The application of mass spectrometry method for the study and identification of medically important viruses (review of literature).

It is difficult to overestimate the urgency of the problem of well-timed diagnosis of viral infections. According to the WHO, dozens of outbreaks of viral diseases are recorded annually, both in developing and developed countries. Moreover, the seasonal flu virus alone is capable of infecting up to 20% of the population, even in European countries with a high level of medicine. And the annual number of deaths due to viral infections, according to official statistics, exceeds 600 thousand people around the world. That's why the provision of a reliable and fairly rapid diagnosis of viruses, along with subsequent therapy, makes a significant contribution to reducing the incidence of mortality. Despite the fact that PCR-based methods currently remain the most common method for identifying viruses in clinical practice, as recent experience shows, in addition to the already known disadvantages, in the event of large outbreaks, such test systems may simply not be in the required amount. In this regard, it is necessary to supplement and improve the existing tools for identification and research of clinically significant viruses. The MALDI-TOF mass spectrometry method combines a degree of accuracy and versatility, sufficient both for the identification of clinical strains isolated from patients, and for the study of the phenotypic properties of viruses in research laboratories and centers. This article presents and summarizes the main data on the existing or potential application of the method of time-of-flight mass spectrometry with matrix-associated laser desorption / ionization for the identification or study of viruses.

[Assessment of the possibility of application in laboratory practice of reagent kit for diagnosis of glanders and melioidosis by real-time polymerase chain reaction.]

The reagent kit AmpligenBurk-mallei/pseudomallei-RT PCR is designed for detecting in vitro diagnostics and differentiate the DNA of glanders and melioidosis pathogens by real-time multiplex PCR in biological (clinical) material and cultures of microorganisms, as well as environmental objects and solid food products (rice). During clinical testing diagnostic value of reagent kit AmpligenBurk-mallei/pseudomallei-RT PCR has been studied. Based on the results obtained, a high analytical sensitivity (1×103 microbe cells/ml) and specificity (100%) of PCR-RT with the developed reagent kit were established, regardless of the type of material being studied. The diagnostic sensitivity of PCR-RT using a set of reagents was at least 98.0% and specificity at least 99%. The stages of state examination have been completed, a registration certificate has been obtained at Roszdravnadzor, production, sale and use of reagent kit in medical laboratory practice have been permitted.

[Preparation for state registration of the reagent kit for the detection and differentiation of the DNA of burkholderia «pseudomallei¼ group.]

The reagent kit designed to detect and simultaneously differentiate the DNA of three species of Burkholderia pseudomallei - causative agents of melioidosis (B. pseudomallei), glanders (B. mallei) and B. thailandensis by the set of genes of ß-lactamases with B and D molecular classes using a multiplex polymerase chain reaction with electrophoretic detection was developed for clinical laboratory diagnosis. The functional properties of the reagent kit were evaluated, tests were carried out, the stages of examination and registration in the Federal Service for Surveillance on Consumer Rights' Protection and Human Well-being were completed. During clinical testing the effectiveness of the reagent kits in the study of various samples of clinical material and isolated cultures of microorganisms was confirmed. It has been established that the indicator of diagnostic sensitivity of the reagent kit for the detection and differentiation of the glanders, melioidosis and B. thailandensis causative agents was less than 99 %, diagnostic specificity - not less than 99 % with a confidence probability of 90 % in the analysis of each of the indicators.

[Zika fever immunodiagnostics: overview of test systems.]

The threat of rapid spread of Zika virus beyond endemic regions has given rise to more research in field of epidemiology and clinic, as well as to the search for Zika fiver new diagnostic and preventive tools. Between 2013 and 2017 in Russia 18 cases of infection transmission by travellers were reported. Fever Zika reference monitoring center in Volgograd Research AntiPlague Institute (Volgograd, Russian Federation) provides counseling and methodological assistance on laboratory diagnosis and monitoring of Zika fever. In this regard, a literature review of commercial test systems for immunodiagnostics of this infection was performed. Currently, a number of test systems for solid-phase enzyme-linked immunoassay method (ELISA), immunochromatography and indirect immunofluorescent method (IIFT) have been developed for immunodiagnostics of Zika fever. Euroimmun Ltd. remains the only manufacturer that has access to detailed information on validation of the specificity of the produced diagnostic kits. Independent studies confirm that Euroimmun test systems have high specificity and high sensitivity, which is proved by the study of the material from various populations, including Europeans travelling to Zika virus endemic regions and people residing in these regions. A detailed overview of characteristics of Euroimmun test systems for immunodiagnostics of Zika fever allows us to conclude that there is a rationale for the use of these test systems for Russian Federation sanitary protection by identification of antibodies in patients, presumably infected with the Zika virus.

