Vol 12, No 1 (2022)

Over the last few years, CRISPR/Cas systems have been extensively studied and used for a wide range of applied purposes. The variety of their applications is accounted for by the ability of Cas-type nucleases to targetly cleave specified nucleic acid sequences. In this case, the researcher might set the necessary sequence of the guiding elements in the CRISPR/Cas system, played by so-called single guide RNAs allowing it to act on select targets. This potential underlies one of the reasons for exerting interest in CRISPR/Cas systems. One of the first areas for applying these systems was its use for genomic editing. Later, the list of potential opportunities has been expanded: e.g., they can be used in gene therapy and epigenetic research. It is possible to create sgRNA libraries which might be used to create a pool of viral vectors applied for bacterial cell transformation with subsequent cas-protein transduction that cause target gene knockout. This approach allows finding genes responsible for resistance or sensibility to diverse substances. Using such systems in molecular diagnostics of infectious diseases is considered as one of the most promising directions allowing to detect even extremely low concentrations of pathogenic organisms in samples due to their specific nucleotide sequences. Simultaneously, such assays turn out to be accurate, rapid and easy to utilize. In addition, some platforms may work without using expensive equipment, because methods for fast and simple sample preparation have already been developed, whereas modern preamplification approaches allow to avoid applying thermocycling devices. Interestingly, a great amount of diverse types of natural CRISPR/Cas systems have been already discovered. Such abundance promotes development of multiple artificial systems, each of which exerting own unique characteristics. Therefore, a variety of diagnostic platforms with different properties are created on their basis that allows researchers and physicians to choose an optimal approach for performing specific tasks. For this reason, insights into structure and operation of CRISPR/Cas systems are necessary for selecting a suitable platform. The current classification of systems is based on such principles serving as the basis, in turn, for convenient evaluation of the very variety of molecular diagnostics platforms and presentation of the typical technical characteristics and nuances for each method. Thus, this review, which is mainly devoted to the platforms for molecular diagnostics of infectious diseases, also touches upon the issues of functioning, devices, and classification of CRISPR/Cas systems.

Streptococcal diseases and their complications are among the global problems of international health. S. pyogenes (group A streptococci — GAS) is a pathogen that causes significant morbidity in different countries and different age groups of the population, occurring both sporadically and epidemically. Due to ineffective antibacterial therapy or its absence, 3–5% of people who have had streptococcal infection may develop complications such as acute rheumatic fever, rheumatic heart disease, acute post-streptococcal glomerulonephritis and invasive complications: necrotizing fasciitis and myositis, septicemia and toxic shock syndrome, highly lethal due to the rapid development of the process and systemic organ damage. According to recent estimates, at least 517 000 deaths occur annually in the world due to diseases caused by GAS. The diverse arsenal of pathogenic factors of this pathogen is manifested in a combination of joint or sequential reactions in the process of microbial colonization of tissue, formation of the focus of infection and overcoming the host's defense mechanisms. It is an important point in the process of studying the pathogenesis of diseases caused by these microbes. The pathogenic factors of GAS can be divided into extracellular and associated with the microbial cell, predominantly with its cell wall. The spectrum activity of pathogenicity factors can be quite wide (M proteins, pyrogenic exotoxins, superantigens) or limited (serine and cysteine proteinases, streptokinase). Information about the specificity of pathogenicity factors, their properties, relationships, regulation and specific function in pathology is the task of scientific, as well as complex researches, leading to understanding the pathogen-host interaction. An integrated approach to the investigation of GAS pathogenicity factors is needed to study the pathogenicity of streptococci, since pathogenicity factors do not manifest themselves in isolation and are not always independently regulated. In many cases, regulators control the expression of more than one of them. S. pyogenes has been studied for about 150 years, but a number of issues related to their pathogenicity remain unknown to this day. Some factors need more in-depth study: for example, endo-β-N-acetylglucosaminidase, arginine deiminase. Immunoglobulin-degrading enzymes require special attention due to their possible participation in the genesis of immunopathological processes of streptococcal etiology. This review summarizes the literature data about most of the pathogenicity factors of S. pyogenes and their role in the infectious process.

