Vol 12, No 3 (2022)

Current review presents a brief overview of the immune system dysregulation during acute COVID-19 and illustrates the main alterations in peripheral blood CD4⁺ T-cell (Th) subsets as well as related target cells. Effects of dendritic cell dysfunction induced by SARS-CoV-2 exhibited decreased expression of cell-surface HLA-DR, CCR7 as well as co-stimulatory molecules CD80 and CD86, suggesting reduced antigen presentation, migratory and activation capacities of peripheral blood dendritic cells. SARS-CoV-2-specific Th cells could be detected as early as days 2–4 post-symptom onset, whereas the prolonged lack of SARS-CoV-2-specific Th cells was associated with severe and/or poor COVID-19 outcome. Firstly, in acute COVID-19 the frequency of Th1 cell was comparable with control levels, but several studies have reported about upregulated inhibitory immune checkpoint receptors and exhaustion-associated molecules (TIM3, PD-1, BTLA, TIGIT etc.) on circulating CD8⁺ T-cells and NK-cells, whereas the macrophage count was increased in bronchoalveolar lavage (BAL) samples. Next, type 2 immune responses are mediated mainly by Th2 cells, and several studies have revealed a skewing towards dominance of Th2 cell subset in peripheral blood samples from patients with acute COVID-19. Furthermore, the decrease of circulating main Th2 target cells — basophiles and eosinophils — were associated with severe COVID-19, whereas the lung tissue was enriched with mast cells and relevant mediators released during degranulation. Moreover, the frequency of peripheral blood Th17 cells was closely linked to COVID-19 severity, so that low level of Th17 cells was observed in patients with severe COVID-19, but in BAL the relative number of Th17 cells as well as the concentrations of relevant effector cytokines were dramatically increased. It was shown that severe COVID-19 patients vs. healthy control had higher relative numbers of neutrophils if compared, and the majority of patients with COVID-19 had increased frequency and absolute number of immature neutrophils with altered ROS production. Finally, the frequency of Tfh cells was decreased during acute COVID-19 infection. Elevated count of activated Tfh were found as well as the alterations in Tfh cell subsets characterized by decreased “regulatory” Tfh1 cell and increased “pro-inflammatory” Tfh2 as well as Tfh17 cell subsets were revealed. Descriptions of peripheral blood B cells during an acute SARS-CoV-2 infection werev reported as relative B cell lymphopenia with decreased frequency of “naïve” and memory B cell subsets, as well as increased level of CD27^hiCD38^hiCD24^– plasma cell precursors and atypical CD21^low B cells. Thus, the emerging evidence suggests that functional alterations occur in all Th cell subsets being linked with loss-of-functions of main Th cell subsets target cells. Furthermore, recovered individuals could suffer from long-term immune dysregulation and other persistent symptoms lasting for many months even after SARS-CoV-2 elimination, a condition referred to as post-acute COVID-19 syndrome.

Recombinant technologies have been long widely used in medicine. This article presents a review on the application of medical technologies based on ESAT-6 and CFP-10 proteins in diagnostics and prevention of tuberculosis. ESAT-6 and CFP-10 are specific proteins whose genes are encoded in the RD-1 zone (region of difference) of M. tuberculosis. M. bovis BCG and in most nontuberculous mycobacteria lack the RD-1 genome fragment. The discovery of ESAT-6 and CFP-10 antigens allowed to make the first and so far, the only breakthrough in improving the diagnostics of latent tuberculosis infection after the first tuberculin skin test (TST) was implemented. The article describes the principle of action and the experience with diagnostic tools based on ESAT-6 and CFP-10 such as in vitro interferon-gamma release assays (IGRA) and in vivo recombinant tuberculosis allergen (RTA, Diaskintest). RTA is inoculated intradermally similar to TST followed by developing delayed-type immune reaction detected in the area closest to M. tuberculosis. Combined use of ESAT-6 and CFP-10 for early detection of tuberculosis infection allowed to ameliorate for many drawbacks related to TST. High sensitivity and specificity was confirmed for ESAT-6- and CFP-10-based tests, so that former BCG vaccination had no more effect on test results and lowered frequency of false positive results due to reaction to non-tuberculous mycobacteria. The results of a large-scale meta-analysis on studies with patients at high risk demonstrated that the risk of developing tuberculosis in subjects with positive vs. negative IGRA was increased by 9.35-fold (95% confidence interval (CI [6.48–13.49]), whereas for TST — by 4.24-fold (95% CI [3.3–5.46]). 95.1%, (95% CI [95.06–95.1]). Analyzing available publications demonstrated sufficient evidence base regarding efficacy of using ESAT-6–CFP-10-based tests in tuberculosis diagnostics. Finally, there are also reviewed the diagnostic tests and vaccines based on using such proteins currently being under development.

