Vol 11, No 4 (2021)

Serological monitoring of the study of population immunity to the SARS-CoV-2 virus in the context of COVID-19 pandemic is a necessary component in epidemiological surveillance, since population epidemiological wellbeing in a context of COVID-19 is determined by state of population immunity to the SARS-CoV-2 virus. The population herd immunity is the limiting factor in spread of the SARS-CoV-2 virus. Information on the state of population immunity is necessary to make a forecast for development of epidemiological situation, as well as to plan measures for specific and non-specific prevention of COVID-19. In this regard, the study of population immunity during the pandemic is necessary to predict development of the epidemic and identify features of epidemic process in any certain region and in the country in general. In May 2020, the Rospotrebnadzor program “Assessment of population immunity to the SARS-CoV-2 virus in the population of the Russian Federation in the context of the COVID-19 pandemic” was developed taking into account WHO recommendations in the format of a longitudinal cohort study with age stratification: at the first stage, a cohort of volunteers is formed; at the second and subsequent stages, the cohort of volunteers formed at the first stage is subject to examination; the number of stages depends on the epidemiological situation. In addition to age-related randomization while forming a cohort, at the first stage, the principle of population coverage uniformity was observed, the participation of volunteers from one institution was excluded, and the use of donated blood or patient blood from medical organizations was excluded. The survey was carried out among 7 age groups of healthy children and adults, regardless present or absent previous COVID-19 disease. Due to the fact that the population study is carried out in the context of COVID-19 pandemic, the optimal timing for collecting biomaterial at each stage is no more than 5–7 days. Questioning, selection of volunteers, their registration for blood donation, as well as processing of the results at the first stage, as well as the implementation of the second and subsequent stages of the Program are carried out only by using cloud service technology. This Program and the technology for its implementation have been successfully tested in 26 regions of the Russian Federation during the implementation of four stages from June 2020 to March 2021. Thus, at present, Rospotrebnadzor has developed a methodology and created an effectively working system of serological monitoring to assess level of herd immunity in different regions of the Russian Federation to predict the epidemiological situation, develop recommendations, and plan vaccination.

Here we discuss the issues for attributing the new coronavirus infection COVID-19 to zoonoses based on the data on probable origin of the SARS-CoV-2 virus, the possible formation of its reservoir in animals (bats) as well as human susceptibility. Today, the dominant point of view is that the outbreak of COVID-19 arose as a result of the SARS-CoV-2 coronavirus overcoming the interspecies barrier, acquiring ability to infect and spread in human population. Comparative phylogenetic analysis at the molecular level showed that SARS-CoV-2 is genetically closest to bat coronaviruses, particularly to the RmYN02 and RaTG13 strains isolated from the horseshoe bat, a species considered to be the main host of SARSCoV and MERS-CoV coronaviruses. The ability of the SARS-CoV-2 coronavirus to infect various wild animal has been revealed. SARS-CoV-2 has been found in minks on farms in the Netherlands with mortality rates ranging from 1.2 to 2.4%. While infecting rhesus monkeys with the SARS-CoV-2, it resulted in productive infection and detected viremia. Cats have been found to be susceptible hosts for the human SARS-CoV-2 virus. A likely explanation for this lies in the high similarity between the human and feline counterpart of the ACE2 receptor. It has been shown that dogs can become infected but transmit no virus to other animals. To date, over the entire period of the pandemic the World Organization for Animal Health provides no information about cases of human infection transmitted from pets. Thus, there is no evidence that animals play a role in the spread of SARS-CoV-2 among people during the current period of the pandemic. Human outbreaks are caused by human-to-human virus transmission, and based on the currently available information, the risk of spreading COVID-19 from animals is considered low. More research is needed to understand how COVID-19 can affect animals of a wide variety of species and how big might be the risks of infection transmission from them to humans.

