childhood tuberculosis comprises genetically divergent strains with variable protective efficacy and rates of BCG-induced adverse events. The whole-genome sequencing (WGS) allowed evaluating the genome stability of BCG strains and the impact of spontaneous heterogeneity in seed and commercial lots on the efficacy of BCG-vaccines in different countries. Our study aimed to assess sequence variations and their putative effects on genes and protein functions in the BCG-1 (Russia) seed lots compared to their progeny isolates available from immunocompetent children with BCG-induced disease (mainly, osteitis). RESULTS: Based on the WGS data, we analyzed the links between seed lots 361, 367, and 368 used for vaccine manufacture in Russia in different periods, and their nine progeny isolates recovered from immunocompetent children with BCG-induced disease. The complete catalog of variants in genes relative to the reference genome (GenBank: CP013741) included 4 synonymous and 8 nonsynonymous single nucleotide polymorphisms, and 3 frameshift deletions. Seed lot 361 shared variants with 2 of 6 descendant isolates that had higher proportions of such polymorphisms in several genes, including ppsC, eccD5, and eccA5 involved in metabolism and cell wall processes and reportedly associated with virulence in mycobacteria. One isolate preserved variants of its parent seed lot 361 without gain of further changes in the sequence profile within 14 years. CONCLUSIONS: The background genomic information allowed us for the first time to follow the BCG diversity starting from the freeze-dried seed lots to descendant clinical isolates. Sequence variations in several genes of seed lot 361 did not alter the genomic stability and viability of the vaccine and appeared accumulated in isolates during the survival in the human organism. The impact of the observed variations in the context of association with the development of BCG-induced disease should be evaluated in parallel with the immune status and host genetics. Comparative genomic studies of BCG seed lots and their descendant clinical isolates represent a beneficial approach to better understand the molecular bases of efficacy and adverse events during the long-term survival of BCG in the host organism.

Proposed rituximab biosimilar BCD-020 versus reference rituximab for treatment of patients with indolent non-Hodgkin lymphomas: An international multicenter randomized trial.

BCD-020 is a proposed rituximab biosimilar, which has shown high similarity to rituximab in quality and nonclinical studies in vitro and in vivo. International multicenter clinical trial was conducted to compare efficacy and safety of BCD-020 and reference rituximab in adult (older than 18 years) patients with indolent lymphomas (follicular lymphoma grade 1-2, splenic marginal zone lymphoma, and nodal marginal zone lymphoma). Pharmacokinetics, pharmacodynamics, and immunogenicity were also studied. Patients with no previous biologic treatment for lymphoma were randomly assigned 1:1 to receive BCD-020 or comparator 375 mg/m2 for 4 weeks. Primary study outcome was day 50 overall response rate defined as complete or partial remission. Equivalence range was -20% to 20% for 95% CI for overall response rates difference. Secondary outcomes included adverse events, pharmacokinetics, pharmacodynamics, and immunogenicity. One hundred seventy-four patients were enrolled, 89 in BCD-020 arm and 85 in comparator arm. The overall response rate was 44.71% in BCD-020 arm and 41.89% in comparator arm. Limits of 95% confidence interval (CI) for difference of overall response rates between arms were (-12.62%-18.24%) showing equivalent efficacy. Sixty-one (68.54%) and 59 (69.41%) patients had at least one adverse event in BCD-020 arm or comparator arm, respectively. No unexpected adverse reactions were reported. Antidrug antibodies with no neutralizing activity were detected in two patients in comparator arm on day 14 further declining below detection threshold. Rituximab concentrations had equivalent pattern after intravenous administration of both drugs. Both drugs caused depletion of B-cells without significant influence on other blood cell lineages. In this study, we showed equivalent efficacy of BCD-020 and reference rituximab when used in patients with CD20-positive indolent lymphomas. We also confirmed pharmacokinetic equivalence of BCD-020 and reference rituximab. Safety profile, pharmacodynamics, and immunogenicity of BCD-020 were also comparable with those of reference rituximab.

Genome-wide sequence analyses of ethnic populations across Russia.

