Vol 12, No 5 (2022)

It is known that the psychrophilic pseudotuberculosis microbe serotype 1 (Y. pseudotuberculosis 0:1b) causing Far East scarlet-like fever (FESLF) — an intestinal infection found in a wide range of invertebrates and vertebrates inhabiting cold regions in the Northern and Central Asia as well as Far East is direct ancestor of the plague causative agent Yersinia pestis. However, the mechanism of Y. pestis speciation remains poorly elucidated. Numerous Y. pestis phylogenies created by using molecular genetic (MG) technologies are largely contradictory, being not in line with reliable data obtained by natural science approaches (e.g., ecology, epizootology, biogeography, and paleontology), which disagree with current evolutionary doctrine (synthetic theory of evolution). The MG approach provides no definitive answer to the questions of where, when, how, and under what circumstances the species Y. pestis arose. One of the reasons for such situation might be due to inadequacy of using the molecular evolutionary model for Y. pestis phylogenetics. Knowledge of the life cycles for the ancestral pseudotuberculosis and derivative plague microbes as well as related unique environmental features allows to create a reliable ecological model for the plague microbe evolution to be further used for assessing patterns of molecular variability and building proper molecular model that might be accepted for MG-reconstruction of plague microbe history. According to the ecological model, the species Y. pestis was formed in a tritopic manner (almost) simultaneously from FESLF clones (populations) in the three geographical populations of the Mongolian marmot-tarbagan (Marmota sibirica) and the flea Oropsylla silantiewi parasitizing on it. The inducer of speciation was coupled to the last maximum (Sartan) cooling in Central Asia occurred 22–15 thousand years ago. Soil cooling and deep freezing resulted in altered behavior of the marmot flea larvae with emergence of facultative hematophagy, which, in turn, led to a unique traumatic (compared to routine alimentary) infection route of sleeping marmots with FESLF and, as a result, a unique way of Y. pestis speciation. The molecular model should predict a Y. pestis peripatric tritope speciation, existing numerous parallelisms in intraspecific variability associated with tritope speciation, and the quantum principle of speciation in the highly variable heterothermic (heteroimmune) stressful marmot-flea (Marmota sibirica — Oropsylla silantiewi) host-vector environment involving stress-induced mutagenesis. Such molecular model of evolution may be useful for improving molecular methodology of phylogenetic constructions for a wide range of parasitic microorganisms.

The international biomedical community has been currently facing a need to find a simple and most accessible type of analysis that helps to diagnose tuberculosis (TB) with the maximum reliability even before the onset of clinical manifestations. Tuberculosis results in more deaths than any other pathogen, second only to pneumonia caused by the SARS-CoV-2 virus, but the majority of infected people remain asymptomatic. In addition, it is important to develop methods to distinguish various forms of tuberculosis infection course at early stages and to reliably stratify patients into appropriate groups (persons with a rapidly progressing infection, chronic course, latent infection carriers). Immunometabolism investigates a relationship between bioenergetic pathways and specific functions of immune cells that has recently become increasingly important in scientific research. The host anti-mycobacteria immune response in tuberculosis is regulated by a number of metabolic networks that can interact both cooperatively and antagonistically, influencing an outcome of the disease. The balance between inflammatory and immune reactions limits the spread of mycobacteria in vivo and protects from developing tuberculosis. Cytokines are essential for host defense, but if uncontrolled, some mediators may contribute to developing disease and pathology. Differences in plasma levels of metabolites between individuals with advanced infection, LTBI and healthy individuals can be detected long before the onset of the major related clinical signs. Changes in amino acid and cortisol level may be detected as early as 12 months before the onset of the disease and become more prominent at verifying clinical diagnosis. Assessing serum level of certain amino acids and their ratios may be used as additional diagnostic markers of active pulmonary TB. Metabolites, including serum fatty acids, amino acids and lipids may contribute to detecting active TB. Metabolic profiles indicate about increased indolamine 2.3-dioxygenase 1 (IDO1) activity, decreased phospholipase activity, increased adenosine metabolite level, and fibrous lesions in active vs. latent infection. TB treatment can be adjusted based on individual patient metabolism and biomarker profiles. Thus, exploring immunometabolism in tuberculosis is necessary for development of new therapeutic strategies.