Cytomegalovirus infection remains one of the leading problems in contemporary healthcare. It belongs to socially and economically significant infections with a high incidence both in children and adults, characterized by polymorphic clinical manifestations and a variety of routes and factors for infection transmission. CMV infection of blood and organ recipients is a serious problem. It should be noted that, despite the great medical and social significance, in the Russian Federation there is no system of CMV epidemiological surveillance and control as it was traditionally developed for other topical infections. The aim of this study is to search for optimal method of cytomegalovirus (CMV) genotyping and estimate genotypic diversity of CMV isolates in Russia for patients underwent solid organ transplantation. The research presents the data after examining blood samples, leukocytes, saliva, urine and lacrimal discharge collected from 160 patients at the Transplantation Department of the Privolzhsky District Medical Center of the FMBA, aged 22 to 64 years, after liver and kidney transplantation. Molecular biological and serological methods were used for testing. Genotyping was carried out by the NGS sequencing of CMV DNA fragments. A high prevalence of CMV was found in patients undergoing solid organ transplantation. For 41.8±3.8% patients, cytomegalovirus DNA was detected in saliva and urine samples, and for 18.1±3.04% of them — in blood samples. In 98.8±3.2% of patients, the diagnosis of Cytomegalovirus infection (CMVI) was confirmed serologically. Based on a summary of reported data, estimation of various methodological approaches for genotyping of clinical CMV isolates was carried out. As a result, a typing option based on genotype determining for two variable genes UL55 (gB) and UL73 (gN) was selected. The spectra and proportional distribution of gB and gN CMV genotypes circulating among adults were determined. It was found that genotype prevalence in the group of patients who underwent solid organ transplantation was as follows: gB2, gN4c, gN4a, gN1. In some cases, a mixed infection was found due to the association of two and three CMV genotypes. The performed phylogenetic analysis of UL55 and UL73 gene nucleotide sequences indicates the genetic heterogeneity found for Russia-wide CMV isolates from in adult patients in the risk group.

Tuberculosis (TB), a chronic bacterial disease caused by Mycobacterium tuberculosis, commonly affects the lung but can also affect other parts of the body (extrapulmonary tuberculosis, EPT). A rapid diagnosis is essential to initiate a specific and effective treatment. Although mycobacterial culture remains the gold standard for EPT diagnosis, molecular tools are attracting increasing interest. GeneXpert MTB/RIF, a rapid automated diagnostic test, allows the detection of Mycobacterium tuberculosis as well as mutations in the hot-spot region of the rpoB gene associated with Rifampicin resistance. The present study was performed to evaluate the performance of the GeneXpert MTB/RIF test for the diagnosis of EPT compared to the standard method. This prospective study was conducted on 304 clinical samples collected from 192 patients attending the Laboratory of Mycobacteria and Tuberculosis of Pasteur Institute of Morocco, between 2016 and 2017. Out of the 304 samples, 113 were pleural f luids decontaminated using the Petroff method and 191 were biopsies (78 lymph nodes and 113 pleural biopsies) decontaminated using the Löwenstein method. Mycobacterium tuberculosis detection and identification were performed on all samples using smear microscopy, Löwenstein–Jensen medium culture and the GeneXpert MTB/RIF test. Our results showed that 54.5 (103/189) were men and 45.5% (86/189) were women. The age of patients ranged from 2–78 years and the majority of patients was in the age group 25–45 years. Extrapulmonary samples were derived from lymph nodes, pleural f luids and pleural tissues, with a percentage of 25.66, 37.17 and 37.17%, respectively. Interestingly, the sensitivity of the GeneXpert was 51.4 for all samples and 83.3% for lymph nodes. In conclusion, the present study revealed that the performance of the GeneXpert test depends highly on the type of sample, with a high sensitivity observed for lymph nodes. Additionally, we clearly showed that the GeneXpert MTB/RIF test presents limitations in the diagnosis of pleural TB. Thus, we recommend the coupled use of the GeneXpert MTB/RIF and the conventional techniques for EPT diagnosis.

Community-acquired pneumonia is a polyetiological infectious disease of bacterial or viral nature. In most cases, it is caused by pathogens such as Streptococcus pneumoniae, Mycoplasma pneumoniae, Haemophilus influenzae, Chlamydophila pneumoniae, Staphylococcus aureus, Legionella spp. However, despite that a wide range of modern diagnostic methods have been introduced into laboratory practice, the etiology of the disease can be determined only in 40–60% of cases. Here, we analyzed the causative agents of community-acquired pneumonia and relevant markers samples of clinical material collected from patients undergoing treatment in the medical facilities of the city of Saratov and the Saratov Region during the period from July to August, 2020, coupled to increase number of cases of new coronavirus infection COVID-19. Clinical material (oropharyngeal swabs, saliva, sputum, blood and blood serum) was obtained from 129 patients and investigated by using microbiological, immunoserological and molecular genetic methods. For this, there were used transport and nutrient media, diagnostic drugs, reagents, discs with antibacterial domestic and foreign drugs registered in the Russian Federation. As a result, 82 (63.6%) patients were found to have markers of the pathogen COVID-19 (viral RNA or specific antibodies of IgM and/or IgG classes), whereas 42% of cases had overlapped RT-PCR and ELISA data. There was a predominance of severe clinical forms of the disease in the group of patients with pneumonia caused by SARS-CoV-2 virus (17.1%), compared with pneumonia of another etiology (12.8%). No marked differences in specific pattern of the accompanying microflora and its sensitivity to antibacterial agents were observed in persons with/without markers of COVID-19. While being examined by bacteriological methods, S. aureus, K. pneumoniae, S. pneumoniae, M. pneumoniae, Acinetobacter spp., both in monoculture and in associations, were isolated from samples of clinical material collected from the majority of patients. In 49.6% of cases, pathogenic microflora, including pathogens causing community-acquired pneumonia, has not been identified, which may be due to the use of antibacterial drugs before the collection of clinical material and treatment applied due to comorbid chronic diseases.

