Vol 11, No 1 (2021)

Issues of accuracy (sensitivity and specificity) of PCR-analysis depending on features of performing preanalytical and analytical stages of laboratory diagnostics of COVID-19, as well as comparing PCR and lung computed tomography (CT) results have been analyzed in the study. Currently, a molecular genetic test based on polymerase chain reaction (PCR) is used for diagnostics of a new coronavirus infection (COVID-19). As of November 1, 2020, more than 750 million PCR tests have been conducted globally. Evidence accumulated by now allows to estimate diagnostic sensitivity and specificity of the SARS-CoV-2-specific PCR as high as 82—91% and 99—100%, respectively. In addition, increased PCR sensitivity may be noted upon performing repeated testing of the upper respiratory tract samples comprising 82.2% during the primary analysis that was further elevated up to 90.6% after two consecutive tests. A whole set of factors affect the PCR accuracy. In particular, false negative data might result from insufficient amount of virus-coupled genetic material in the sample, timeframe and mistakes made upon selecting biological samples. It was found that SARS-CoV-2 virus RNA was detected at the maximum diagnostic sensitivity in the upper respiratory tract 1—3 days before the onset of symptoms and sustained within the 5—6 days after disease onset. Such period of time is associated with the peak risk of SARS-CoV-2 transmission. On week 2 after disease onset, there have been noted elevated rate of detecting viral RNA in bronchopulmonary samples. The duration of detecting virus-related markers (including those found in the absence of viable virus forms) correlates with disease severity and may last for as long as 1—2 months. Another real-world issue related to PCR analysis is posed by an opportunity of obtaining false positive data, which solution requires high level organized laboratory research, especially in case large-scale studies. Upon that, it is worth noting that positive PCR results may account for detecting solely certain RNA-related fragments present in any sample, rather than a viable virus. It was noted that PCR in comparison to CT analysis demonstrates higher specificity, but does not allow to distinguish pneumonia caused by SARS-CoV-2 from pneumonia caused by other etiological agents (up to 25% false positive results). However, the diagnostic CT sensitivity was 97.2% that exceeds such parameter for PCR by 10—15%. It was concluded that the approach combining use of both PCR and CT by taking into account their own features as well as factors affecting the accuracy of the data obtained, allows us to correctly interpret the diagnostical results.

A supportive homeostatic function of neutrophilic granulocytes is accomplished in the physiology of diverse tissues and body systems. Neutrophils are found along the entire female reproductive tract (FRT), gradually declining in numbers from the upper parts towards the vagina. At the same time, both quantity and activity of FRT mucosal neutrophils are controlled by hormonal changes at different phases of menstrual cycle. Tissue neutrophils serve as an important source of broad-spectrum proteolytic enzymes such as matrix metalloproteinases and elastase necessary for extracellular matrix remodeling as well as vascular endothelial growth factor (VEGF) required for physiological FRT angiogenesis. During pregnancy, decidual neutrophils play a prominent role in vascular remodeling in pregnant uterus as well as development of maternal-fetal immune tolerance. The influx of neutrophils into the intestinal mucosa due to its trauma or infection not only ensures defense against pathogens, but also leads to increased proliferation of intestinal epithelial cells. Neutrophilic granulocytes elicit signals and events protective for the epithelium by marking them with a “hypoxic signature” to trigger transcription of the gene set responsible for production of mucins, mucin-modifying peptides, antimicrobial proteins, в-defensins, ultimately contributing to lesion healing and recovery of epithelial barrier function. “Inflammatory hypoxia” initiated by neutrophils and subsequent stabilization of the transcription factor hypoxia-induced factor (HIF) in intestinal epithelial cells trigger mechanisms of self-limited and resolved inflammation, which prevent excessive accumulation of neutrophils in the intestinal lumen and development of chronic inflammatory process. Neutrophilic granulocytes dominate in the oral cavity mucosa and comprise more than 95% of total leukocyte population recruited into the gingival sulcus and gingival fluid. Neutrophils maintain physiological amount and stability of symbiotic microflora composition in dental and gingival biofilms, counteracting pathogenic bacteria via phagocytosis, degranulation and extracellular trap formation, thereby ensuring healthy state in periodontal structures. Finally, similar to some other congenital disorders affecting neutrophil quantity and functions it was shown that in case of leukocyte adhesion deficiency type 1 (LAD-1) pathogenesis of periodontitis may not only be associated with a defect in their protective effector activity, but also with altered immunoregulatory function of tissue neutrophils.