Currently, vaccination is one of the most efficient ways to control and prevent influenza infection. Vaccine production largely relies on the results of laboratory assays, including hemagglutination inhibition and microneutralization assays, which are time-consuming and laborious. Viruses can escape from the immune response that results in the need to revise and update vaccines biannually. The hemagglutination inhibition assay can measure how effectively antibodies against a reference strain bind and block an antigen of the test strain. Various computer-aided models have been developed to optimize candidate vaccine strain selection. A general problem in modeling of antigenic evolution is the representation of genetic sequences for input into the research model. Our motivation stems from the well-known problem of encoding genetic information for modeling antigenic evolution. This paper introduces a two-fold encoding approach based on reduced amino acid alphabet and amino acid index databases called AAindex. We propose to apply a simplified amino acid alphabet in modeling of antigenic evolution. A simplified alphabet, also called a sub-alphabet or reduced amino acid alphabet, implies to use the 20 amino acids being clustered and divided into amino acid groups. The proposed encoding allows to redefine mutations termed for amino acid groups located in reduced alphabets. We investigated 40 reduced amino acid sets and their performance in modeling antigenic evolution. The experimental results indicate that the proposed reduced amino acid alphabets can achieve the performance of the standard alphabet in its accuracy. Moreover, these alphabets provide deeper insight into various aspects of the relationship between mutation and antigenic variation. By checking identified high-impact sites in the Influenza Research Database, we found that not only antigenic sites have a significant influence on antigenicity, but also other amino acids located in close proximity. The results indicate that all selected non-antigenic sites are related to immune responses. According to the Influenza Research Database, these have been experimentally determined to be T-cell epitopes, B-cell epitopes, and MHC-binding epitopes of different classes. This highlighted a caveat: while simulating antigenic evolution, the model should consider not only the genetic information on antigenic sites, but also that of neighboring positions, as they may indirectly impact antigenicity. Additionally, our findings indicate that structural and charge characteristics are the most beneficial in modeling antigenic evolution, which is in agreement with previous studies.

In our study, we aimed to evaluate the significance of specific cytokines in blood plasma as predictive markers of COVID-associated mortality. Materials and methods. In plasma samples of 29 patients with PCR-confirmed COVID-19 we measured the concentrations of 47 molecules. These molecules included: interleukins and selected pro-inflammatory cytokines (IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-12 (p40), IL-12 (p70), IL 13, IL-15, IL-17A/CTLA8, IL-17-E/IL-25, IL-17F, IL-18, IL-22, IL-27, IFNα2, IFNγ, TNFα, TNFβ/Lymphotoxin-α(LTA)); chemokines (CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL7/MCP-3, CCL11/Eotaxin, CCL22/MDC, CXCL1/GROα, CXCL8/IL-8, CXCL9/MIG, CXCL10/IP-10, CX3CL1/Fractalkine); anti-inflammatory cytokines (IL-1Ra, IL-10); growth factors (EGF, FGF-2/FGF-basic, Flt-3 Ligand, G-CSF, M-CSF, GM-CSF, PDGF-AA, PDGFAB/BB, TGFα, VEGF-A); and sCD40L. We used multiplex analysis based on xMAP technology (Luminex, USA) using Luminex MagPix. As controls, we used plasma samples of 20 healthy individuals. Based on the results, we applied Receiver Operating Characteristic (ROC) analysis and Area Under Curve (AUC) values to compare two different predictive tests and to choose the optimal division point for disease outcome (survivors/non-survivors). To find optimal biomarker combinations, we as used cytokines concentrations as dependent variables to grow a regression tree using JMP 16 Software.Results. Out of 47 studied cytokines/chemokines/growth factors, we picked four pro-inflammatory cytokines as having high significance in evaluation of COVID-19 outcome: IL-6, IL-8, IL-15, and IL-18. Based on the results received, we assume that the highest significance in terms of predicting the outcome of acute COVID-19 belongs to IL-6 and IL-18. Conclusion. Analyzing concentrations of IL-6 and IL-18 before administering treatment may prove valuable in terms of outcome prognosis.

