Vol 13, No 1 (2023)

The role of neutrophil granulocytes (NG) in the pathogenesis of COVID-19 is associated with the NG recruitment into inflammatory foci, activation of their functions and enhanced formation of neutrophil extracellular networks (NETs). In this review, we analyzed a large body of scientific literature devoted to the features of developing NETs, their role in the COVID-19 pathogenesis, a role in emerging immunothrombosis, vasculitis, acute respiratory distress syndrome, cytokine storm syndrome, and multi-organ lesions. Convincing data are presented clearly indicating about a profound role of NETs in the COVID-19 immunopathogenesis and associated severe complications resulting from intensified inflammation process, which is a key for the course of SARS-CoV-2 virus infection. The presented role of NGs and NETs, along with that of other immune system cells and pro-inflammatory cytokines, is extremely important in understanding development of overactive immune response in severe COVID-19. The scientific results obtained available now allow to identify an opportunity of regulatory effects on hyperactivated NGs, NETosis at various stages and on limiting a negative impact of pre-formed NETs on various tissues and organs. All the aforementioned data should help in creating new, specialized immunotherapy strategies designed to increase the odds of survival, reduce severity of clinical manifestations in COVID-19 patients as well as markedly reduce mortality rates. Currently, it is possible to use existing drugs, while a number of new drugs are being developed, the action of which can regulate NG quantity, positively affect NG functions and limit intensity of NETosis. Continuing research on the role of hyperactive NG and NETosis as well as understanding the mechanisms of regulating NET formation and restriction in severe COVID-19, apparently, are of high priority, because in the future the new data obtained could pave the basis for development of targeted approaches not only for immunotherapy aimed at limiting education and blocking negative effects already formed NETs in severe COVID-19, but also for immunotherapy, which could be used in combination treatment of other netopathies, primarily autoimmune diseases, auto-inflammatory syndromes, severe purulent-inflammatory processes, including bacterial sepsis and hematogenous osteomyelitis.

Introduction. The rapid spread of a new coronavirus infection among populations in many countries worldwide has contributed to the genetic evolution of the virus, resulting in the emergence of multiple genetic variants of the SARS-CoV-2 coronavirus. Mutations in the viral genome can affect the ability of the virus to bypass the immune system and complicate development of diagnostic and prophylactic drugs. Data on the neutralizing activity of the sera obtained against previously circulating genetic variants of the virus in relation to current SARS-CoV-2 strains may serve as a scientific basis for the selection of the antigens in vaccine development. The aim of this work was to study cross-reactivity of SARS-CoV-2 coronavirus strains belonging to different genetic variants, which were isolated in the territory of the Russian Federation during 2020–2022 in the neutralization reaction using mouse hyperimmune sera. Materials and methods. Ten strains of SARS-CoV-2 coronavirus belonging to different genetic variants were used (three non-VOC strains, alpha, beta, gamma, delta, delta+AY, omicron 1 and omicron 2). The hCoV-19/Australia/VIC01/2020 strain (Wuhan) was included in the study as a prototypical variant. BALBc mice were immunized with inactivated concentrated antigen mixed with a 1:1 adjuvant, which was a virus-like immunostimulatory complex based on Quillaja saponaria (Quillaja saponaria). The antibody titer was determined in the neutralization reaction. Results. Essential decrease of neutralizing ability of antibodies specific to non-vOC genetic variants of SARS-CoV-2 coronavirus was revealed against beta VOC and to a lesser degree against alpha and gamma VOC variants. The differences in the neutralizing activity level of antibodies for alpha and beta VOC variants are not significant among themselves, and with gamma VOC variants — there are no significant differences. Neutralizing ability of antibodies specific to delta VOC against alpha and beta VOC variants decreased 4-fold. Neutralizing activity of sera obtained to omicron 1 and 2 variants in relation to the prototype coronavirus variant was reduced 18-fold, to the gamma variant — 12-fold, to delta variants — more than 30-fold; for other variants it was even lower. Conclusions. The results obtained testify to the presence of cross-reactivity between strains of coronavirus belonging to genetic lines Wuhan, alpha, beta, gamma; it is weaker for delta variants. Mutations in the genome of VOC omicron variants led to a significant decrease in antigenic cross-links with earlier genetic variants of the coronavirus. These findings explain the low efficacy of vaccines based on the Wuhan strain, synthetic immunogens, and recombinant proteins based on it against omicron VOC variants, which have caused a rise in morbidity since early 2022, as well as cases of re-infection of humans with new genetic variants of the coronavirus.

