Vol 12, No 5 (2022)

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Full Issue


Ecological scenario of the plague microbe Yersinia pestis speciation underlying adequate molecular evolutionary model

Suntsov V.V.


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.

Russian Journal of Infection and Immunity. 2022;12(5):809-818
pages 809-818 views

Evolutionary aspects of gastrointestinal tract microbiome-host interaction underlying gastrointestinal barrier integrity

Loskutov S.I., Proshin S.N., Ryabukhin D.S.


In the host sustenance and homeostasis, the microbiome is a key component in the functional system. Throughout ontogenetic development, microbiome including that of the gastrointestinal tract (GIT) is the vital factor that ensures not only host functioning, but also its interaction with environment. To uncover the mechanisms underlying GIT microbiome showing a decisive influence on host organism, a systematic approach is needed, because diverse microorganisms are predominantly localized in different parts of the GIT. Recently, a new interdisciplinary direction of science, nanobioinformatics that has been extensively developed considers “gene networks” as the major object of study representing a coordinated group of genes that functionally account for formation and phenotypic “disclosure” of various host traits. Here, an important place should be provided to the genetically determined level of the gastrointestinal tract microbiome, its interaction at the level of the host food systems. There have been increasing evidence indicating that the microbiome is directly involved in the pathogenesis of host diseases showing a multi-layered interaction with host metabolic and immune systems. At the same time, the microbial community is unevenly distributed throughout the gastrointestinal tract, and its different portions are variously active while interacting with the host immune system. The “architecture” of interaction between the microbiome and host cells is extremely complex, and the interaction of individual cells, at the same time, varies greatly. Bacteria colonizing the crypts of the small intestine regulate enterocyte proliferation by affecting DNA replication and gene expression, while bacteria at the tip of the intestinal villi mediate gene expression responsible for metabolism and immune response. Enterocytes and Paneth cells, in turn, regulate the vital activity of the community of microorganisms through the production of polysaccharides (carbohydrates) and antibacterial factors on their surface. Thus, the integrity of the gastrointestinal barrier (GIB) is maintained, which protects the body from infections and inflammation, while violation of its integrity leads to a number of diseases. It has been shown that depending on the dominance of certain types of bacteria the microbiome can maintain or disrupt GIB integrity. The structural and functional GIB integrity is important for body homeostasis. To date, at least 50 proteins have been characterized as being involved in the structural and functional integrability of tight junctions between gastrointestinal tract epithelial cells. The current review comprehensively discusses such issues and presents original research carried out at various facilities of translational biomedicine.

Russian Journal of Infection and Immunity. 2022;12(5):819-826
pages 819-826 views

Metabolic biological markers for diagnosing and monitoring the course of tuberculosis

Korotetskaya M.V., Rubakova E.I.


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.

Russian Journal of Infection and Immunity. 2022;12(5):827-836
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Reduced amino acid alphabet-based encoding and its impact on modeling influenza antigenic evolution

Forghani M., Firstkov A.L., Alyannezhadi M.M., Danilenko D.M., Komissarov A.B.


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.

Russian Journal of Infection and Immunity. 2022;12(5):837-849
pages 837-849 views

Evaluation of the effects in the in vitro system of synthetic thymic hexapeptide on the expression levels of NF-κB, IFNα/βR and CD119 neutrophilic granulocytes in patients with chronic herpes viral co-infections

Nesterova I.V., Khalturina E.O., Nelubin V.N., Khaidukov S.V., Chudilova G.A.