Congenital viral infectious diseases are characterized by polyetiologic pathology holding an important place in the structure of perinatal losses. Due to the wide distribution and lack of specific prophylaxis, the problem of herpesvirus infections is of greatest interest, namely of herpes infection caused by herpes simplex virus type 1 and 2, human herpes simplex virus type 6 and cytomegalovirus infection, as well as parvovirus infection B19. The opportunities to investigate a relation between manifestations of the infectious process and host molecular genetic characteristics have been expanded after developing full genome sequencing methods and creating genetic data international banks. It has been proven that herpes virus genetic variations can account for related neurovirulence, showing that diverse cytomegalovirus genotypes are associated with hepatosplenomegaly, hearing impairment and the symptoms of the central nervous system diseases. Nevertheless, the data on correlation between genotypes and clinical manifestations are still scarce and contradictory, whereas high level of variability becomes extremely evident while comparing genomic sequences of viral strains. The herpesvirus type 6 has been proven to integrate into germ cells with potential for subsequent vertical transmission of chromosomally integrated virus to the offspring and its further intergeneration inheritance. А direct relationship between B19V genospecies and disease manifestations including congenital infections has not yet been identified. Taking into account possible differences in the geographical distribution of such viruses on the territory of the Russian Federation, ethnic populational characteristics, and high frequency of related congenital infectious diseases with a wide range of clinical manifestations, it seems promising to expand scientific research on the genotyping of herpes simplex viruses, cytomegalovirus, herpes viruses type 6 and parvovirus B19V in Russia. The results of such studies will be demanded by practical healthcare in order to develop and use more effective etiotropic drugs and specific prophylaxis in the light of trends to develop personalized and preventive medicine.

The study of new antimicrobial compounds includes determining the mechanism of their effect on the microbial cell. As a rule, an effect for the majority of current synthetic antimicrobials is associated either with suppressed DNA synthesis, or with inhibiting bacterial protein production at translational or transcriptional level. A number of sensitive and easy-todo methods are available for screening and monitoring potential genotoxic activity of a wide range of natural and synthetic compounds. To date, the Ames test has been widely used, which is based on the sensitivity of Salmonella strains to carcinogenic chemicals, although some compounds resulting in Ames negative reactions could actually be carcinogenic to animals. Likewise, the SOS chromotest represents a SOS transcriptional analysis able to assess DNA damage caused by chemical and physical mutagens by measuring the expression of a reporter gene (β-galactosidase) encoding the β-galactosidase enzyme that metabolizes ortho-nitrophenyl galactopyranoside resulting in emerging a yellow-colored compound detected at wavelength 420 nm. Next, the induction of β-galactosidase is normalized by the activity of alkaline phosphatase, an enzyme expressed constitutively by Escherichia coli. SOS chromotest is also widely used for genotoxicological studies providing a quick answer (several hours) and requiring no survival of the test strain. Dose-response curves for various chemicals consist of a linear region, which slope corresponds to the SOS induction. Therefore, the SOS chromotest was selected for the study allowing to identify DNA-mediated effects of the analyzed compounds. The aim of the study was to evaluate the SOSinducing activity for 1H-indol-4-, -5-, -6-, -7-ylamines-substituted antimicrobial compounds. The Escherichia coli PQ 37 with the genotype F-thr leu his-4 pyrD thi galE lacΔU169 srl300::Th10 rpoB rpsL uvrA rfa trp::Mis+ sfiA:: Mud (Ar, lac) cts was used as a test strain. Due to the link of the sfi A::lac Z genes, lacZ β-galactosidase gene expression in the strain PQ 37 is controlled by the sfiA gene promoter, one of the components in the E. coli SOS regulon. Activity of β-galactosidase assessed relative to constitutive microbial alkaline phosphatase reflects SOS-inducing activity triggered by examined compounds in the SOS chromotest that also allows to control their toxic effects on bacterial cells. The data showed that 4,4,4-trifluoroN-(6-methoxy-1,2,3-trimethyl-1H-indol-5-yl)-3-oxobutanamide (1), 4,4,4-trifluoro-N-(6-methyl-2-phenyl-1H-indol-5-yl)- 3-oxobutanamide (2) and N-(1,5-dimethyl-2-phenyl-1H-indol-6-yl)-4,4,4-trifluoro-3-oxobutanamide (3) exerted no SOSinducing activity at the examined concentrations. In contrast, 4-Hydroxy-8-phenyl-4-(trifluoromethyl)-1,3,4,7-tetrahydro- 2H-pyrrolo [2,3-h]-quinoliN-2-one (4), 9-hydroxy-5-methyl-2-phenyl-9-(trifluoromethyl)-1,6,8,9-tetrahydro-7Н-pyrrolo- [2,3-f]-quinoliN-7-one (5), 6-hydroxy-2,3-dimethyl-6-(trifluoromethyl)-1,6,7,9-tetrahydro-8H-pyrrolo[3,2-h]quinoliN-8- one (6) and 1,2,3,9-tetramethyl-6-(trifluoromethyl)-1,9-dihydro-8H-pyrrolo [3,2-h]-quinoliN-8-one (7) displayed a dosedependent SOS-inducing activity at bactericidal concentrations. The data obtained allowed us to identify compounds 4, 5, 6, 7, which mechanism of action relies on affecting microbial cell DNA.

