Molecular epidemiological monitoring of the tuberculosis pathogen in the Arkhangelsk region

Introduction. Against the background of improvement of the main epidemiological indicators (morbidity and mortality) for tuberculosis in the Arkhangelsk region, the proportion of newly diagnosed tuberculosis patients with multidrug-resistant pathogen (MDR-TB) increased from 18.7% in 2002 to 33.8% in 2018. The purpose of this study was the genotypic characterization of Mycobacterium tuberculosis strains obtained from newly diagnosed tuberculosis patients in the Arkhangelsk region in 2018. Materials and methods. 89 M. tuberculosis strains isolated in 2018 from newly diagnosed tuberculosis patients were studied. Beijing genotype, its clusters B0/W148 and Central Asian/Russian were determined by PCR detection of the specific markers: IS6110 insertions in the dnaA-dnaN region, mutations in codons 48 of the mutT4 gene (CGG GGG) and 58 of the mutT2 gene (GGA CGA), IS6110 insertions in the Rv2664 region-Rv2665 and Rv1359-Rv1360, substitutions G A in the sigE gene. Non-Beijing strains were spoligotyped. Results. Drug resistance was detected in 41.6% (37/89), MDR — in 33.7% of strains. In 90% (27/30) of MDR strains, resistance to rifampicin and isoniazid was due to rpoB Ser531Leu and katG Ser315Thr mutations. Following M. tuberculosis genotypes were identified: Beijing (67.4%), T (14.6%), Ural (4.5%), Haarlem (4.5%), LAM (2.3%) and CAS1-Delhi (1.1%). Among the Beijing strains, clusters Central-Asian/Russian (60%; 36/60) and B0/W148 (30%; 18/60) prevailed. The majority of MDR strains belonged to the Beijing family (93.3%; 28/30), of which 64.3% (18/28) and 21.4% (6/28) belonged to clusters B0/W148 and Central-Asian/Russian, respectively. Conclusion. In heterogeneous population of the causative agent of tuberculosis in the Arkhangelsk region, the most common strains were those of the Beijing genotype; in 2018 its share increased to 67.4% (40.4% in 1998–1999). Among MDR strains, the proportion of Beijing reached 93.3%, of which more than half (64.3%) belonged to the epidemiologically and clinically significant in Russia cluster B0/W148

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

Allele-Specific rpoB PCR Assays for Detection of Rifampin-Resistant Mycobacterium tuberculosis in Sputum Smears

We describe an allele-specific PCR assay to detect mutations in three codons of the rpoB gene (516, 526, and 531) in Mycobacterium tuberculosis strains; mutations in these codons are reported to account for majority of M. tuberculosis clinical isolates resistant to rifampin (RIF), a marker of multidrug-resistant tuberculosis (MDR-TB). Three different allele-specific PCRs are carried out either directly with purified DNA (single-step multiplex allele-specific PCR), or with preamplified rpoB fragment (nested allele-specific PCR [NAS-PCR]). The method was optimized and validated following analysis of 36 strains with known rpoB sequence. A retrospective analysis of the 287 DNA preparations from epidemiologically unlinked RIF-resistant clinical strains from Russia, collected from 1996 to 2002, revealed that 247 (86.1%) of them harbored a mutation in one of the targeted rpoB codons. A prospective study of microscopy-positive consecutive sputum samples from new and chronic TB patients validated the method for direct analysis of DNA extracted from sputum smears. The potential of the NAS-PCR to control for false-negative results due to lack of amplification was proven especially useful in the study of these samples. The developed rpoB-PCR assay can be used in clinical laboratories to detect RIF-resistant and hence MDR M. tuberculosis in the regions with high burdens of the MDR-TB

Novel IS6110 Insertion Sites in the Direct Repeat Locus of Mycobacterium tuberculosis Clinical Strains from the St. Petersburg Area of Russia and Evolutionary and Epidemiological Considerations

A modification of spoligotyping with primers derived from the direct repeat (DR) and IS6110 sequences was used to identify IS6110 insertions in the DR locus of Mycobacterium tuberculosis clinical strains from the St. Petersburg area of Russia. Novel IS6110 insertions were identified: (i) in two epidemiologically unlinked Beijing family strains, an asymmetrical direct insertion in DR37; (ii) in a non-Beijing strain, an asymmetrical insertion in the opposite orientation in DR38; (iii) in another non-Beijing strain, a direct insertion in DR38 and one in the opposite orientation in DR14 (DR numbering is according to standard spoligotyping). Our results strengthen an observation that the DR locus structure is extremely conserved in the Beijing genotype. Asymmetrical insertions prevented detection of the adjacent spacer by standard spoligotyping. This, therefore, should be taken into consideration when similar spoligoprofiles that differ only in signals 37 and 38 are interpreted

Detection of Ethambutol-Resistant Mycobacterium tuberculosis Strains by Multiplex Allele-Specific PCR Assay Targeting embB306 Mutations

We describe a multiplex allele-specific (MAS)-PCR assay to detect simultaneously mutations in the first and third bases of the embB gene codon 306ATG. These mutations are known to confer ethambutol (EMB) resistance in the majority of clinical Mycobacterium tuberculosis isolates worldwide. The mutated bases are revealed depending on the presence or absence of the respective indicative fragments amplified from the embB306 wild-type allele. Initially optimized on purified DNA samples, the assay was tested on crude cell lysates and auramine-stained sputum slide DNA preparations with the same reproducibility and interpretability of the generated profiles in agarose gel electrophoresis. Since EMB resistance is generally linked to multiple-drug resistance (MDR), the MAS-PCR assay for EMB resistance detection can be used in clinical laboratory practice in areas with a high prevalence and a high transmission rate of MDR-EMB-resistant tuberculosis

Molecular Characterization of Mycobacterium tuberculosis Isolates from Different Regions of Bulgaria▿

Mycobacterium tuberculosis isolates from different regions of Bulgaria were studied by a variety of molecular typing tools. Based on spacer oligonucleotide typing (spoligotyping), the 113 strains were subdivided into 35 spoligotypes: 5 unique profiles and 15 profiles shared by two to 29 strains; the Hunter-Gaston diversity index (HGI) was 0.9. Comparison with the international database SITVIT2 at the Institut Pasteur de Guadeloupe showed the presence of two globally distributed shared types, ST53 (25.7%) and ST47 (6.2%). Nineteen (16.8%) and six (5.3%) strains belonged to the ST125 (LAM/S subfamily) and ST41 (LAM7_TUR subfamily) types described in SITVIT2 as ubiquitous/rare and ubiquitous/common types, respectively. Seven spoligoprofiles (12 strains) were not found in the database; two of them constituted new shared types. The Beijing genotype strains were not found in the studied collection in spite of close contacts with Russia in the recent and historical past. Additional subtyping by IS6110-restriction fragment length polymorphism (RFLP) and 12-locus mycobacterial interspersed repetitive unit (MIRU)-variable number of tandem repeat analyses were performed within selected spoligotypes. In particular, MIRU typing showed better discrimination within ST125 than IS6110-RFLP typing (HGI = 0.83 versus 0.39). A high gradient for ST125 in Bulgaria compared to its negligible presence in the global database and neighboring countries leads us to suggest a Bulgarian phylogeographic specificity of this spoligotype. To conclude, this first study of the Bulgarian M. tuberculosis population demonstrated its heterogeneity and predominance of several worldwide-distributed and Balkan-specific spoligotypes