Vol 91, No 2 (2014)

Aim. Carry out comparative analysis of survival of typical strains and genovariants of V. cholerae biovar El Tor imported in different years to the territory of Russian Federation, in the absence of nutrients and under the conditions of temperature stress. Materials and methods. 24 V. cholerae biovar El Tor strains isolated in 1970 - 2011 were studied, 8 of those were typical isolates and 16 - genetically altered variants. Strain survival was studied in 0.9% sodium chloride solution and autoclaved river water at various temperature modes (5, 25, 37 and 42°C). Protein composition and exopolysaccharide production were determined by electrophoresis method by U.K. Laemmli. Results. Genovariants as well as typical strains were shown to be able to exist for a long time (up to 5 months) in the absence of nutrients at the temperature of 25°C. However, unlike typical eltor vibrios, genovariants were more resistant to temperature stress. As a result of adaptation to high temperature (42°C) biosynthesis of porin proteins of outer membrane OmpU and/or OmpT is increased in genovariant cells, and at lower temperatures (5°C) - exopolysaccharide. Conclusion. V. cholerae biovar El Tor genovariants are able to adapt to temperature change better, that may facilitate their higher survival in the environment.

Aim. Establishment of ratios that would allow to execute recalculation of mycoplasma concentration from CFU/ml and/or CCU/ml into units obtained during PCR analysis - geq/ml. Materials and methods. Pure cultures of Mycoplasma hominis, Ureaplasma parvum and Ureaplasma urealyticum were studied by cultural and molecular-biological methods with quantitative evaluation. Studies of initial cultures as well as series of 10-fold dilutions were carried out. 32 experiments in total were carried out. Results. Ratio between geq/ml and CFU/ml for M. hominis was 3.5; geq/ ml and CCU/ml ratio - 4.4. Ratio between geq/ml and CCU/ml for U. parvum was 7.1; for U. urealyticum - 11.2. Conclusion. Ratios between indexes obtained during quantitative study of pure genital micoplasma cultures by using 2 methods were established.

Aim. Development of an algorithm of genetically altered Vibrio cholerae biovar El Tor strain identification that ensures determination of serogroup, serovar and biovar of the studied isolate based on pheno- and genotypic properties, detection of genetically altered cholera El Tor causative agents, their differentiation by epidemic potential as well as evaluation of variability of key pathogenicity genes. Materials and methods. Complex analysis of 28 natural V. cholerae strains was carried out by using traditional microbiological methods, PCR and fragmentary sequencing. Results. An algorithm of toxigenic genetically altered V. cholerae biovar El Tor strain identification was developed that includes 4 stages: determination of serogroup, serovar and biovar based on phenotypic properties, confirmation of serogroup and biovar based on molecular-genetic properties, determination of strains as genetically altered, differentiation of genetically altered strains by their epidemic potential and detection of ctxB and tcpA key pathogenicity gene polymorphism. The algorithm is based on the use of traditional microbiological methods, PCR and sequencing of gene fragments. Conclusion. The use of the developed algorithm will increase the effectiveness of detection of genetically altered variants of the cholera El Tor causative agent, their differentiation by epidemic potential and will ensure establishment of polymorphism of genes that code key pathogenicity factors for determination of origins of the strains and possible routes of introduction of the infection.

Aim. Study features of epidemic process and etiology of oral cavity and limb enterovirus exanthema group diseases in a number of territories of Northwestern Russia. Materials and methods. Isolation and identification of non-poliomyelitis enteroviruses from material of patients was carried out according to WHO recommendations. Phenotyping and phylogenetic analysis of enteroviruses was carried out. Results. In 3 territories of Northwestern Russia oral cavity and limb enterovirus group diseases were registered. Children aged less than 14 years, predominately aged less than 3 years, were shown to be involved in the epidemic process. Coxsackie A16 enteroviruses from 27 samples of patients were isolated in cell cultures and identified by using specific sera. Coxsackie A16 enteroviruses from 16 samples were identified by using partial sequencing of VP1 genome area. Phylogenetic analysis has shown that the identified Coxsackie A16 viruses distributed among 2 phylogenetic groups. Conclusion. Coxsackie A16 enteroviruses that had never been detected in the region previously were established to be the etiologic factor of oral cavity and limb enterovirus exanthema group disease in the 3 territories of Northwestern Russia. The data obtained give evidence on the necessity of epidemiologic and virological control for enterovirus infection with the aim of obtaining novel information on the circulation of non-poliomyelitis enteroviruses in the population and the establishment of development patterns for epidemic process of this infection.