The aim of the study was to characterize toxigenic strains of Corynebacterium diphtheriae by examining 12 toxigenic strains of C. diphtheriae isolated in Russia between January, 2017 to June, 2019. The morphological, toxigenic and biochemical properties of C. diphtheriae was studied. Genotyping of C. diphtheriae strains was performed using MLST and dtxR gene sequencing with subsequent phylogenetic analysis. Results. Toxigenic strains of C. diphtheriae were isolated in the Novosibirsk, Samara and Chelyabinsk Regions, the Khanty-Mansi Autonomous Okrug — Yugra as well as the Republic of Northern Ossetia — Alania. Among these strains, 5 were isolated from diphtheria patients (moderate disease found in one case, mild course — remaining patients) and 7 strains were isolated from bacterial carriers. In two cases C. diphtheriae from diphtheria patients were identified as ST25 sequence type, gravis variant; in one case — ST8 type, gravis variant; two cases — ST67 sequence type, mitis variant. In asymptomatic carriers of tox-positive C. diphtheriae strains they belonged to ST25 sequence type, gravis variant — in two cases, ST67 type, mitis variant — in four cases. A sequencing type was not identified in one case. All sequence types were widespread globally being presented by a large number of isolates in the PubMLST and characterized by a substantial amount of derivative sequence types. At the same time, they belonged to different clonal complexes and differed markedly from each other contributing to their reliable difference as assessed by MLST. Study of gene dtxR sequence diversity showed that all allelic variants were typical for the representatives of these sequence types. New alleles of gene dtxR were not revealed in strains examined. It was shown that non-synonymous substitution C440T leading to A147V amino acid substitution was found solely in one allele distributed in ST8, ST185, ST195 and ST451 types suggesting at late mutation. In contrast, the polymorphism C640A resulting in the amino acid substitution L214I was found not only in the same allele, but also in the basal tree branches indicating that isoleucine was in the ancestral sequence of the protein.

The aim of the study was to identify INDEL markers and study geographical origin of regional H. pylori strains circulating in the European part of the Russian Federation. The study included 56 strains of H. pylori isolated in three regions of the Russian Federation: Saint Petersburg, Astrakhan and Rostov Regions. Genomic DNA was isolated using a set of Probe NA (DNA Technology, Russia), according to the manufacturer’s instructions. Detection of INDEL markers hp5605, hp6405, hp340, hp1390, hp3660 was performed using PCR. Clustering of the identified INDEL genotypes and building a phylogenetic tree were performed using the BioNumerics 7.6 and GrapeTree software packages. 21 strains from the GenBank database with known geographical origin were used as reference strains. In 20 strains from Saint Petersburg, 13 individual genotypes were identified, while 17 strains belong to the European cluster (hpEurope), 2 strains belong to the hspEAsia cluster and one strain belongs to the hspWAfrica cluster. The most common genotype identified in the European cluster includes six strains from Saint Petersburg and two strains from the GenBank database. For further differentiation of these strains, the VNTR typing method was used, which allowed identifying eight individual genotypes in eight strains. Fifty-six studied russian strains are represented by thirty individual genotypes, which reflects the high heterogeneity of strains circulating in the European part of the Russian Federation. The most frequent genotype is represented by two hpEurope strains, one strain from the Astrakhan region, as well as 5 and 6 strains from the Rostov Region and Saint Petersburg, respectively. The vast majority of Russian strains (52/56) belong to the hpEurope population, while two strains from Saint Petersburg are included in the hspEAsia population, and one strain from Saint Petersburg and the Astrakhan Region is included in the hspWAfrica population. Total, 77 H. pylori strains are represented by 37 individual genotypes with a high diversity index (DI = 0.95), which allows us to consider the proposed INDEL typing method as an independent method for genotyping H. pylori strains. Taking into consideration the complexity of the problem of accurately determining the geographical origin of H. pylori strains, the proposed simple and convenient method of INDEL typing of H. pylori strains, based on an available PCR method becomes very relevant and allows us to conduct an adequate primary analysis of the geographical origin of Russian H. pylori strains.