Leptospirosis holds one of the leading places among zoonoses due to the breadth of distribution of natural and economic foci, as well as the severity of the clinical manifestation and mortality rate. A relatively low incidence rate in some regions is usually accounted for by unsatisfactory level of differential diagnostics, including insufficient coverage by diagnostic tests. Leptospira are characterized by marked phenotypic and genotypic polymorphisms, affecting epidemiological features and clinical manifestation of leptospirosis. Therefore, laboratory methods are important tool for epidemiological surveillance and diagnostics of leptospirosis. Serological methods primarily the microscopic agglutination test (MAT) currently remain the leading tool in investigating leptospirosis. To study a near 60 year-long contribution of various leptospira serogroups to etiological pattern of leptospirosis, we have analyzed archived MAT serum data from patients suspected of leptospirosis retrieved from our laboratory journals during from the years 1962 to 2020. It was shown that antibodies to the Grippotyphosa serogroup (25.1%) were found most often in patients, which might be associated with the wide range of reservoir host animals and resistance of this leptospira serogroup to environmental factors. At the same time, it should be noted that at different periods antibodies to various leptospira serogroups prevailed in the sera of patients, and in many cases this data matched with the contribution of these pathogens to the etiological morbidity pattern. In particular, dominance of pathogens of the Sejroe serogroup is currently the “global trend” in the etiological pattern of leptospirosis that was also observed by us at some years. The data obtained might be useful for epidemiological surveillance of leptospirosis infection, including predicting potential outbreaks, as well as for developing approaches to its diagnostics, treatment and prevention.

Since 2017, the incidence rate of rubella in the Russian Federation has been below 1 case per million total population. In addition, no circulation of endemic strains of the rubella virus is recorded evidencing about achieving infection elimination phase. In modern conditions, it is important to constantly monitor the level of herd immunity to the rubella virus to identify epidemically significant population groups, especially in countries lacking rubella vaccination or featured with insufficient disease control. Purpose: to study herd immunity to the rubella virus in selected countries in Eurasia and Africa. Materials and methods. Between 2018 and 2021, 15,594 samples of blood sera were tested for IgG and IgM antibodies to the rubella virus from subjects of different ages obtained from regional measles and rubella surveillance centers in the Northwestern Federal District (NWFD) of the Russian Federation, the Republic of Serbia, South Vietnam, and the Republic of Guinea. The “Anti-Rubella Virus ELISA (IgМ)” and “Anti-Rubella Virus ELISA (IgG)” (Euroimmun, Germany) test kits were used. Statistical data processing was carried out using the MS Excel, Prizm 5.0 (GraphPad Software Inc.), and Statistica 8.0 (StatSoft Inc.) software package. Results. During the observation period (2018–2020) the population seroprevalence of the to the rubella virus in the NWFD of the Russian Federation was 96.6–97.7% and fluctuated slightly both in separate years and among individual age groups evidencing about high coverage of rubella vaccination. In the Republic of Serbia conducting two-fold immunization against rubella the overall seroprevalence rate was lower than in the Russian Federation and comprising 86.8%. The minimum number of IgG-positive sera was recorded in the 2–4-year-old age group pointing to the shortcomings of routine vaccination. In South Vietnam, children aged 1–3 years (41.9%) predominated among those recovering from rubella, i.e. the age cohort that should be protected by vaccination at the age of 18 months. No rubella vaccination is carried out in Guinea. The total proportion of seropositive individuals was 75%; herd immunity to the rubella virus was established mainly among children and adolescents, reaching 90% only in the older age group. 30% of unprotected subjects of the most active reproductive age were identified among the females surveyed in Guinea. Conclusion. Insufficient herd immunity to the rubella virus, identified in a number of countries, may contribute to the maintenance of the infectious process and the spread of infection. Globalization contributes to the virus importation into regions being at the stage of measles and rubella elimination. The results obtained suggest about a need to continue efforts aimed at maintaining epidemiological wellbeing regarding rubella in diverse countries of the world.