The Russian Federation is the largest and one of the most ethnically diverse countries in the world, however no centralized reference database of genetic variation exists to date. Such data are crucial for medical genetics and essential for studying population history. The Genome Russia Project aims at filling this gap by performing whole genome sequencing and analysis of peoples of the Russian Federation. Here we report the characterization of genome-wide variation of 264 healthy adults, including 60 newly sequenced samples. People of Russia carry known and novel genetic variants of adaptive, clinical and functional consequence that in many cases show allele frequency divergence from neighboring populations. Population genetics analyses revealed six phylogeographic partitions among indigenous ethnicities corresponding to their geographic locales. This study presents a characterization of population-specific genomic variation in Russia with results important for medical genetics and for understanding the dynamic population history of the world's largest country.

Primary efficacy of netakimab, a novel interleukin-17 inhibitor, in the treatment of active ankylosing spondylitis in adults.

OBJECTIVES: Netakimab (NTK) is a humanised monoclonal antibody targeting interleukin-17A, previously investigated in a phase 1 trial in healthy volunteers. Here, we report the results of a phase 2 trial, conducted to assess safety and pharmacokinetics (PK), to establish a therapeutic dose of NTK in a target population of patients with active ankylosing spondylitis (AS). METHODS: 89 patients with active AS, despite non-steroidal anti-inflammatory (NSAID) drug treatment, were randomised to receive 40, 80 or 120 mg of subcutaneous NTK or placebo at weeks 0, 1, 2 and q2wk thereafter until week 12. The primary endpoint was to achieve a proportion of patients with ≥20% improvement in Assessment of Spondyloarthritis. RESULTS: Rates of ASAS20 response at week 16 for NTK with 95%CI for difference in ASAS20 rates NTK vs. placebo were 72.73% [1.69%;58.05%], 81.82% [12.36%;65.56%], 90.91% [23.71%;72.39%] at doses of 40, 80 and 120 mg. The response rate in the placebo arm was 42.86%. The pre-specified margin of clinically non-meaningful difference was 10%. Superiority to placebo was confirmed for doses 80 and 120 mg. The most frequent adverse events (AEs) were lymphocytosis, neutropenia, and asymptomatic bacteriuria. No dose-dependent toxicity or serious adverse events (SAEs) were observed. The most effective dose with the fastest response onset and favourable safety profile was 120 mg. CONCLUSIONS: The data obtained demonstrate the efficacy and favourable safety profile of NTK in active AS. Clinical development of NTK will be continued in a phase 3 trial aimed to evaluate the efficacy of 1-year treatment with NTK 120 mg in patients with AS.

Genomic Variations in Drug Resistant Mycobacterium tuberculosis Strains Collected from Patients with Different Localization of Infection.

Mycobacterium tuberculosis is a highly studied pathogen due to public health importance. Despite this, problems like early drug resistance, diagnostics and treatment success prediction are still not fully resolved. Here, we analyze the incidence of point mutations widely used for drug resistance detection in laboratory practice and conduct comparative analysis of whole-genome sequence (WGS) for clinical M. tuberculosis strains collected from patients with pulmonary tuberculosis (PTB) and extra-pulmonary tuberculosis (XPTB) localization. A total of 72 pulmonary and 73 extrapulmonary microbiologically characterized M. tuberculosis isolates were collected from patients from 2007 to 2014 in Russia. Genomic DNA was used for WGS and obtained data allowed identifying major mutations known to be associated with drug resistance to first-line and second-line antituberculous drugs. In some cases previously described mutations were not identified. Using genome-based phylogenetic analysis we identified M. tuberculosis substrains associated with distinctions in the occurrence in PTB vs. XPTB cases. Phylogenetic analyses did reveal M. tuberculosis genetic substrains associated with TB localization. XPTB was associated with Beijing sublineages Central Asia (Beijing CAO), Central Asia Clade A (Beijing A) and 4.8 groups, while PTB localization was associated with group LAM (4.3). Further, the XPTB strain in some cases showed elevated drug resistance patterns relative to PTB isolates. HIV was significantly associated with the development of XPTB in the Beijing B0/W148 group and among unclustered Beijing isolates.

A phase III study of BCD-055 compared with innovator infliximab in patients with active rheumatoid arthritis: 54-week results from the LIRA study.

