[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.

[Imperfect and Compound Microsatellites in the Genomes of Burkholderia pseudomallei Strains].

Evolution of microsatellites (or simple sequence repeats, SSRs) is a complex process that converts perfect repeats to novel structural elements with functions poorly understood, such as imperfect and compound microsatellites. An in silico analysis often Burkholderia pseudomallei genomes revealed 215683 micro-satellites, and more than 98% of them proved imperfect. The density of microsatellites in the genome ranged from 2922.7 to 3022.6 per Mbp. Approximately 10.20-10.67% of the repeats were parts of compound micro-satellites. The of compound microsatellite density varied from 144.7 to 150.6 per Mbp. Between-strain differences in microsatellite distribution were explained by a direct correlation of the SSR density with the GC content and an inverse relationship between the SSR density and the genome size. For each B. pseudomallei chromosome, the SSR density similarly correlated with its size and GC content. Chromosome 2 showed a significant correlation between the SSR and compound microsatellite densities (r = 0.93, p < 10^(-3)). The association of imperfect and compound microsatellite densities with the structural features of each chromosome and the fact that motifs are degenerate and occur in few copies in the majority of B. pseudomallei microsatellites agree with the previous hypothesis of negative selection affecting extended SSRs. The mechanism of selection possibly involves an accumulation of point mutations, which lead to an interruption of the repeat during replication because easily passable secondary structures may form to stabilize the microsatellite length.

[The development and application of reagents kit for detection RNA of the causative agents of melioidosis and glanders by transcription-based amplification (NASBA) in real-time.]

The polymorphism of clinical manifestations of melioidosis and glanders and their high mortality require improvement of diagnostics for detection of this agents. The perspectivity of development of transcription-based amplification real-time NASBA diagnostic kits is determined by high analytical sensitivity and the opportunity to perfom the verification of the results of other methods for pathogenic Burkholderia species detection. The fragment of 23S rRNA gene was selected as the target for development of real-time NASBA kit. The high specificity of the constructed oligonucleotides was confirmed during the analysis of wide range of heterological strains of microorganisms and sequencing of amplified fragments of 23S rRNA gene. The analytical sensitivity of the developed kit allowed to detect Burkholderia pseudomallei and Burkholderia mallei in concentration of 1×101 microbial cells per ml. The high functional characteristics of developed kit as well as the possibility to use it in case of appereance of discordant result during the detection of pathogenic Burkholderia species were demonstrated while studying biological samples.

[GENOTYPING OF THE BURKHOLDERIA MALLEI STRAINS BASED ON DIFFERENT REGION ANALYSIS].

Development of the genotyping methods of glanders agent is urgent due to its high pathogenicity, lack of effective preventive measures and threat of the use of Burkholderia mallei as a biological weapon. In this work we proposed a scheme for the typing of the B. mallei strains based on different region analysis (DFR). The choice of variable loci differentially presented in various strains of glanders agents was performed by analyzing annotated whole-genome sequences of the B. mallei strains. Primers and fluorescence probes were designed for 9 selected loci. The amplification conditions for different regions were optimized in two variants: with electrophoretic detection and hybridization-fluorescence detection in the strip format. The possibility of applying the DFR analysis to genetic characterization of strains was assessed in 14 B. mallei strains. The genetic profiles of the studied B. mallei strains revealed that the developed DFR-typing scheme was characterized by high discrimination power (Hunter-Gaston index value was 0.92), reproducibility, rapidity, easy interpretation, and applicability for epidemiological surveillance of glanders.

[IDENTIFICATION OF CAUSATIVE AGENTS OF GLANDERS AND MELIOIDOSIS BASED ON PRINCIPLES OF POLYPHASE TAXONOMIC APPROACH].

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.

[Characteristics of isogenic variants of Bacillus anthracis with various content of virulence plasmids].