Epstein–Barr virus (EBV) and human herpesviruses 6A and 6B (HHV6A and HHV6B) are ubiquitous, infecting representatives of all social groups, starting from early childhood. Currently, information on the genetic heterogeneity of EBV, even at the level of the main types (EBV1 and EBV2), as well as HHV6A and HHV6B, their prevalence and clinical significance are limited mainly by foreign data. In Russia, there are not so many publications devoted to this issue. In this case, the objects of study are mainly plasma and leukocytes of peripheral blood, scrapings or swabs from the oropharynx are used much less often. Saliva is the main factor in the transmission and spread of EBV and HHV6A/B infections. Saliva testing is an affordable, inexpensive, and non-invasive method for detecting viral DNA. The purpose of this work is to improve the methodological base for differential detection of HHV6A/HHV6B and the main types of EBV in saliva. The material for the study was unstimulated mixed saliva of children aged 1–17 years with acute infectious mononucleosis (n = 22) and no clinical symptoms of this disease (n = 26), as well as conditionally healthy adults (n = 9). Samples were collected once and dynamically (daily for 14 days). The detection and quantification of EBV DNA and HHV6A/B DNA was performed using real-time PCR. For the differential determination of EBV1/EBV2 and HHV6A/HHV6B, an optimized one-round PCR variant with electrophoretic detection of amplification products in an agarose gel was used. Statistical data processing was carried out using the R programming language and the RStudio environment. According to the results of our own research, the frequency of detection of EBV, HHV6A/B and EBV+HHV6A/B DNA in acute infectious mononucleosis was 95, 91 and 86%, and among conventionally healthy children — 69, 85 and 61.5%, respectively. It was found that among the examined children of the Nizhny Novgorod Region, EBV1 and HHV6B prevail in the viral population, which is consistent with existing ideas about their geographical distribution in the adjacent territories. EBV2 and HHV6A were not detected in any of the examined saliva samples. According to the results of 14-day dynamic monitoring of saliva virus secretion in healthy virus carriers (adults and children), it was shown that a single EBV DNA study does not allow to reliably assess the infection of individuals or the intensity of EBV secretion. In this case, HHV6A/B is characterized by a more constant and uniform release. The methodological approach optimized in this work makes it possible to separately detect EBV1/EBV2 and HHV6A/HHV6B according to a single laboratory protocol, and in combination with an additional stage of saliva sample preparation increases the diagnostic sensitivity of PCR analysis, minimizes the proportion of discordant and false negative results. Such an integrated approach can be applied for diagnostic, epidemiological and research purposes.