Melioidosis is a particularly dangerous infection with endemic distribution caused by the Gram-negative microorganism from the pathogenicity group II Burkholderia pseudomallei. In endemic countries, melioidosis holds one of the leading places in mortality rate after HIV, tuberculosis and, in recent years, COVID-19. The natural ecological pathogen niches are located in tropical and subtropical climate zones, primarily in Southeast Asia and Australia, where its existence as a species is maintained in moist soil and water in a certain temperature environmental range. However, at present, more and more often cases of melioidosis are registered outside endemic territories, which emphasizes the relevance of improving the means and methods of laboratory diagnostics of this disease both for countries located in the zone of natural foci as well as for local healthcare of the countries after importation of this poorly known infection into their territory. In such countries, including the Russian Federation, the population has no natural immunity to the pathogen, and therefore this infection acquires even greater clinical and epidemiological significance. In the Volgograd Plague Control Research Institute, an erythrocyte antigenic melioidosis diagnostic agent for IHA was designed allowing to detect the presence of serum melioidosis antibodies. The diagnostic agent was obtained on the basis of a biological carrier — ram erythrocytes sensitized with isolated protein antigenic complexes of B. pseudomallei. The high analytical characteristics of the diagnostic agent were confirmed on sera models of immunized and recovering experimental animals. Using the obtained set of reagents, the level of serum antibodies against the causative agent of melioidosis was studied in residents from the 3 provinces of Vietnam (Ha Giang, Lang Son and Quang Ninh), as well as in control group composed of residents of the Volgograd region. In samples obtained from a non-endemic region, not more than 25% of cases contained IHA titers at lower than 1:10 dilution, which is apparently due to cross-reactivity of serum immunoglobulins. Positive serum samples from clinically healthy residents of Ha Giang, Lang Son and Quang Ninh provinces were at a titer of 1:10 detected in 71.5%, in dilutions of 1:20–1:80 — in 28.5% of cases. Thus, we believe that serum antibody titer of 1:80 established in the IHA results, has a diagnostic significance, reflecting the intensity of the anti-melioidosis populational immunity.

Introduction. Infective endocarditis (IE) is a bacterial disease with frequent pathogen localization on the heart valve apparatus. IE is characterized by rapid development of heart failure and frequent thromboembolic complications (TEC). IE features are accounted for by foreign pathogen nature and state of human immune system (IS). The imbalanced IS in infective endocarditis is manifested by impaired cytokine-mediated interactions. This confirms the rationality of studying cytokines to advance understanding of the pathogenesis for various conditions. Most cytokine genes are characterized by polymorphism and existing isoforms underlying disease predisposition. Genetic polymorphism of vascular endothelial growth factor — A (VEGF-A) plays an important role in the induction of vasculogenesis and angiogenesis. The pathogenetic VEGF role in IE has not been thoroughly studied. Research objective — analysis of polymorphic nucleotide sequence variants in the vascular endothelial growth factor gene, taking into account a relation with its serum concentration in patients with infective endocarditis. Materials and methods. 86 patients treated with verified diagnosis of infective endocarditis at the Scientific Research Institute — “Regional Clinical Hospital No. 1” of Krasnodar were divided into two clinical groups in accordance with the IE course: Group 1 — IE with TEC (n = 44), group 2 — IE without TEC (n = 42), and the control group consisted of 20 apparently healthy individuals. The concentration of serum VEGF-A (pg/mL) was measured by ELISA on day 1 of hospitalization. Genomic DNA was isolated from whole blood leukocytes and used to determine the frequency of genotypes of VEGF gene polymorphic variants. Results. Significant differences in the frequency distribution of VEGF-rs2010963 genotypes between patients with infective endocarditis and control group were revealed: G/G (OR = 0.25; p = 0.012) and G/C (OR = 4.28; p = 0.022), as well as differences between VEGF concentrations for various SNP-rs2010963 genotypes (p = 0.0001). A study of VEGF genotype frequency distribution between patients of clinical groups showed a significantly decreased frequency of the genotype G/G (rs2010963) in the IE group with TEC (OR = 0.21; p = 0.014) and increased frequency of G/C (OR = 4.72; p = 0.024) compared with the control group, whereas in patients with IE without TEC, significant (p = 0.0003) differences in serum concentrations of VEGF-rs2010963 were found in accordance with genotypes GG/CC (p = 0.01) and GG/GC (p = 0.003). Conclusion. The relationship between the VEGF genotypes (G/G and G/C of rs2010963 polymorphism) and related serum concentration among patients with IE was revealed. Carriers of the minor C allele (rs2010963) had higher serum VEGF levels. The results obtained complement and systematize current scientific data on the disease pathogenesis, as well as focus on the genetic determinant of the developing complications.