BCD-055 is a biosimilar of innovator infliximab (IFX). Here we present the 54-week results from phase 3 clinical study in patients with rheumatoid arthritis (RA). The aim of this study was to demonstrate the equivalent efficacy and safety of BCD-055 and IFX in patients with active rheumatoid arthritis. 426 adults with active RA were enrolled. Patients were randomized into 2 study arms in 2:1 ratio to receive BCD-055 or IFX innovator in dose of 3 mg/kg at week 0, 2, 6 and then every 8 weeks up to week 54. Primary efficacy endpoint was the rate of American College of Rheumatology (ACR) 20 response at week 14. The equivalence margin was set as 15%. Immunogenicity and safety were also assessed. Rate of ACR20 at week 14 in PP (Per-Protocol) population was 71.2% in BCD-055 group and 67.9% in IFX group. Difference in ACR20 rates between groups was 3.2% with 95% CI [- 7.0%; 13.5%] (р = 0.587). Throughout 54-week study period, both groups were characterized by similar rates of ACR20/50/70 response at all timepoints without significant differences (p > 0.05). The rates of adverse events (AE) were similar in groups (74.64% in BCD-055 arm vs 66.67% in IFX arm, p = 0.111). Antibodies to infliximab were detected in 28.46% patients for BCD-055 arm and 26.56% for IFX arm (p = 0.786). BCD-055 and IFX were comparable in efficacy (including radiographic progression), safety and immunogenicity throughout the 54-week study.Trial registration ClinicalTrials.gov ID, number NCT02762838.

Sequencing of Supernumerary Chromosomes of Red Fox and Raccoon Dog Confirms a Non-Random Gene Acquisition by B Chromosomes.

B chromosomes (Bs) represent a variable addition to the main karyotype in some lineages of animals and plants. Bs accumulate through non-Mendelian inheritance and become widespread in populations. Despite the presence of multiple genes, most Bs lack specific phenotypic effects, although their influence on host genome epigenetic status and gene expression are recorded. Previously, using sequencing of isolated Bs of ruminants and rodents, we demonstrated that Bs originate as segmental duplications of specific genomic regions, and subsequently experience pseudogenization and repeat accumulation. Here, we used a similar approach to characterize Bs of the red fox (Vulpes vulpes L.) and the Chinese raccoon dog (Nyctereutes procyonoides procyonoides Gray). We confirm the previous findings of the KIT gene on Bs of both species, but demostrate an independent origin of Bs in these species, with two reused regions. Comparison of gene ensembles in Bs of canids, ruminants, and rodents once again indicates enrichment with cell-cycle genes, development-related genes, and genes functioning in the neuron synapse. The presence of B-chromosomal copies of genes involved in cell-cycle regulation and tissue differentiation may indicate importance of these genes for B chromosome establishment.

Whole-Genome Analysis of Mycobacterium tuberculosis from Patients with Tuberculous Spondylitis, Russia.

Whole-genome analysis of Mycobacterium tuberculosis isolates collected in Russia (N = 71) from patients with tuberculous spondylitis supports a detailed characterization of pathogen strain distributions and drug resistance phenotype, plus distinguished occurrence and association of known resistance mutations. We identify known and novel genome determinants related to bacterial virulence, pathogenicity, and drug resistance.

Mouse chromocenters DNA content: sequencing and in silico analysis.