Enteroviruses comprise highly diverse group of single-stranded positive RNA viruses belonging to Enterovirus genus, Picornaviridae family. They are the most prevalent viruses worldwide highlighted by high resistance to environmental cues. Enteroviruses normally cause seasonal self-limiting infections, but also known as causative infectious agents of encephalitis, myocarditis, poliomyelitis, acute heart failure and sepsis. Enterovirus genetic plasticity contributes to widespread epidemics and sporadic outbreaks (e. g., outbreaks of Enterovirus D68 and Enterovirus 71). Type B Coxsackieviruses of Enterovirus B species is one of commonly identified infectious agents associated predominantly with mild upper respiratory and gastrointestinal illnesses. Nevertheless, Coxsackieviruses B3 infection can result in severe myocarditis leading ultimately to heart failure. The pathogenesis of Coxsackievirus B3-induced myocarditis is well known being mediated by both direct damage due to viral proteases and indirectly via secondary host immune responses. Despite success in preventive vaccination of some enterovirus infections that allowed to control some of them direct antiviral agents for treatment of enteroviral infection particularly Coxsackieviruses B3 myocardial infection are still in demand. In addition, no ongoing clinical trials for therapy or prevention of Coxsackieviruses B3 infection are available. Current treatment strategies are mainly aimed to stabilize patient condition and relieve discomfort condition. It seems that relatively small market for anti-enteroviral drugs prevents pharma industry from developing new drugs. The Coxsackieviruses B3 lifecycle have been extensively studied and potential targets for drug design have been identified. The aim of our review was to describe current state in the field of antiviral drug design combating Coxsackieviruses B3 infection emphasizing direct-acting antivirals, albeit paying some attention to host factor-targeting inhibitors (including compounds from medicinal plant extracts) as well. The following categories of direct Coxsackieviruses B3 inhibitors are discussed in detail: capsid binders (pleconaril and its derivatives), viral 3C protease inhibitors (rupintrivir and its analogs), drugs targeting viral replication (both nucleoside analogs and non-nucleoside inhibitors). Results of drug repurposing screens for amiloride, benzerazide, dibucaine and fluoxetine are also discussed.

The first reports about HIV (human immunodeficiency virus) were appeared to the 1980s. By 2017 more than 37 million people were living with HIV. Human papilloma virus (HPV) is universally spread, with some estimates showing that about 1% of the sexually active population having genital warts. Human papilloma virus (HPV)-induced infection frequently accompanies the clinical course of HIV and can manifest itself in a full spectrum of clinical-pathologic forms ranging from common warts to malignant neoplasia. Due to the widespread use of antiretroviral therapy, the number of patients with a combined infection (HIV+HPV) is steadily increasing. Here we review current clinical treatment options for HPV manifestations. High-dose antiretroviral therapy does not impede HPV treatment, and can even improve its efficacy in some cases. The topical administration of imiquimod, an immune response modifier, is an effective conservative treatment in HIV-infected patients with HPV. The immunomodulation therapy of imiquimod can serve as an effective alternative of aggressive chemical and mechanical procedures. Maximum efficacy with the lowest replaces rates may be expected from combined use of mechanical ablation methods with a subsequent follow up treatment with imiqui-mod. The best therapeutic result is expected in HIV-positive patients who are received high-dose antiretroviral treatment. The advantages of Vartocid, the modified Russian equivalent of the generic imiquimod.

Dendritic cells as the most active and highly specialized antigen presenting cells, play a key role in initiating immune responses. Currently, generation of medications activating dendritic cells for development of anti-infective and anticancer vaccines is of highly relevance. Preparations of microbial origin are promising to augment activity of dendritic cells, because they carry innate immune ligands for Toll-like receptors. Such preparations include experimental protein-containing pneumococcal preparations, obtained from acetone-inactivated microbial mass of the deposited S. pneumoniae 6B vaccine strain No. 296, followed by aqueous extraction and separation of 30—100 kDa fraction. Dendritic cells were obtained from bone marrow cells of CBA mice (n = 15), and cultured in complete growth medium RPMI-1640 added with recombinant GM-CSF and IL-4 (Biosource, USA). On day 6, experimental protein-containing pneumococcal preparations (50 pl/ml) were administered to the cultured immature dendritic cells. Commercial TNFa (20 ng/ml, Biosource, USA) was used as a standard maturation inducer (positive control). Immunophenotyping of dendritic cells was conducted by using flow cytometry with FITC- and PE-labeled monoclonal antibodies against cell surface receptors: CD34, CD38, CD83, CD86, CD80, CD11c, MHC II, CD14, CD282 (TLR2), CD284 (TLR4), (eBioscience, USA). Studying an effect of preparations on maturation of dendritic cells revealed that morphological characteristics of dendritic cells generated by using experimental protein-containing preparations did not differ significantly between each other as well as those induced by TNFα. The cells were characterized by large sizes, oval or irregular shape, veiled cytoplasm, eccentrically located nucleus and numerous long thin protrusions. Experimental proteincontaining preparations induced in cultured dendritic cells decrease in count of CD34+ immature and TLR2/TLR4+ cells, increased count of cells expressing markers of adhesion (CD38+), activation (MHC II+), costimulation (CD80/ CD86+) and terminal differentiation (CD83+), which may evidence about events of differentiation upon dendritic cell maturation. The 30—100 kDa fraction increased count of cells expressing adhesion molecules to a greater extent than aqueous extract that more pronouncedly stimulated rise in count of dendritic cells bearing costimulatory molecules (p < 0.05). The activity of the examined proteins regarding their effect on CD83+ cells was comparable. Experimental protein-containing antigens derived from pneumococcal vaccine strain were shown to induce maturation of dendritic cells from bone marrow precursors, induce a decrease in the count of TLR2 and TLR4-expressing cells accounting for activating effect on innate immune effectors.