AIM: Production and characteristics by main cultural-morphologic and antigenic properties of isogenic variants Bacillus anthracis, that differ by the presence of virulence plasmids. MATERIALS AND METHODS: B. anthracis 81/1, 575/122 virulent and B. anthracis STI, 55, Sterne vaccine strains were used in the study. Isogenic variants, that differ by the presence of virulence plasmids, were obtained by temperature elimination of plasmids, as well as during cultivation of anthrax strains in medium with kanamycin. The strains were characterized by cultural-morphologic, biochemical properties. The presence of virulence plasmids was determined by polymerase chain reaction method. Antigenic properties were studied in immune diffusion reaction with growing cultures with sera against protective antigen and S-layer proteins, electrophoresis, immune blotting. RESULTS: Isogenic variants were produced from virulent strains B. anthracis 81/1, 575/122 and vaccine strains STI, 55, Sterne: mono-plasmid toxin-producing (81/1 R01, 575/122 R01) and capsule-containing (81/1 R02, 575/122 R02), and plasmid-less (81/1 R00, 575/122 R00, STI R00, 55 R00, Sterne R00), that differ by the presence of virulence plasmids. Strains had typical cultural-morphologic properties, differed by biochemical and antigenic properties. Cultural filtrates of toxin-producing strains had protein of anthrax toxin; plasmid-less strains--had proteins, that had molecular masses corresponding to molecular masses of S-layer EA1 and Sap proteins. CONCLUSION: These strains may be used to study variability and proteomic analysis of anthrax causative agent, as well as for isolation of antigens with the aim of evaluating their immune diagnostic significance.

[Persistence of antibodies specific to West Nile virus in blood of reconvalescents of Volgograd region].

AIM: Determine the duration of persistence of IgM and IgG in reconvalescents of West Nile fever (WNF) 1 year after the disease in southern regions of Russia and evaluate effectiveness of PCR method for acute infection diagnostics. MATERIALS AND METHODS: Blood sera of 87 patients with WNF diagnosis was studied for the presence of West Nile virus (WNV) RNA and IgM and IgG by PCR and EIA. Samples of the first sera were collected in 2010 at days 2 - 30 after the onset of the disease, samples of the second sera--at days 5 - 23 and third--264 - 385 days later. RESULTS: During the first 2 weeks of the disease WNV RNA was detected in more than 50% of patients. In all the first sera IgM at titers of ≥ 1:800 were detected. Seroconversion of IgG titers of 4 and more times was observedin 83% (30/36) of patients. In 2011 IgG were detected in 91% of reconvalescents (79/87), IgM--in 57% (50/87), and in 25% (22/87) IgM titers were ≥ 1:800. CONCLUSION: The results obtained give evidence on the necessity of using several diagnostic criteria simultaneously for the confirmation of WNF clinical diagnosis.

[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 genetic analysis of Pseudomonas aeruginosa strains isolated from environment and patients in health care facilities].

AIM: Analysis of genetic heterogeneity of Pseudomonas aeruginosa strains isolated in and out of hospitals. MATERIALS AND METHODS: To study the genetic diversity of 36 strains of P. aeruginosa plasmid analysis, random amplified polymorphic DNA (RAPD) technique as well as polymerase chain reaction for detection of virulence genes algD, lasB, toxA, plcH, plcN, exoS, nan1, and nan2. RESULTS: Epidemically important strains were found in different ecological niches. It was shown that these virulence factors could play important roles in pathogenesis of infection. CONCLUSION: RAPD technique was effective for analysis of P. aeruginosa isolates. Number of studied typing bands differed between related isolates for each random primer.

[Phenotypic and genotypic identification of bacteria from the Burkholderia cepacia complex].

AIM: Evaluation of the diagnostic value of pheno- and genotypic characteristics of B. cepacia strains collection. MATERIALS AND METHODS: Phenotypic and genetic methods of identification and differentiation of 25 strains of the B. cepacia complex. RESULTS: Polyphasic taxonomic approach utilizing multiple diagnostic tests was used for accurate identification of Burkholderia species. Algorithm for identification of microorganisms from the B. cepacia complex was developed. CONCLUSION: Combined use of phenotypic and molecular genetic tests, such as recA gene PCR, is recommended for differentiation of the B. cepacia complex genomovars.

[Comparative assessment of DNA extraction methods for identification of glanders and melioidosis etiological agents by PCR].

Pathogenic Burkholderia are considered as a cause of dangerous infections and potential agents of bioterrorism. Comparative assessment of different methods of extraction and purification of DNA for PCR analysis of pure cultures and samples contaminated by etiological agents of glanders and melioidosis was performed. Samples of soil and food artificially contaminated by pathogenic Burkholderia as well as organs of infected animals were tested. DNA was extracted by methods of boiling, nucleosorption with presence of guanidine thiocyanate, guanidine thiocyanatephenol extraction, guanidine thiocyanate-phenol extraction with additional purification of DNA by nucleosorption. Amplification was performed by "Flash" technique and detector of fluorescence was used for analysis of PCR products. Utilization of the recommended methods of preparation depending on the nature of sample let to detect by the "Flash" technique the etiological agents of glanders and melioidosis in concentration =10(3) microbial cells per ml. Choice of DNA extraction and purification methods is determined by type of a sample and presence in it of admixtures inhibiting PCR.

