REVIEWS
ORIGINAL ARTICLES
Assessment of a particular territory by its epidemiological significance is very relevant in the light of widespread occurrence of new coronavirus infection, COVID-19, in the Russian Federation.
Objective. Detection of the epidemiological features and revealing trends in the epidemic process of new coronavirus infection in the Irkutsk Region and forecasting of its spread.
Materials and methods. An operational analysis of the epidemiological situation on COVID-19 in the Irkutsk Region as of August 16, 2020 was performed. Restrictive measures were substantiated based on the calculation of the infection spread rate (Rt).
Results and discussion. Irkutsk Region remained one of the adverse territories as regards human COVID-19 incidence in the Siberian Federal District and the Russian Federation as of August 16, 2020. The gradual dynamics of the epidemic process was revealed: at the first stage of the epidemic development it was facilitated by the imported cases from affected countries and regions of the Russian Federation, and subsequently by local transmission. The incidence increase was registered 2 months later than in central regions of the Russian Federation. Currently there is a consistent downward trend. The increase in the cases prevailed among rotation workers staying for shift and seasonal activities in the administrative center and northern districts of the Irkutsk Region. The epidemic pattern was determined by gender, age and social characteristics. It is demonstrated that the risk of the new coronavirus infection is determined by the intensity of contacts in family foci, the spread of infection in medical organizations and a significant proportion of asymptomatic carriers. High lethality rates (0.7 %) and mortality rates (31.1 o / oooo) were established among persons over 65 years. The stationary time series observed since June for changing the Rt-indicator with fluctuations ranging from 0.92 to 1.01 requires maintaining control of restrictive measures with prompt management decisions making based on the evolving epidemiological situation in the Irkutsk Region and taking into account the assessment of its possible complication risks.
Objective. Selection of indicators of lymphocyte populations in rhesus macaques determined by flow cytometry to evaluate variations of cellular constituent of their immune status.
Materials and methods. Blood of 11 healthy rhesus macaque males, 2,0–2,5 years old, weighing 2,5–3,0 kg, was used. Monkeys were examined simultaneously in each of 7 months of observation (since May till November). Immunophenotyping was conducted by FC500 cytofluorimeter using Affymetrix eBioscience monoclonal antibodies. The following cellular constituent indicators of immune status were differentiated: total T lymphocytes (phenotype CD2+CD20-); total B lymphocytes (phenotype CD2-CD20+); natural killer cells (phenotype CD2+CD56+); T helper cells (phenotype CD2+CD4+); cytotoxic T lymphocytes (phenotype CD2+CD8+); double-positive T lymphocytes (phenotype CD4+CD8+) and T lymphocytes positive by antigens CD2 and CD20 (phenotype CD2+CD20+).
Results and discussion. Statistical analysis of the obtained results revealed the absence of the effect of research time factor on the stated indicators. To assess changes in the cellular constituent of immune status of rhesus macaques, it is possible to use indicators that are less variable: total T and B lymphocytes, T helper cells, cytotoxic T lymphocytes and T lymphocytes with phenotype (CD2+CD20+). The use of CD56 as a marker of natural killer cells of rhesus macaques is impractical due to its low expression and a small size of the population bearing this marker. The research results may form the basis of the normative indicators of the subpopulation cell composition of immune system in rhesus macaques, which will allow the study of infected animals when assessing the quality of medical products in relation to dangerous and particularly dangerous infections.
Aim. Production of recombinant viral antigens of the main immunodominant proteins: glycoprotein (GPΔMLD), nucleoprotein (NP) and matrix protein (VP40) of the Marburg virus, as well as the study of their antigenic and immunogenic properties.
Materials and methods. To create recombinant proteins GPΔMLD, NP and VP40 of the Marburg virus, synthesized nucleotide sequences encoding these proteins cloned into the pET21a expression vector were used. The immunogenic and antigenic properties of the obtained recombinant proteins were tested using a number of biomodels (mice, chickens, and guinea pigs).
