Introduction. People living with human immunodeficiency virus (HIV) are more likely to experience Epstein–Barr virus (EBV) reactivation and develop EBV-associated diseases. In Russia, the clinical significance of EBV genetic diversity in HIV-infected patients has not been assessed. The aim was to analyze a relationship between the major EBV types and LMP-1 genovariants with clinical and laboratory parameters in HIV-infected persons. Materials and methods. Peripheral blood leukocytes were collected from 138 HIV(+) individuals aged 20–69 years. Association between EBV types, LMP-1 variants and subvariants with clinical and laboratory parameters (CD4+ T-lymphocyte count, HIV and EBV viral load, use and adherence to antiretroviral therapy (ART)), was performed using the principal component analysis method and the Mann–Whitney U test. Results. It has been shown that detectable HIV viral load increases in patients with low CD4+ T-lymphocyte counts, high EBV viral load, and low or no ART adherence. In general, infection with EBV-2 or the LMP-1 B95-8 alone resulted in lower EBV and HIV viral loads compared with other variants. Significant EBV-1 LMP-1 subvariants were identified, the biological potential of which was enabled in immunodeficiency state (CD4+ T-lymphocyte count ≤ 200 cells/μl). In “naive” patients, EBV-1/LMP-1 (S309N)+HIV co-infection occurred with a higher, and EBV-1/LMP-1(E328Q)+HIV with the lowest HIV viral load. The highest EBV DNA concentrations were observed with EBV-1/LMP-1(Q334R)+HIV. In “experienced” patients, the level of EBV DNA was significantly lower when infected with EBV-1/LMP-1(E328Q)+HIV and, conversely, higher in case of detected EBV-1/LMP-1(H358P)+HIV. Conclusion. The features of clinical and laboratory parameters EBV+HIV co-infection caused by different EBV-1 LMP-1 subvariants (at the level of amino acid substitutions S309N, E328Q, Q334R, H358P) have been identified. It is necessary to study the functional role of such mutations in vitro and in vivo. In the context of assessing a clinical significance of EBV molecular genetic diversity, it is advisable to conduct larger-scale studies in different territories of Russia.

Laboratory confirmation of the diagnosis of tick-borne infections is often hampered due to delayed seroconversion. Objective. Assessment of clinical and laboratory predictors to develop a differential diagnosis model between the mixed infection of Lyme borreliosis with tick-borne encephalitis and the monoinfection of Lyme borreliosis non-erythemal form at the onset of the disease. Patients and methods. In the first week of the disease, 54 clinical and laboratory parameters were studied in 27 patients with the mixed infection of Lyme borreliosis with tick-borne encephalitis (mean age: 47.2 years) and 65 patients with borreliosis monoinfection (mean age: 45.3 years). The logistic regression model was developed using Statistica 12.0. Results. Predictive factors for diagnosis of the mixed infection included painful eye movement, photophobia, nausea, meningism, intoxication syndrome intensity (AUC = 0.69) fever intensity (AUC = 0.79), neutrophil to lymphocyte ratio (AUC = 0.75), neutrophil to monocyte ratio (AUC = 0.73), neutrophils count (AUC = 0.76), immature granulocytes count (AUC = 0.80), lymphocytes count (AUC = 0.73) and reactive lymphocytes count (AUC = 0.70). A logistic regression model was developed with very good prognosis of mixed infection (AUC = 0.88). Conclusion. The model is allowed to assess the probability of a preliminary diagnosis of the mixed infection of Lyme borreliosis and tick-borne encephalitis with good sensitivity and specificity values. Key words: Lyme borreliosis, tick-borne encephalitis, mixed infection, non-erythemal form, clinical predictors, neutrophil to monocyte ratio, hemogram parameters, logistic regression model