A search for new targeted therapeutic strategies based on examining immunopathogenetic mechanisms for emerging co-infections is relevant and may further contribute not only to optimizing choice of immunotropic drugs, but also to achieving positive clinical and immunological remission for abnormal infectious processes. Previously, our studies found that recurrent viral-bacterial respiratory infections are associated with dysfunction of neutrophilic granulocytes (NG) with varying degree of intensity in altered effector properties. NG dysfunctions are often associated with diverse phenotypic profiles characterized by varying density for expression level of functionally significant trigger receptors. The aim of the study was to pinpoint phenotype transformation in CD64^-CD32⁺CD16⁺CD11b⁺, CD64⁺CD32⁺CD16⁺CD11b⁺ neutrophilic granulocytes in experimental model of viral-bacterial infection in vitro. We examined 52 peripheral blood samples collected from 13 healthy adult volunteers. Viral, bacterial and virus-bacterial infection was modelled in vitro by incubating blood-derived cell samples with formyl-methionyl-leucyl-phenylalanine (fMLP), double-stranded RNA (dsRNA) or in combination followed by assessing changes in immunophenotyping of CD64^-CD32⁺CD16⁺CD11b⁺NG, CD64⁺CD32⁺CD16⁺CD11b⁺NG by using using MAbs CD16-ECD, CD64-FITC, CD32-PE, CD11b-PC5 conjugates (Beckman Coulter International SA, France). It was demonstrated that NGs from healthy adult volunteers were dominated by CD64^-CD32⁺CD16⁺CD11b⁺NG as well as minor subset СD64⁺CD32⁺CD16⁺CD11b⁺ NG varying in expression density of membrane molecules. Percentage of the minor subset СD64⁺CD16+CD32⁺CD11b⁺ NG was significantly increased after exposure with dsRNA, fMLP and dsRNA+fMLP compared to untreated samples. Comparative analysis revealed that various immunotropic agents differed in affecting expression of surface receptor molecules CD16, CD32 and unidirectional effects, but of varying magnitude altering CD11b marker both in major and minor subsets. Preincubation with dsRNA followed by adding fMLP allowed to find that they co-stimulated expression of surface receptors in both NG subsets. We generated an experimental model of viral-bacterial co-infection in vitro by using fMLP and dsRNA and observed types of phenotype transformation in CD64^-CD32⁺CD16⁺CD11b⁺ NG and CD64⁺CD32⁺CD16⁺CD11b⁺ NG subsets. This model can be used to evaluate transformation of other NG subset phenotypes, NG functional activity, features of NET formation as well as impact of various immunotropic agents on NG.