[The problems of identification of various strains of vegetative form of Bacillus anthracis using MALDI-ToF MS technique.]

The article considers experience of application of mass-spectrometry with matrix-activated laser desorption/ionization for fast and reliable identification of Bacillus anthracis and heterologous species of microorganisms. The particular interesting characteristics and difficulties occurred during identification are covered.

[ON DEVELOPMENT OF TOOLS OF IMMUNE DIAGNOSTIC OF INFECTIOUS DISEASES: PROBLEMS OF DIAGNOSTIC IN VIVO AND IN VITRO].

The article considers a number of problematic issues concerning development of effective means of immune diagnostic of infectious diseases of bacterial and mycotic etiology related to approaches of choosing appropriate diagnostic targets.

[The diagnostic significance of particular immune-dominating proteins of isogenic variants of Bacillus Anthracis.]

The polyclonal mono-specific sera to proteins of toxin and antigens of S-layer of Bacillus Anthracis separated in preparative electrophoresis. While fluorescent antibody technique was applied, immunoglobulins of sera to protein of S-layer of Bacillus Anthracis m.m. 94 kDa had specificity and specific activity in presented number of strains. In the soil samples spores in concentration of 1x104 KOE/ml were detected that permits to recommend the given immunoglobulins for indexation of Bacillus Anthracis. The proteins m.m. 90 kDa relevant to antigens of toxin of Bacillus Anthracis reacted in immuno-blotting with sera from patients with skin form of anthrax and guinea pigs immunized and survived after infection. Thew sera to them are species specified and can be applied for diagnostic of disease (detection of protective antigen) and detection of production of proteins in reaction of immunodiffusion with growing cultures.

[SPREAD OF ESPECIALLY DANGEROUS MYCOSES IN THE WORLD].

Contemporary information on the spread in the world of especially dangerous mycoses - coccidioidomycosis, histoplasmosis, blastomycosis and paracoccidioidomycosis are presented in the review. Sources and infection routes of causative agents of these diseases are examined, clinical forms of mycoses are briefly characterized. An increase of morbidity due to them over the last decade is noted. A necessity of timely diagnostics of imported mycoses outside endemic regions is underscored.

[CONJUGATIVE INTEGRATIVE ELEMENTS (ICEs) OF MICROORGANISMS].

Integrative conjugative elements (ICEs) are an extensive group of mobile genetic elements found in the Gram-positive and Gram-negative bacteria. These genetic elements are replicated being incorporated into host chromosome, but retain the ability for excision and conjugative transfer. Given a set of the genes of the conjugative transfer, control of removal and integration, ICEs are directly involved in the processes of horizontal transfer of genetic determinants, which increase the adaptive potential of the bacterial species, as well as act as a mobilizing factor for other genetic elements.

[The comparative evalution of informativeness of immunologic and molecular genetic methods and means during stages of specific indication of melioidosis agent].

The reference-center of monitoring of agents of glanders and melioidosis carried out testing of reagents kits for diagnostic of agent of melioidosis and other close-related species of Burkholderiae in vitro. At the stage of specific identification of pathogenic Burkholderiae the diagnostic possibilities of commercial and experimental kits of reagents for express- and rapid analysis were evaluated. The criteria of evaluation of diagnostic value of kits of reagents were sensitivity, specificity and time of implementation of studies. The analysis with application of mono- and multi-locus amplification systems, including real-time polymerase chain reaction permitted during 5-6 hours to implement identification and differentiation of Burkholderia pseufomallei, B. thailandensis and B. cepacia.

[Molecular typing of the Burkholderia pseudomallei strains with different resistance to antibiotics].

Wild type strains of Burkholderia pseudomallei, spontaneous mutants with high resistance to fluoroquinolones and ceftazidime, and Tn5-induced mutants with reduced resistance level were studied using polymorphic and gene-specific DNA fingerprinting. Cluster analysis of genomic DNA patterns obtained using PCR with arbitrary primer (5'-GTTTCGCTCC-3') and primer specific to the class I integrase intll gene (5'-CCTCCCGCACGATGATC-3') was performed. According to the DNA pattern conformity, the distinct groups submitted by high-level resistant B. pseudomallei derivatives were revealed by both typing approaches. The obtained results may be useful in searching for molecular markers associated with different types of antimicrobial resistance among pathogenic and related burkholderiae.

[Properties of the Burkholderia pseudomallei insertional mutants deficient in membrane proteins production].