The aim of the study was to establish the concentration of the circulating immune complexes (CICs) with high (CIC 2,5%), medium (CIC 4,2%) and low (CIC 8.0%) molecular weights in patients with pulmonary tuberculosis (TB) as biomarkers of the endogenous intoxication and the degree of severity. We investigated 56 patients diagnosed with pulmonary TB and 36 healthy persons (control group). The patients were assigned to 2 groups: the study group 1 (I SG) included 29 patients in which the test established the drug susceptible TB and in the study group 2 (II SG) that included 27 patients with multidrug-resistant TB (MDR-TB). The inclusion criteria were: primary detected pulmonary TB, age older than 18 years, confirmed drug susceptibility (I SG) and confirmed MDR-TB (II SG). The exclusion criteria were: anti-TB treatment in anamnesis, extrapulmonary TB, age below 18 years, HIV infection, severe somatic diseases. The method of CIC quantification consisted in placing 50 μl serum into 3 tubes followed by adding 100 μl borate buffer and solution of 2.5%, 4.2% and 8.0% polyethylene glycol. The tubes were incubated 15 minutes at 25°C and the optical density was assessed at the wavelength 340 nm. The results were compared with the general blood count and leucocital intoxication index Kalf-Kalif (LII_KK) as an alternative method to identify endogenous intoxication. As the results of the research we established that the peak level was observed for CICs with high and medium molecular weight, wrereas less pronounced it was for CICs with low molecular weight. The high severity of the endogenous intoxication predominated in the I SG and middle degree in the IISG, without achieving the statistical significance. LII_KK was lower in study groups comparing with the control healthy subjects due to increased count of eosinophils.

SARS-CoV-2 can cause a decrease in the number of lymphocytes and their functional activity. In patients with COVID-19, especially with a severe course of the disease, a decrease in the quantitative composition of the main populations of lymphocytes is recorded. These indicators do not recover for a long time after recovery. However, it is still not known how long the quantitative and qualitative changes in the composition of lymphocytes will last and whether the severity of the infection affects this process. The aim of our study was to assess the quantitative and qualitative composition of the main populations and subpopulations of lymphocytes in patients with a new coronavirus infection of varying severity. 65 patients with a new coronavirus infection were examined. The average age of the subjects was 47.3±14.5 years. The volunteers were divided into three groups according to the severity of the infection: asymptomatic (n = 21); moderate (n = 32); severe (n = 12). The relative and absolute number of T-lymphocytes (CD3⁺), T-helper cells (CD3⁺CD4⁺), cytotoxic lymphocytes (CD3⁺CD8⁺), natural killers (CD3^–CD16⁺CD56⁺), TNK-lymphocytes (CD3⁺CD16^–CD56⁺) and B-lymphocytes (CD45⁺CD19⁺) were determined. To calculate the percentage of lymphocytes, whole blood was lysed with OPTILYSE C solution (Beckman Coulter, USA) and stained with monoclonal antibodies CD3⁺-FITC/CD8⁺-ECD/CD4⁺-PC5/CD16⁺CD56⁺PE and CD45⁺-Pacific Blue/CD19⁺-PC5 (Beckman Coulter, USA). Cytofluorometry was performed on a flow cytofluorometer “Navios™” (Beckman Coulter, USA). The results of our studies showed that there were no statistically significant changes in the relative and absolute number of immunocompetent cells in asymptomatic patients with a new coronavirus infection, compared with the control group. With an average severity of the disease, a decrease in the relative and absolute content of CD3⁺ and CD8⁺ lymphocytes was revealed, these indicators were restored to control values six months after recovery. In patients with severe infection, there was a decrease in the relative and absolute number of T cells and cytotoxic lymphocytes. These values remained significantly below normal for six months and returned to normal only seven to eight months after recovery. Also, the volunteers of this group were found to have a decrease in the number of natural killers and TNK cells. Thus, the data obtained by us indicate that pathological changes concerning the relative and absolute number of the main populations and subpopulations of lymphocytes in patients with COVID-19 depend on the severity of the disease. The number of T-lymphocytes, cytotoxic lymphocytes, natural killer cells, that is, populations of cells that play a crucial role in effective antiviral protection, has been reduced in those who have undergone a new coronavirus infection in moderate and severe form. This information can be useful for expanding methodological approaches to the diagnosis, treatment of a new coronavirus infection, as well as measures for the rehabilitation of those who have suffered it.