BACKGROUND: Chromocenters are defined as a punctate condensed blocks of chromatin in the interphase cell nuclei of certain cell types with unknown biological significance. In recent years a progress in revealing of chromocenters protein content has been made although the details of DNA content within constitutive heterochromatin still remain unclear. It is known that these regions are enriched in tandem repeats (TR) and transposable elements. Quick improvement of genome sequencing does not help to assemble the heterochromatic regions due to lack of appropriate bioinformatics techniques. RESULTS: Chromocenters DNA have been isolated by a biochemical approach from mouse liver cells nuclei and sequenced on the Illumina MiSeq resulting in ChrmC dataset. Analysis of ChrmC dataset by the bioinformatics tools available revealed that the major component of chromocenter DNA are TRs: ~ 66% MaSat and ~ 4% MiSat. Other previously classified TR families constitute ~ 1% of ChrmC dataset. About 6% of chromocenters DNA are mostly unannotated sequences. In the contigs assembled with IDBA_UD there are many fragments of heterochromatic Y-chromosome, rDNA and other pseudo-genes and non-coding DNA. A protein coding sfi1 homolog gene fragment was also found in contigs. The Sfi1 homolog gene is located on the chromosome 11 in the reference genome very close to the Golden Pass Gap (a ~ 3 Mb empty region reserved to the pericentromeric region) and proves the purity of chromocenters isolation. The second major fraction are non-LTR retroposons (SINE and LINE) with overwhelming majority of LINE - ~ 11% of ChrmC. Most of the LINE fragments are from the ~ 2 kb region at the end of the 2nd ORF and its' flanking region. The precise LINEs' segment of ~ 2 kb is the necessary mouse constitutive heterohromatin component together with TR. The third most abundant fraction are ERVs. The ERV distribution in chromocenters differs from the whole genome: IAP (ERV2 class) is the most numerous in ChrmC while MaLR (ERV3 class) prevails in the reference genome. IAP and its LTR also prevail in TR containing contigs extracted from the WGS dataset. In silico prediction of IAP and LINE fragments in chromocenters was confirmed by direct fluorescent in situ hybridization (FISH). CONCLUSION: Our data of chromocenters' DNA (ChrmC) sequencing demonstrate that IAP with LTR and a precise ~ 2 kb fragment of LINE represent a substantial fraction of mouse chromocenters (constitutive heteroсhromatin) along with TRs.

Emerging peak on the phylogeographic landscape of Mycobacterium tuberculosis in West Asia: Definitely smoke, likely fire.

To date, a major attention was justly given to the global lineages of Mycobacterium tuberculosis. Here, we demonstrated an importance of the minor ones, on an example of intriguing and underestimated NEW-1 family that belongs to Euro-American lineage (lineage 4). Analysis of the global WGS/NGS datasets (5715 strains) identified 2235 strains of Lineage 4 and 66 strains of sublineage L4.5. This latter is marked with RD122 genomic deletion and includes NEW-1 family. Phylogenomic analysis confirmed a separate position of the NEW-1 family that we tentatively designate L4.5.1/Iran. We propose an evolution/migration scenario starting with origin of L4.5 1000-1300 ya in China, subsequent origin of the pre-NEW-1 intermediate genotype in Tibet, further migration to Xinjiang/Uyghur, and finally to Iran since 800 ya (origin of NEW-1), possibly, via expansion of the Mongol Yuan empire. Analysis of longitudinal phylogeographic datasets revealed a sharp increase in prevalence of NEW-1 strains in Iran and its eastwards neighbors in the last 20years; most alarmingly, it is accompanied with significant association with multidrug resistance (MDR). Ongoing migration, especially, Afghan refugees flows to developed countries emphasize a risk of the wider spread of the epidemic MDR subtype within NEW-1 family that we coin as emerging resistant clone of M. tuberculosis in West Asia.

Draft genome of Prochlorothrix hollandica CCAP 1490/1T (CALU1027), the chlorophyll a/b-containing filamentous cyanobacterium.

Prochlorothrix hollandica is filamentous non-heterocystous cyanobacterium which possesses the chlorophyll a/b light-harvesting complexes. Despite the growing interest in unusual green-pigmented cyanobacteria (prochlorophytes) to date only a few sequenced genome from prochlorophytes genera have been reported. This study sequenced the genome of Prochlorothrix hollandica CCAP 1490/1T (CALU1027). The produced draft genome assembly (5.5 Mb) contains 3737 protein-coding genes and 114 RNA genes.

Contrasting origin of B chromosomes in two cervids (Siberian roe deer and grey brocket deer) unravelled by chromosome-specific DNA sequencing.