Background. Strategies used by herpes viruses with human cells are complex and multifaceted. On one hand, inborn defects in antiviral immune defense have been unveiled, which also affect interferon (IFN) system underlying development of chronic recalcitrant relapsing viral infections such as remittent respiratory viral infections, herpesvirus infections, and papillomavirus infections. On the other hand, numerous viruses are able to damage both immune system and IFN network. During inborn and acquired defects in IFN network, inborn or induced mutation in gene products involved in signaling cascades aimed at upregulating gene expression responsible for IFN production are observed. One of the strategies used by diverse viruses is altering some signaling pathways resulting in activated transcription factors including nuclear factor NF-kB. However, antiviral mechanisms executed by neutrophilic granulocytes (NGs), particularly affecting NF-kB expression have not been elucidated. Aim of the study: to study in vitro features of NF-kB expression and number of neutrophilic granulocytes (NG) expressing membrane IFNα/βR and IFNγR in patients with atypical chronic active herpes virus infections (AChA-HVI), followed by assessing an effect of arginyl-alpha-aspartyl-lysyl-valyl-tyrosyl-arginine — hexapeptide (HP), a synthetic analogue of the active center of the thymopoietin (active substance of drug “Imunofan”, Russia), on the expression of NG NF-kB and IFNα/βR and IFNγR. Materials and methods. We observed 25 patients of both sexes aged 23 to 64 years with AChA-HVI, manifested by chronic fatigue syndrome and cognitive disorders. Study design: stage 1 — clinical, ELISA, PCR methods, FC was used. Stage 2 — the in vitro experiment: 32 blood samples from 8 healthy adults and 375 blood samples from 25 patients with AChA-HVI were analyzed: % NG expressing NF-kB, IFNα/βR, IFNγR and the relevant MFI levels by using FC before and after incubation with HP. Results. Our study demonstrated low level (MFI) of NF-kB expression in 100% NG associated with decreased % of NG expressing IFNα/βR and IFNγR in all patients with AChA-HVI and low serum level for IFNα and IFNγ in comparison with healthy individuals. In the in vitro experiment there was shown that 100% of NG expressed NF-kB after exposure to HP. However, only 48% patients (SG 2) restored NF-kB expression level (MFI) to normal range and 52% of cases (SG 1) had no response. HP increased % of NG expressing IFNα/βR in SG 2 and increased % of NG expressing IFNγR in SG 1. Conclusions. It was shown, that influence of HP “in vitro” has ambiguous effects on the expression of NF-kB, % of NG expressing IFNα/βR and IFNγR in patients with AChA-HVI. We assume that different NF-kB response to HP is associated with inborn or secondary NF-kB deficiency.

Russian Journal of Infection and Immunity. 2022;12(5):850-858
pages 850-858 views

Predictive value of specific cytokines for lethal COVID-19 outcome

Arsentieva N.A., Liubimova N.E., Batsunov O.K., Korobova Z.R., Kuznetsova R.N., Rubinstein A.A., Stanevich O.V., Lebedeva A.A., Vorobyev E.A., Vorobieva S.V., Kulikov A.N., Gavrilova E.G., Pevtcov D.E., Polushin Y.S., Shlyk I.V., Totolian A.A.


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.

Russian Journal of Infection and Immunity. 2022;12(5):859-868
pages 859-868 views

Associations between serum levels of C3, C4, and total classical complement activity in COVID-19 patients at the time of admission and clinical outcome

Razi A., Azimian A., Arezumand R., Solati A., Ahmadabad H.N.


In the present study, we investigated the association between complement system status at the time of admission and clinical outcomes in COVID-19 patients. This single-center study was carried out with sixty-one adult patients with COVID-19 who were hospitalized at Imam Hassan Hospital of North Khorasan University of Medical Sciences (Bojnurd, Iran) with less than three days passage since onset of COVID-19 symptoms. Twenty-three healthy volunteers with demographic features similar to the patient group (matched by age and gender) were included in the study as a control group. Patient information including demographic information, demographic data, clinical characteristics, and clinical outcomes were obtained from electronic medical records. Of 61 hospitalized patients with COVID-19, 28 (47.54%) were female, and the average age was 48.7±8.8 years. The healthy control group included 23 cases (11 (47.8%) female, 12 (52.1%) males, mean age 46.4±4.4 years). Twenty-one of the 61 patients (34.4%) were admitted to the ICU, and sixteen of them (26.2%) died. Thirty-three (54.10%) patients with COVID-19 were hospitalized for less than 7 days, and 28 (45.90%) of them were hospitalized for ≥ 7 days. Our results show that length of hospital stay in the no-ICU group was significantly lower than the ICU admission or death groups (6.49±0.24 vs. 8.85±1.59 and 10.53±1.80, p = 0.0002). The levels of C3, C4, and CH50 were determined through the immunoturbidimetric method and single-radial-haemolysis plates, respectively, on serum samples obtained from patients at the time of admission or those in the control group. Our results indicate that C3, C4 and CH50 levels were markedly lower in COVID-19 patients than in the control group. We also found that complement parameter levels in COVID-19 patients who died or were admitted to ICU were significantly lower than in non-ICU COVID-19 patients. In general, it seems that serum level of C3, C4, and CH50 at admission may predict disease progression or adverse clinical outcome in COVID-19 patients.

