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The paper describes a developed comprehensive system for molecular-genetic identification of Yersinia pestis strains according to their appurtenance to certain subspecies, biovars, phylo-geographic populations, using realtime PCR (RT-PCR), allele-specific RT-PCR, and multiplex PCR with hybridization fluorescent registration of results on a solid substrate. Application of this system makes it possible to establish the appurtenance of Y. pestis strains to the following phylogenetic branches: 0.ANT1, 0.ANT2, 0.ANT3, 0.ANT5, 3.ANT, 4.ANT of antique biovar of the main subspecies; 2.MED0, 2.MED1, 2.MED2, 2.MED3, 2.MED4 of medieval biovar of the main subspecies; 1.IN1, 1.IN2, 1.IN3 of intermedium biovar of the main subspecies; 1.ORI1, 1.ORI2, 1.ORI3 of oriental biovar of the main subspecies; 0.PE3 (angolica subspecies), 0.PE7 (tibetica subspecies) and 0.PE10 (qinghaica subspecies). The first stage of the studies within the frames of the developed system is indication of plague agent using registered diagnostic drugs. The second stage is the determination of belonging to individual subspecies through RT-PCR or by the method of multiplex PCR system with hybridization-fluorescent registration of results on a solid substrate, which also allows for establishing to which biovars of the main subspecies and the main phylogenetic lines of the ancient biovar the strains belong. The third stage is the identification of strain appurtenance to phylogenetic branches by the AS-RT-PCR method. The designed complex system for molecular-genetic identification of Y. pestis strains can be applied at the regional and federal levels of the laboratory network of the Russian Federation for diagnostics of infectious diseases. Its use will considerably facilitate and increase the efficiency of intraspecific differentiation of Y. pestis strains within the framework of the epidemiological investigation of outbreaks or importation of strains of plague pathogen into the territory of the Russian Federation or during the certification of strains in collection activities.

Emergence of various genovariants of the SARS-CoV-2 virus, which are characterized by a higher ability to spread and a more severe clinical manifestations compared to the initial variants, require molecular-genetic monitoring of strains circulating in the Russian Federation.The aim of the work was to identify the VOC SARS-CoV-2 genovariants in the territory of the Republics of Bashkortostan, Tatarstan, Udmurtia, and Samara, Penza, Saratov, Ulyanovsk, and Orenburg Regions.Materials and methods. The identification of genovariants and the determination of the type of mutations was carried out by the Sanger fragment sequencing method.Results and discussion. The study examined 298 samples of clinical material obtained from the Centers for Hygiene and Epidemiology in the Republics of Bashkortostan, Tatarstan, Udmurtia, Samara, Penza, Saratov, Ulyanovsk, and Orenburg Regions. In 17 % of cases, the variability of the SARS-CoV-2 virus was observed for one or more markers: in three samples, a new coronavirus of the B. 1.1.7 line (“British”) was detected; in a number of cases, only one mutation was detected in the virus found in samples – deletion Y144 or substitution D138Y, E484K, N501Y, and very rarely two mutations – deletion Y144 and substitution E484K. The presence of the L141-G142-V143 deletion localized in the recurrent deletion region RDR2 of the S-gene was shown in 10 % of the cases. The data obtained indicate the heterogeneity in macroorganism of the population of the new coronavirus with the deletion L141-G142-V143, which leads to a change in the antigenic structure of the virus, which probably allows the virus to evade the immune response.

For a reliable assessment of the presense of the immune layer to cholera agent, it is necessary to determine the level of specific antibodies in human blood serum. To detect specific anticholera antibodies serological methods are used, aimed to identify agglutinating, vibriocidal and toxin neutralizing antibodies. At the same time, the stated methods have several drawbacks which can be eliminated when using biological microarrays to detect specific antibodies. Object of work. Assessment of the level of the immune layer to cholera agent in individuals residing in the territory of the Republic of Guinea, using a biological microchip. Materials and methods. 190 blood serum samples of people living on the territory of three provinces of the Republic of Guinea, collected over the period of May-October 2016 were studied. The detection of specific antibodies to antigens of V. cholerae was performed using the immunochip for serodiagnosis in the indirect analysis. V. cholerae O-antigens and cholera toxin were used as specific antigens for sensibilization of the immunochip surface. Results and discussion. As a result of the analysis, using immunochip, specific antibodies to O1 and O139-antigens of V. cholerae at the titer of 1:100 were not detected in any of the cases. At the same time, antibodies to cholera toxin were found in 66 samples (34.7 %); titers varied from 1:100 to 1:1600, being 1:100 in 59 samples, 1:400 - in 1 sample, 1:800 - in 2 samples, 1:1600 - in 4 samples. The absence of statistically significant differences depending on the gender of the examined people and the territory of their residence was noted. The obtained results can be explained by the fact that antibodies to cholera toxin are more resistant and circulate longer in human serum than antibodies to O-antigens. Studies have demonstrated the presence of IgG antibodies complementary to cholera toxin in sera, which may indicate both the contact of the population with the cholera pathogen and the formation of post-vaccinal immunity.