Coinfection with the human immunodeficiency virus (HIV) and Epstein–Barr virus (EBV) represents a current biomedical problem. The purpose of the study was to evaluate blood leukocyte EBV detection rate and viral load in adult HIV-infected patients. Materials and methods. There were examined blood leukocytes collected from 138 HIV(+) and 68 HIV(–) individuals aged 20–69 years. Statistical analysis was carried out differentiated according to the stages of HIV infection, the CD4+ T-lymphocyte count, and adherence to antiretroviral therapy. Results. It was shown that EBV DNA was detected significantly more often in HIV(+) vs HIV(–) individuals (70.3±3.9% and 48.5±6.1%, p = 0.008), with EBV viral load comprising 18 [5; 139] versus 2 [1; 3] copies/105 cells (р 0.001), respectively. It has been shown that the group of HIV(+) patients is heterogeneous in the frequency of EBV detection and viral load, with the peak EBV frequency (86.7±6.2%) and DNA (121 [34; 252] copies/105 cells) level observed in “naive” patients with severe immunodeficiency. Among “experienced” patients receiving therapy, the relative risk of detecting EBV DNA with low treatment adherence was significantly higher compared to those who developed high adherence (р 0.05). When the HIV viral load reached undetectable level, EBV DNA concentration was significantly lower where HIV RNA was detectable (1 [0; 8] versus 15 [1; 162] copies/105 cells, р 0.001). Detection of EBV DNA is associated with higher HIV viral load level and lower CD4+ T-lymphocyte count compared to patients with undetected EBV DNA. A relationship has been established between the CD4+ T-lymphocyte count in HIV(+) patients and the likelihood of active EBV infection. A threshold cut-off of 200 cells/μl was determined. CD4+ T-lymphocyte count 200 cells/μl vs ≥ 200 cells/μl (р 0.001) is associated with a 3.3-fold higher risk of detecting active EBV infection. Conclusion. It is necessary to continue interdisciplinary research to improve early diagnostics of EBV-associated diseases in HIV-infected individuals.

Background. Early diagnosis of the mixed infection of ixodic tick-borne borreliosis and tick-borne encephalitis can be difficult without laboratory confirmation of the diagnosis.The aim. To assess clinical and laboratory predictors in patients for building the models of early differential diagnosis between mixed infection of erythema form of ixodic tick-borne borreliosis and febrile form of tick-borne encephalitis and monoinfection of erythema form of ixodic tick-borne borreliosis.Materials and methods. The clinical study included 26 patients with mixed infection of the erythema form of ixodic tick-borne borreliosis with tick-borne encephalitis (mean age – 46.7 years) and 30 patients with monoinfection of the erythema form of ixodic tick-borne borreliosis (mean age – 48.5 years). 59 clinical, epidemiological and laboratory parameters, including 6 leukocyte blood indices, standard and extended hemogram profiles were studied in these patients the first week of the disease. For statistical analysis, we used Pearson and Mann – Whitney goodness-of-fit tests, ROC analysis with calculation of the area under curve (AUC) and logistic regression. Results. Patients with mixed infection, compared to the patients with monoinfection, had increased odds of having febrile fever, weakness, headache and orbital pain; meningism symptoms occurred only in patients with mixed infection. Early predictors of a preliminary diagnosis of mixed infection were elevated values of fever (AUC = 0.87), intoxication syndrome score (AUC = 0.71), leukocyte count (AUC = 0.64) and neutrophils (AUC = 0.71) in the hemogram, levels of leukocyte intoxication index according to V.K. Ostrovsky (AUC = 0.67) and neutrophil-leukocyte index (AUC = 0.65), as well as a decrease in the number of platelets (AUC = 0.77), eosinophils (AUC = 0.72) and basophils (AUC = 0.79) in peripheral blood. Two prognostic models which included clinical and laboratory parameters demonstrated excellent prediction of mixed infection (AUC = 0.90 and AUC = 0.91).Conclusion. Early clinical and laboratory predictors have been established and models have been developed that make it possible, with excellent prognosis quality, to calculate in patients the probability of a preliminary diagnosis of mixed infection of the erythema form of ixodic tick-borne borreliosis with tick-borne encephalitis in comparison with borreliosis monoinfection.