General vaccination of population with vaccinia virus leaded to the eradication of smallpox, then it was finished because of the danger of adverse events. The recurrence of research interest in smallpox vaccine is due to the research of using the virus as a weapon of bioterrorism and the increased frequency of orthopoxvirus infections whereas the population immunity decline. The vaccinia virus is also used as a vector for creating recombinant vaccines. Understanding the pathway and predicting the immune response it will be able to avoid possible adverse events and excessive immunization. The aim of the study was to assess the correlations between humoral immunity, clinical signs during a vaccination period, sex and age characteristics of adults who had received several doses of vaccinia virus. We studied a vaccination clinical data of 135 subjects revaccinated with a smallpox vaccine from twice to 10 times. A total of 95% and 5% vaccine recipients experienced mild or moderate vaccination period, respectively. Inoculation skin lesions was noted at 127 subjects (94.1%). Among them more than 22% vaccine recipients experienced local or systemic adverse events. Compared to mild group moderate group had larger hyperemia (p = 0.04), scab (p = 0.01), healing time (p = 0.001). The age subjects with a moderate vaccination period is less than mild (p = 0.03), the chance of lymphadenopathy development is higher within moderate vaccination period (p < 0.001). Vaccinia neutralizing antibody titers were determined for 54 subjects using plaque reduction neutralization tests. There was a noted tendency of higher protective antibody values at women compared with men. Negative correlation between the antibody titers and the hyperemia size was revealed. Frequently axillary adenopathy is assotiated with higher protective antibody values. Vaccinia neutralizing antibody titers value are not associated with the presence and size of the lesion, the scab falling time, age and the number of previous vaccinations. The clinical variability and the immune response using the same vaccine and the same pattern vaccination would be explained by individual genetic differences that should be further explored.

Influenza and other acute respiratory viral infections are the most common infectious diseases of our time, causing a significant harm to human health as well as great economic damage. At least five groups of viruses, including more than 300 subtypes, are currently related to ARVI pathogens. Such infectious agents are characterized by a high degree of variability resulting in replaced virus antigenic characteristics augmenting their contagiousness, immunoevasion, and resistance to chemotherapeutic drugs. Of relevance, influenza and other ARVIs also pose a threat due to subsequent rapid formation of bacterially-associated respiratory diseases as well as their continuous variability and emergence of new pathogenic species. In recent years, subtype A (H1N1) and A (H3N2) with predominance of pandemic strain, as well as type B influenza viruses have been simultaneously found in circulation. Most common among the causative agents of noninfluenza ARVIs, are respiratory syncytial virus, rhino- and adenoviruses, as well as I/III parainfluenza viruses. Here we present the results of virological and serological studies of clinical samples collected during the 2018—2019 epidemic season in the territory of the Republic of Kazakhstan after analyzing 2794 clinical samples (2530 nasopharyngeal swabs and 264 blood serum samples) of patients diagnosed with ARVI, ARI, bronchitis, and pneumonia. Examining nasopharyngeal swabs by using RT-PCR showed that the mixed etiology influenza viruses with predominant A/H1N1pdm virus circulated in Kazakhstan. In particular, influenza virus genetic material was found in 511 swabs (20.20% of total examined samples), so that influenza A virus RNA was detected in 508 biological samples such as A/H1N1 — in 289, A/H3N2 — 209, unverified virus subtype — 10 samples. Type B influenza virus was detected in 3 samples. Analyzing 264 blood serum samples by the HAI assay and ELISA showed the presence of antibodies specific to influenza A/H1N1, A/H3N2, and B viruses in the population of various regions of Kazakhstan, thereby indirectly confirming their co-circulation. 42 influenza virus strains were isolated in chicken embryos, of which 28 were assigned to A/H1N1pdm virus, 13 — A/H3N2 virus, and one isolate was identified as influenza B virus. The laboratory diagnostics of clinical samples for ARVIs revealed that respiratory syncytial virus prevailed among identified non-influenza agents, whereas rhino- and adenoviruses were less common. Metapneumoviruses, bocaviruses, coronaviruses, and type I parainfluenza viruses were detected in few cases. Comparison of our study data with the data on 2017—2018 circulation of influenza pathogens showed that in Kazakhstan influenza A and B viruses continued to circulate, with the dominance of A/H1N1pdm virus as it was in the previous epidemic season. Identification of non-influenza viruses, the causative agents of 2018—2019 respiratory infections, showed the predominance of respiratory syncytial virus that correlated with the aforementioned results.

The state of the interferon system in 38 children with acute infectious mononucleosis (IM) caused by the Epstein—Barr virus was analyzed. Interferon status was examined in accordance with F.I. Ershov method based on assessing related biological activity by measuring interferon level in the blood serum or produced by blood cells. The aim of the study was to gain scientifically justified data for use of interferon preparations or interferonogens in IM combination therapy. For this, interferon status in children with acute IM was compared with that one not only in control group (30 healthy children, aged 3—6 and 20 children, aged 7—14 years, examined earlier to create an intra-laboratory interferon normal range), but also in children with lacunar angina or acute respiratory viral infection, hospitalized in the same department of the clinic and comparable with the main group in severity of the condition. In addition, we assessed changes in IFN-status in IM patients receiving no interferon preparations, one month after the disease onset. The study showed that patients with moderate acute IM were featured with decreased potential of blood leukocytes to virus-induced IFNa and mitogen-induced IFNy production observed with almost similar or some lower rate as in the control group of children hospitalized with angina or acute respiratory viral infection. Peripheral blood cells from moderate acute IM patients in the 3-6-year age group were found to produce virtually unaltered interferon level, whereas almost sole IFN-alpha production was affected in 7-14-year-old patients. Moreover, in 7-14-year old patients the level 1 and level 2 of IFNa deficiency was observed in 38% and 6% of cases, respectively. It is likely it was just this patient group requiring administration of any IFNa replacement therapy. As few as 12 children were re-examined after discharge from the clinic. Initially, prevalence and severity level of impaired interferon production in this subgroup did not differ from that one for total patient sample, whereas 1 month later a host potential to produce both IFNa and IFNy even without therapy acting on interferon system was noted to be moderately augmented.