Currently available Russia-made preparations intended for serological plague diagnostics are usually aimed at detecting antibodies to single bacterial antigens in the blood serum. To improve reliability of the data obtained, it is rational to use test systems to simultaneously quantify antibodies to several immunodominant Y. pestis antigens. An opportunity of using biochip technology for quantifying specific antibodies to Yersinia pestis antigens was investigated. To do this, 5 commercially available sera, 35 blood sera obtained from individuals vaccinated with live plague vaccine collected 1, 4, 5, 18 months after immunization, as well as 5 sera obtained from healthy donors were analyzed. The objective of this work was to develop a biological microchip (immunochip) for detecting antibodies specific to Y. pestis-derived antigens. In particular, amino-modified slides were sensitized by immunodominant Yersinia pestis-derived antigens: capsule antigen F1, lipopolysaccharide (LPS), main somatic antigen (MSA), fibrinolysin, and pestin PP. Diagnostic specificity and sensitivity of the immunochip were assessed by using the approved homoand heterologous immune-biological preparations and experimental animal sera. It was found that the immunochip demonstrated a 100% diagnostic efficiency. An opportunity of applying this immunochip to determine specific antibody profile in individuals vaccinated with live plague vaccine was estimated. A commercially available ELISA-AB-F1 of Yersinia pestis kit was used for comparison that allowed to detect antibodies to Y. pestis F1 antigen in 77.1% of vaccinated individuals within the examined time period covering between 1 to 18 months post-vaccination, at titer 1:160–1:2560. In contrast, using the immunochip resulted in detecting F1 antigen-specific antibodies in 91.4% of samples post-vaccination at titer 1:320–1:2560. Moreover, such immunochip additionally allowed to detect antibodies specific to the remainder of Y. pestis-derived LPS, MSA, pestin PP in 54.3%, 20%, 42% of vaccinated individuals, respectively. The percentage of positive seroconversion in individuals vaccinated with live plague vaccine was 77.1% based on the ELISA data, 91.4% — to the F1 antigen according to the immunochip data, and 94.3% — by analyzing an extended antigen panel. Combining multiple antigenic markers in our immunochip allowed to identify greater seroconversion among vaccinated people compared to a standard ELISA. Thus, the data obtained suggest that the proposed immunochip technology might be promising in assessing developing humoral immunity.

The review presents an analysis of modern methods and instruments for performing nonspecific detection of pathogenic biological agents in environmental objects. Discussed are technological characteristics of application of these methods for the detection of biological substances of protein nature in samples. The spectrum of means for non-specific PBA detection includes home-produced and foreign field devices based on protein contamination indication using various colorimetry variants. Technologies for remote and direct monitoring of environment for the presence of aerosols of biological nature are represented by hybrid lidar systems (biolidars) and biodetectors. For PBA nucleic acids tracing, the complexes based on DNA molecule binding with fluorophore with further fluorescence detection are described. Given are the examples of chemiluminescent analysis application in the developed automatic impurity detectors, as well as systems using bioluminescence. Based on the literature data analysis, put forward is a possible algorithm for indication of pathogenic biological agents when carrying out monitoring of the environment in zones of possible emergency situation occurrence and mass events holding.

Objective of the study is to design the DNA-microarray for differentiation of Y. pestis strains of the main and non-main subspecies and biovars of the main subspecies. Materials and methods. Efficiency analysis for the devised means was conducted using 62 Y. pestis strains of various subspecies and biovars, isolated in the natural foci of Russia and neighboring countries. Results and conclusions. Selected have been the DNA-targets, probes and primers – calculated. Enhanced is the method of sub-specific and biovar differentiation of Y. pestis strains by means of DNA-microarray. DNA-chip with “Med24”, “glpD(-93)”, and “45” targets allows for prompt differentiation of the strains of the main and non-main subspecies and biovars of the main subspecies based on the presence and absence of fluorescent signal by the specific for the main subspecies and its biovars DNA-targets.

Objective of the study was to demonstrate practicability of data base creation, containing reference mass-spectra of agents of particularly dangerous infections, using MALDI-TOF mass-spectrometry, by the example of plague agent strains. Materials and methods. MALDI-TOF mass-spectrometry was deployed for the obtainment of mass-spectra of ribosomal proteins from the microorganisms under investigation with the help of mass-spectrometers - Microflex LT. Results and conclusions. Carried out was comparative analysis of the obtained mass-spectra of 10 Y. pestis strains and reference spectra of Y. tuberculosis , contained in commercial data base of MALDI Biotyper 3.1 (Bruker Daltonics, Germany). Developed data base was validated in the process of identification of plague microbe strains, isolated in the territory of natural plague foci of the Russian Federation. That data base provided for correct identification of Y. pestis strains up to a species.