The aim of the study was to develop an experimental version of a DNA microarray for parallel detection of community-acquired pneumonia bacterial pathogens. Materials and Methods:We studied the samples of the pharyngeal mucosa smears taken from children aged 1-15 years with X-ray confirmed pneumonia. The selection of DNA probes for specific detection of community-acquired pneumonia pathogens (S. pneumoniae, H. influenzae, M. pneumoniae, C. pneumonia, and L. pneumophila) and development of the microarray design were carried out using the disprose program. The nucleotide sequences of pathogens were obtained from NCBI Nucleotide database. In the research we used CustomArray microarrays (USA). For a pooled sample containing S. pneumoniae and H. influenzae DNA, we performed a sequential selection of the best combinations of hybridization parameters: DNA fragment size, DNA amount, hybridization temperature. The selection criteria were: the percentage of effective probes with a standardized hybridization signal (SHS) ≥3 Z, and the excess of SHS levels of effective specific probes compared to SHS of effective nonspecific probes. We selected the probes to detect of S. pneumoniae and H. influenzae characterized by an effective hybridization signal under optimal conditions. The developed microarray was tested under the selected conditions on clinical samples containing S. pneumoniae or H. influenzae DNA. Using ROC analysis there were established threshold values for the signals of specific probes at optimal sensitivity points and the test specificity, the excess of which was interpreted as the evidence of pathogen presence in a sample. Results:A microarray design included 142 DNA probes to detect S. pneumoniae, H. influenzae, M. pneumoniae, C. pneumoniae, and L. pneumophila, the probes being synthesized onto slides. Using the example of clinical samples containing S. pneumoniae and/or H. influenza DNA, we selected optimal parameters for DNA hybridization on microarrays, which enabled to identify bacterial pathogens of community-acquired pneumonia with sufficient efficiency, specificity and reproducibility: the amount of hybridized DNA was 2 μg, the DNA fragment size: 300 nt, hybridization temperature: 47°C. There was selected a list of probes for specific detection of S. pneumoniae and H. influenzae characterized by an effective hybridization signal under the identified conditions. We determined the threshold values of standardized probe signals for specific detection of S. pneumoniae (4.5 Z) and H. influenzae (4.9 Z) in clinical samples. Conclusion:A DNA microarray was developed and synthesized for parallel indication of bacterial pathogens of community-acquired pneumonia. There were selected the optimal parameters for DNA hybridization on a microarray to identify bacterial pathogens - S. pneumoniae and H. influenzae, and determined the threshold values of significant probe signals for their specific detection. The interpretation of the microarray hybridization results corresponds to those obtained by PCR. The microarray can be used to improve laboratory diagnostics of community-acquired pneumonia pathogens.

Lyme borreliosis is a group of transmissible infectious diseases that are similar in etiology, but diverse in clinical manifestations. The development of Lyme disease symptoms is due not only to the activity of the pathogen itself, but also to the result of its interaction with immune system of macroorganism. The first line of defense, represented by a variety of cellular and humoral components of innate immunity, is most rapidly involved in immune response, and it is it that seeks to limit dissemination of the causative agent from the initial site of infection. However, a wide range of protective surface proteins of Borrelia and a number of other structures aimed at avoiding immune mechanisms prevent the destruction of the pathogen. Not the last place in this dynamic process is occupied by ixodid ticks themselves, since the secret of their salivary glands has an inhibitory effect on a number of cells and complement system. In parallel with innate immunity, adaptive immune response factors are activated, serving as a second line of defense. The synthesis of specific antibodies in the early period of Lyme disease has its own ambiguous features, but this does not exclude their importance in fight against borreliosis infection. To date, the issues of interaction with dendritic cells and cytotoxic T-lymphocytes remain less studied. Research of all aspects, including little-studied ones, is extremely important for both practical healthcare and fundamental medicine.

Aim. The aim of the study was to provide clinical and laboratory characteristics of infectious mononucleosis caused by Epstein-Barr virus (EBV) genotype 1 in hospitalized children. Materials and methods. The material of the study was blood leukocytes and saliva of children aged 1-17 years, hospitalized with a diagnosis of infectious mononucleosis caused by EBV (n=67). For differential detection of EBV-1/ EBV-2, we used an optimized one-round PCR variant with electrophoretic detection of amplification products in agarose gel. Clinical symptoms and laboratory data of patients were analyzed separately and combined into groups of signs. Statistical data processing was carried out using the R programming language and the RStudio environment. Results. In all children, only one type of virus (EBV-1) was detected in blood leukocytes and saliva. The first data on clinical and laboratory signs of EBV-1 infectious mononucleosis, characterized by a typical symptom complex, have been obtained. The age criterion for dividing patients into groups of younger (1–5 years) and older (6–17 years) age, which were characterized by the most pronounced differences in the manifestations of overt EBV-1 infection, was determined. In young children, the leading was the syndrome of intoxication, in older children, signs of cytolytic syndrome were more pronounced. According to laboratory data, in the age group of 1–5 years, monocytopenia and a decrease in hemoglobin were more often observed, and in children of 6–17 years, lymphocytosis, monocytosis, elevated levels of hepatic transaminases and hemoglobin. Conclusion. For the first time, EBV typing was performed in children with infectious mononucleosis. The dominant genotype of the virus in infectious mononucleosis in children living in the territory of a large city in the European part of Russia is EBV-1. For the first time, the characteristics of EBV-1 infection are given. The data obtained on the different severity of clinical and laboratory signs of the disease are prerequisites for continuing research aimed at finding the relationship between the characteristics of the clinical course of the infection and the genetic heterogeneity of the EBV population.