The relationship between the incidence of COVID-19 in pregnant women who have had a coronavirus infection at different gestational ages and the health status of paired neonates is of great interest. However, no sufficient convincing data fully reflecting features of subsequent neonatal period, the state of the immune system in this category of children, affecting characteristics of postnatal period have been accumulated. Based on this, it underlies the relevance of the current study aimed at investigating parameters of clinical and immunological state of neonatal health after paired mothers recovered from COVID-19 at different gestational ages. The prospective study included 131 women and 132 children. The main group consisted of women (n = 61) who had COVID-19 during pregnancy and paired newborns (n = 62) at gestational age (GA) of 37–41 weeks, the comparison group — women without laboratory-confirmed COVID-19 during pregnancy (n = 70) and paired newborns (n = 70) of similar gestational age. While analyzing the anamnesis of the patients, no significant differences in somatic and obstetric-gynecological diseases were found. Analyzing course of pregnancy revealed that low molecular weight heparins were significantly more often applied in the main group. The term and frequency of delivery by caesarean section in pregnant women in the main group did not significant differ from that of the control group. No significant difference in the frequency of causes accounting for the severity of the condition of neonates in paired mothers with COVID-19 at different trimester of gestation was found. Investigating lymphocyte subset composition, neutrophil phagocytic activity, and IgG class antibodies specific to SARS-CoV-2 was carried out. It was found that lymphocyte subset profile in newborns from paired mothers with COVID-19 at different trimesters of gestation differed only in the level of NK cells (CD56⁺) in children born to mothers recovered from COVID-19 in the first trimester. In this study, in general, no severe perinatal outcomes in newborns from paired mothers with COVID-19 during pregnancy were documented. No cases of moderate or severe maternal COVID-19 were observed. Therefore, further prospective studies are needed to assess an impact of COVID-19 severity on maternal and fetal birth outcomes and clarify optimal management of pregnant women in such cases.

Background. The COVID-19 pandemic has become a substantial global health crisis, unparalleled in world history. Infection dynamics can have specific characteristics in different countries due to social, economic, climatic, or geographic factors. Aim: to study features of SARS-CoV-2 collective immunity among the Armenian population. Materials and methods. A cross-sectional, randomized study of collective immunity was carried out according to a program developed by Rospotrebnadzor and the St. Petersburg Pasteur Institute, taking into account WHO recommendations. The study was approved by the ethics committees of the National Center for Infectious Diseases (Armenia) and the St. Petersburg Pasteur Institute (Russia). A volunteer cohort was formed (N = 6057), randomized by age and region. The study’s analysis included: shares and distributions of antibodies (Abs) to nucleocapsid (Nc) antigen (Ag) and receptor binding domain (RBD) S-1 Ag in the cohort; and quantitative determination of these Abs by ELISA. During the survey, a history of vaccination was indicated by 4395 people. Results. Overall seropositivity formed in the whole cohort (by April 14, 2022) was 98.6% (95% CI: 98.1–98.7). It did not depend on age, place of residence, or occupation. When quantifying Nc and RBD Abs, the proportions of volunteers with Nc Ab levels of 1–17 BAU/ml and RBD Ab levels of 22.6–220 BAU/ml were the smallest, amounting to 6.9% (95% CI: 6.2–7.5) and 20.4% (95% CI: 19.4–21.4), respectively. With increasing serum concentrations (Nc > 667 BAU/ml, RBD > 450 BAU/ml), the proportions of individuals with the corresponding levels were 20.2% for Nc (95% CI: 19.2–21.3) and 54.2% for RBD (95% CI: 52.9–55.5). Vaccination coverage was 72.6% (95% CI: 71.5–73.7). The most frequently used were Sinopharm/BIBP (32.4%), AZD1222 (22.3%), and Gam-COVID-Vac (21%). The remaining vaccines (CoronaVac, mRNA-1273, BNT162b2, CoviVac) were used by 24.3% of vaccinated individuals. When summing vaccines by platform, it was found that: vector vaccines were used in 40.34% (95% CI: 33.57–42.39) of cases; whole-virion vaccines were used in 26.83% (95% CI: 24.76–32.20); and mRNA vaccines were used in 6.33% (95% CI: 4.84–8.91). Conclusion. The epidemic situation in Armenia by April 2022 was characterized by a high level of collective immunity, independent of age or regional factors. Vector and whole-virion vaccines have been used most widely.

