Biosafety of personnel of microbiological laboratories in the context of the Federal Law of the Russian Federation № 492-FZ of December 30, 2020 «On the biological safety of the Russian Federation¼.

One of the most important requirements for the personnel of microbiological laboratories working with pathogenic and infectious agents is the observance of precautionary measures and the implementation of a set of preventive measures, collectively interpreted as biological safety (biosafety). To a large extent, biosafety problems are also relevant for all clinical laboratories working with biosubstrates, with the potential threat of containing pathogens of bloodborne infections in them. On December 30, 2020, the President of the Russian Federation signed Federal Law № 492 «On the Biological Safety of the Russian Federation¼ (№ 492-FZ), which regulates the basic legal norms and regulation of biosafety issues, as well as a list of measures to prevent the risks of the spread of infections due to accidents, bioterrorist acts and sabotage. The current pandemic of the coronavirus infection COVID-19 has demonstrated, on the one hand, the epidemiological vulnerability of the single world space, and on the other hand, the decisive influence of biological emergencies on the emergence of negative political and economic processes in the world community. In this regard, the issues of ensuring biosafety in the work of microbiological laboratories in the context of protecting personnel and the environment from accidental or unintentional spread of infections are relevant. Working with pathogenic biological agents in microbiological laboratories is constantly associated with the risk of accidents and possible laboratory infection (laboratory-acquired infections) of employees, environmental pollution if the requirements of regulatory documents on biological safety are not met. In accordance with the requirements of № 492-FZ, in order to prevent biological threats, it is necessary to create a system for monitoring biological risks in microbiological laboratories when working with any infected material.

Ultrastructure and Morphological Variability of Non-Culturable Forms of Yersinia pseudotuberculosis Bacteria.

One of the mechanisms underlying the appearance of chronic infections is transition of pathogens into a non-culturable state, which is largely associated with the use of antibiotics. We studied ultrastructure of dormant bacteria Yersinia pseudotuberculosis obtained from the vegetative form of strain 512 by inhibition with kanamycin. On the model of the causative agent of pseudotuberculosis we showed that transition of prokaryotes to a dormant state occurs through apoptosis of bacteria. Fragmentation and condensation of chromatin with the formation of electron-dense fibrils, clumps and large conglomerates characteristic of apoptosis were found in the nucleoid zone of the cytoplasm of inhibited bacterial cells. These results are of great importance for understanding the mechanisms of the existence of pathogens in different conditions, as well as for identifying the causative agents of infectious diseases.

Learning from Nature: Bacterial Spores as a Target for Current Technologies in Medicine (Review).

The capability of some representatives of Clostridium spp. and Bacillus spp. genera to form spores in extreme external conditions long ago became a subject of medico-biological investigations. Bacterial spores represent dormant cellular forms of gram-positive bacteria possessing a high potential of stability and the capability to endure extreme conditions of their habitat. Owing to these properties, bacterial spores are recognized as the most stable systems on the planet, and spore-forming microorganisms became widely spread in various ecosystems. Spore-forming bacteria have been attracted increased interest for years due to their epidemiological danger. Bacterial spores may be in the quiescent state for dozens or hundreds of years but after they appear in the favorable conditions of a human or animal organism, they turn into vegetative forms causing an infectious process. The greatest threat among the pathogenic spore-forming bacteria is posed by the causative agents of anthrax (B. anthracis), food toxicoinfection (B. cereus), pseudomembranous colitis (C. difficile), botulism (C. botulinum), gas gangrene (C. perfringens). For the effective prevention of severe infectious diseases first of all it is necessary to study the molecular structure of bacterial spores and the biochemical mechanisms of sporulation and to develop innovative methods of detection and disinfection of dormant cells. There is another side of the problem: the necessity to investigate exo- and endospores from the standpoint of obtaining similar artificially synthesized models in order to use them in the latest medical technologies for the development of thermostable vaccines, delivery of biologically active substances to the tissues and intracellular structures. In recent years, bacterial spores have become an interesting object for the exploration from the point of view of a new paradigm of unicellular microbiology in order to study microbial heterogeneity by means of the modern analytical tools.

Biosensor Technologies in Medicine: from Detection of Biochemical Markers to Research into Molecular Targets (Review).