The contribution of the antimicrobial activity of sIgA, lactoferrin, α-lactalbumin, serum albumin, and lysozyme to the total antimicrobial activity of whey was evaluated. 66 breast milk samples from healthy women aged from 23 to 45 years collected at different periods of lactation were studied. It was found that along with proceeding the lactation period, not only the total antimicrobial activity (r = –0.944) decreased, but also the activity of the low molecular weight fraction containing the sum of antimicrobial peptides (r = –0.950). The median lactoferrin level varied from 3.46 mg/ml in colostrum down to 0.94 mg/ml one year after the onset of lactation that correlated with whey activity (r = 0.616). The concentration of sIgA was peaked in the whey of colostrum (5.01 mg/ml) and significantly reduced in mature milk, remaining approximately at the same level in subsequent periods of lactation (1.0 mg/ml). Serum albumin concentration significantly decreased while proceeding through lactation period (from 5.52 to 4.68 mg/ml) and correlated with whey activity (r = 0.589). Evaluating effects of the purified α-lactalbumin on C. albicans cells within the range of 5–20 mg/ml by spectrophotometry and microscopy showed the absence of fungicidal activity. At the same time, purified lactoferrin, IgA, lysozyme, and serum albumin demonstrated a direct fungicidal effect. The activity of the IgA preparation at a concentration relevant to the sIgA content in the whey of the first day colostrum was 50.0%, and in the whey after 12 months — 31.0%; lactoferrin — 26.7% and 3.4%; serum albumin — 15.0% and 17.7%; lysozyme — 0.1% and 1.8%. Thus, the sum of the activities of these polypeptides at concentrations typical for the first-day colostrum was 91.7%, which is comparable to the total activity of this whey — 82.4%. The sum of the activities of these polypeptides, typical for 12 months of lactation, was 53.9%, and the total whey activity relevant to this period was 64.5%. Therefore, IgA and lactoferrin contribute the most significant antimicrobial activity in the whey of colostrum, whereas after 12 month-breastfeeding IgA and serum albumin become most significant.

Microbiological monitoring after infectious diseases in the system of epidemiological surveillance implies simultaneous pathogen identification both among patients and in hospital environment. Our aim is to assess potential hospital environmental hazard for the two in-patient infectious disease hospitals of the Khabarovsk city by using bacteriological and epidemiological analysis during new coronavirus disease pandemic. Materials and methods. Bacteriological assessment of nasopharyngeal microflora in 241 patients suffering from community-acquired pneumonia that were hospitalized in the two prevention and treatment facilities of the Khabarovsk city was performed. Sanitary-bacteriological control of hospital environment (428 hospital environment samples and 91 air samples) was carried out in parallel. Bacteriological assessment was performed with classical methods. Identification of isolated bacteriological pathogens and evaluation of drug-resistant strains were carried out by utilizing bacteriological analyzer Vitek 2 Compact. Results. Nine different pathogens (Pseudomonas aeruginosa, Pseudomonas stutzeri, Acinetobacter baumannii, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Pantoea, Enterococcus faecium, Staphylococcus haemolyticus) were isolated in 20 out of 428 samples — 4.7% [2.7–6.7]. Half of isolated agents — 2.3% [0.9–3.8] — were represented by drug-resistant isolates (10 out of 20 isolates) including 5 carbapenem-resistant isolates (Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae) and 5 isolates with multiple drug resistance (Enterobacter cloacae, Pantoea, Enterococcus faecium, Staphylococcus haemolyticus). Air samples contained pathogenic biological agents found in 6 out of 91 samples — 6.6% [1.5–11.7], and half of them — 3.3% [0.6–7.9] — were identified as drug-resistant variants, including S. aureus и S. haemolyticus. One of the surveyed hospitals was recognized as more hazardous due to microflora isolated from intensive care unit (A. baumannii and P. aeruginosa were resistant to 3^rd–4^th generation cephalosporins and carbapenems). Conclusion. Revealed circulation of wide range of microorganisms isolated from environment of two in-patient hospitals indicates high risk of healthcare-associated infections formation. Intensive care units can serve as a reservoir of healthcare-associated infections due to high percentage of patients with severe disease cases (“main reservoir” of drug-resistant strains).