Results and discussion. Recombinant plasmids containing genes encoding proteins GPΔMLD, NP, VP40 of the Marburg virus, as well as Escherichia coli producing strains, with the yield of purified preparations of recombinant proteins GPΔMLD, NP, VP40 from one liter of culture fluid – 5, 10, and 10 μg were obtained, respectively. When mice are immunized, recombinant proteins GP, NP, and VP40 MARV induce the synthesis of high titer antibodies (recombinant proteins NP and VP40 – more than 409600, and recombinant protein GPΔMLD – 12800). Mouse antibodies specific to recombinant proteins interact in an enzyme-linked immunosorbent assay (ELISA) with the antigen of inactivated MARV. Antibodies of chickens immunized with virus-like particles containing the surface glycoprotein of the Marburg virus and antibodies of guinea pigs immunized with an experimental DNA vaccine containing the GPΔMLD MARV gene recognize the recombinant GPΔMLD protein and the viral protein in the inactivated MARV. The resulting recombinant proteins are immunogenic/antigenic and can be used for the development of enzymelinked immunosorbent assay systems.
The aim was to evaluate the effectiveness of using the “Sterius 60” microwave disinfection system (Russia) for decontamination of objects infected with PBA of groups I–IV emerging as a result of working with infected laboratory animals.
Materials and methods. Effectiveness verification of disinfection of biological waste generated as a result of the life of laboratory animals by SHF radiation was carried out in the microwave system “Sterius 60”, recommended by the manufacturer for disinfection of epidemiologically hazardous and extremely dangerous medical waste, including biological ones (classes B and C), by volumetric SHF heating. Carcasses of uninfected laboratory animals (white mice, Guinea pigs, suckling rabbits), granulated feed and bedding material (wood shavings), which are objects directly in contact with biomodels, were used as vivarium waste to be decontaminated. The following microorganisms were utilized as model test ones: Bacillus subtilus VKM B-911, Bacillus stearothermophilus VKM B-718, Bacillus licheniformis G VKM B-1711-D, Alcaligenes faecalis 415, Yersinia pestis EV, Bacillus anthracis STI. Laboratory utensils (plastic Petri dishes, porcelain mortars and pestles) were used as a mock-up chamber filler for model test microorganisms.
Results and discussion. As a result of the study, data were obtained indicating that the microwave system for disinfection of medical waste “Sterius 60” is ineffective for decontamination of biological waste in laboratories working with biomodels infected with PBA of groups I–II. The established standard mode of disinfection of this system was effective only for non-spore forms of microorganisms, pathogenicity groups III–IV. Therefore, in our opinion, it is advisable to use it for decontamination of laboratory utensils infected with PBA of groups III–IV, directly at sites of waste generation.
The aim of the study was molecular-genetic identification and analysis of the phylogenetic relationship of Yersinia pestis strains isolated on the Ukok Plateau in 2020, in order to establish the current boundaries of the natural mega focus of plague in the Altai Mountains in Russia and Mongolia.
Materials and methods. 37 strains of Y. pestis of the main subspecies isolated in the Tuva mountain and Gorno-Altai high-mountain plague foci and adjacent territories of Mongolia in 1971–2020 were studied. The whole genome sequencing of the strains was performed using the Ion S5 XL System (Thermo Fischer Scientific). Ion Torrent Suite software package 5.12 and Newbler gsAssembler 2.6 were used to process the data and assemble de novo the sequences of raw reads. The average size of the collected genome was 4.55 million base pairs. Core SNPs were detected by aligning the contigs of Y. pestis strains on the CO92 genome using the Snippy 4.6 program, then 28 SNP homoplasies were removed. The resulting set of SNPs contained only the core region of the genome (955 SNPs). The dendrogram was constructed using the Maximum Likelihood method applying the PhyML 3.1 program.