Infections are a major cause of premature death. Fast and accurate laboratory diagnostics of infectious diseases is a key condition for the timely initiation and success of treatment. Potentially, it can reduce morbidity, as well as prevent the outbreak and spread of dangerous epidemics. The traditional methods of laboratory diagnostics of infectious diseases are quite time- and labour-consuming, require expensive equipment and trained personnel, which is crucial within limited resources. The fast biosensor-based methods that combine the diagnostic capabilities of biomedicine with modern technological advances in microelectronics, optoelectronics, and nanotechnology make an alternative. The modern achievements in the development of label-free biosensors make them promising diagnostic tools that combine rapid detection of specific molecular markers, simplicity, ease-of-use, efficiency, accuracy, and cost-effectiveness with the tendency to the development of portable platforms. These qualities exceed the generally accepted standards of microbiological and immunological diagnostics and open up broad prospects for using these analytical systems in clinical practice directly at the site of medical care provision (point-of-care, POC concept). A wide variety of modern biosensor designs are based on the use of diverse formats of analytical and technological strategies, identification of various regulatory and functional molecular markers associated with infectious pathogens. The solution to the existing problems in biosensing will open up great prospects for these rapidly developing diagnostic biotechnologies.

COVID-19 diagnostic laboratory strategies: modern technologies and development trends (review of literature).

The COVID-19 pandemic, associated with the new coronavirus SARS-CoV-2, has caused a surge in incidence worldwide, as well as a severe crisis in global health and economy. Therefore, fast and accurate diagnosis of infection is key to timely treatment and elimination of the spread of the virus. Currently, the standard method for detecting coronavirus is reverse transcription polymerase chain reaction (RT-PCR). However, this method requires expensive equipment and trained personnel, which limits the conduct of mass testing and lengthens the time to obtain a research result. Serological tests for antibodies against SARS-CoV-2 and the determination of protective immunity in various populations are used to retrospectively identify patients with asymptomatic and mild forms of infection, monitor the course of infection in hospitalized patients, and also track contacts and epidemiological surveillance. The use of standard methods for diagnosing COVID-19 in conditions of mass morbidity, especially in conditions of insufficient resources and lack of appropriate infrastructure, is associated with a number of limitations. Therefore, the search and development of new, fast, inexpensive, simple, device-free and no less sensitive and specific tests is an urgent task. Therefore, the search and development of new, fast, inexpensive, simple, device-free and no less sensitive and specific tests is an urgent task. The review examines new laboratory technologies for diagnosing a new infection - loop isothermal amplification (LAMP) and immunochromatographic analysis (ICA), which can become a real alternative to the used molecular and enzyme immunoassay methods. The dynamic development of these methods in recent years expands the prospects for their use both for diagnosing COVID-19 and monitoring a pandemic.

Ultrastructural Changes of Bacteria in Static Cultures of Yersinia pseudotuberculosis under Long Storage under Conditions of Low Temperature.

Electron microscopy study revealed changes in the ultrastructure of bacteria of Yersinia pseudotuberculosis strains characterized by significantly reduced reproductive ability and virulence potential after long-term storage at low temperature of 4-8°C. Most bacterial cells contained dark cytosol with reduced cellular material or empty cytosol, while the cell wall was preserved. The revealed ultrastructural changes in the bacterial cells of the static culture of Y. pseudotuberculosis suggest that storage of strains under low positive temperatures could induce the transition of the majority of bacterial cell population to a dormant, non-cultivated state with a decrease in their virulence. This fact is of great scientific and applied importance in studies of causative agents of saprozoonoses, including pseudotuberculosis, which has the etiopathogenetic background of persistent infection.

Simplified formats of modern biosensors: 60 years of using immunochromatographic test systems in laboratory diagnostics.

Immunochromatographic test systems known to foreign laboratory diagnostic experts as lateral flow immunoassay (LFIA) are simplified tape formats of modern biosensors. For 60 years, they have been widely used for the rapid detection of target molecules (ligands) in biosubstrates and the diagnosis of many diseases and conditions. The growing popularity of these test systems for providing medical care or diagnostics in developing countries, medical facilities, in emergency situations, as well as for individual home use by patients while monitoring their health are the main factors contributing to the continuous development and improvement of these methods, the emergence of a new generation of formats. The attractiveness and popularity of these fast, easy-to-use, inexpensive and portable diagnostic tools is associated primarily with their high analytical sensitivity and specificity, as well as the ease of interpretation of the results. These qualities have passed the test of time, and today LFIA test systems are fully consistent with the modern world concept of «point-of-care testing¼, finding wide application not only in medicine, but also in ecology, veterinary medicine, and agriculture. This review will highlight the modern principles of designing the most widely used formats of immunochromatographic test systems for clinical laboratory diagnostics, summarize the main advantages and disadvantages of the method, as well as current achievements and prospects of LFIA technology. Modern innovations aimed at improving the analytical characteristics of LFIA technology are interesting, promising and can bring additional benefits to immunochromatographic platforms that have gained popularity and attractiveness for six decades.

