Pecularities of Candida yeast identification by mass spectrometric analysis (MALDI-ToF MS).

To carry the comparative analysis of sample preparation methods for the most effective identification of Candida yeast by mass spectrometric analysis. 265 strains of yeast and yeast-like fungi isolated from the sputum of patients with pneumonia were investigated. The selected strains were identified by conventional methods (cultural, morphological, tinctorial, enzymatic properties) and MALDI-ToF MS using the Autoflex speed III Bruker Daltonics mass spectrometer (Germany) and Flex Control software. To evaluate the effectiveness of fungi species determinination, the comparative analysis of sample preparation was performed using 4 methods: direct application to the target, an extended direct application method, protein extraction using ethanol/formic acid or trifluoroacetic acid. The accelerated scheme of identification of fungi by the culture method does not provide clear and unambiguous results. When using mass spectrometric analysis, the reliability of the results depended on the sample preparation. A comparative study of the effectiveness of fungi species determination by various methods of the sample preparation of 50 clinical isolates was carried out. It was revealed that the extraction of cells using TFC acid does not lead to the appearance of the recordable protein spectra. The use of direct and extended direct application methods made it possible to establish the species only in 32-44% of the strains. The most effective method of sample preparation was the method using formic acid and ethanol, which allowed us to determine the species affiliation in 100% of the studied fungi (Score 2.0). Depending on the yeast species, a high statistical indicator (Score≥2.3) was registered for 42-100% of samples. The results of present study show that the use of MALDI-ToF MS is the most reliable and informative method of Candida spp.identification.

[ROLE OF VARIOUS ANTIGENIC PREPARATIONS OF FRANCISELLA TULARENSIS IN FORMATION OF ALLERGY REACTION IN HUMANS AND ANIMALS].

AIM: Study the role of LPS in induction of anti-tularemia immunity in humans and animals. MATERIALS AND METHODS: Activity of various antigenic preparations of tularemia microbe, including highly purified from protein and S- and R-LPS, was studied using leukocytolysis reaction with blood of vaccinated humans and guinea pigs and skin allergy test (guinea pigs). RESULTS: Only the whole cells of Francisella tularensis, killed in protein non-denaturating conditions and conserving full S-LPS structure (tularin⁺) were shown to be inductors of delayed-type hypersensitivity reaction. Alterations in LPS structure (tularin⁻) results in a significant decrease, and denaturation of bacterial proteins (during boiling) results in a complete loss of immune stimulating properties of the preparations. Purified LPS preparations and O-polysaccharide fraction of S-LPS are not able to activate cell-mediated immunity. CONCLUSION: The presence of LPS with the full structure affects the ability of antigenic preparations of F. tularensis to cause allergic reactions, and thus, form cell-mediated antitularemia immunity. LPS of F. tularensis can not be excluded as an adjuvant and provides the most effective presentation of epitopes of protein molecules for interaction with receptors of T-lymphocytes.

[Comparative analysis of parameters of humoral and cell specific immunity in individuals immunized with a live tularemia vaccine].

AIM: Comparative analysis of parameters of humoral and cell immunity in individuals, vaccinated against tularemia. MATERIALS AND METHODS: Sera and blood samples of 258 immunized individuals were studied by indirect hemagglutination and leukocytolis with tularin reaction. RESULTS: 73% of the examined individuals had both specific antibodies and positive values of cell immunity. The presence of anti-tularemia immunity was registered in 26 of 30 individuals immunized 10 - 20 years ago. However in 76 individuals (26%) we have detected discrepancies of the results of the 2 methods that complicate the evaluation of specific immunity status. As such, the use of only 1 method characterizing either humoral or cell immunity does not give objective information. CONCLUSION: The use of 2 methods directed on detection of both specific antibodies and delayed-type hypersensitivity reaction is reasonable for the increase of validity of results of anti-tularemia immunity status evaluation. Only positive immunologic parameters of both tests confirm the presence of immunity against Francisella tularensjs and the possibility of revaccination period delay.

[Phase variations of Francisella tularensis lipopolysaccharide in human infection and immunization].

The comparative study of the specificity of antibodies in human sera after tularemia infection and immunization with live tularemia infection was carried out with the use of passive hemagglutination and immunoblotting techniques. The sera of tularemia patients contained two different types of immunoglobulins: strictly specific to the antigenic epitopes of F. tularensis Iipopolysaccharide (LPS) and strictly specific to F. tularensis subsp. novicida LPS. Such phenomenon may be due to phase variations of the antigenic structure of F. tularensis LPS in the body of a slightly susceptible host. The immune sera of vaccinated were found to contain antibodies, strictly specific only to F. tularensis LPS. At the same time in one vaccinee by the presence of pronounced postvaccinal reactions was found sharply defined interaction between serum imunoglobulins and F. tularensis subsp. novicida LPS. As the result, the data on the possibility of the antigenic modification of F. tularensis in tularemia infection in humans were obtained. At the same time antigenic epitopes, characteristic of faintly pathogenic and closely related F. tularensis novicida LPS, appeared in the structure of F. tularensis LPS.