Results and discussion. The current population structure of Y. pestis of the main subspecies, antique biovar, phylogenetic line 4.ANT, endemic to the foci of the Altai Mountains in Russia and Mongolia has been determined. The presence of 4.ANT-21 clone, which became widespread in the territory of these natural foci of plague at the begining of the XXI century, was revealed. It is shown that three strains isolated on the Ukok Plateau in 2020 belong to clone 4.ANT-21. According to phylogenetic analysis, evidence of 4.ANT circulation on the Ukok Plateau before 2018 was obtained. The lesson that has been learned is that it is necessary to study the territories of Mongolia, Kazakhstan and China bordering the Ukok Plateau in order to establish the current boundaries of the 4.ANT mega focus.
The purpose of the study was to determine the effect of Yersinia pestis EV NIIEG on the process of neutrophil extracellular traps formation in vivo when modeling plague infection and assess their contribution to antiplague protection.
Materials and methods. BALB/c mice, which were immunized subcutaneously with the Y. pestis EV NIIEG vaccine strain, were used in the study. Animals were infected with a virulent strain Y. pestis 231 at a dose of 20 LD50 (103 CFU). To evaluate the contribution of neutrophil extracellular traps (NETs) to antibacterial protection, an experimental model was used based on fermenting NETs in the abdominal cavity of mice with nuclease. To calculate the number of NETs in peritoneal exudate (PE) fluorescent microscopy was applied. Phagocytic activity of PE cells was determined by flow cytometry. Bactericidal effect of NETs was recorded using bacteriological method.
Results and discussion. In pre-immunized mice, the process of NETs formation in response to the reintroduction of plague microbe living cells was 5 times more intense than in intact animals and was accompanied by a significant increase in the killing of Y. pestis cells in PE. The use of micrococcus nuclease in the experiment for fermentation of the NETs, produced in the body of immunized animals, provided evidence of NET participation in conferring anti-infective protection against plague infection. Thus, the established fact of the NET formation in case of Y. pestis infection of mice immunized with Y. pestis EV NIIEG vaccine strain and the influence of this process on the effectiveness of protection against plague is the basis for further clarifying the immunopathogenetic role of neutrophil granulocytes in plague.
Objective of the study was to determine the modern epidemiological peculiarities of Crimean-Congo hemorrhagic fever in the south of the Russian Federation.
Materials and methods. Data of statistical documentation (epidemiological survey of the infectious disease focus, annual summary reports dated 2010–2019) and epizootiologic monitoring data submitted by the Rospotrebnadzor Administrations and the Centers of Hygiene and Epidemiology in the constituent entities of the Southern and the North Caucasian Federal Districts were used. Descriptive, genetic, analytical methods and retrospective epidemiological analysis were applied.
Results and discussion. The СCHF natural focus in the European south of Russia has an area of 815 thousand square kilometers and it is characterized by persistence, expansion of the territory, and circulation of CCHF virus European genotypes. CCHF virus genetic variants form the local overlapping populations. Over the period of 2010–2019 997 CCHF cases were registered. Moderate forms of the disease clinically prevailed (74.2 per cent). Hemorrhagic symptoms were noted in 29.3 % of patients. The lethal outcome of the disease was registered in 31 CCHF patients (3 %). Two cases of nosocomial infection were identified, nine health workers were infected by CCHF pathogen. The expansion of epizootically active territory of the CCHF natural focus is associated with the expansion of the Hyalomma marginatum ticks area northward due to the aridization of the steppes, in part because of global climate change. The stability of the CCHF virus population was noted. The epidemic core of the CCHF natural focus remains on the adjacent territory of the Rostov Region, Stavropol Territory and the Republic of Kalmykia. The higher efficiency of CCHF prevention measures on the territory of the core is stemming from the targetness and intensity of measures, contributing to the early seeking of medical care by patients, their early hospitalization which in its turn influenced the development of clinical manifestations and outcome of the disease, and also made it possible to minimize the CCHF nosocomial infections number, including those among the medical personnel.
Currently, hemorrhagic fever with renal syndrome (HFRS) remains the most common natural focal disease of viral etiology in the Russian Federation. Effective antiviral drugs have not yet been developed. Pathogenetic and symptomatic drugs are mainly used in the treatment of HFRS.
The aim of the study was to evaluate the clinical and laboratory effectiveness of intravenous gabriglobin in the treatment of moderate forms of HFRS.