Russian Journal of Infection and Immunity. 2022;12(5):869-874
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Analyzying the documented results by using microscopic agglutination test to examine sera from patients suspected of leptospirosis

Samsonova A.P., Petrov E.M., Savelyeva O.V., Ivanova A.E., Sharapova N.E.


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.

Russian Journal of Infection and Immunity. 2022;12(5):875-890
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Seroprevalence of tick-borne diseases in the Northwest Federal District of the Russian Federation

Tokarevich N.K., Blinova O.V., Stoyanova N.A., Baimova R.R., Siuziumova E.A., Lomonosova V.I., Tronin A.A., Buzinov R.V., Sokolova O.V., Gnativ B.R., Buts L.V., Bubnova L.A., Safonova O.S., Stankevich A.I., Kalinina E.L., Vikse R., Andreassen A.K.


Introduction. Knowledge about tick-borne disease (TBD) distribution is necessary to improve prevention, whereas detection of human serum IgG antibodies against relevant pathogens is a method for monitoring TBD prevalence in local population. The study objective was to estimate seroprevalence of IgG antibodies against tick-borne encephalitis virus (TBEV), Borrelia burgdorferi sensu lato, Coxiella burnetii, Anaplasma phagocytophilum, and Ehrlichia chaffeensis/E. muris in healthy residents from the five territories of the Northwestern Federal District of the Russian Federation (Arkhangelsk Oblast, Leningrad Oblast, Pskov Oblast, the Republic of Komi and the Republic of Karelia). Materials and methods. In 2017–2019, a total of 1244 serum samples from healthy residents, not vaccinated against TBDs or other flavivirus-caused infections was studied by ELISA. Results. 21.7% of the sera samples contained IgG antibodies against a single TBD pathogen, whereas 2.1% showed signs of coinfection with two or more pathogens. The most common were IgG antibodies against TBEV (5 territories, 12.2%), followed by Borrelia burgdorferi sensu lato (5 territories, 3.5%), C. burnetii (4 territories, 2.9%), Anaplasma phagoсytophilum (3 territories, 1.6%), E. chaffeensis/E. muris (5 territories, 1.5%). The IgG antibodies were more common in men (55.2%) than in women (44.8%), being found virtually evenly in age-independent manner (from juniors under 18 to seniors over 60). Conclusion. The results of this first comprehensive serosurveillance study in the Northwestern Federal District of the Russian Federation assessing serum IgG antibodies against tick-borne diseases indicate a wide distribution of such pathogens. Moreover, infections caused by C. burnetii, Anaplasma phagocytophilum, and Ehrlichia chaffeensis/E. muris might be highly underdiagnosed.

Russian Journal of Infection and Immunity. 2022;12(5):891-901
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A herd immunity to rubella virus in selected geographical regions

Lavrentieva I.N., Bichurina M.A., Antipova A.Y., Camara J., Hoang M., Bancevic M.D., Zheleznova N.V., Egorova S.A., Totolian A.A.


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.

Russian Journal of Infection and Immunity. 2022;12(5):902-908
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State of anti-rubella virus population immunity in the Republic of Belarus and Russian Federation

Tsvirkun O.V., Samoilovich E.O., Tikhonova N.T., Gerasimova A.G., Turaeva N.V., Ermolovich M.A., Semeiko G.V.