High adjuvant reactogenicity is the main limitation for increasing the effectiveness of vaccine therapy. The aim was to reduce the immunotoxicity effects of complete Freund’s adjuvant (CFA) in warm-blooded animals. Materials and methods. The study examined Wistar rats by dividing animals into negative control (solvents); positive control (single subcutaneous CFA injection of 0.1 ml/200 g body weight (b.w.)); the “minimum” and “maximum” (per os administration of 1:4 citric and succinic acids in ratio of 17 and 88 mg/kg b.w. during 4 weeks after immunization of CFA) experiment. Body weight, hematological (complete blood count) and biochemical (hydroperoxides, malondialdehyde, catalase activity, mitochondrial dehydrogenase activity) parameters were dynamically investigated. At the end of the experiment, necropsy was performed and the relative internal organ mass coefficients were calculated. The spleen and connective tissue (knee joint) were examined histologically. The median, C₂₅–C₇₅ quartiles, Mann–Whitney U-test were calculated. Results and discussion. it was found that parameters examined were within normal range in animals of negative control group. Immunization of warm-blooded animals with CFA was accompanied by transition of acute-to- chronic inflammatory reaction (week 3 and week 7, respectively). The total leukocyte count increased from 12.5 × 10⁹ (negative control) up to 26.6 × 10⁹/L (P = 0.01) on week 3 followed by its decline down to 19.2 × 10⁹/L (P = 0.01) by week 7. Platelet count also increased significantly: from 506 × 10⁹ (negative control) up to 656 × 10⁹/L (P = 0.01, week 3) followed by decrease down to 610 × 10⁹/L by week 7 (P = 0.01). Activation of lipid peroxidation was manifested by malondialdehyde (MDA) level elevated by 55.8–61.8% (P = 0.01); the general CFA-related toxic effect resulted in 11.7% weight loss (P = 0.01), spleen swelling and thymus reduction. Administration of antioxidant acids led to a dose-dependent decline in inflammation (leukocyte count at the minimum dosage — 19.6 × 10⁹–20.9 × 10⁹/L; at the maximum 16.6 × 10⁹–16.0 × 10⁹/L), as well as normalized the “platelet/leukocyte” index up to 29.5–36.3 (positive control 24.6, negative control 40.5). The acid-related protective effect was also manifested as maintained body weight, activated catalase and inhibited lipid peroxidation. The therapeutic effect in alleviated degenerative changes in the spleen and connective tissue were revealed: reduced hemorrhagic focuses and swelling as well as preserved histoarchitectonics. Conclusion. The use of citric and succinic acids contributes to profoundly lowered CFA toxicity due to increased total antioxidant status, inhibited lipid peroxidation, improved mitochondrial metabolic activity, which ultimately lead to a decline in general systemic inflammation and allows to recommend such acids as immunoprotectors from oil adjuvant-coupled effects.