Influenza and other acute respiratory viral infections lead to a substantial incidence of severe cases and hospitalizations and so remain a global health problem. Within the frame of the Global Influenza Hospital Surveillance Network (GIHSN), we assessed the contribution of influenza and other respiratory viruses to severe cases of influenzalike diseases in patients hospitalized to the Novosibirsk infectious hospitals in the years 2018–2019. We analyzed 484 nasopharyngeal swabs collected from patients admitted to the hospitals with acute respiratory infections (ARI) using real-time polymerase chain reaction commercial kits. We confirmed viral etiology of ARI in 69.8% cases. Influenza viruses were detected in 47.1% cases, wherein concomitant circulation of influenza A(H1N1)pdm09 and A(H3N2) viruses was observed in 20.7% and 26% of patients, respectively, whereas influenza B virus was detected only in one sample. All analyzed influenza A viruses were antigenically similar to vaccine strains. Genetically, the Novosibirsk strains were closely related to influenza A viruses distributed in Russia and worldwide. Influenza A(H1N1)pdm09 virus was detected in all patients aged 0 to 14 years and required intensive care. Other respiratory viruses were detected in 36.4% of children and 5.8% of adults, and 8.3% of children had viral coinfection, whereas no cases of coinfection were detected in adults. The most common viruses in children were metapneumovirus — 12.8%, rhinovirus — 9.3% and respiratory syncytial virus — 8.0%. In adults, metapneumovirus, adenovirus, parainfluenza virus and rhinovirus were detected with a detection rate no exceeding 2%. In this study, we found no differences in the detection rate of the influenza virus due to concomitant chronic diseases, pregnancy, or smoking habits. At the same time, the detection rate of other respiratory viruses in non-smokers vs. smokers was significantly lower than in smokers and former smokers (26.15%, 66.67% and 62.50%, respectively). In addition, the level of detection of respiratory viruses in children with vs. without chronic pathology was significantly higher (55.3% and 38.7%, respectively). Thus, our and similar studies are important for monitoring and control of the infection.

Objective is to study the features of impaired activation of T and B lymphocytes in order to predicting severe cytomegalovirus infection in newborns. Materials and methods. 133 newborns with cytomegalovirus infection were examined. Immediately after diagnosing cytomegalovirus infection, all patients observed were immunologically ex amined, including assessing count of peripheral blood T and B lymphocytes, as well as their intercellular interaction by using flow cytometry immunostaining for CD3, CD3⁺CD28^–, CD3⁺CD28⁺, CD3^–CD28⁺, CD4, CD8, CD20, CD20⁺CD40⁺, CD28, CD40. The test was performed by using a Beckman Coulter Epics XL laser flow cytofluorometer. Depending on the condition severity, all children were divided into two groups: 1 — cytomegalovirus infection, severe form — 60 subjects (45.1%); 2 — cytomegalovirus infection, moderate form — 73 subjects (54.9%). Results of the entire set of studied indicators for cellular and humoral arms of immune system revealed statistically significant differences for the prognosis of severe cytomegalovirus infection: CD3⁺CD28^–, CD20, CD20⁺CD40⁺, CD4. T lymphocytes with CD3⁺CD28⁺ activation markers, through which costimulating signals necessary for the activation of T helper cells are exerted cell-intrinsic features, serving as an important factor ensuring immune response. Using the “classification trees” method, we developed a differentiated approach to forecast severe cytomegalovirus infection in newborns. Systems of inequalities were obtained, four of which classify a subgroup of newborns with severe cytomegalovirus infection. The consistent application of the obtained inequalities makes it possible to isolate from the input stream of sick patients with a prognosis of the development of severe cytomegalovirus infection. The proposed diagnostic rules can be considered as screening markers for predicting a severe cytomegalovirus infection in newborns, which makes possible the timely onset of specific therapy.