According to the WHO Strategic Plan, measles should be eradicated in 2020 in the five WHO Regions including European Region. However, large measles outbreaks are being periodically registered in diverse European countries. In the Republic of Serbia (SRB), 5,076 measles cases were detected in 2018, among which 15 cases were fatal.

Aim of the study was to examine herd immunity to measles and rubella viruses in the population of the Republic of Serbia.

Materials and methods. Blood serum samples obtained in 2018 and 2019 from conditionally healthy residents of the Republic of Serbia were tested for the presence of IgG antibodies to measles and rubella viruses in five age groups: I — children from 2 to 6 years old, II — children from 8 to 14 years old, III — 15 to 24 years old, IV — 25 to 49 years old and V — over 50 years old. A total of 1000 samples were obtained, 200 sera in each group. Enzygnost® Anti-Measles virus/IgG and Enzygnost® Anti-Rubella virus/IgG ELISA test systems (Siemens Healthcare Diagnostics Products GmbH, Germany) were used according to the manufacturer's instructions.

Results. Overall, around 23.0% and 33.7% of the surveyed persons had no or low level of anti-measles IgG antibody (≥ 275.0 — ≤ 1000.0 IU/1). In age group I, 60% children contained no or “low” anti-measles antibodies titer (29.5% and 30.5%, respectively). In addition, low antibody titer level was mainly detected in individuals from age group II and III (p < 0.05). A third of children under 8—14 contained high IgG-antibodies titer against measles (> 3000.0 IU/l) that might serve as an evidence that such subjects recently recovered after measles. Similar results were obtained for IgG antibodies to rubella in the same age groups.

Discussion. The study results evidence about altered routine immunization against measles and rubella in children aged 12—15 months (first vaccination) and those at age of 6—7 years (revaccination) with MMR vaccine. The data obtained correlate with official data on coverage with measles and rubella vaccines in the Republic of Serbia.

Urinary tract infections (UTIs) pose a topical problem in current pediatrics, pediatric nephrology and urology. UTI-related clinical picture in childhood is polymorphic, sometimes being rather subtle and undergoing age-related alterations. Often, typical UTI symptoms in infants and early children occur subclinically. Microbe-related properties colonizing renal tissues dominate among multiple factors involved in developing UTI. In recent years, etiological importance of Enterococcus faecalis in development of such pathology has been increased. Our study was aimed to determine E. faecalis-associated UTI clinical signs in children and unveil their biological characteristics to assess related clinical significance.

Materials and methods. A nine-year pediatric UTI etiological pattern was analyzed at the multi-field pediatric clinical hospital. The data of clinical and laboratory examination of 181 UTI children aged 3 days — 17 years as well as microbiological study of 60 E. faecalis strains isolated from patient urine were obtained.

Results. Clinical and laboratory characteristics of E. faecalis-associated UTIs, age-related symptom variability were presented. Intoxication syndrome and fever dominated in the clinical picture. A key sign of UTIs was gastrointestinal dysfunction (in neonates and one-year old children) and pain in the lumbar region (in older children). The identified clinical symptoms may be associated with the upper urinary tract damage, concomitant diseases, and the pathogenic properties of E. faecalis. Other symptoms were less common, consistent with the age of the patients, although abdominalgia was equally common for children in all age groups. Data of laboratory examination also depended on patient age. It was noted that leukocytosis and thrombocytosis were more prominent in neonates, whereas leukocyturia and proteinuria — in children above one year of age, although clinical symptoms in this group were less overt. Specific features and clinical significance of E. faecalis-related biological properties, their heterogeneity related to patient age were noted. An inter-connected relationship between pathogenic properties and certain clinical symptoms was revealed.

Conclusion. The dominant clinical symptoms (intoxication, hyperthermia), indicative of damaged upper urinary system is related, among other things, to the set of E. faecalis biological properties exerting tissue-damaging and cytolytic effects.