Despite many advances in the diagnosis, screening, and rapid treatment of tuberculosis, it is still a public health concern in the world. Due to the importance of this issue in diagnosis and reduction of transmission of infection and treatment of the disease especially where this study was conducted due to the high prevalence of tuberculosis, the aim of our study was to determine the relationship between sputum smear positivity grade and chest X-ray findings in pulmonary tuberculosis patients in a hospital in southeast of Iran. This cross-sectional study was performed on all patients with pulmonary TB referencing the health centers in the Zabol city from January 1, 2015 to December 30, 2020. Sputum smear and radiographic findings of the chest X-ray were evaluated. Data was collected using a form of information and finally analyzed by SPSS 22. Out of 101 patients examined in the present study, 71 were women and 30 were men. The mean age of the patients was 62.68±13.61 years. The frequency of opacity in patients with grades 1, 2, and 3 was 71.4, 78.5, and 76.5%, respectively. Frequency of cavitation in patients with grade 1, 2 and 3 was 11.5%, 28.5% and 52.9% respectively (P value 0.001). The frequency of reticulonodular presentations in patients with grade 1, 2, and 3 was 24.2, 7.1, and 0%, respectively. In general, the results of this study showed that, with increasing grading of smears (1+, 2+, and 3+), the frequency of cavitation presentation increased significantly and the frequency of reticulonodular presentations decreased significantly. The findings of the present study can help physicians to improve TB diagnostics.

Multidrug-resistant K. pneumoniae bacterial strains producing extended range of beta-lactamases or carbapenemases are of serious clinical concern. The aim of the study was to determine the resistance factors of K. pneumoniae strains isolated from the lower respiratory tract of patients diagnosed with community-acquired pneumonia during the COVID-19 pandemic. Materials and methods. The study of resistance to antimicrobial drugs included 138 strains of K. pneumoniae isolated from the sputum of patients treated in infectious diseases monohospitals in the city of Tyumen and the Tyumen region within the period from May 2020 to June 2021. Among the strains examined, 51.4% of them were isolated from SARS-CoV-2 positive patients. The presence of resistance genes was determined by PCR in 71 strains of K. pneumoniae (34 strains from COVID-19-positive and 37 strains from COVID-19-negative patients). Identification of isolated bacterial strains was carried out according to the protein spectra by using a desktop time-of-flight mass spectrometer with matrix laser desorption MALDI-TOF MS (Bruker, Germany). The belonging of the strains to the hypermucoid phenotype was determined using the string test. Sensitivity to antimicrobial drugs was assessed in the disk diffusion method on Muller–Hinton medium. The sensitivity of culture strains to bacteriophage preparations was determined by the drop method (spot-test). In the study, we used “Polyvalent Sextaphage Pyobacteriophage” and “Purified Polyvalent Klebsiella Bacteriophage” (JSC NPO Microgen, Russia). Detection of resistance genes to beta-lactam antibiotics by real-time PCR was carried out using the BakRezista kit (OOO DNA-technology, Russia). Results. The results of the study evidence that K. pneumoniae bacteria isolated from COVID-19-positive and COVID-19-negative patients diagnosed with community-acquired pneumonia displayed a high resistance to antimicrobial drugs and commercial phage-containing drugs. Resistance of K. pneumoniae strains was recorded from 50% (to aminoglycosides and carbapenems) to 90% (to inhibitor-protected penicillins). Sensitivity to bacteriophages was noted on average in no more than 20% of strains. It is important to emphasize that strains isolated from COVID-19-positive patients more often showed a hypermucoid phenotype, suggesting a high bacterial virulence, and also showed greater resistance to all groups of antibacterial drugs examined in the study, which is confirmed by the presence of resistance genes of the ESBL group and carbapenemase. The results of the study suggest that the high level of resistance of K. pneumoniae strains isolated from COVID-19-positive patients is associated with immunosuppression provoked by the SARS-CoV-2 virus, which contributes to their colonization by more virulent strains.