[Long-term impact of breast reconstruction on quality of life among breast cancer patients].

Psychological impact of modified radical mastectomy carried out simultaneously with breast reconstruction (n=45) was compared with that in controls who underwent similar surgery for cancer alone (n=50). After 12 months, depression was less frequent in group 1 and indices such as being a good mixer, joyfulness, activity, happiness, job satisfaction and access to leisure and entertainment were significantly higher than among controls (p< or =0.05). However, deep depression persisted in group 1 (13%) as long as for 12 months and intense angst in 17.8%.

[Identification of the agents of coccidioidomycosis using polymerase chain reaction].

Two pairs of primers for diagnosis of coccidioidomycosis using the method of PCR were constructed. One pair was used for identification of the two species of Coccidioides (C. immitis and C. posadasil) on the basis of MBP-1 gene. The other pair was chosen on the basis of SOWgp82 gene, which encodes an immunodominant, spherule outer wall glycoprotein for detecting only C. posadasii. The used primers allowed the agents of coccidioidomycosis to be detected using PCR with high sensitivity and specificity. The effective method of isolation of fungus DNA from soil contaminated with arthroconidia of Coccidioides spp. was developed. It includes guanidinthiocyanate-phenol-chloroform deproteinization followed by DNA purification using nuclear sorption.

[The use of polymerase chain reaction for detection of the agents of glanders and melioidosis using experimental infection].

Glanders and melioidosis are severe infectious diseases of people and animals. The causative agents of these infections refer to the potential agents of bioterrorism of group B. In this work the possibility of use of flagellin-based primers for the identification of B. mallei and B. pseudomallei and for diagnosis of experimental glanders and melioidosis was studied. The obtained results permit to make a conclusion that PCR using the developed primers may be recommended for the incorporation in the scheme of laboratory diagnosis of glanders and melioidosis both for the identification of clean cultures and in experimental clinical material.

[Use of PCR for identification of Burkholderia mallei]. / Ispol'zovanie PTsR dlia identifikatsii Burkholderia mallei.

Stimuli of glanders belong to the potential agents of biological terror. The possibility to use various primers in the identification of B. mallei was investigated and the significance of polymerase chain reaction (PCR) was defined within the scheme of laboratory glanders diagnosis in the offered paper. The constructed amplifying test-systems can be used to detect the glanders both in the environmental objects contaminated with B. mallei and in experimental clinical material.

[Identification of the causative agents of glanders and melioidosis by polymerase chain reaction]. / Identifikatsiia vozbuditelei sapa i melioidoza s pomoshch'iu polimeraznoi tsepnoi reaktsii.

Burkholderia mallei and B. pseudomallei are causative agents of glanders and melioidosis, respectively, i.e. severe and fatal infection diseases of man and animal. The computer-based analysis of the 23S rRNA gene sites was used for selecting the primers. Two pairs of primers were chosen for the identification of B. mallei and Bpseudomallei. DNAs from 48 B. pseudomallei and 15 strains of B. mallei, unlike from other geterological bacteria, were positively amplified. Therefore, the method of polymerase chain reaction can be used in laboratory diagnosis of glanders and melioidosis.

[The clinical picture and course of the nonerythematous form of ixodid tick-borne borreliosis]. / Klinika i techenie bezéritemnoi formy iksodovykh kleshchevykh borreliozov.

The territory of the Perm region is highly endemic in Ixodes (B. garinii and B. afzelii)-borne borreliosis. The clinical manifestations of erythema-less Ixodes-borne borreliosis were studied in the 1991-1994 epidemiological seasons. A total of 54 patients were examined in the acute stage of infection and 4-6 months later. There were common symptoms of damage to the central nervous system in early infection and pronounced signs of damage to the central and peripheral nervous systems in the late period which was characterized by cardiovascular diseases as disturbances of automatism functions, conduction, diffuse and local muscle changes.