Functional State of Mesenchymal Stem Cells upon Exposure to Bioactive Coatings on Titanium Alloys.

Bioactive coatings on implants affect osteogenic differentiation of mesenchymal stem cells (MSC). We studied the morphofunctional state of bone marrow MSC cultured on the surface of calcium phosphate coatings on titanium formed by plasma electrolytic oxidation (PEO). The biocompatible properties of the coatings manifested in the absence of the cytotoxic effect on cells. High expression of receptors (CD90, CD29, and CD106), enhanced synthesis of osteocalcin and osteopontin, and changes in surface architectonics of MSC adherent to the samples confirmed osteoinductive properties of the calcium phosphate PEO coating.

[A comparative evaluation of differential and diagnostic properties of the Serov's agar medium and the base of selective agar for isolating enteropathogenic yersinia.]

The interest in the problem of enterоpathogenic yersinioses in the world is associated with the ubiquitous distribution of these infections and the clinical significance of their pathogenic agents, Yersinia enterocolitica and Y. pseudotuberculosis. In spite of the novel diagnostic tools, microbiological cultivation on nutrient media still remains the main method of their verification. As an agar of choice, American and European practical guidelines recommend a selective agar for Yersinia, which is widely used in many countries for isolating pathogenic Yersinia from clinical material. The authors compare the differential and diagnostic properties of the base of selective agar with those of the Serov's agar medium, as one of the substrates for Yersinia recommended by the current Russian regulatory documents. Based on the studies conducted, a conclusion has been made that the Serov's agar medium is not inferior to its foreign analogue in the differential and diagnostic properties for identification of enteropathogenic Yersinia.

THE STRUCTURAL CHANGES OF MACROPHAGES INFECTED WITH TICK-BORNE ENCEPHALITIS VIRUS.

Macrophages belong to the innate immune cells and play a key role in the pathogenesis of viral infections. The results of ultrastructural study of macrophages infected with tick-borne encephalitis virus (TBEV), the Flavivirus family, pathogens of human infections, affecting the nervous system, were presented. With the assistance of virological methods was found that the TBEV are absorbed by macrophages and replication in them. An ultrastructural study has shown that the virus enters into the cytoplasm by local destruction of plasmalemma and newly synthesized virus particles exited from the cell by same. Simultaneously there is a seal of perinuclear cytoplasm space, where found in a large number of ribosomes, microfilaments, ribonucleoprotein fibers and viral special structure: nucleocapsids, tubular formations and viral layers (fabrics). On the surface of last structures the newly synthesized virus particles were visualized. Thus, the evidence shows that macrophages play a role in the spread of TBEV, being for their the target cell. As active antigen presenting cells the macrophages can modulate the protective response of the body and influence on the pathogenesis of tick-borne encephalitis.

Morphogenesis of Experimental Infection Caused by Plasmid Variants of Yersinia pseudotuberculosis.

The dynamics of pathomorphological changes in response to infection with plasmid variants of Yersinia pseudotuberculosis was studied in experimental animals. Variability of cell injuries in pseudotuberculosis histopathology depended on the plasmid-associated virulence of the infection agent. Infection with highly virulent two-plasmid strain pYV48:pVM82 MDa and Y. pseudotuberculosis strain with low virulence with the only plasmid pVM82 MDa led to the development of cell destruction (necrosis and apoptosis) in the target organs. Apoptosis predominated in response to infection by plasmid variant pVM82 MDa with low virulence.

[Pathomorphism of Hantavirus infection under prolonged heat stress]. / Patomorfoz khantavirusnoi infektsii na fone prolongirovannogo teplovogo stressa.