Transposon-induced B. pseudomallei mutants deficient in membrane proteins production were obtained for evaluation of the functional role of these cell components. In comparison with the wild type strain B. pseudomallei 57576, mutant clones TTM6, TTM7 and TTM9 carrying Tn5 chromosome insertions were characterized by lost or decreased production of outer membrane proteins 27, 48, 52, 150, 200 kDa. Alterations in outer membrane protein spectra were accompanied by twofold increase in susceptibility of bacteria to fluoroquinolones (pefloxacin, ofloxacin) and cephalosporins (ceftazidime) and noticeable reduction of virulence for white mice and guinea pigs in contrast to the initial strain, the obtained mutants were also less resistant in in vitro phagocyte killing.

[Ultrastructural immunochemical study of pathogenic Burkholderia capsule].

The capsular structures of Burkholderia pseudomallei, B. mallei, B. cepacia and their avirulent noncapsular mutants were studied with the use of electron ahd immunocytochemical techniques. For this purpose, antimelio-idosis monoclonal antibodies (McAb) G11 and 1 G2, epitope-aimed at capsular glycopyotein of 200 kD and outer-membrane proteins of 42 and 39 kD, were used. As revealed in this study, the typical causative agents of melioidosis and glanders formed the capsule and exhibited high virulence due to the antiphagocytic activity of 200 kD glycoprotein, whose epitopes were found to be incorporated into the capsule, in contrast to avirulent variants and B. cepacia, found to have no such structure. The recognition of the membrane determinants of McAb 1 G2 on the outer-membrane surface of the non-capsular variants of microbes known to be the causative agents of melioidosis and glanders was indicative of absence of the capsule in these microbial cells. These data concerning the role of 200 kD antigen in virulence, its structural and functional characteristics may be efffectively used in the study of the pathogenetic mechanisms of melioidosis and glanders, as well as in the construction of preparations for their immunodiagnostics and prophylaxis.

[Comparative analysis of total cell protein electrophoregram of pathogenic Burkholderia].

Whole-cell proteins of 22 strain of Burkhoderia pseudomallei, including 13 B. mallei, 5 B. cepacia strains and 14 strains of opportunistically pathogenic Pseudomonas defined by 1D SDC-PAAG electrophoresis. Electrophoregrams contained 35 to 45 protein fractions sized 19 to 130 kDa, which were highly reproductive. On the basis of computer-aided comparative analysis of protein patterns the interspecies and intraspecies grouping of studied microorganisms was made. The cluster analysis of the similarity matrix of protein spectra made it possible to allocate two groups of strains at the level of similarity of 78%. Group I was formed by Burkholderia species that previously belonged to the II RNA-DNA homology group of Pseudomonas: B. pseudomallei, B. mallei, B. cepacia. All Pseudomonas species were added to the 2nd Group: P. aeruginosa, P. stutzeri, P. testosterone, P. fluorescens, P. putida, P. mendocina. Four phenons were isolated among the strains of B. pseudomallei and 2 phenons--among the strains of B. mallei at the threshold similarity level (89%). The authors conclude that the comparative analysis of electrophoregrams of whole-cell proteins can be useful in the identification and typing of pathogenic Burkholderia.

[Cross-reacting antigens of pathogenic Burkholderia and some dangerous causative agents of infectious diseases].

Cross-reacting antigens in B. mallei, B. pseudomallei, B. thailandensis, Francisella tularensis, Yersinia pestis and Mycobacterium tuberculosis were studied with the use of immuno- and electrophoretic techniques. The set of antigens was shown to be almost identical in the causative agents of glanders, melioidosis, as well as in B. thailandensis, though in the latter organism 200-kD glycoprotein was absent. The analysis of immuno- and proteinograms demonstrated the presence of cross-reactions in the representatives of the genus Burkholderia with the causative agents of plague, tularemia and tuberculosis, which served as the basis for making the scheme of their antigenic relationships. The use of immunosorption techniques with subsequent analysis of the preparations by means of the SDS polyacryl gel electrophoresis and immunoblotting made it possible to characterize cross-reacting antigens of the pathogenic microorganisms under study, to establish their molecular weights (81-15 kD) and to show that some detected antigens are analogous to B. pseudomallei outer membrane proteins (34 and 30 kD).

[Immune response in experimental animals immunized with Burkholderia pseudomallei surface antigens]. / Immunnyi otvet éksperimental'nykh zhivotnykh pri immunizatsii poverkhnostnymi antigenami Burkholderia pseudomallei.