BACKGROUND: B chromosomes are dispensable and variable karyotypic elements found in some species of animals, plants and fungi. They often originate from duplications and translocations of host genomic regions or result from hybridization. In most species, little is known about their DNA content. Here we perform high-throughput sequencing and analysis of B chromosomes of roe deer and brocket deer, the only representatives of Cetartiodactyla known to have B chromosomes. RESULTS: In this study we developed an approach to identify genomic regions present on chromosomes by high-throughput sequencing of DNA generated from flow-sorted chromosomes using degenerate-oligonucleotide-primed PCR. Application of this method on small cattle autosomes revealed a previously described KIT gene region translocation associated with colour sidedness. Implementing this approach to B chromosomes from two cervid species, Siberian roe deer (Capreolus pygargus) and grey brocket deer (Mazama gouazoubira), revealed dramatically different genetic content: roe deer B chromosomes consisted of two duplicated genomic regions (a total of 1.42-1.98 Mbp) involving three genes, while grey brocket deer B chromosomes contained 26 duplicated regions (a total of 8.28-9.31 Mbp) with 34 complete and 21 partial genes, including KIT and RET protooncogenes, previously found on supernumerary chromosomes in canids. Sequence variation analysis of roe deer B chromosomes revealed a high frequency of mutations and increased heterozygosity due to either amplification within B chromosomes or divergence between different Bs. In contrast, grey brocket deer B chromosomes were found to be more homogeneous and resembled autosomes in patterns of sequence variation. Similar tendencies were observed in repetitive DNA composition. CONCLUSIONS: Our data demonstrate independent origins of B chromosomes in the grey brocket and roe deer. We hypothesize that the B chromosomes of these two cervid species represent different stages of B chromosome sequences evolution: probably nascent and similar to autosomal copies in brocket deer, highly derived in roe deer. Based on the presence of the same orthologous protooncogenes in canids and brocket deer Bs we argue that genomic regions involved in B chromosome formation are not random. In addition, our approach is also applicable to the characterization of other evolutionary and clinical rearrangements.

Trends in molecular epidemiology of drug-resistant tuberculosis in Republic of Karelia, Russian Federation.

BACKGROUND: Russian Republic of Karelia is located at the Russian-Finnish border. It contains most of the historical Karelia land inhabited with autochthonous Karels and more recently migrated Russians. Although tuberculosis (TB) incidence in Karelia is decreasing, it remains high (45.8/100 000 in 2014) with the rate of multi-drug resistance (MDR) among newly diagnosed TB patients reaching 46.5 %. The study aimed to genetically characterize Mycobacterium tuberculosis isolates obtained at different time points from TB patients from Karelia to gain insight into the phylogeographic specificity of the circulating genotypes and to assess trends in evolution of drug resistant subpopulations. METHODS: The sample included 150 M. tuberculosis isolates: 78 isolated in 2013-2014 ("new" collection) and 72 isolated in 2006 ("old" collection). Drug susceptibility testing was done by the method of absolute concentrations. Spoligotyping was used to test genotype-specific markers of a Latin-American-Mediterranean (LAM) family and its sublineages as well as a Beijing B0/W148-cluster. RESULTS: The largest spoligotypes were SIT1 (Beijing family, n = 42) and SIT40 (T family, n = 5). Beijing family was the largest (n = 43) followed by T (n = 11), Ural (n = 10) and LAM (n = 8). Successful Russian clone, Beijing В0/W148, was identified in 15 (34.9 %) of 43 Beijing isolates; all В0/W148 isolates were drug-resistant. Seven of 8 LAM isolates belonged to the RD115/LAM-RUS branch, 1 - to the LAM RD174/RD-Rio sublineage. MDR was found in Beijing (32/43), Ural (3/10), and LAM (3/8). In contrast, all T isolates were pansusceptible. Comparison of drug resistant subgroups of the new and old collections showed an increasing prevalence of the B0/W148 clonal cluster, from 18.0 % (mainly polyresistant) in 2006 to 32.6 % in 2014 (mainly MDR and pre-XDR). The West-east increasing gradient is observed for the Ural genotype that may be defined a 'Russian' strain. In contrast, the spoligotype SIT40 of the T family appears to be a historical Karelian strain. CONCLUSIONS: Circulation of the MDR M. tuberculosis isolates of the Beijing genotype and its B0/W148 cluster continues to critically influence the current situation with the MDR-TB control in northwestern Russia including the Republic of Karelia. Revealed phylogeographic patterns of some genotypes reflect a complex demographic history of Karelia within the course of the 20(th) century.

Consortium of the 'bichlorophyllous' cyanobacterium Prochlorothrix hollandica and chemoheterotrophic partner bacteria: culture and metagenome-based description.