The aim of our study was to evaluate the clinical-anamnestic, serological, immunological and biochemical tests used for early diagnostics of gastric cancer associated with Helicobacter pylori infection in the adult population of the Krasnoyarsk Territory. Materials and methods. The control group consisted of 104 apparently healthy blood donors, the comparison group – 97 patients with chronic atrophic gastritis as well as a group of patients with early gastric cancer comprising 98 subjects. Assessment of monocyte and neutrophil spontaneous and induced chemiluminescence (CL) was carried out on a 36-channel biochemiluminometer BLM-3607. Phagocytosis was measured by using a Beckman Coulter FC 500 flow cytometer. A Varian Cary Eclipse spectrofluorometer was used to study lipid peroxidation and factors of the antioxidant defense system. Results and discussion. While studying the phagocytic arm of immunity, it was found that all patients with early gastric cancer were reported to have parameters of the maximum intensity for neutrophil spontaneous CL from 17 831 c.u. and lower, whereas induced CL reached at least 30 000 c.u. Phagocytic activity of neutrophilic granulocytes in patients with early gastric cancer was 36% or less. While studying the indicators of monocytes, it was found that spontaneous and induced CL decreased from 454 c.u. and 1186 c.u., respectively, in the patients with early gastric cancer. Monocytic activity in early gastric cancer was 34% or less. In the study of lipid peroxidation, an antioxidant defense in patients with chronic atrophic gastritis and gastric cancer had increased malondialdehyde (MDA) level. Patients with gastric cancer had decreased activity of the enzyme catalase (CAT), whereas subjects with chronic atrophic gastritis had reduced glutathione peroxidase (GPO) level. In contrast, patients with early gastric cancer were featured with increased GPO activity. We have proposed coefficients for assessing the factors of the antioxydant defence system in patients: the ratio for superoxide dismutase to catalase activity (SOD/CAT) as well as the ratio for superoxide dismutase to glutathione peroxidase activity (SOD/GPO). Conclusion. During the study, threshold values of parameters were obtained for assigning groups at high risk of developing early gastric cancer, which can be used for screening in adult population.

To assess an effect of vaccination on the level of humoral anti-influenza herd immunity, 2955 sera were collected and analyzed by HIT in the 2019–2020 and 2020–2021 epidemiological seasons. All sera were obtained from healthy adult donors residing in various cities of the Russian Federation. Among them, 1057 volunteers were vaccinated with seasonal influenza trivalent inactivated vaccine. Characteristics of humoral anti-influenza immunity (average geometric antibody titers and the proportion of individuals seropositive for the vaccine viruses) obtained in autumn 2019 and 2020 (1–2 months after vaccination) in vaccinated individuals vs. unvaccinated subjects were found to be markedly higher evidencing about a positive vaccination-related contribution to developing herd immunity against influenza in the preepidemic periods. After the 2019–2020 influenza epidemic, in spring 2020 (6–7 months after vaccination), the levels of antibodies to all vaccine components decreased by 2.6–3.5-fold in vaccinated donors compared to the pre-epidemic period in 2019 autumn. Antibody titers became substantially lower than the protective level (titer by HIT < 1/40). At the same time, no significant differences between the groups of vaccinated vs. unvaccinated individuals were observed afterwards. This indicates instability of post-vaccination anti-influenza humoral immunity. As a result, it may decrease an influenza-resistant population cohort of working age on the eve of new epidemic season. The immunogenicity of the inactivated trivalent seasonal influenza vaccine was estimated by HIT while analyzing paired sera obtained from 295 and 112 healthy individuals of various ages vaccinated in autumn 2019 and 2020, respectively. The response to the vaccine was found to be age-related. Children aged 3–14 years vs. older subjects showed a more efficient response. Insufficient immunogenicity of influenza B virus vaccine components was shown. In all age groups, average geometric titers for influenza B virus antibodies were lower (2–8-fold) than for current A(H1N1)pdm09-like strains and influenza A(H3N2) viruses 1–1.5 months post-vaccination. Analyzing vaccine immunogenicity showed a significant inverse relationship between the level of preexisting strain-specific serum antibodies before vaccination and formation of antibodies to the corresponding vaccine virus 1–1.5 months after vaccination. Seroconversion to each vaccine component was remarkably more frequent in individuals with a low preexisting level of antibodies specific to the corresponding virus.