Introduction. Infectious mononucleosis (IM) is an high priority viral infection in children. Epstein-Barr virus (EBV) is the main etiological agent of IM. EBV is classified into two main types EBV-1 and EBV-2. In addition, different variants of the virus are isolated based on individual genes, among which the LMP-1 gene and the oncoprotein it encodes are the most well known. So far, the study of the clinical significance of EBV genetic diversity in EBV-IM in children in Russia has not been conducted. The aim of the study was to evaluate a relationship between EBV LMP-1 molecular genetic variants and clinical and laboratory manifestations of IM in children. Materials and methods. The material of the study was presented by blood leukocyte and saliva samples of children aged 117 years with EBV-IM (n = 69). A total of 132 EBV isolates were studied. For differential detection of EBV-1/EBV-2, we used a previously optimized one-round PCR variant with electrophoretic detection of amplification products in agarose gel. The nucleotide sequences of the C-terminal region of the LMP-1 gene were determined by Sanger sequencing followed by analysis of the obtained sequences using the MEGA X software. Multiple Factor Analysis was used to search for the relationship between LMP-1 variants and clinical and laboratory manifestations of IM (32 signs and 8 groups of signs). Results. It was established that only one type of virus, EBV-1, was identified in all children. At the same time, the severity of clinical manifestations of EBV-IM in children varied significantly (from 15.5 to 35.5 scores in total). Molecular genetic analysis of the sequences of the LMP-1 C-terminal region in Nizhny Novgorod region EBV isolates demonstrated a significant heterogeneity of the viral population, which was not limited only to their grouping according to known variants. According to the frequency of detection, B95-8 was the dominant variant of LMP-1 (60.66.0% of cases), other variants were less common (China 1, NC, Med and China 1+В95-8). It was found that EBV-IM proceeded more easily and with less severity of the intoxication syndrome in cases of infection with a virus having the molecular genetic profile of EBV-1/B95-8, in particular EBV-1/B95-8/E214D. Conversely, EBV-1/Med, as well as EBV-1/Med/L338S, EBV-1/Med/S229T, EBV-1/China 1/L338S and EBV-1/NC/S229T profiles were associated with more severe infection. Conclusion. For the first time, the influence of the genetic diversity of EBV on the clinical manifestations of IM in children was revealed. In the context of the tasks to be solved in this study, it is necessary to conduct a larger-scale and systemic studies in different territories of Russia.

Objective: is to assess clinical and laboratory prognostic factors to develop a differential diagnostic model between the monoinfection of tick-borne encephalitis febrile form and the mixed infection of tick-borne encephalitis with Lyme borreliosis non-erythemal form at the onset of the disease. Materials and methods. The clinical examination involving 56 patients with tick-borne encephalitis febrile form (mean age: 46.1±3.1 years) and 27 patients with the mixed infection of tick-borne encephalitis with Lyme borreliosis non-erythemal form (mean age: 47.2±3.2 years) has resulted in the assessment of 65 clinical and laboratory parameters in the first week of the disease including 14 indicators of standard and extended hemogram profiles and 6 blood leukocyte indices. Pearson’s goodness-of-fit test was used for statistical analysis. The predictive values of the parameters were determined by the odds ratio and ROC analysis with AUC. The logistic regression model was developed using STATISTICA 12.0. Results. To make differential diagnosis between mono- and mixed infection at the onset of the disease the following hematological parameters with “average” or “good” predictive values can be used: band neutrophil count (AUC=0.65), the index of leukocytes and erythrocyte sedimentation rate ratio (AUC=0.66), erythrocyte sedimentation rate (AUC=0.70), neutrophil granularity intensity (AUC=0.66), neutrophil reactivity intensity (AUC=0.72) and reactive lymphocytes count (AUC= 0.72). A logistic regression model with a “very good” predictive value (AUC=0.83) is developed which includes the following four predictors: band neutrophil count, erythrocyte sedimentation rate, NEUT-RI and NEUT-GI in peripheral blood. Conclusion. The model is allowed to make a differential diagnosis between the mono- and the mixed infection of tick-borne encephalitis with good sensitivity and specificity values in the first week of disease.