To confirm a progress towards the rubella elimination, immunization coverage indicators and the results of serological studies are used. At the same time, the most objective method of assessing collective immunity is a serological examination. The purpose of this work was to compare the results of assessing state of population immunity against rubella virus in the Republic of Belarus and the Russian Federation using various vaccines and different approaches to study organization and cohort selection — screening and monitoring. 2019 laboratory studies to determine IgG antibodies against rubella virus were carried out by ELISA method of the unified enzyme immunoassay system “Ecolab” (Russia). The serum study results of 701 subjects aged 2–45 years from all the 7 regions of the Republic of Belarus (screening study) and 55 082 subjects aged 3–49 years (monitoring) from 85 regions of the Russian Federation were analyzed. The results were evaluated in three age groups — children, adults, total cohort by using a threshold level of 83–85% seropositive individuals sufficient to prevent virus transmission. The antigenic composition of the rubella component in the mono- and complex vaccines respectively used in the Russian Federation and the Republic of Belarus is identical, which allows to compare the state of population immunity against rubella virus therein without taking into account an influence of the vaccine strain. Studies have shown that while using varying vaccine preparations and approaches to organization of serological examination, both in the Republic of Belarus and the Russian Federation, rubella virus-specific IgG level was detected at rather high level in all age groups comprising more than 90%. The portion of rubella seropositive subjects in entire country was 95.44% in the Republic of Belarus and 97.14% in the Russian Federation. Moreover, the level of specific antibodies remains high, despite the low incidence rate and absence of booster effect in both countries. The mean group antibody concentration in seropositive patients in all age groups was quite high and exceeded the minimum protective concentration (11 IU/ml) by 10 or more times. The data obtained indicate the legitimacy of using a serological study of collective rubella immunity as a routine or seromonitoring as well as periodic or screening investigation.

Russian Journal of Infection and Immunity. 2022;12(5):909-918
pages 909-918 views

Obtaining and using erythrocyte antigenic meliidosis diagnostic agent in the analysis of serum samples from meliidosis-endemic provinces Ha Giang, Lang Son and Quang Ninh of the Socialist Republic of Vietnam

Tereshko D.L., Novitskaya I.V., Zakharova I.B., Trien D., Kuznetsov A.N., Kulakov M.Y., Budchenko A.A., Pushkar V.G., Viktorov D.V., Toporkov A.V.


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.

Russian Journal of Infection and Immunity. 2022;12(5):919-928
pages 919-928 views

CDR1, CDR2, MDR1 and ERG11 expression in azole resistant Сandida albicans isolated from HIV-infected patients in city of Moscow

Voropaev A.D., Yekaterinchev D.A., Urban Y.N., Zverev V.V., Nesvizhsky Y.V., Voropaeva E.A., Likhanskaya E.I., Afanasiev M.S., Afanasiev S.S.


Candida fungi are common opportunistic microorganisms capable of causing infections of various localization, as well as life-threatening conditions in immunocompromised patients, such as HIV-infected individuals, oncology patients, subjects undergoing HSCT, which number has been steadily increasing in recent years. In addition, resistance to anti-fungal drugs has been spreading as well. Naturally sensitive to azoles, C. albicans possess a variety of mechanisms of acquired resistance, including efflux transporters and target protein-encoding gene amplification. This study was conducted to assess a prevalence of such mechanisms in the isolates sample obtained from HIV-infected patients in the Moscow region of the Russian Federation, characterize a relationship between these mechanisms and patterns of developing drug resistance. 18 strains of C. albicans resistant to fluconazole and voriconazole were isolated from HIV-infected patients with recurrent oropharyngeal candidiasis in the Moscow region. The expression levels of the ERG11, MDR1, CDR1, CDR2 genes involved in the formation of acquired azole resistance were measured using quantitative PCR, the –2ΔΔCT method with ACT and PMA genes as control genes and reference values of sensitive isolates. Expression levels exceeding the average values of sensitive isolates by more than 3 standard deviations were considered significantly elevated. In most of the isolates, elevated levels of CDR1 and CDR2 gene expression were found: 89% and 78%, respectively. The expression level of the MDR1 gene was increased only in 28% of cases. ERG11 expression levels were significantly elevated in 78% of the isolates. Expression levels of all resistance genes studied were significantly increased in 4 strains. In this sample of C. albicans isolates, acquired resistance is mainly associated with efflux vectors encoded by the CDR1 and CDR2 genes. Also, in most isolates, an increased expression level for the azole target protein gene — ERG11 was detected. The expression level of the efflux transporter gene MDR1 was increased in the smallest number of samples. It is also impossible to exclude a potential role of other mechanisms in developing acquired resistance, such as mutations in the ERG11 gene. It can be assumed that the identified mechanisms of resistance result from long-term, widespread, and sometimes uncontrolled use of azoles, including those in treatment and prevention of candidiasis in HIV-infected patients.