Materials and methods. The study included 22 patients with a diagnosis “HFRS of moderate severity” in the febrile stage of the disease. In the febrile period of HFRS, patients of the main group (n=10) were given gabriglobin via intravenous route at a dose of 0.1 g/kg per day along with pathogenetic therapy. The course of treatment was 2–3 infusions. The control group (n=12) received only pathogenetic therapy.
Results and conclusions. As a result of the studies, the clinical and laboratory efficacy of intravenous administration of gabriglobin in the treatment of patients with moderate-grade HFRS was revealed, which was manifested by a decrease in the duration of the febrile period by 2.1 days, the severity of oliguria and a decrease in the duration of thrombocytopenia by 3.5 days compared with the group of patients receiving only pathogenetic therapy.
The aim was to study the possibility of inducing Marburg-neutralizing chicken antibodies (MARV) using various immunogens.
Materials and methods. Recombinant vaccinia virus expressing the surface glycoprotein (GP) transgene MARV of Musoke strain and pseudovirus particles exhibiting GP of three strains of MARV – Popp, Musoke and DRC2000 based on lentivirus and recombinant strain of vesicular stomatitis virus (VSV) were used as immunogens. Two groups of birds were involved in the study. Chickens were immunized 9 times: first time they were injected with the recombinant vaccinia virus, and then 8 times – with pseudovirus particles (based on lentivirus and a recombinant strain of the vesicular stomatitis virus). The accumulation of specific antibodies was evaluated by enzyme-linked immunosorbent assay (ELISA). We used recombinant VSV exhibiting GP MARV, and natural MARV strain Popp for the analysis of accumulation of neutralizing antibodies.
Results and discussion. We have developed an effective immunization schedule for chickens with three recombinant constructs presenting GP MARV, which results in the induction of chicken IgY antibodies against Marburg virus with a titer in ELISA from 1:100 to 1:1 million. The obtained IgY neutralize MARV pseudoviruses (Popp, DRC2000, Musoke) at a dilution of 1/256 to 1/1024 and the natural MARV virus of the Popp strain at a dilution of 1/8. More stable results were demonstrated by immunization using Freund’s incomplete adjuvant.
Objective: comparative study of the etiological structure of community-acquired pneumonia in SARSCoV-2 “+”and SARS-CoV-2 “-“ patients who sought help from medical organizations in the Rostov Region.
Materials and methods. Biological material from 508 patients diagnosed with community-acquired pneumonia who were on outpatient treatment or in hospitals in Rostov-on-Don was studied. Verification of respiratory viruses, including SARS-CoV-2 RNA, as well as M. pneumoniae, C. pneumoniae, and L. pneumophila was performed by polymerase chain reaction in nasopharyngeal smears. Bacteriological analysis of sputum was carried out using differential diagnostic media, identification of isolated pathogens was carried out using time-of-flight mass spectrometry on Autoflex (Bruker Daltonics) with BioTyper 3.0 software.
Results and discussion. During the spread of a new coronavirus infection in the Rostov Region, the main etiological agent of community-acquired pneumonia is the new SARS-CoV-2 coronavirus. Specific character of pneumonia in patients with laboratory-confirmed COVID-19 is a higher incidence of mixed infection of both viral and bacterial etiology. Against the background of detection of a new coronavirus infection in patients with pneumonia, cases of detection of other types of coronaviruses have been registered (HKU-1,OC43, HL-63 and 229Е). The most common etiological agent of bacterial pneumonia in patients was Streptococcus spp., both in patients with COVID-19-associated pneumonia and in patients who tested negative for SARS-CoV-2. Coronavirus patients represent a high risk group for the development of mycotic lung lesions.
The global community is experiencing one of the largest infectious disease outbreaks in the 21st century. In the Saratov Region, the first case of new coronavirus infection was confirmed on March 19, 2020.The maximum increase in cases was noted between May 15 and June 30, during that time the total number of infected people in the region increased from 1526 to 6444. Since July 2020, a stable incidence level of new coronavirus infection has been observed in the Saratov Region, without a steady decline.