Recently, there have been increasing an interest to study a role of polyamines in intercellular interactions, especially in the focus of inflammation, where accumulation of such polycations is observed. In this regard, products of microbial origin — cadaverine and putrescine — are of particular attention. The role of polyamines as «scavengers» of free radicals has been described, but no data of their effect on the leukocyte radical-producing activity have been obtained so far. The aim of the investigation was to study features of hydroxyl radical generation by human leukocytes exposed to microbe-derived polyamines. Materials and methods. Peripheral venous blood samples were obtained from 20 healthy donors. To assess radical production, a luminol-dependent chemiluminescence reaction was carried out with blood leukocytes pre-incubated with cadaverine (0.01 M) and putrescine (0.01 M), measured on a Luminoskan Ascent^® Thermo Labsystems luminometer (USA) for 180 min. For statistical analysis, an integral chemiluminescence index was used for the entire measurement period (RLU). Results and discussion. It was shown that cadaverine has a stimulating effect on the leukocyte potential to produce radicals (averaged area under the curve is 6.7±0.7 r.u., p < 0.05). Putrescine had little effect on the radical-producing activity of human cells (2.8±0.4 r.u., p < 0.05). This might be due to the direct influence of polycations on the mechanisms of radical generation, as well as increased activity of leukocyte diamine oxidase catalyzing the conversion of diamines into aminoaldehyde, which is accompanied by the release of hydrogen peroxide reacting with luminol. In addition, the enzyme inactivates other compounds, such as histamine leading to formation of a less pronounced clinical picture. Polyamines, particularly cadaverine and putrescine, can be referred to the factors remodeling the metabolic activity of the host leukocytes, which is aimed at maintaining the viability and increasing the number of microorganisms. Conclusion. Thus, cadaverine and putrescine produced by microorganisms, depending on the conditions of the microenvironment, might be evidently considered as mediators of the mild or asymptomatic course of inflammatory diseases, which contributes to underlying persistent potential of bacteria.

Since the spread of the novel coronavirus infection, most researchers have noted a low proportion of sick children in general pediatric cohort compared to adults, who had a mild disease course and rare complications. The most frequent clinical manifestations of the disease are respiratory and, some less frequently diarrheal syndromes. The disease has predominantly mild or asymptomatic course. The risk of adverse outcomes in children, similar to adults, clearly correlate with the presence of background chronic pathology. The need for respiratory support prevails in children with a severe premorbid burden. Here, a clinical case of ongoing novel coronavirus infection in adolescent patient comorbid with chronic kidney pathology is described. In adolescence, the patient was diagnosed with mesangioproliferative glomerulonephritis (IgA-nephropathy), and further registered at the dispensary receiving a combination therapy with angiotensin converting enzyme inhibitors and disaggregation drugs. The epidemiological history contained no established contacts with infectious patients. The clinical manifestations of COVID-19 in the patient are represented by catarrhal and diarrheal syndromes, transient renal dysfunction in the acute period of the disease. The onset of coronavirus infection was clinically characterized by symptoms of damaged gastrointestinal tract and was considered as acute gastroenteritis of infectious etiology. Empirically prescribed antibacterial therapy in combination with antiplatelet agents and symptomatic drugs had no effect. The diagnosis of the novel coronavirus infection was verified only on day 4 of hospitalization, clinical and laboratory signs of lung damage emerged. The inflammatory process developed in the patient lungs was secondary to the main pathology. The severity of the patient’s condition was determined by the presence of respiratory and renal insufficiency. Lung damage with minimal severity complaints and clinical data had a bimodal pattern and required respiratory support. A comprehensive approach to treatment, including respiratory, antiviral, enterosorption, anticoagulation, anti-inflammatory, antihypertensive, hepatoprotective, symptomatic therapy with change in antibacterial drugs allowed to achieve positive dynamics. On day 12 of the illness, the patient required no respiratory support. The presence of symptoms of gastrointestinal tract damage in COVID-19 necessitates the mandatory inclusion of PCR assay for SARS-CoV-2 into diagnostic protocol in patients with diarrheal syndrome to perform etiological disease interpretation.