'Bacterial consortium' sensu lato applies to mutualism or syntrophy-based systems consisting of unrelated bacteria. Consortia of cyanobacteria have been preferentially studied on Anabaena epibioses; non-photosynthetic satellites of other filamentous or unicellular cyanobacteria were also considered although structure-functional data are few. At the same time, information about consortia of cyanobacteria which have light-harvesting antennae distinct from standard phycobilisome was missing. In this study, we characterized first, via a polyphasic approach, the cultivable consortium of Prochlorothrix hollandica CCAP 1490/1 (filamentous cyanobacterium which contains chlorophylls a, b/carotenoid/protein complex in the absence of phycobilisome) and non-photosynthetic heterotrophic bacteria. The strains of most abundant satellites were isolated and identified. Consortium metagenome reconstructed via 454-pyro and Illumina sequencing was shown to include, except for P. hollandica, several phylotypes of Proteobacteria and Bacteroidetes. The ratio of consortium members was essentially stable irrespective of culture age, and restored after artificially imposed imbalance. The consortium had a complex spatial arrangement as demonstrated by FISH and SEM images of the association, epibiosis, and biofilm type. Preliminary data of metagenome annotation agreed with the hypothesis that satellite bacteria contribute to P. hollandica protection from reactive oxygen species (ROS).

Evaluation of a low-density hydrogel microarray technique for mycobacterial species identification.

In addition to the obligatory pathogenic species of the Mycobacterium tuberculosis complex and Mycobacterium leprae, the genus Mycobacterium also includes conditionally pathogenic species that in rare cases can lead to the development of nontuberculous mycobacterial diseases. Because tuberculosis and mycobacteriosis have similar clinical signs, the accurate identification of the causative agent in a clinical microbiology laboratory is important for diagnostic verification and appropriate treatment. This report describes a low-density hydrogel-based microarray containing oligonucleotide probes based on the species-specific sequences of the gyrB gene fragment for mycobacterial species identification. The procedure included the amplification of a 352-nucleotide fragment of the gene and its hybridization on a microarray. The triple-species-specific probe design and the algorithm for hybridization profile recognition based on the calculation of Pearson correlation coefficients, followed by the construction of a profile database, allowed for the reliable and accurate identification of mycobacterial species, including mixed-DNA samples. The assay was used to evaluate 543 clinical isolates from two regions of Russia, demonstrating its ability to detect 35 mycobacterial species, with 99.8% sensitivity and 100% specificity when using gyrB, 16S, and internal transcribed spacer (ITS) fragment sequencing as the standard. The testing of clinical samples showed that the sensitivity of the assay was 89% to 95% for smear-positive samples and 36% for smear-negative samples. The large number of identified species, the high level of sensitivity, the ability to detect mycobacteria in clinical samples, and the up-to-date profile database make the assay suitable for use in routine laboratory practice.

Genome-wide Mycobacterium tuberculosis variation (GMTV) database: a new tool for integrating sequence variations and epidemiology.

BACKGROUND: Tuberculosis (TB) poses a worldwide threat due to advancing multidrug-resistant strains and deadly co-infections with Human immunodeficiency virus. Today large amounts of Mycobacterium tuberculosis whole genome sequencing data are being assessed broadly and yet there exists no comprehensive online resource that connects M. tuberculosis genome variants with geographic origin, with drug resistance or with clinical outcome. DESCRIPTION: Here we describe a broadly inclusive unifying Genome-wide Mycobacterium tuberculosis Variation (GMTV) database, (http://mtb.dobzhanskycenter.org) that catalogues genome variations of M. tuberculosis strains collected across Russia. GMTV contains a broad spectrum of data derived from different sources and related to M. tuberculosis molecular biology, epidemiology, TB clinical outcome, year and place of isolation, drug resistance profiles and displays the variants across the genome using a dedicated genome browser. GMTV database, which includes 1084 genomes and over 69,000 SNP or Indel variants, can be queried about M. tuberculosis genome variation and putative associations with drug resistance, geographical origin, and clinical stages and outcomes. CONCLUSIONS: Implementation of GMTV tracks the pattern of changes of M. tuberculosis strains in different geographical areas, facilitates disease gene discoveries associated with drug resistance or different clinical sequelae, and automates comparative genomic analyses among M. tuberculosis strains.