The aim of the study was to assess the status of population immunity to the SARS-CoV-2 virus among residents of Saratov and the Saratov Region under the COVID-19 epidemic.
Materials and methods. In the period from June 23 to July 26, 2020, a serological study of blood samples from 3372 volunteers of different age groups was conducted. The content of antibodies to SARS-CoV-2 was determined by ELISA using a set of reagents “ELISA anti-SARS-CoV-2 IgG” produced by the State Scientific Center of Applied Microbiology and Biotechnology of the Rospotrebnadzor (Russia).
Results and discussion. In general, the incidence of COVID-19 in the Saratov Region is taking place against the background of moderate seroprevalence to the SARS-CoV-2 virus, accompanied by a high incidence of non-apparent (asymptomatic) forms of the infectious process. The absence of clinical symptoms of the disease, in the context of the limited use of methods for determining the RNA of the SARS-CoV-2 virus in PCR (11 % of the region’s population) makes it difficult to assess the real spread of the virus in the population and to establish the timing of the formation of persistent herd immunity. A low rate of antibody response among individuals with a positive result of PCR analysis, as well as among volunteers who had an infection in May, June 2020, indicates a weak formation of the immune response, or the prevalence of individuals reacting mainly by activating the cellular link of the immune system in the population. The obtained results, although they need to be explained in a number of respects, can be applied to the organization of preventive measures, including vaccination, in the region.
By August 2020, more than 850000 cases of new coronavirus infection (COVID-19) caused by SARSCoV-2 were confirmed in the Russian Federation, with the Rostov Region as one of the ten most affected regions in Russia. The spread of the disease is largely determined by the state of population immunity in a certain area. Our research focuses on specific humoral immune response and estimates the level of herd immunity to SARS-CoV-2 virus among the population of the Rostov Region.
Materials and methods. The study involved 3,048 people; the volunteers participating in the study were divided into seven age groups. The content of antibodies to SARS-CoV-2 was determined applying ELISA using a kit for the analysis of human serum or blood plasma for the presence of specific IgG to the nucleocapsid of the SARS-CoV-2 virus, manufactured by the State Scientific Center of Applied Microbiology and Biotechnology (Obolensk) in accordance with the instructions for use.
Results and discussion. The assessment of seroprevalence to SARS-CoV-2 in the Rostov Region showed that the proportion of people positive for IgG to the new coronavirus was 16.5 %, the range of seropositive individuals in the general population was between 13.9 % and 19.1 % (p<0.05). There were no significant gender differences in the degree of seroprevalence with a positive result registered in 16.6 % of women and 16.5 % of men. A high level of humoral immunity to SARS-CoV-2 was established in individuals aged 1–17 against the background of low incidence rates, which may indicate the dominance of asymptomatic forms of the disease in this age group. The highest level of seropositivity was found in preschool children (33.6 %), students (29.3 %), employees (17.3 %), and education professionals (15.3 %).
The aim of the study was to develop a unified approach to a comprehensive risk assessment of the anthrax situation deterioration based on the ranking of territories by epidemiologically significant factors.
Materials and methods. For a comprehensive risk assessment of the situation complication, the statistical principles of ranking of the estimated indicators were used. These indicators comprise the presence and activity of soil foci of anthrax, the prevailing soil types and their main characteristics, as well as social risk factors associated with the peculiarities of animal husbandry and the possibility of infection of the population. The proposed assessment method has been tested on a national scale, and its content and results are described in this paper by the example of the Volga Federal District. The materials of the study were federal and regional cadastres of stationary hazardous as regards anthrax areas, an atlas and maps of soils, data of statistical accounting and reporting of Federal Service for Veterinary and Phytosanitary Surveillance, Federal Service for State Registration, Cadastre and Cartography and Rospotrebnadzor.