The aim of the study was to verificate of the causative agent of Siberian tick-borne typhus (STT) upon ixodic tick mixed infection on the territory of the Altai Republic and Khabarovsk Krai using an integrated molecular biological approach. Materials and methods. The material for the study was presented as 304 imago of ixodic mites of six species. The ticks were collected on the territory of the Altai Republic and the Khabarovsk Krai in the years 2014–2022. Rickettsia DNA was identified by two-round PCR using genus- and species-specific primers for gltA and ompA genes, followed by sequencing and using PCR reagent kits “RealBest DNA R. sibirica/R. heilongjiangensis” (Vector-Best, Novosibirsk). Results. The detection of rickettsia in ticks in the Altai Republic was 82.6% (CI: 69.1–96.1), in the Khabarovsk Krai — 53.1% (CI: 44.9–61.3). The species composition of rickettsias in various species of ixodic mites in these territories, endemic to Siberian tick typhus, is characterized by the presence of mixed forms. Rickettsia sibirica and R. raoultii were found in ticks Dermacentor nuttalli, Haemaphysalis concinna, D. silvarum. Only Candidatus R. tarasevichiae were detected in Ixodes persulcatus, I. pavlovsky mites and I. pavlovsky/persulcatus hybrids. The DNA of R. heilongjiangensis was found in H. mites. concinna in the Khabarovsk Krai. The DNA of Candidatus R. tarasevichiae is also present in the ticks of D. nuttalli and H. concinna from the Altai Republic. The DNA of the “classic” pathogenic species, R. sibirica, was detected in ticks D. nuttalli and H. concinna in the Altai Republic, ticks D. silvarum, H. concinna and H. japonica douglasi in the Khabarovsk Krai. In addition, in the Khabarovsk Krai, the DNA of R. heilongjiangensis was detected in H. concinna ticks, and a DNA fragment common to R. sibirica subsp. mongolitimonae and R. conorii was detected in H. japonica douglasi ticks, which requires further study. Candidatus R. tarasevichiae is common in Ixodes mites (I. persulcatus, I. pavlovsky and their hybrids), in the Altai Republic in D. nuttalli and H. concinna. R mites. raoultii was detected in ticks of the genus Dermacentor — D. nuttalli in the Altai Republic and D. silvarum in the Khabarovsk Krai. Conclusions. During molecular biological monitoring of rickettsias in endemic territories, the detection of R. raoultii “masks” the presence of the etiological agent of STT — R. sibirica. This phenomenon makes it possible to explain the high incidence of STT in the studied territories, with the rare detectability of R. sibirica DNA during molecular biological screening in ixodic mites.

Background. Intestinal parasites are among the most important infectious agents with an impact on human health. Indeed, in the lack of an available treatment option, these parasites could constitute a real health problem in the population. In the present study, we investigated for the first time the effect of a novel synthetic heterocyclic ((C₁₅H₁₂N₅OCL)2-(benzo(d)(1,2,3)triazol-1-yl)-N-benzylideneacetohydrazine) compound on two intestinal parasites (Entamoeba histolytica and Giardia lamblia). Methods. The parasite isolates tested were collected from outpatients at the General Pediatric Hospital in Kirkuk, Iraq, between September 2019 and January 2020. Thus, we studied the in vivo and in vitro pharmaceutical activity of the ingredient on both parasites. The toxicological effects of the substance on some blood parameters and liver and kidney function tests were also studied. Results. After five days of treatment, the drug’s in vivo action on G. lamblia resulted in an inhibition rate of 88.2% at a dose of 1 mg/kg. On the other hand, we observed that the influence of this synthetic substance on cultured E. histolytica was very close to the metronidazole effect. The maximum result was at a concentration of 1 g/ml and was obtained after 72 hours of incubation with an inhibition rate of 89.4%. The substance did not affect the blood parameters or the studied liver and kidney functions. Conclusion. It can be concluded that this substance is highly effective against both E. histolytica and G. lamblia, and that it has no toxic effects on the studied parameters. Therefore, it could be a promising pharmacophore for intestinal protozoan parasites including E. histolytica and G. lamblia and an alternative or competitor to the current medications available.