The aim of the study was to obtain and characterize a recombinant vaccine for the immunoprophylaxis of infections caused by Pseudomonas aeruginosa. A set of two highly immunogenic P. aeruginosa recombinant proteins was used to create related vaccine. The first vaccine component was presented by recombinant outer membrane protein F (OprF), whereas the second one — by recombinant atoxic exotoxin A (toxoid). These antigens allowed to develop vaccine inducing immune response against the surface bacterial cues and promote production of neutralizing antibodies against exotoxin A, one of the most dangerous P. aeruginosa pathogenicity factors. Recombinant proteins were synthesized in Escherichia coli cells and isolated by two-step purification. In case of recombinant OprF protein, it was initially (stage 1) isolated as a precipitate containing hydrophobic fraction of producer cell-derived proteins, whereas for recombinant toxoid we purified inclusion bodies. At stage 2, a Ni Sepharose column affinity chromatography was performed. Next, purified recombinant proteins were dialyzed against buffer solution containing 50 mМ Tris-HCl (pH 9.0) and 0.01% Tween 20 followed by filtration sterilization. Three lots of the Pseudomonas Recombinant Vaccine (PRV) were obtained for the study, wherein the recombinant antigens were absorbed with aluminum hydroxide. Recombinant OprF and toxoid protein in vaccine formula were used at a dose of 25 μg and 50 μg, respectively. Absorption completeness of the recombinant antigens within the vaccine was evaluated by polyacrylamide gel electrophoresis with PRV preparations concentrated by ultrafiltration in spin columns. Authenticity of recombinant vaccine was assessed by using customized method by desorbing antigens followed by ultrafiltration concentration in spin columns. Final concentrated desorbed vaccine preparations were analyzed by polyacrylamide gel electrophoresis and immunoblotting, which allowed to confirm presence of specific recombinant antigens in the vaccine. The experimental PRV series demonstrated specific activity (protective properties) after inoculation in animals (mice) by using a dual vaccination protocol, wherein mice were immunized intraperitonially twice with a two-week interval. Next, two weeks later mice were infected by toxigenic P. aeruginosa strain PA-103 culture cells. The index of protective efficiency (IE) for experimental vaccine series was at least a value of three (IE: 3.0–3.3) that was by 1.5-fold higher than that for using single vaccine components (IE: 2.0–2.3). Thus, we confirmed an additive effect of using a set preparation to protect against infection caused by toxigenic Pseudomonas aeruginosa strains.

The rapid formation of microbial resistance to modern antibacterial drugs requires to search for new, alternative therapies. It is known that some organisms, such as plants, algae, fungi, are able to convert inorganic metal ions into metal nanoparticles due to the recovery process carried out by proteins, sugars and metabolites contained in the tissues and cells of these organisms. At the same time, many plants (e.g., plantain, yarrow, wormwood, turmeric long, calendula, marsh bagulnik, etc.) and metals (copper, silver, gold, zinc, etc.) themselves have antibacterial properties, so that metal nanoparticles obtained by biological method, or via “Green” synthesis method, from extracts of such plants can become a current alternative to many modern antibacterial drugs. The antibacterial mechanism of action of nanoparticles depends on the type of microorganisms affected, as well as on the type of nanoparticles, their concentration, size, and how they are obtained. Based on this, the study of the antibacterial effect of nanoparticles is one of the promising directions of solving the problem of microbial antibiotic resistance. There was examined antibacterial effect of metal nanoparticles containing silver, copper and gold obtained by biological method from the salts of AgNO₃, CuSO₄, H[AuCl₄] metals, respectively, and the extract of the plant — turmeric long (lat. Curcuma longa) — related to the following bacteria strain collection: E. coli (ATCC 25922), S. aureus (ATCC 25923), MRSA (ATCC 38591) and polyresistant clinical strains isolated from patients of the Regional clinical hospital (Krasnoyarsk) — К. рneumoniae, strain 104, P. аeruginosa, strain 40, P. аeruginosa, strain 215, А. baumannii, strain 210, А. baumannii, strain 211. Study allowed to identify the minimum suppressive concentration of nanoparticles by the method of serial dilutions (MUK 4.2.1890-04) with azurin dye. It was proved that metal nanoparticles exhibit different antibacterial efficacy depending on the type of nanometals used and bacterial cultures. Copper nanoparticles have the highest antibacterial activity, and gold nanoparticles have the lowest. The most marked antibacterial effect was observed against clinical polyresistant strains. Metal nanoparticles can become an alternative to the currently known antibacterial drugs, but despite the high efficiency of nanoparticles against polyresistant to antibacterial drugs microorganisms in vitro, it is necessary to take into account their possible toxic effect on live tissues, which requires further study in experiments in vivo.

