Search

The article is about methods of chikungunya fever laboratory diagnosis. An algorithm for the study of biological material for the presence of antibodies against chikungunya virus and virus antigens is presented. The overview describes the information about commercial immunodiagnostic and genodiagnostic kits and their detailed specifications. The information presented in the review will be useful for doctors of clinical laboratory diagnostics to choose a method and an acceptable test system for laboratory confirmation of Chikungunya fever diagnosis, as well as differential diagnosis with other fevers, which have similar symptoms, common geographical distribution and carriers of infection., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: The authors declare no conflict of interest.

Aim: Determine an optimal set of the most effective methods of identification and intraspecies typing ofcausative agents ofglanders and melioidosis. Materials andmethods. Bacteriologic, immunochemical, molecular-genetic methods were used., Results: A possibility to identify collection strains of pathogenic and closely related Burkholderia in semiautomatic systems is studied. Means of detection of informative variable genome segments ofthe specified microorganisms were developed, methods of their genetic typing were selected. Effectiveness of application of precipitating mAbs for differentiation of Burkholderia was established. Data on diagnostic possibilities of immunoglobulins fluorescing based on monoclonal antibodies of various etiotropic directionality for detection and identification of B. mallei and B. pseudomallei are generalized. Experimental series of amplification test-systems for identification of glanders and melioidosis causative agents in real-time PCR format are created., Conclusion: A number of methods for identification and typing of glanders and melioidosis causative agents is proposed.

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.

A new method for obtaining the liposomal formulation of streptomycin and tetracycline is described in the present work. The physicochemical properties of this formulation were evaluated. We compared the effects of free and liposomal formulations from streptomycin and tetracycline on activity of liver enzymes in albino mice. Immobilization of antibiotics in liposomes was followed by a decrease in the inhibitory effect on protease, alkaline phosphatase, and phosphorylase. The influence of this preparation on ATPase was reduced by 2 times.

Aim: Extraction of complex of Burkholderia pseudomallei antigens 6+d (Ag6+d) and study of its immunogenic and protective characteristics., Materials and Methods: Studied antigens were obtained from acrtone-dried cells of B. pseudomallei 57576. Experiments were performed on white mouse model. Microbiological, immunochemical as well as immunological methods were used in the study., Results: It was shown that antigenic complex 6+d has a glycoprotein nature and corresponds to high-polymeric catode-moving antigen 6 with molecular mass 500 kDa and anode-moving antigen d with molecular mass 40 - 55 kDa. Immunogenic and protective characteristics of surface antigenic complex 6+d was studied. It was noted that Ag6+d caused reliable stimulating effect on cellular arm of immune system of white mice appeared in activation of delayed-type hypersensitivity reactions, enhanced phagocytic activity of polymorphonuclear macrophages as well as increased expression of Fc-receptors on macrophages and neutrophils., Conclusion: Observed stimulation of cellular immunity by surface antigens was confirmed during study of their protective characteristics on the model of experimental meloidosis in white mice.

Preparation and physicochemical properties of liposome-incorporated tetracycline are described. Comparison of the effects of tetracycline hydrochloride in free and liposome-incorporated forms on enzymatic functions of the liver showed that immobilization of the antibiotic into liposomes protects liver cells from functional disturbances.

Study showed that five (C3, C6, C9, C10, C11) out of ten chromatographic fractions of surface and capsular antigens of B. mallei significantly stimulated cell-mediated immunity that manifested in activation of delayed hypersensivity reactions (DHS) and phagocyteability of noncapsulated avirulent strain of B. mallei with added surface and capsular antigenic complexes. Other fractions did not stimulate cell-mediated immunity, furthermore, fraction C8, which contained capsular biopolymer with mass of 200 kD (Ar8), was characterized by immunosuppressive effect on DHS and phagocytosis. Observed stimulation of cell-mediated immunity by fractions referred above has been confirmed by assessment of their protective effects on the model of experimental melioidosis in white rats. Relationship between markers of humoral and cell-mediated immunity, including markers of specific response, was not observed.

The authors have developed a homogenous method for the detection of the cells, surface antigen, and Ag8 of glanders and melioidosis pathogens and antibodies to them, based on the compliment-dependent lysis of Ag8-sensitized liposomes. The method is highly specific; its sensitivity to antibodies to glanders and melioidosis pathogens is 24 to 440 times higher than that of RID: its sensitivity to Ag8 is 100 ng/ml corresponding to 1.25x10-10 M, and its sensitivity to cells is 10(5) to 10(6) cells/ml).

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.

The results of the evaluation of the toxicity of bacterial antigens obtained from the causative agents of plaque, glanders, melioidosis, cholera on infusoria of the species P. caudatum, as well as on cell lines L-929, CHO K-1 and peritoneal macrophages of BALB/c mice, are presented. As revealed in this study, the method of toxicity determination on infusoria is similar in its sensitivity to the methods of testing on. CHO K-1 and L-929 cells, but the former is simpler, more available and permits the determination of toxic doses producing disturbances in the vital activity of the infusoria, but not leading to their death.