At present EVD epidemic, which claimed the lives of more than 10000 people, is still underway in West Africa Countries. Promptness and quality of laboratory diagnostics, alongside with delivered anti-epidemic measures predetermine efficacy of Ebola response operation. Due to a lack of means for the specific prophylaxis and treatment of the disease, asymptomatic patients are discharged from hospitals, based on criteria recommended by WHO, which might be insufficient. Viral RNA is detected in different clinical samples taken from the patients even at the advanced stages of convalescence, which requires essential investigation of peculiarities of Ebola persistence in various biological fluids. The article contains the data on the studies of biological samples, obtained from a female patient diagnosed with Ebola virus disease, applying various methods and techniques.

Given is an account of the EVD epidemics that started in the Republic of Guinea in December, 2013 and spread over West African countries within 2014. Established have been the grounds for the Rospotrebnadzor SAET deployment in the Republic of Guinea, objectives, goals and stuffing of the mission, and mobile complex technical performance. Described are the key stages of the work, including the process of integration into the UNMEER. Outlined are priority areas of collaboration with the National Public Health Ministry and international partner organizations. Represented are the results of work on the Ebola fever and other dangerous infectious diseases diagnostics, carried out at the mobile facility. Provided is molecular-genetic characteristics of the Ebola virus.

Mass spectrometry is a modern physical-chemical analytical method that provides for qualitative and quantitative assessment of the substance composition. It is based on pre-ionization of the atoms and molecules included into it. One of the advanced methods of ionization, due to which mass-spectrometry investigation of macromolecules has become a frequent practice, is matrix-assisted laser desorption/ionization (MALDI). The essence of it is the pulsed laser irradiation of the matter under study, mixed with the matrix. The review discusses current data on MALDI mass-spectrometry application for the performance of species-specific and genus-specific identification of microorganisms at the premises of diagnostic laboratories. Considered are the basic advantages of MALDI-TOF identification as compared to bacteriologic, immunologic, and molecular-genetic methods of assessment. Allocated is the mass-spectrometry position in the system of laboratory diagnostics of infectious diseases, including particularly dangerous ones, in the territory of the Russian Federation.

Taking into consideration experience in utilization of “AmpliSens EBOV (ZAIRE)-FL” in the setting of SAET mobile laboratory complex, carried out has been the improvement of the panel with a view to elapsed time reduction and analytical sensitivity enhancement. It provides for the analytical sensitivity increase up to 200 copies per ml, which is 10 times higher than the previous measurement, that accounted for 2·103 copies per ml. The timing of the assay has been reduced up to 1 h 25 min, which is 25 % less than the time needed for the standard study. Modification of “AmpliSens EBOV (ZAIRE)-FL” panel benefits to the enhancement of epidemiological surveillance activities intended to stop the spread of Zaire ebolavirus in West Africa and prevent its importation into the Russian Federation.

The risk of occurrence of emergency situations different in their character is still a pressing issue for all the states of the world's community, including the Russian Federation. Nowadays, development of the modern methods and tools for rapid toxin detection and identification holds a high position in biological safety system. Specified means for biological toxin detection do not comply with the requirements of rapidness. One of the prospective means for rapid toxin indication is immune-chromatographic, immunochip, and immunosensor test-systems with high sensitivity, differentiation capacity and expression. In this review described are some up-to-date foreign and home-grown technologies for rapid toxin indication.

The review presents data on application of UV, IR and optical spectroscopy methods for non-specific indication of pathogenic biological agents, and IR Fourier spectroscopy, and Raman spectroscopy - for their identification. Considered are advantages, disadvantages and prospects of different spectroscopy methods application for monitoring of the environment for the presence of pathogenic biological agents.

The review presents the functional characteristics of the main types of biosensors: electrochemical, piezoelectric and optical. Shown are the examples of biosensors application for pathogenic biological agents detection. The prospects of biosensors development for laboratory diagnostics of particularly dangerous infectious diseases are discussed.

The atomic power microscopy (APM) is used to study the cell surface structure of particularly dangerous infectious diseases agents and to carry out the morphometric analysis. APM shows similar results with scanning electron microscopy. However, its application makes it possible to avoid time-consuming and labour-intensive procedures of samples preparing for testing by fixation, dehydration and sputtering of conducting layer. Methodological approach has been elaborated for preparing and analysis of samples of agents of particularly dangerous infectious diseases by means of APM. This approach includes a selection of optimal substrate, mode of disinfection and scanning of samples.