Russian Journal of Infection and Immunity. 2022;12(5):929-937
pages 929-937 views

VEGF gene polymorphism in complicated infective endocarditis

Samoylenko E.S., Kolesnikova N.V., Baklay V.I., Maydannikova E.Y., Omelchenko E.V.


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.

Russian Journal of Infection and Immunity. 2022;12(5):938-946
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Role of Toxoplasma gondii in thyroiditis in pregnant women

Valizadeh G., Khamseh M., Kashaniyan M., Rafiei-Sefiddashti R., Hadighi R.


Toxoplasmosis (acute and latent) is the most prevalent parasitic infection worldwide and can be associated with some problems in pregnant women. Thyroid diseases are the most common endocrine disorders secondary to diabetes among pregnant women. Previous studies proposed a relationship between latent еoxoplasmosis (LT) and autoimmune thyroiditis diseases (AITDs). This study intended to investigate the frequency and correlation between toxoplasmosis and AITD in pregnant women. In this cross-sectional study, the statistical population included 1248 pregnant women at the gestational age of 9–16 weeks and in Tehran. The Toxoplasma IgM and IgG tests were assessed with enzyme-linked immunosorbent assay (ELISA). The diagnostic criteria for toxoplasmosis were abnormal IgG and IgM titers. In addition, FT4, TPO Ab, and TSH were evaluated using enzyme-linked fluorescence immunoassay (ELFA). TPO Ab was used to distinguish thyroid patients with autoimmune origin from those with other thyroiditis diseases. The analysis showed no significant relationship between keeping a house cat and acute toxoplasmosis. Acute and latent toxoplasmosis represented 3.4% and 29.6%, respectively. The frequency of thyroid diseases was 18.8% (hypothyroidism 15.8% versus hyperthyroidism 3%). The frequency of autoimmune thyroiditis diseases (AITDs) was 5.5%, and 27.9% of subjects with latent toxoplasmosis (LT) had a thyroiditis disease, but 13.8% of pregnant women with LT had only AITD with a significant correlation (p < 0.001). Results show that Toxoplasma IgG+ can increase the risk of AITD by 10.39-fold and a higher TPO Ab titer in people with LT. It seems Toxoplasma gondii may cause thyroiditis in pregnant women likely because antigenic similarity of Toxoplasma and thyroperoxidase leads to cross-reactivity in the immune system, potentially causing AITD. It might be said that the high prevalence of LT among pregnant women may have a potential role in the stimulation of the immune system to the development of autoimmune diseases, such as AITD. So future studies could be conducted with a focus on discovering molecular similarities between thyroperoxidase and Toxoplasma antigens.

Russian Journal of Infection and Immunity. 2022;12(5):947-952
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Improving safety of oil adjuvant-based vaccines

Skupnevskii S.V., Pukhaeva E.G., Badtiev A.K., Rurua F.K., Batagova F.E., Farnieva Z.G.


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.

Russian Journal of Infection and Immunity. 2022;12(5):953-962
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Cytokine profile in in vitro mouse macrophage culture infected with Bacillus anthracis spores with varying plasmid composition

Koteneva E.A., Tsygankova O.I., Shcherbakova V.Y., Kalinin A.V., Rodionov I.S., Serdyukov V.V., Abramovich A.V., Kulichenko A.N.


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.

Russian Journal of Infection and Immunity. 2022;12(5):963-970
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Immune response evaluation in the guinea pigs after immunization with the experimental Puumala virus vaccine

Kurashova S.S., Balovneva M.V., Ishmukhametov A.A., Teodorovich R.D., Popova Y.V., Tkachenko E.A., Dzagurova T.K.