The influence of the chromatographic fractions of B. pseudomallei surface antigenic complex (C, C1, D, H) on immune response in white rats and white mice was under study. These antigenic complexes were noted to produce perceptible stimulating effect on the immune system of white rats, in contrast to that of white mice. The immunization of the mice the above-mentioned fractions suppressed the phagocytic activity of peritoneal macrophages (PM) and slightly enhanced cell-mediated immunity. In experiments on white rats, fraction C induced the growth of specific antibody titers and stimulated the phagocytic activity of PM, as well as the indices of delayed hypersensitivity (DH). Fraction D showed a lower level of the induction of the phagocytic activity of PM and was inactive in the manifestation of cell-mediated immunity, but induced a high level of humoral immunity. Antigenic complexes C1 and H increased the phagocytic activity of PM and DH characteristics with a low level of antibody production. The studied fractions of the causative agent of melioidosis decreased the content of bactericidal cationic proteins (BCP) in rat blood neutrophils, and in mice a decreased content of BCP in phagocytes was registered. The fractions increased the activity of myeloperoxidase in blood neutrophils in mice and rats. As revealed with the use of immunoelectrophoresis, SDS PAAG electrophoresis and immunoblotting, the surface antigenic complex contained proteins of 18, 22, 39 kD and glycoproteins 42, 55, 90 kD. The latter glycoprotein was found in all the fractions under study, having protective properties.

[Cloning and expression of 7.55 kb KPNI fragment of Burkholderia pseudomallei PPM1 plasmid in Escherichia coli]. / Klonirovanie i ékspressiia 7,55 t.p.h. KPNI-fragmenta plazmidy PPM1 Burkholderia pseudomallei v Escherichia coli.

BamHI, SalI, PstI, and KpnI fragments of pPM1 (B. pseudomallei 12.95 kb plasmid) were cloned in E. coli. The recombinant clones carrying a 7.55 kb KpnI fragment of pPM1 were highly resistant to several aminoglycosides (streptomycin, kanamycin, and gentamycin) and fluoroguinolones (perfloxacin, ofloxacin). Two outer membrane proteins (23 and 27 kDa) absent in E. coli and capable to form 120 kDa oligomer complex were detected by the Western blot method in the strain carrying recombinant pS19 plasmid. The integration of a cloned 7.55 kb sequence in the chromosome was observed by the dot and Southern hybridization analysis in the clones carrying recombinant plasmids pS12 and pS14.

[Immunobiological properties of Burkholderia pseudomallei and Burkholderia mallei capsular substances]. / Immunobiologicheskie svoistva kapsul'nogo veshchestva Burkholderia pseudomallei i Burkholderia mallei.

The biopolymer composition, immunotropic and immunogenic properties of the fractions of B. pseudomallei and B. mallei were under study. The first two capsular fractions of these agents were found to be similar in their biopolymer composition that was indicative of their close relations. At the same time the causative agents of glanders proved to have decreased content of high molecular glycoproteids and LPS fragments. In the causative agents of melioidosis, capsular fractions K3 and K4 were characterized by the domination of proteins with a molecular weight of 42-25 kD. Fraction K4 in B. pseudomallei and fraction K1 in B. mallei had pronounced immunosuppressing properties ensuring the protection of encapsulated microbial cells in the body. The biopolymers forming fractions K1, K2, K3 in B. pseudomallei and fraction K2 in B. mallei were characterized by immunomodulating properties.

[Active membrane transport and bacterial multiple antibiotic resistance]. / Aktivnyi membrannyi transport i mnozhestvennaia antibiotikoresistentnost' bakterii.

Multiple drug resistance can form in bacteria by functioning the membrane transport systems, responsible for release of antibacterial compounds from the cell into the environment. These transport mechanisms activated in the majority of cases by energy of proton transmembrane gradient are presented by solitary membrane transporting proteins and by functionally related transporter groups, periplasma proteins, and external membrane porines. Many bacterial drug transporters can bind and transfer a number of structurally heterogeneous substrates. Drug transporters known today have different origin and primary physiological functions. The genetic system of transporter type drug resistance is as a rule characterized by a cluster structure and related to mobile genetic elements. Transport mechanisms of drug resistance create an extra adaptation potential of microorganisms under conditions of selective pressure.

[Immunotropic and immunogenic properties of Burkholderia pseudomallei surface and membrane antigens]. / Immunotropnye i immunogennye svoistva poverkhnostnykh i membrannykh antigenov Burkholderia pseudomallei.

The immunotropic and immunogenic properties of some chromatographic fractions of B. pseudomallei surface antigenic complex, as well as the preparations of B. pseudomallei outer and cytoplasmic membranes, were studied. The difference between the biopolymers under study in cytotoxicity, humoral and cell-mediated immunity characteristics, phagocytic activity were established. Some antigenic fractions (B, C, C1, H) showed perceptible protective activity (25-60%) in experiments on mice infected with B. pseudomallei virulent strain. One of the preparations of cytoplasmic membrane (CM-1) was also found to have protective properties (30%). Complex immunization with the antigenic complexes under study, introduced in combination with the immunomodulating agent Bromantan, was shown to enhance the protective effect.