Bacillus anthracis, the causative agent of anthrax, is able to exist both in environmental conditions (soil) and in the macroorganism. The manifestation of pathogenic properties of B. anthracis strains is determined by relevant plasmid composition, because the main toxin and the capsule-related virulence factors are located in bacterial plasmid. Modeling anthrax infection in vitro in macrophage culture might reveal an influence of individual B. anthracis strain characteristics on infection and development of infectious process. The aim of this study was to analyze cytokine level during infection of in vitro macrophage cell cultures with spores of anthrax microbe strains bearing varying plasmid composition. The dependence of the macrophage cell cytokine profile on the plasmid composition of B. anthracis strains was revealed while modeling anthrax infection in vitro. The presence of the toxin-producing plasmid pXO1 in anthrax microbe strains has a powerful stimulating effect on the production of macrophages J774A cell line cytokines. B. anthracis strains lacking the pXO1 plasmid virtually stimulated no production of IL-1β, caused very low secretion of IL-1α, IL-6, MCP-1, MIP-1α, MIP-1β, IL-12 (p70) and active G-CSF products. The low cytokine response of macrophage cells infected with monoplasmid strains bearing only the capsule-forming plasmid was due not only to the absence of a binary toxin, but also to disturbed regulation of capsule production associated with the absence of the atxA gene. The capsule, along with lethal and edematous toxins, belongs to the main virulence factors of B. anthracis, but strains lacking the pXO1 virulence plasmid, had its production impaired, because the main regulator of capsule synthesis is the atxA gene localized on the pXO1 plasmid being positively regulated by the acpA and acpB genes, so that strains lacking the toxin-forming plasmid, even in the presence of the encapsulation plasmid, elicit a weak cytokine response in infected cells. Diplasmid strains of B. anthracis, due to produced main virulence factors — a two-component toxin and a capsule, enforce macrophages (in the experiment) to actively produce IL-1β, IL-6, MCP-1, G-CSF, MIP-1α; MIP-1β, IL-12 (p70). Strains with moderate virulence and capable of capsulation in air virtually did not differ from highly virulent strains in terms of their effect on in vitro macrophage culture.

Introduction. Sequencing of the 16S rRNA gene is the predominant method for assessing microbial communities and strain molecular identification. The short reads (2^nd generation sequencing)-based technology does not allow analysis beyond the 16S rRNA gene. The taxonomic verification level of samples usually remains at the genus or even family level. Currently, there have been proposed the latest versions of long-read technologies (Oxford Nanopore MinION, PacBio) for amplicon sequencing of near-complete ribosomal operon, including genes 16S, 23S, 5S, and internal transcribed spacer (ITS). At the moment, this approach has not been sufficiently studied, in addition, it involves PCR amplification of a very extended DNA region (more than 4000 bp-long). Materials and methods. The collection of non-tuberculous mycobacteria strains and their primary identification was carried out in the years 2019–2021. The strains were obtained by inoculation of positive cultures from the Bactec MGIT 960 bacteriological analyzer lacking MPT64 antigen in the MGIT TB Identification Test (Becton Dickinson, USA) on Lowenstein-Jensen medium. Preliminary species strain identification was carried out with the Speed-oligo Mycobacteria kit (Vircell, Spain) according to the manufacturer’s protocol. In this work, both known and newly developed universal bacterial primers flanking the near-complete 16S rRNA gene, ITS, and the beginning of the 23S rRNA gene are used. In the present study, both known and newly developed universal bacterial primers are used to flank the near-complete 16S rRNA gene, ITS, and start of the 23S rRNA gene. Results and discussion. Sanger sequencing of the amplicons (about 2000 bp) obtained shows the taxonomic level sufficient to determine species up to 8 strains of non-tuberculous mycobacteria isolated from humans that caused clinically and bacteriologically confirmed diseases. The method proposed for PCR amplification of a bacterial operon a fragment containing most of the 16S rRNA gene, ITS, and the beginning of the 23S rRNA gene is considered by us as an approbation of a methodological approach to study microbial communities in material with a high degree of degradation (necrotic foci, etc.). The results obtained indicate a significantly higher resolution of the approach used than the classical 16S rRNA gene sequencing.