Background. There is no current generally accepted classification and clinical approaches to assess the clinical course severity of tick-borne encephalitis. The aim is to develop models for the quantitative severity scores of the febrile form of tick-borne encephalitis using the most informative predictors. Materials and methods. In accordance with clinical criteria of the height and duration of fever, two groups of 9 and 30 patients (52.2 ± 4.3 and 49.5 ± 3.9 years old) with severe and non-severe febrile form of tick-borne encephalitis were formed. The study design was a retrospective case-control study. Statistical significance assessment of variables was carried out using the chi-square test. The odds ratios were also calculated. The development of logistic regression models was carried out using Statistica 12.0 modules. Results. A questionnaire of febrile form of tick-borne encephalitis severity score was developed using the height of fever and 11 most informative clinical predictors including arthralgia, chills, sleep disturbance, dizziness, orbital pain, nausea, photophobia, myalgia, severe headache, vomiting and meningism. To predict the severe clinical course of febrile form of tick-borne encephalitis, two logistic regression models were developed with or without severity score assessment of fever syndrome (coefficients α and β were –12.13 and 0.94 and –5.78 and 0.75 for models 1 and 2, respectively). Models 1 and 2 demonstrated a high predicted probability for cut-off values equal to 14 and 9 points and excellent quality of prediction test (AUC are equal to 0.99 and 0.98, respectively). Conclusions. The models of severity score demonstrated high predicted probabilities to predict severe clinical course of febrile form of tick-borne encephalitis, which can be used in clinical practice and for scientific purposes.

Objective: is to develop a model for early diagnosis of mixed infection of the non-erythema migrans form of Lyme borreliosis and tick-borne encephalitis using the assessment of the complete blood count and the blood leukocyte indices of patients in the first week of the disease.Materials and methods. The retrospective clinical study involved Group 1 of 27 patients with the mixed infection of the non-erythema migrans form of Lyme borreliosis and the febrile form of tick-borne encephalitis and Group 2 of 29 patients with the monoinfection of the non-erythema migrans form of Lyme borreliosis, who were hospitalized no later than in the 7th day of the disease. The average age of patients in Groups 1 and 2 was 50.6±3.4 and 49.9±2.3. We analyzed 14 parameters of the complete blood count as well as calculated the leukocyte intoxication index and the body resistance index. Statistical significance assessment was carried out using the chi-square test and ROC analysis. Logistic regression model was developed using STATISTICA 12.0 modules.Results. The levels of the band and polymorphonuclear neutrophils (<0,001 и p=0,002) and the leukocyte intoxication index (p<0,001) were significantly higher and the levels of the body resistance index (p<0.001), lymphocytes (p<0.001) and platelets (p=0.004) were lower in Group 1 than in Group 2. Informative predictors of mixed infection included the body resistance index (AUC=0.77), leukocyte intoxication index (AUC=0.75), the band and polymorphonuclear neutrophils (AUC=0.74), lymphocytes (AUC=0.77), and platelets (AUC=0.70). Logistic regression model has a “very good” predictive value (AUC=0.85) and include two parameters: body resistance index and platelets (×109/L).Conclusion. The developed model has a “very good” predictive value for early diagnosis of the mixed infection of the non-erythema migrans form of Lyme borreliosis and tickborne encephalitis before laboratory diagnosis confirmation.

В 2022 г. отмечается 100-летие образования государственной санитарно-эпидемиологической службы России. 15.09.1922 Декретом Совета Народных Комиссаров РСФСР «О санитарных органах Республики» был установлен перечень задач, а также права санитарно-эпидемиологической службы как государственного санитарно-контрольного органа. Федеральная служба по надзору в сфере защиты прав потребителей и благополучия человека (Роспотребнадзор, Служба) относится к федеральным органам исполнительной власти, осуществляющим разработку государственных санитарно-эпидемиологических правил и гигиенических нормативов, а также реализующим мероприятия в сферах санитарно-эпидемиологического надзора и надзора в области защиты прав потребителей. Решение возложенных на Роспотребнадзор задач обеспечивают 85 территориальных управлений и 85 центров гигиены и эпидемиологии в субъектах РФ, 21 научно-исследовательский институт, 18 противочумных станций и организации дезинфекционного профиля.