In the Russian Federation, the vaccine against hemorrhagic fever with renal syndrome is at the stage of preclinical and clinical trials. The aim of the study was to analyze an effect of vaccine schedule on neutralizing antibodies (nAB) dynamics in guinea pig models applied with experimental Puumala virus based hantavirus vaccine (HV). Quantitative evaluation of neutralizing antibodies was presented as antibody titer geometric mean expressed in binary logarithms (log2) by the 50% reduction of focus-forming units (FRNT50) in Vero cell in the focus reduction neutralization test. The HV dual inoculation to guinea pigs was carried out in 14 day intervals, booster injection was applied on day 182 after the onset, in the thigh muscle tissue by using 0.3 ml undiluted (HV-u/d) and in the 1/10 dilution (HV-1/10). nAB titer on day 14 after the first HV-u/d and HV-1/10 injection was measured to be at titer of 5.5±0.3 and 4.8±0.3, respectively. After the second injection, the nAB peak was as high as 9±0.2 on day 42 after the first HV-u/f injection, and 6.5±0.2 on day 14 after the HV-1/10 injection. nAB decreased down to 6.2±0.3 and 5±0.3, respectively, on day 364 after the first injection. The booster HV-u/d and HV-1/10 injection induced increase in nAB up to 9.5±0.3 and 6.5±0.3, respectively. After the booster injection, it induced significantly higher nAB observed on day 238 after the first HV-u/d injection and delayed up to the 294 day for the HV-1/10. The results of the study indicated the early formation of the immune response, long-term nAB persistence and significantly enhanced immune response after the booster injection on day 182, which indicated a potential for the booster injection a year later. The immunological efficacy and protective activity of the vaccine schedule may be finally assessed according to the results of clinical trials.

Russian Journal of Infection and Immunity. 2022;12(5):971-975
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16S-ITS-23S rRNA operon segment sequencing provides necessary and sufficient conditions for bacterial species-specific identification

Ogarkov O.B., Zhdanova S.N., Orlova E.A., Khromova P.A., Belkova N.L., Sinkov V.V., Kondratov I.G.


Introduction. Sequencing of the 16S rRNA gene is the predominant method for assessing microbial communities and strain molecular identification. The short reads (2nd 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.

Russian Journal of Infection and Immunity. 2022;12(5):976-980
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Modified quantitative approach for assessing peripheral blood TREC and KREC levels in immunodeficient patients

Saitgalina M.A., Ostankova Y.V., Liubimova N.E., Semenov A.V., Kuznetsova R.N., Totolian A.A.


Introduction. The immune status is a multifaceted parameter quantitatively and qualitatively analyzing functional activity immune system state in immune organs as well as some non-specific mechanisms of antimicrobial protection. Peripheral blood level of T-receptor excision rings (TREC) and B-cell excision rings (KREC), respectively, can serve as surrogate markers of T- and B-cell maturation. Currently, the diagnostic kits available on the market have two significant disadvantages: i) the kits are aimed at diagnosing immunodeficiency conditions only in newborns and children, while keeping adult patients uncovered; ii) essentially, use solely single reference normalization gene for data normalization resulting in increased variability and decreased sensitivity of the assay data. The aim: to develop a highly sensitive method for laboratory assessment of the state of immunity in immunodeficient patients by using real-time PCR for assessing TREC and KREC level in children and adults. Materials and methods. There were used whole blood and dry blood spot samples obtained from newborns and adults, apparently healthy individuals as well as patients with verified PID and HIV-infection. A total of 2577 samples were examined. Commercial kits were used as comparison methods. Results. Multiplex PCR was carried out, analyzing the number of target molecules TREC and KREC, as well as fragments of the HPRT and RPP30 normalization genes analyzed with the developed series of plasmid calibrators. The established analytical range of TREC/KREC DNA measurements comprised 103 to 109 copies/mL. The accuracy of measurements on a tablet-type instrument (CFX) was 95.84%, on a rotary-type instrument (Rotor-Gene 3000) — 95.11%, which corresponds to the standard indicator. The equivalence between the data obtained after assessing whole blood samples and dry blood drops was shown. The data analysis allowed to find out 100%-diagnostic specificity and sensitivity of the method proposed. Conclusion. The method developed by us allows to diagnose decline in T- and/or B-cell immunity in children and adults and can be used to detect TREC and KREC molecules both in peripheral whole blood samples and dry blood spots using Guthrie cards. Moreover, the uniform values of reference norms can be used regardless of the type of analyzed clinical material. The study data evidence about potential for effective use of multiplex PCR diagnostics both for complex primary testing/screening of newborns and assessing state of immunity to identify adult patients with PID and as a part of the diagnostic monitoring of patients with secondary immunodeficiencies, e.g., HIV infection.

Russian Journal of Infection and Immunity. 2022;12(5):981-996
pages 981-996 views

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