Examining a role of fungal allergy in the development of atopic dermatitis (AD) and psoriasis (PS) is of particular interest and determines the relevance of the current study. The purpose of the study is to analyze and carry out a comparative analysis for multiple sensitizations to fungal allergens in patients with AD and PS. Materials and methods. The study involved patients with atopic dermatitis (group 1, n = 53, mean age 33.0±1.3 years) and psoriasis (group 2, n = 53, mean age 40.0±1.8 years) aged from 18 to 66 years old. Sensitization to fungal allergens was assessed by skin prick testing with standardized fungal allergens: Candida albicans, Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum, Penicillium notatum (Allergopharma, Germany) and nutritional yeast (Microgen, Russia). The concentration of allergen-specific IgE to a mixture of fungal allergens: Penicillium notatum, Cladosporium herbarum, Aspergillus fumigatus, Mucor racemosus, Alternaria alternata (Alkor Bio, Russia) was determined by the method of indirect immunofluorescence analysis on a semiautomatic analyzer Multiskan FC (ThermoFisher Scientific, Finland). The test was considered positive at an IgE level of ≥ 0.35 kE/L. Statistica 8.0 software package was used for statistical analysis. Results and discussion. When studying the features of sensitization to fungal allergens, it was determined that in the group of patients with AD was the highest frequency of sensitization to nutritional yeast, as well as fungi of the genus Alternaria alternata and Cladosporium herbarum. In the group of PS patients, the highest frequency of sensitization to fungi of the genus Cladosporium herbarum, Aspergillus fumigatus and Alternaria alternata was found. When studying intergroup differences, it was determined that the rate of sensitization to fungi was insignificantly higher in the group of patients with PS in comparison with AD. Sensitization to a mixture of fungal allergens based on the concentration of allergen-specific IgE was more often observed in the group of AD patients vs. patients with PS: 15% versus 5,3%. Most likely, reagins of a different class (IgG4) can participate in the development of AD and PS, or other immunopathological mechanisms of allergy triggering (according to the Gell and Coombs classification) can be involved. Thus, fungal allergy may play an important role in the etiopathogenesis of atopic dermatitis and psoriasis. Our research can guide clinicians in terms of personalized approaches to the diagnosis and treatment of these pathologies, including specific allergological examination, not only for patients with AD, but also for those with PS.

Currently, the assessment of the immunogenic properties of influenza viruses as a part of influenza vaccines is carried out by using seroprotection, seroconversion as well as the rate of increases in post-vaccination antibodies.    At the same time, significant differences in the immunogenicity of vaccines related to dynamic formation of high antibody titers responsible for long-term protection of the vaccinated, are neglected. Influenza viruses such as A(H1N1)pdm09 that caused 2009–2010 pandemic continue to circulate in the population, therefore, the assessment of the immunogenic activity of vaccine viruses prepared during the pandemic period is interesting in for the methodology to prepare pandemic vaccines to be used in various groups (adults, children, elderly people). Analyzing immunogenicity of influenza vaccines used during the 2009–2010 swine influenza pandemic and the post-pandemic period up to the year 2014 was carried out by applying the graphical method for assessing immunogenicity (immunographs) measured as follows: for each group of vaccinated subjects (depending on the vaccine used), an increased rate in antibody level was calculated and the graphs of immunogenicity were plotted. An increased rate of serum antibodies magnitude from vaccinated subjects and the number of sera (in %) with a given fold increase rate in antibody level from 1 to the maximum magnitude were plotted on the x- and y-axis, respectively. The proposed method for assessing immunogenicity allows to plot immunogenicity graphs regardless of the serum antibodies level found in volunteers. The assessment described above revealed a several features for developing immune response to the pandemic virus A(H1N1)pdm09 such as the lack of immune response in a substantial number of adult volunteers (25–27%) and young children (60–70%) after monovaccine administration. The reason for such immune response can be both an insufficient dose of vaccine-containing viral antigen and suppressed immune response caused by the influenza A(H1N1)pdm09. A study on the immunogenic properties for seasonal influenza vaccines containing the influenza A(H1N1)pdm09 virus antigen in the years 2010–2014 revealed a variety in emerging humoral immunity ranging from a short-term, low-frequency increase in antibodies from vaccinated children to the formation   of high antibody titers in elderly. Practically, immunographic analysis of influenza vaccines particularly those derived from the influenza A(H1N1)pdm09 virus, may result in proposing recommendations to increase an antigenic load at  the beginning of a pandemic cycle and/or block the suppressive properties of vaccine-contained viruses in pediatric vaccines, because escalating virus dose in the vaccine may not always be achievable in this case.