[Comparative analysis of the immune response of a rabbit to antigens to live and killed Francisella species bacteria]. / Sravnitel'nyi analiz immunnogo otveta krolika na antigeny zhivykh i ubitykh bakterii roda Francisella.

Serum antibodies were analyzed in rabbits immunized with live and formalin-killed Francisella (F. tularensis, F. novicida, F. novicida-like, and F. philomiragia). Passive hemagglutination test with erythrocytes sensitized by these bacteria' LPS showed much higher titers of species-specific antibodies in all sera to live microorganisms than sera to killed bacteria. The results of immunoblotting with purified LPS and bacterial lysates indicate that sera to live bacteria contained mainly immunoglobulins to species-specific antigenic epitopes of LPS O-polysaccharide chain and few antibodies to the protein component of the cell. By contrast, killed bacterial cells induced weak production of antibodies to S-LPS and a pronounced antibody response to protein antigens. Besides the quantitative differences, live and killed bacteria differed by the qualitative spectrum of immunodominant proteins. Serum to live F. tularensis 15/10 contained antibodies to at least 3 immunodominant antigens of the cell, while serum to killed bacteria contained antibodies to only two of these. Immunoglobulins to protein antigens, absent in homologous sera to live bacteria, were detected in the sera to killed F. novicida and F. novicida-like bacteria. Both sera to F. philomiragia had antibodies reacting with LPS epitopes and immunodominant complex containing protein. In contrast to other Francisella, F. philomiragia was found to synthesize an uncommon LPS representing two major lipooligosaccharides with different molecular weights and antigenic specificity. Therefore, immune response of the host to live and killed Francisella is different: live cells more effectively induce the production of antibodies to S-LPS epitopes, while killed ones to protein antigens.

[Species- and genus-specific antigenic epitopes of Francisella tularensis lipopolysaccharides]. / Vido- i rodospetsificheskie antigennye épitopy lipopolisakharidov Francisella tularensis.

Lipopolysaccharide (LPS) antigenic epitopes of natural virulent and isogenic avirulent Francisella tularensis strains and other species of the Francisella genus (F. novicida, F. novicida-like, and F. philomiragia) were studied by dot and immunoblotting. Polyclonal rabbit and human sera to virulent F. tularensis strains and monoclonal antibodies to F. tularensis LPS O-side chain were used for detecting species- and genus-specific LPS epitopes. Typical virulent F. tularensis strains produce two types of S-LPS with different antigenic specificity simultaneously. Antigenic determinants of two LPS types were located in LPS O-polysaccharide but not in the core oligosaccharide. The epitopes of the first LPS type were characterized by species specificity for F. tularensis in contrast to determinants of the second LPS type, which had epitopes common with F. novicida. Cross exhaustion of human and rabbit antitularemic sera by F. tularensis and F. novicida LPS showed that F. novicida LPS molecules contained at least two epitopes--highly specific for F. novicida and common with the second type of F. tularensis LPS. The immune response of rabbits and humans to F. tularensis LPS epitopes was different in principle. Sera from rabbits immunized with vaccine and virulent F. tularensis strains contained antibodies "recognizing" antigenic epitopes of two S-LPS forms of the bacterium: type 1 species-specific (in high titers) and type 2 epitopes common with F. novicida LPS (in low titers). In addition to these, sera from patients with tularemia contain immunoglobulins to species-specific epitopes of F. novicida LPS in high titers. Experiments on avirulent mutants showed that in some cases attenuation of F. tularensis can involve loss of species-specific LPS form, while S-LPS with epitopes common with F. novicida LPS will be retained. The difference in specificity of human and rabbit antitularemic antibodies is due to individual features in the host immune system.

[The use if Francisella tularensis lipopolysaccharide in the dot solid phase enzyme immunoassay]. / Ispol'zovanie preparatov lipopolisakharida Francisella tularensis v tochechnom tverdofaznom immunofermentnom analize.

To determine antitularemia antibodies in the sera of humans and animale, the possibility of using dot immunoassay with the use of F. tularensis lipopolysaccharide (LPS) as antigen-containing preparation was ascertained. Experiments demonstrated that this method made it possible to determine specific antitularemia antibodies in the sera of sick and immunized humans and animals. Investigetions carried out with the use of heterologous antisera to F. novicida, F. novicida-like and F. philomiragia, as well as Brucellf abortus, Vibrio cholerae and Yersinia enterocolitica, revealed that F. tularensis S-LPS was highly specific. The results obtained in this investigation are indicative of good prospects of using F. tularensis LPS in dot blotting for the laboratory diagnostics of tularemia in humans.