AIM: to provide the morphological characteristics of experimental Hantavirus infection under heat stress conditions to identify the possibilities of its modeling in resistant laboratory animals. MATERIAL AND METHODS: Experiments were carried out on outbred albino mice that were divided into 4 groups: 1) intact mice unexposed to heating; 2) those exposed to heating; 3) Hantavirus-infected animals unexposed to preheating; 4) those exposed to preheating. The animals in Groups 2-4 were long exposed to heat stress at a temperature of 30 °C for 3 hours during 3 days. Strain Aa 60343 (PM-79-95) of the Far East genovariant of Hantaan virus (the genus Hantavirus, family Bunyaviridae) from the collection of the G.P. Somov Research Institute of Epidemiology and Microbiology was used to induce infection. The animals in Groups 3 and 4 were intraperitoneally injected with 700 FFU of Hantavirus per mouse. Materials (lung, liver, kidney, and spleen) taken from Groups 2-4 animals were collected for histological examination on days 1, 3, 7 and 14 of observation. RESULTS: The intact albino mice in Group 1 showed no histopathological changes in the organs. After heat exposure, Group 2 animals were found to have an immunomorphological response in the interstitial tissues of the lung, liver and kidney in partial lymphoid hypoplasia of the spleen. There were no signs of inapparent infection in the presence of marked immunomorphological changes in the organs in Group 3 of hantavirus-infected animals unexposed to preheating. In Group 4, those exposed to preheating exhibited dystrophic and destructive changes in the target organs (lungs, kidneys) in the presence of immunodeficiency manifestations of manifestations that were more pronounced in dead animals. CONCLUSION: In an unresponsive model (adult albino mice), Hantavirus infection caused only obvious immunomorphological changes in the organs. Prolonged preheat stress in the hantavirus-infected animals promoted inapparent infection and morphological manifestations of induced secondary immunodeficiency that was responsible for the manifestation of an infectious process in some animals.

[PLASMID-ASSOCIATED VIRULENCE OF YERSINIA PSEUDOTUBERCULOSIS AND INFECTIOUS PROCESS].

Literature data regarding genetically-determined pathogenicity factors of Y pseudotuberculo- sis and associated manifestations of this infection caused by various plasmid types of the causative agent are generalized. Principal attention is given to features of cell-tissue alterations mediated by virulence plasmid pYV, as well as effects of pathogenicity of an understudied pVM82 plasmid present only in Y pseudotuberculosis sttains causing clinical-epidemic manifestation of the infec- tions as Far East scarlet-like fever (FESLF). The data obtained on the ability of far-eastern strains to produceYPMa super-antigenj Ypseudotuberculosis-derivative mitogenA, probablygive evidence on its key role in FESLF pathogenesis. Variability of damage of innate immunity cells and target- organs caused by various plasmid types of Y pseudotuberculosis by virulence could determine polymorphism of clinical-morphological manifestations of this infection. In-depth understanding of dependency of immune pathogenesis mechanisms of the disease on molecular characteristics of the causative agent opens up-perspectives of enhancement of diagnostics and prognosis of the severity of the course of pseudotuberculosis and yersiniosis in human in general.

Effects of immunomodulators on functional activity of innate immunity cells infected with Streptococcus pneumoniae.

Low activity of bactericidal enzymes was found in innate immunity cells infected with S. pneumonia. The death of these cells was fastened under these conditions. On the contrary, treatment with antibiotic maxifloxacin was followed by an increase in activity of bactericidal enzymes in phagocytes and induced their death via necrosis. Analysis of the therapeutic properties of immunomodulators tinrostim and licopid in combination with maxifloxacin showed that these combinations correct functional activity of cells infected with S. pneumonia.

Pathomorphosis of experimental infection in mice, infected by Streptococcus pneumoniae, under the effect of immunotropic drugs.

Pathomorphological changes in the organs of animals intranasally infected with Streptococcus pneumoniae were studied under conditions of immunotropic therapy added to antibiotic therapy. The pathomorphosis in the lungs, spleen, and thymus in animals treated with likopid, tinrostim, and roncoleukin was described. A positive time course of the pathological process in experimental animals in comparison with intact animals and animals receiving no immunotropic drugs was demonstrated.

Neutrophil apoptosis induction by tick-borne encephalitis virus.

Tick-borne encephalitis virus infects neutrophils and induces their apoptosis, judging from moderate increase of succinate dehydrogenase activity and a trend to anaerobic energy production in neutrophils infected with the virus (shown by an increase of lactate dehydrogenase activity).