Aim. To determine the threshold value of the Epstein-Barr virus (EBV) viral load (VL) in blood leukocytes to improve the laboratory diagnostics of infectious mononucleosis in children.Materials and methods. EBV DNA quantification in blood leukocytes in children aged 1-17 years (n=163) was determined by real-time polymerase chain reaction. VL were compared in groups of EBV mononucleosis (n=67), non-EBV mononucleosis (n=25) and healthy donors (n=25). Threshold was determined based on VL data from children with active and latent EBV infection. The R program and the RStudio environment were used for satistic analysis.Results. EBV DNA is found in blood leukocytes in infectious mononucleosis not associated with EBV and in healthy virus carriers, however VL in these groups is significantly lower than in patients with EBV mononucleosis (p<0.001). The threshold value was determined – 41 copies/105 cells (or 1.6 lg of EBV DNA/105 cells), which was characterized by acceptable values of specificity and sensitivity (0.90 and 0.85, respectively) of laboratory diagnostics. High EBV VL (equal to or above the set threshold) is associated with an 8.5-fold increased risk of detecting active EBV infection compared to children who have a low VL (below a set threshold) (RR 8.5; 95% CI: 3.7–19.7, p<0.001).Conclusion. In general, the results obtained create prerequisites for more intensive implementation of quantitative studies of EBV DNA in blood leukocytes, both in the context of improving the early diagnosis of infectious mononucleosis and its etiological interpretation, and in terms of detailing the features of the course of EBV infection.

The aim of the study was to develop an algorithm for the selection of discriminating probes to identify a wide range of causative agents of human infectious diseases. Materials and Methods The algorithm for selecting the probes was implemented in the form of the disprose (DIScrimination PRObe SElection) computer program written in the R language. Additionally, third-party software was used: the BLAST+ and ViennaRNA Package programs. The developed algorithm was tested by selecting specific probes for detecting Chlamydophila (Chlamydia) pneumoniae — an atypical bacterial pathogen causing community-acquired pneumonia (CAP). Nucleotide sequences for analysis were downloaded from the NCBI databank. Results An algorithm for the selection of specific probes capable of detecting human infectious pathogens has been developed. The algorithm is implemented in the form of the disprose modular program, which allows for performing all stages of the probe selection process: loading the nucleotide sequences and their metadata from available databanks, creating local databases, forming a pool of probes, calculating their physicochemical parameters, aligning the probes and sequences contained in local databases, processing and evaluating the alignment results. The algorithm was successfully tested and its performance was confirmed by selecting a set of probes for the specific detection of Chlamydophila pneumoniae. The specificity of the selected probes calculated in silico indicated a low risk of their nonspecific binding and a high potential of using them as molecular genetic diagnostic tools (DNA microarrays, PCR). Conclusion An algorithm for the selection of specific probes detecting a wide range of human pathogens in clinical biomaterial has been developed and implemented in the form of the disprose modular program. The probes selected using this program can serve as the functional basis of DNA-oriented microarrays able to identify causative agents of polyetiological diseases, such as CAP. Due to the flexibility and openness of the program, the scope of its application can be expanded.

EpsteinBarr 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 117 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.

Данная работа посвящена изучению развития судебной экспертизы под влиянием преступлений разных эпох. Анализ этой темы проводится на примере двух видов судебной экспертизы, которые появились первыми в России: судебно-медицинская экспертиза и экспертиза документов.

Introduction. The search for specific molecular and genetic markers of the risk of developing infectious disease complications is a current area of research in modern medical and biological science. Materials and methods. In order to solve this issue, we developed a MiDA software that implements an integrated approach allowing for selection of potential markers on the basis of indicators of expression fold change of a number of genes in the comparison groups and the feature importance for classification, i.e. the assignment of samples to the analyzed groups. Results. Using the MiDA software, we searched for molecular and genetic markers of the risk of developing severe dengue fever and chronic brucellosis. As a result of the study, the HSPA6 gene was proposed as a risk marker for the dengue complication. HSPA6 expression was reduced in the peripheral blood samples of severe dengue cases. Markers of chronic brucellosis included a decrease in the expression of miRNA hsa-miR-198 and hsa-miR-501-3p, as well as an increase in the expression of miRNA hsa-miR-618 in CD4+ T-lymphocytes. Conclusion. We demonstrated the possibility of applying the MiDA software to the analysis of big data obtained using modern techniques (sequencing, biochips, etc.). It is possible to expand the scope of the software application in order to analyze the expression of genes, transcripts and proteins in diseases of various origins, to determine molecular mechanisms of the pathological process, to search for diagnostic and prognostic markers of the disease, as well as potential targets for the development of specific therapies.