Introduction. Candida is the cause of most systemic fungal infections that plays a role in the pathophysiology of sepsis in newborns, especially in premature infants with late-onset sepsis. The Candida score can be used to assess the occurrence of systemic candidosis where a Candida score > 2.5 can accurately identify patients who are at high risk for candidiasis infection. Monocytes also play an important role in preventing candida invasion. Materials and methods. This study used a cross sectional research design. Data was collected from premature infants with late-onset sepsis being suspected of systemic candidosis in neonatology inpatient ward. It was submitted from the period of November-December 2021. It takes a minimum of 31 samples to meet the criteria to process and analyze the data. The data obtained were processed and analyzed using the Receiver Operating Characteristic (ROC) method to obtain the Area Under Curve (AUC) value. Based on the AUC curve, the search for the most optimal intersection is carried out to obtain the sensitivity and specificity values. Results. Of the 31 research subjects, the number of subjects with positive PCR results was 27 (76.93%) while negative were 4 respondents (12.9%). The mean value of PCR density in the positive group of subjects was 76.93 and the range was 40.23–122.78. Meanwhile, in the group of subjects who were negative, the PCR density value was 0. The results of the Candida score diagnostic test showed that the sensitivity obtained was 81%. Higher sensitivity and specificity > 70% were found in the combined examination of Candida scores and monocyte counts according to cut-off compared with separate examinations. Conclusions. The combined examination of Candida score and monocyte count can be used as a diagnostic test for systemic candidosis in premature infants with late-onset sepsis, and could be used to consider the initiation of empirical antifungal therapy, either prophylactically or therapeutically, especially in limited laboratory facilities in Indonesia.

Background. Rheumatoid arthritis (RA) is characterized by synovial membrane inflammation that results in joint damage. Many earlier studies have measured cytokines for a better diagnosis of RA. In the present study, three bone biomarkers [osteopontin, stromelysin-1 (MMP3), and vascular endothelial growth factor-A (VEGF)] are examined for their ability to estimate the severity of disease by using artificial neural network (NN) analysis and binary logistic regression analysis. Methods. The study enrolled 87 RA patients and 44 healthy control subjects. The biomarkers were measured by the enzyme-linked immunosorbent assay technique. Disease Activity Score (28 joints) and C-reactive protein (CRP) (DAS28-CRP) was calculated by using DAS28-CRP calculator. The patients with DAS28-CRP ≥ 5.1 are considered as having high disease activity (HDA). While patients’ group with DAS28-CRP < 5.1 are considered as moderate disease activity (MDA). The neural network (NN) analysis was used for the differentiation between groups. Results. Results showed that the most sensitive predictor for high disease activity (HDA) of RA is MMP3, followed by osteopontin and VEGF. These three biomarkers can differentiate significantly between HDA and MDA with a relatively high size effect (Partial η² = 0.323, p < 0.001). The HDA group has a significantly higher MMP3, CRP, RF, and anti-citrullinated protein antibodies (ACPA) than the MDA group. MMP3 is strongly associated with two inflammatory indicators; CRP and ESR. Conclusion. There was a significant elevation in the serum level of MMP3 in RA patients with HDA compared to the MDA and control groups. High DAS28, RF, CRP, and ACPA were found in HDA patients compared with the MDA group. The use of the NN analysis indicated that the measured biomarkers help predict the HDA state in RA patients. MMP3 and osteopontin are diagnostic biomarkers for the severity of RA and are related to many disease-related characteristics with a sensitivity of 88.9% and specificity of 68.4%.