The significance of chronic hepatitis is determined not only by the disease per se, but also by the increased risk of the formation of long-term adverse effects such as liver cirrhosis. Viral cirrhosis of the liver (as the outcome of chronic hepatitis B, C, B + D) comprise from 10% to 24.5% of all liver cirrhosis cases. Cirrhosis of the liver is considered as an irreversible stage of chronic hepatitis, more often observed in men above 40 years, but recently more and more its cases are found in young, working ones leading to disability. Due to the high liver regenerative potential, a prognosis for liver lesions of different etiologies may be rather favorable. As a result, early diagnostics, dynamic monitoring and the use of promising methods for treating liver pathology to stimulate its regeneration that compensates for lost liver functions are necessary. In the clinical and morphological case, pathomorphological changes in organs with liver cirrhosis, which developed as a result of viral hepatitis are described. The aim of the study was to describe pathomorphological changes in organs during liver cirrhosis, which developed due to viral hepatitis, which led to multiple organ failure in a young patient. Materials and methods. The analysis of the obtained accompanying medical documentation (outpatient card, medical history) was performed. The standard methodology of autopsy was used. To process histological sections of autopsy material, hematoxylin-eosin staining was used. Results. Histological examination in the liver shows development of portal tract fibrosis with lymphohistio cytic infiltration, formation of monolobular regenerated nodes, signs of edema, areas of demyelination, dystrophic changes in neurocytes, signs of acute renal failure, a combined profound change in the kidneys detected in the lungs and brain, which resulted in multiple organ failure and subsequent lethal outcome in a young patient. The early formation of portal hypertension with liver cirrhosis, esophageal and gastric varicose veins dilatation lead to fatal bleeding in half of patients. Therefore, timely diagnosed cirrhosis and hepatitis as their predecessors is one of the most relevant issues in medicine.

Among infectious diseases, opportunistic mycoses hold a special place. There has been accumulating a lot of evidence regarding the clinical and epidemiological aspects of infection caused by Fusarium spp., which global incidence rate among microbial keratitis ranges from 2 to 40% depending on the geographical location of the country. Colonizing mucous membranes, fungi can exist not only in the form of plankton, but form biofilms after surface attachment, which leads to elevated resistance to multiple antifungal agents. Here we describe a clinical case of fungal keratitis due to Fusarium solani by determining profile of the antifungal sensitivity for isolated fungal strains, by taking into account their potential for biofilm formation. We used an F. solani culture isolated from the patient as well as F. solani test culture obtained from the Russian National Collection of Microorganisms. While determining the sensitivity of fungal planktonic cultures to antifungal agents from the azole group (fluconazole, voriconazole), amphotericin B and terbinafine, it was revealed that antimycotics amphotericin B and voriconazole exerted a marked antifungal activity against clinical isolate, whereas the plankton F. solani test culture was more sensitive to all groups of antifungal agents. Due to a long-lasting progressive course of the infectious process and the high biofilm-forming ability of the clinical strain F. solani, the activity of antifungal agents on biofilm cells was modeled and examined in vitro. It was shown that regarding to the fungal biofilms, value of the minimally inhibitory concentration exceeded those for planktonic cultures by 100-fold. The mechanisms of action for antifungal agents on vital parameters of fungal cell structures were analyzed by using confocal laser scanning microscopy after staining samples with propidium iodide and acridine orange for 15 min to detect changes between intact and damaged cell surface. It was found that within the biofilm fungal cells preserved viability even after exposure to high concentrations of antifungals. In addition, despite the fungicidal drug activity at substantial concentrations acting on the biofilm cell membrane, the cell nuclei remained viable. Owing to the presence ot the mechanism of resistance in mycelial fungi shown in the study, it is necessary to take into account and investigate characteristics of biofilms in terms of drug sensitivity that will allow to optimize a choice of antimicrobial therapy.