We previously found that the number of CCR6+ T-helpers with the phenotype of effector/effector memory T cells increases in the blood of patients with H. pylori-associated peptic ulcer. The mature phenotype and the expression of the chemokine receptor CCR6, which is involved in migration of lymphocytes to the inflamed mucous membrane of the gastrointestinal tract, suggests that these cells are involved in the immune response observed in this clinical condition. To better understand the pathogenetic role of these cells, it is necessary to study their functional activity, specifically, the production of pro-inflammatory cytokines involved in the pathogenesis of the disease. The aim of the study was to evaluate changes in the blood level of pro-inflammatory types of mature CCR6+ T-helpers in H. pylori-associated peptic ulcer disease.MATERIALS AND METHODS:CCR6+ T-helpers were isolated from the blood by using immuno-magnetic separation adapted to this study. The number of T-helpers of types 1 and 17 (Th1 and Th17) and cells with mixed properties of Th1 and Th17 (Th1/Th17) was determined by intracellular cytokine assay.RESULTS:Initially, we planned to activate unseparated peripheral blood mononuclear cells ex vivo and evaluate the number of cytokine producers among mature CCR6+ T-helper cells by gating them during the flow cytometry. However, dramatic changes in the phenotype of T-helpers upon activation did not allow us to reliably identify the cells of interest. Subsequently, we used a two-stage immunomagnetic separation procedure to obtain functionally active mature CCR6+ T-helpers with a purity of >90%. The quantitative yield of these cells from the blood of patients with gastric and duodenal peptic ulcer associated with H. pylori was 9 times higher than that from the blood of healthy donors. Activation of CCR6+ T-helpers purified from blood of ulcer patients revealed an increased content of Th1, Th17, and Th1/Th17. One ml of the patient's blood yielded 18.1 times more CCR6+ Th1, 19.4 times more CCR6+ Th17, and 21.1 times more CCR6+ Th1/Th17 compared with the blood of healthy subjects.CONCLUSION:The content of mature CCR6+ T-helper cells with pro-inflammatory activity significantly increases in the blood of patients with peptic ulcer associated with H. pylori infection.

Epstein—Barr virus (EBV) is one of the etiological agents causing infectious mononucleosis. A severe form of the disease can result in developing serious complications, which risk might also depend on the state of patient’s immune system. To date, no specific tests for assessing a risk of developing severe disease form are available. Our study was aimed at identifying molecular genetic markers of severe EBV-infectious mononucleosis (EBV-IM) in immunocompetent peripheral blood cells. Expression of 483 genes and gene transcripts regulating apoptosis, proliferation and differentiation of immunocompetent cells was measured in the peripheral blood leukocytes from patients with severe and moderate EBV-IM as well as apparently healthy donors. A DNA-microarray designed by us and subsequent data processing were carried out by using custom-made “MiDA” software. To identify markers of a severe form of the pathology, expression of each gene and transcript was compared in EBV-IM patients and apparently healthy donors. For each gene and transcript, the level of expression fold change and significance for binary classification were determined. Genes and transcripts, characterized by the maximum values of two determined parameters while comparing patients with severe infection and healthy donors, as well as patients with severe and moderate EBV-IM forms, were selected as markers of severe EBV-IM. Genes and transcripts with differed expression in patients with moderate EBV-IM and healthy donors, were excluded from the list of markers. In addition, sex- and age-linked markers with differed expression were excluded as well. The markers for severe EBV-IM consisted of apoptosis regulators (BCL2L11, BIRC3 genes and XIAP.NM_001167 transcript) and splicing factors (CELF6 gene and SF1.NM_201995 transcript). Compared with donors and patients with a moderate form of the disease, a decreased expression of BCL2L11, BIRC3 genes, transcripts SF1.NM_201995 and XIAP.NM_001167, as well as increased expression of the CELF6 gene were detected in the blood of patients with severe EBV-IM. The functional role of identified molecular markers suggests that severe EBV-IM is characterized by suppressed mitochondrial and activated TRAF-dependent apoptosis pathways in immunocompetent cells. The expression pattern for select markers is specific for severe EBV-MI, not associated with patient sex and age. Thus, study data may be used to develop specific tools for assessing a risk of developing complications of EBV mononucleosis.