The progress in investigating microbiome in children and adolescents as well as its impact on maintaining health is considered one of the most significant achievements of modern medicine. In children with various diseases, the qualitative and quantitative indicators of the intestinal microflora change, which causes the development of dysbiosis. The assessing changes in intestinal microflora in children during treatment with anti- tuberculosis drugs has retained its importance and relevance due to insufficient data in the field, which requires a more detailed understanding of this problem. The presented article assesses the state of gut microbial biocenosis in children receiving anti-tuberculosis treatment. The purpose of the study: to study the microbial landscape of the colonic contents in children receiving anti-tuberculosis therapy. Materials and methods. The study included 24 children with verified tuberculosis of the respiratory organs receiving treatment in a 24-hour hospital of the Kuzbass Clinical Phthisiopulmonological Medical Center named after I.F. Kopylova. The study of intestinal microflora was carried out by a quantitative bacteriological method. The material of the study was feces collected in a sterile disposable container, which was delivered to the Department of Microbiology, Immunology and Virology of the Federal State Budgetary Educational Institution of the Russian Ministry of Health. Results. Dysbiosis with predominance of microecological disorders of II (41.7%) and III (37.3%) degrees was detected in all children receiving anti-tuberculosis therapy. The microbial landscape of the intestine was characterized by a decrease in the quantitative content of representatives of the obligate flora Bifidobacterium spp. in 66.7% of children and Lactobacillus spp. in 45.8%, an increase in the number of facultative microorganisms: E. coli lac+ and Citrobacter freundii in 12.5%. Maintaining a full-fledged and active intestinal normobiota is possible by ensuring its dynamic control in children with tuberculosis during treatment. The results of studying the characteristics of the microbiota suggest that patients need probiotic therapy during the treatment of tuberculosis. Conclusion. Microecological disorders in children with tuberculosis therapy were characterized by a decrease in the quantitative content of representatives of obligate flora and an increase in the number of facultative microorganisms. Also in the material, various types of fungi of the genus Candida were identified.

The severity of complications in cystic fibrosis are determined by microorganisms colonizing the lower airways. Paranasal sinuses can be a reservoir of aggressive pathogens. We have developed a method for collection and primary inoculation of nasal lavage fluid from cystic fibrosis patients for microbiological investigation. As a clinical case illustrating the feasibility of this technique, we describe the dynamics of the microflora composition in a patient with cystic fibrosis. The patient had a clinical and microbiological picture of P. aeruginosa eradication from the lung tissue, owing to which the antibacterial therapy was stopped. Six months later, the microflora in the nasal lavage fluid and sputum were assessed in parallel. The growth of P. aeruginosa (10² CFU/mL) but not P. aeruginosa in sputum was detected. To determine origin of this strain, the degree of genetic relationship between 5 strains obtained from the patient from 2008 to 2016 was assessed based on bacterial protein profiling. A typical strain of P. aeruginosa ATTS 27853 was used as a control. Strains isolated from the patient in 2009 and 2016 were identical suggesting that the antibacterial therapy led to eradication of P. aeruginosa in the lungs, but not in the upper airways. Four months later, the growth of P. aeruginosa was found in sputum. The patient was prescribed to use antibacterial drugs inhaled into paranasal sinuses. Repeated test performed 3 months later resulted in growth of P. aeruginosa 10¹ CFU/mL from nasal lavage fluid, but not from sputum. The patient was referred to a risk group on airway colonization by pathogen strains derived from the upper airway tract. The clinical example illustrates relevance of conducting a regular microbiological study of nasal lavage fluid in order to early identify clinically significant pathogens to prevent their spread to the lower airway tract.