The data on preparation of liposome-entrapped gentamicin sulfate and cefoperazone and their investigation on albino mice with staphylococcal destructive pneumonia are presented. Comparative study of the efficacy of gentamicin sulfate and cefoperazone in free and liposome-entrapped forms showed that immobilization of the antibiotics in phospholipid vesicles provided a 2-fold increase of their efficacy.

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.

The dynamics of the antigen 8 synthesis in Burkholderia pseudomallei and B. mallei under conditions of their submerged was studied. Differences in the intensity of this antigen synthesis in two pathogenic Burkholderia species were established and the producer strains, most effective with respect to this sign, were selected.

In this work conditions for the reproduction of hybridoma technology, specially adapted to C. neoformans, for obtaining monoclonal hybridomas (McAb) to diagnostically significant antigens of C. neoformans, the infective agent of cryptococcosis, are presented. The advantages of using the short-time cycle of stimulation of mouse B lymphocytes with low doses of C. neoformans capsular polysaccharide and the effectiveness of the hybridization of mouse spleen cells with myeloma cells, line Sp2/0, are shown. Four lines of stable hybridomas, producing McAb to different epitopes of C. neoformans surface antigens, have been obtained. The specific activity of McAb has been studied in the indirect immunofluorescence assay, the cytochemical and solid-phase enzyme immunoassays (EIA). McAb 3E2, Cr2 and 2G9 have been shown to be suitable for use in diagnostic EIA systems.

Phosphatase, phospholipase C and a proteolytic complex, including casein- and hemoglobin-hydrolases, have been isolated from cell-free extracts of B. pseudomallei cultivation medium. A set of monoclonal antibodies (McAb) to the antigenic complexes of this infective agent has been obtained. Conditions for the study of the interaction of the enzymes and McAb have been worked out, and most of the 14 immunoglobulins under study have been shown to neutralize 2-3 hydrolases each. Three types of McAb, selectively inhibiting only the proteolytic complex, only casein-hydrolase and only phospholipase C, have been identified.

The paper summarizes the data concerning the production and study of monoclonal antibodies (MAb) to the diagnostically significant glanders and melioidosis bacillus antigens. It evaluates the efficiency of using MAb in the gel immunodiffusion and agglutination tests as a basis of new-generation preparations for fluorescent antibody assay, indirect hemagglutination test which are used while detecting and identifying pathogenic pseudomonads. The paper defines the quality indices for monoclonal luminescent immunoglobulins and provides evidence for the benefits of monoclonal diagnostic agents over polyclonal analogues.

The indirect immunoperoxidase method with the use of monoclonal antibodies has been employed to determine the localization of antigens of melioidosis agents. The formed reaction products were then detected by the electron microscopic analysis. The most important in melioidosis pathogenesis antigens 6 and 8 (AG 6 and AG 8) were determined to localize on the cell wall and in the capsule-like mucous layer, respectively. Monoclonal antibodies to AG 8 due to their marked cross-linking properties promote stabilization, preservation and detection of labile melioidosis agent. At the same time the mucous layer prevents monoclonal antibodies to AG 6 to access to the corresponding antigen, localized on the cell wall and makes its detection difficult.

As established with the use of electron-immunochemical techniques, glycoprotein antigen 6 is the outer membrane component of P. pseudomallei cell wall, while glycoprotein antigen 8 is localized on the cell surface as a capsule-like formation. Antigen 6 plays no perceptible role in the realization of the pathogenic properties of the infective agent, but serves as a reliable sign in the differentiation of P. pseudomallei strains into serovars. Subcultures, defective in the synthesis of antigen 8, have sharply reduced virulence for laboratory animals. As revealed in this study, the pathogenetic action of antigen 8 is linked with its pronounced antiphagocytic function. Thus, antigen 8 is considered to be one of the key pathogenicity factors of P. pseudomallei.

A highly effective and specific solid-phase enzyme immunoassay system has been developed. The enzyme immunoassay is a highly sensitive technique for the detection and identification of C. immitis cellular and metabolic antigens. This technique is suitable for the study of strain differences in the antigenic composition of C. immitis, rendered harmless by different methods. The expediency of the preliminary sonification of cell suspensions of C. immitis, the causative agent of coccidioidomycosis, has been experimentally confirmed.

The use of IHAT for rapid assay of antibiotic sensitivity of bacteria causing malleus and melioidosis was shown to be possible. Pure bacterial cultures and suspensions of the spleen of infected animals were used as the investigation objects. The materials were additionally grown in meat-peptone broth with 5 per cent of glycerol at 37 degrees C with addition of an antibiotic or without it (control). Antibiotic efficacy was estimated by the difference in the titers in the main and control experiments. The test not requiring isolation of pure bacterial cultures provided a 2-3-fold decrease in the investigation time.