An interaction between decreasing host anti-infective defense due to long-term invasion with Opisthorchis felineus in the hepatobiliary system, duct bile colonization by microflora and revealing the endogenous mutagenesis (carcinogenesis) factor in bile — secondary bile acids — is considered in the article. The role of organism genotype in pathogen-related immune response to Opisthorchis felineus trematode and helminth development in the hepatobiliary system has been shown. The role of dysregulated mechanisms of tissue homeostasis in induction of compensatory chronic homeostatic proliferation and somatic cell oncogenesis is discussed. The study results evidence that disturbed functioning of regulatory T cells, inhibition of the NK cell effector function and very high functional activity of memory B cells are of great importance in imbalanced host immunobiological reactivity, caused by chronic opistorchis invasion. Decreased host anti-infective protection causes intrahepatic bile duct infection with different bacterial species. Presence of secondary bile acids in hepatobiliary system was associated with biliary bacterial strains inhabiting intestinal tract: Proteus vulgaris, Proteus mirabilis, Citrobacter freundii, Bacteroides alcaligues faecalis, Clostridium, Streptococcus faecalis, Еscherichia coli — gut microflora agents. Participation of microbiota in bile acid biotransformation immediately in the duct bile has been confirmed in experiments in vitro. Experimental methods on Drosophila melanogaster and Salmonella typhimurium strains: TA100, TA98 allowed to find out that bile from chronic opistorchiasis patients exerts higher mutagenic activity compared to control groups. Mutational events in somatic and bacterial cells depend on the presence of secondary bile acids (deoxycholic, lithocholic) in duct bile, as well as the level of total bile acid concentration. The study data confirm the concept by Professor A.A. Shain about the presence of endogenous risk factor for developing primary cholangiocellular liver cancer such as secondary bile acids in the bile of chronic opistorchiasis patients. A concept of cholangiocarcinogenesis, based on mutational events, is added up with disturbance of generative cycle in tissue cells and their differentiation due to decreased chalone factor activity, as well as sensitivity threshold to it. Level of investigation and understanding of mechanisms underlying cholangiocarcinogenesis during chronic opisthorchis invasion will allow to develop pathogenetic approaches to correct homeostasis regulation and prevention of cholangiocarcinomas.

Microbiological monitoring, being an integral component of epidemiological surveillance of healthcare associated infection (HAI), is one of the most important components in hospital patient safety. The purpose of monitoring is the etiological interpretation of pathogens, the identification of microbial hospital strains and development of strategies as well as tactics for combating them. In modern conditions development of healthcare and humanity as a whole, the most important problem is the steady increase in the number of HAI caused by antimicrobial drug-insensitive pathogens and decreased drug effectiveness used in therapy. The structure of infectious conditions in the hospital is specific and is determined by the profile and nature of the treatment, nosology and age of patients. The main pathogens in the etiological structure of HAI are Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumanii, Enterococcus faecalis and Enterococcus faecium. The study estimated the prevalence of bloodstream infections pathogens resistant to antimicrobial agents in healthcare facilities in St. Petersburg. We retrospectively analyzed data on the antimicrobial resistance for the six clinically significant HAI pathogens that were detected in the blood samples at the 50 hospitals of St. Petersburg during the years 2016–2019. It was found that the use of β-lactam antibiotics is not effective in the treatment of patients with bloodstream infections (BSI) caused by S. aureus (in 21.9% of BSI cases), E. coli (2.1%), Klebsiella spp. (39.6%), P. aeruginosa (40.5%), Acinetobacter spp. (66.7%), Enterococcus spp. (4.4%). This article shows that targeted microbiological monitoring of HAI caused by resistant pathogens should be considered as an effective organizational mechanism for timely detection and prevention of complications that are observed in medical care to patients at high risk for bloodstream infections. Identification of the features of the epidemic process for purulentseptic infections in medical organizations contributes to the improved preventive measures in order to improve the quality of medical care. Microbiological monitoring data are necessary for the development and implementation of measures to curb the further growth of antimicrobial resistance of infectious disease pathogens.