The pandemic of coronavirus infection is characterized by a low percentage of complications and severe forms in sick children compared to the adult population. However, there have been described cases of severe clinical course of COVID-19 in children with comorbidities among which is obesity. The aim of this study was to analyze the severe course of a new coronavirus infection paralleled with morbid obesity in a pediatric patient. Materials and methods. All accompanying patient medical documentation was examined. Results and discussion. From the anamnesis of life it is known that the patient was long time complained of intensively increased body weight, on which she repeatedly underwent examinations. In 2018, hypothalamic pubertal syndrome was diagnosed for the first time, for which the patient received hypoglycemic and antihypertensive drugs, hepatoprotectors on an ongoing basis. In the epidemiological anamnesis, the intrafamilial COVID-19 contact with mother was established. The main disease began acutely with a rise in body temperature up to 39–39.5°C, cough and weakness. During the first week of illness, the patient did not seek medical help and receive self-treatment, but the positive effect was not achieved. Saturation measurement showed low oxygen level (SpO₂ 71%). In this regard, the patient underwent chest computed tomography, which revealed a bilateral interstitial polysegmental lung lesion with signs of consolidation. After emergency hospitalization, the patient was prescribed empiric antibiotic therapy, anti-inflammatory and antithrombotic treatment, as well as respiratory support. A positive PCR result of a throat and nasal swab for SARS-CoV-2 was obtained in the hospital. Due to a poor response to therapy, the patient was transferred to a respiratory hospital. At the time of hospitalization, the condition was considered severe due to severe respiratory failure and premorbidity. The range of treatments included oxygenotherapy, antibacterial and anticoagulation therapy, as well as surfactant and the nucleoside analogue Remdesivir. During treatment, the clinical picture gained a positive trend, and after 6 days of hospitalization the patient no longer needed respiratory support. According to the results of repeated computed tomography, bilateral interstitial polysegmental pneumonia was diagnosed with damage to the lung tissue up to 95%. The patient remained stable and showed no signs of respiratory failure during the following days of hospitalization. On the 20th day of ilness, the patient was discharged from hospital with full clinical recovery. Conclusion. This clinical case demonstrates the role of premorbid background in aggravating the clinical picture of a new coronavirus infection in a child. Careful study of anamnestic characteristics is necessary in patients of any age, even with an uncomplicated disease course.

The aim of the study is to increase the selectivity of isolating Acinetobacter baylyi from river water. For developing a selective culture medium, we used 5 strains of A. baylyi, 4 of which were isolated from the water of the river Neva, 1 — from clinical material. A. baylyi strains were identified by a combination of phenotypic features and/or using the mass spectrometric method (MALDI-ToF MS). In preliminary studies we found that the bacteria A. baylyi utilizes no L-phenylalanine as a single source of carbon and nitrogen, as well as the only source of carbon in a medium with mineral nitrogen sources, but utilizes L-phenylalanine as the only source of nitrogen only if it is supplemented with ethanol as the only source of carbon. Based on this A. baylyi property, a selective medium was developed for the isolation of these bacteria from river water. Composition and preparation of liquid selective medium were as follows: 1 liter of distilled water added with L-phenylalanine (CAS 63-91-2) 2.0 (g/l); NaCI 5.0; Na₂SO₄ 2.0; KH₂PO₄ 1.0; K₂HPO₄ 2.5; MgSO₄ 0.1; dissolved while heating, boiled for 5 minutes, 4 ml of 96-degree ethanol was added to a warm medium, pH 7.2±0.2 assessed; 40 ml were poured into sterile vials. Dense selective medium: the same ingredients and extra 15.0 g of agar are added to 1 liter of distilled water; with similar preparation technique, the medium is poured into sterile Petri dishes. Method of application of selective nutrient medium for isolation of A. baylyi from river water: water from the river Neva is seeded in a volume of 10 ml into a vial with 40 ml of liquid selective medium, incubated in aerobic conditions at 28°С for 48 h, then the enriched material from the flask is placed with a bacteriological loop onto the surface of same dense selective medium in two flasks; incubated in aerobic conditions at 28°С for 48 h. 10 isolated colonies are selected for subsequent identification, they are transplanted into sectors of nutrient agar, incubated at 28°С 24 h and isolates are identified by a complex of phenotypic characteristic of A. baylyi, and/or by the MALDI-ToF MS method. In this case, 19 isolates from 20 were identified as A. baylyi (95% of isolates). A study using this technique of three more water samples from the river Neva showed the results of the same narrowly selective orientation — 90–95% of the isolates were bacteria A. baylyi.