Results and discussion. It has been established that in all constituent entities of the district there are potential risks of the situation deterioration that are associated with the presence of soil foci. In terms of the sum of ranks, the greatest potential disadvantage is noted in a number of republics and regions of the Volga Region. At the same time, the real danger is mainly due to social factors, which include the characteristics of animal husbandry, as well as the degree of susceptibility of the population. The maximum risks are currently identified in the territories of the republics of Bashkortostan and Tatarstan, as well as in the Saratov Region. The study is aimed at improving the efficiency of anthrax epidemiological surveillance in the Russian Federation, and the proposed methodology can be used at all levels of surveillance.
The aim of the work was development of a monoclonal antibody-based latex agglutination test to identify the causative agent of melioidosis, and the approbation of a freeze-dried experimental preparation for screening of environmental bacterial isolates in Vietnam.
Materials and methods. The carriers of specific antibodies were polyacrolein latex particles with active aldehyde groups on the surface. Typical strains of the causative agents of melioidosis and glanders with a full-fledged antigenic structure, as well as the strains Burkholderia thailandensis, Burkholderia cepacia, Pseudomonas aeruginosa, and Pseudomonas putida were used to control the test specificity. The latex agglutination reaction was carried out on plastic Petri dishes with daily bacterial cultures, from which suspensions were prepared at a concentration of 1–2·109 m.c./ml. The results of the reaction were registered visually for 5–8 min using a 4-cross system against a dark background under lighting. The reaction to 3–4 crosses was recorded as positive. Colonies suspected of belonging to pathogenic Burkholderia from primary inoculations were transferred to L-agar with polymyxin B and grown for 36 hours at (37±1) °C. The species of the selected suspicious colonies was determined by multiplex PCR.
Results and discussion. With collection strains, latex test demonstrated high sensitivity agglutinating 97.7 % of B. pseudomallei and all B. mallei strains. At the same time, it was negative with B. thailandensis, B. cepacia, P. aeruginos and P. putida. In microbiological screening of bacterial cultures isolated from environmental objects, the latex test had a diagnostic sensitivity of 89.4 %. Using the latex test at the stage of primary screening, it is possible to significantly reduce the time when processing a lot of samples received for analysis, as well as to reduce the consumption of reagents used at the subsequent stages of identification.
The aim of the study was to improve the procedure for the Yersinia pestis EV strain cell concentration using the system for tangential flow microfiltration with the ASF-020 filter support unit.
Materials and methods. The study used the vaccine Y. pestis EV strain derived from NIIEG cell line. Submerged cultivation of the native culture was performed using BIOR-0.25 reactor with automated control system. Microbial suspension concentrate was produced through microfiltration applying (Adaptive filtration system) AFS-009 and AFS-020 installations. The content of live microbial cells was determined by cytorefractometry. Assessment of the resistance of Y. pestis EV strain cells to technological factors was performed by photometric registration of changes in the optical density of bacterial suspension during the lytic response of cells to sodium dodecyl sulfate. Physical-chemical and immunobiological properties of the dry live plague vaccine were determined in accordance with the pharmacopoeial item.3.3.1.0021.15 of the State Pharmacopoeia of the Russian Federation, 14th edition.
Results and discussion. The design features of the equipment introduced made it possible to carry out membrane filtration of microbial suspension, using BIOR-0.25 reactor as an intermediate storage unit, thereby excluding three technological stages. The total concentration of microbes in the suspension obtained by routine and improved methods was more than 150 billion microbial cells per ml. A comparative study of the effect of various hydrodynamic regimes in the working cavities of AFS-009 and AFS-020 filter units did not significantly affect the morphometric properties and resistance of microbial cultures to extreme (technological) factors. Based on the experimental data, the mass balance of the membrane filtration process has been determined. The optimized technology gave 0.13 liter yield of concentrate from 1 liter of native culture, and the process duration was reduced to 5 hours, the yield of the finished product in one production cycle was increased by 3 times. Thus, the process of concentrating Y. pestis EV strain cells during the production of the tablet form of live plague vaccine has been enhanced. A comparative study of the morphometric properties and resistance of plague microbe cultures to technological factors in the process of their concentration using optimized technology did not reveal any significant differences as compared to the routine one. Technological stage of concentrating has been reduced to 5 h term with a three-fold increase in the yield of finished product.
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