Online Service with Automated Interpretation of Sequencing Data and Prediction of Pyrazinamide Resistance in Mycobacterium tuberculosis.

Pyrazinamide plays an important role in the treatment of tuberculosis. However, the microbiological test for pyrazinamide resistance is more complex and less reliable than testing of susceptibility to other anti-tuberculosis drugs due to the need to grow the pathogen at pH 5.5. Identification of mutations that cause resistance to anti-tuberculosis drugs can replace microbiological methods. Mutations in the pncA gene are responsible for the main mechanism of the resistance to pyrazinamide and are found in more than 90% of resistant strains. However, the genetic method for determining drug susceptibility is very complex, because mutations leading to pyrazinamide resistance are diverse and scattered throughout the gene. We have developed a software package for automatic data interpretation and prediction of the resistance to pyrazinamide based on Sanger sequencing results. The effectiveness of detection of pyrazinamide resistance in 16 clinical samples was compared using the BACTEC MGIT 960 automated system and pncA gene Sanger sequencing with automated analysis of the results. A significant advantage of the developed method over a single microbiological study was shown, due to greater reliability of the results irrespective of the purity of isolates.

A Method for Evaluation of the Level of Circulating Mitochondrial DNA by ND1 and ND2 Genes.

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.

SNP rs657152 Is Not Associated with the Level of Viral Load in COVID-19 or the Probability of Disease in the Population of Caucasians in Eastern Siberia.

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 detection of highly-transmissible genotypes of agent in clinical samples for prognosis of unfavorable course of tuberculosis.]

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; p < 0,01) 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; p < 0,05). 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; p < 0,05) 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.

GENETIC DIVERSITY OF THE MYCOBACTERIUM TUBERCULOSIS ISOLATES IN THE REPUBLIC SAKHA (YAKUTIA), RUSSIA.

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; p < 0.001) 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;p<0.001) 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.

[Analysis of complete sequence of cryptic plasmid pTP33 from Yersinia pestis isolated in Tuva natural focus of plague].

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 toxin­antitoxin 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 microorganisms­endosymbionts of insects (Sodalis glossinidius) and endosymbionts of entomopathogenic nematodes (Photorhabdus luminescens, P. asymbiotica, Xenorhabdus bovienii).