Aim. Genotyping of noroviruses that had circulated in the territory of Nizhny Novgorod during 6 epidemic seasons (2006 - 2012), detection of dominating genovariants and analysis of their change. Materials and methods. Feces samples from children hospitalized in an intestinal infection department of one of the infectious disease hospitals of Nizhny Novgorod served as material for the study. Noroviruses were detected by reverse transcription polymerase chain reaction. Genotypes and gene variants were determined by analysis of nucleotide sequences of viral genome regions coding capsid protein and RNA-dependent RNA-polymerase. Results. During examination of 6589 children with an acute intestinal infection between July 2006 and June 2012 noroviruses were detected in 17.55% of cases. Nucleotide sequences of capsid and/or polymerase gene regions were determined for 114 norovirus isolates. Genotyping has shown that noroviruses of 8 various genotypes had circulated in the territory of Nizhny Novgorod - GII.1, GII.2, GII.3, GII.4, GII.6, GlI.7, GII.12, GII.13 with the domination of GII.4 noroviruses for the whole observation period. A dynamic of change of epidemic variants of genotype GII.4 noroviruses that had been accompanied by an increase of frequency of detection of norovirus in children hospitalized with acute intestinal infection similar to global was established. A short-term circulation of GII.4 2006b-NN 2008 norovirus subvariant in spring of2008 and spread of genotype GII.12 norovirus during 2009, 2010 epidemic season were also shown. Conclusion. The data obtained give evidence to the necessity of norovirus circulation monitoring with the aim of early detection of novel virus variants that may determine an increase of norovirus infection morbidity.

Aim. Study some immunological indexes in children with allergic diseases depending on body weight and clinical manifestations of allergy. Materials and methods. A correlation analysis of relationship of indexes of natural resistance (phagocytosis, complement), immunoglobulin level and main lymphocyte populations with body weight in 214 children aged 12 - 17 years with various allergic diseases (rhinitis/ rhinoconjunctivitis, atopic dermatitis, bronchial asthma) was carried out. The children were divided into groups based on body mass index (BMI): 73 (34%) children with normal weight, 74 (35%) overweight and 67 (31%) obese. Results. The analysis has shown that the frequency of detection of children with obesity is the highest for age 12 - 14 years. With the increase of age the number of obese children decreases (OR - 9.0; 95% CI: 1.56 - 51.87; p=0.008 and OR - 0.27; 95% CI: 0.08 - 0.94; p=0.04, respectively). An interrelation of BMI with clinical exacerbations of allergy was detected. Out of 166 patients with allergic diseases combined with bronchial asthma excessive weight was detected in 62 (37%), obesity - in 57 (34%) and normal weight - in 47 (28%). In a group of 48 children with allergy without asthma excessive weight was noted in 12 (25%), obesity - in 10 (21%) and normal weight - in 26 (54%) of patients. In children with bronchial asthma excessive weight occurs almost 3 times more frequently than in children with allergy and without asthma. Differences could not be detected in 3 groups of children when immune status indexes were compared, except total IgE and NK cell levels. Total IgE level was the highest in obese children (2.7 log), differed significantly from the level in obese (2.46 log) and normal weight (2.37 log, r=0.32, p<0.05) children. The relative content of NK cells in blood of obese children was significantly higher than in children with normal and excessive weight (r=0.41). The analysis of significant correlation coefficient indexes detected correlative associations of some immunological indexes with BMI. In overweight children a negative relation between the level of complement and BMI (r=-0.61) and positive relation with phagocytosis index (r=0.58) were detected. Conclusion. Obesity in children with allergic diseases is associated with an increase of conjugation of immunological indexes manifesting in an increase of number of natural killers (NK), phagocytosis indexes, increased total IgE level against the background of negative interrelation with the main populations of lymphocytes, that in general influences aggravation of allergopathology in the form of a higher frequency of detection of atopic bronchial asthma.

Aim. Study features of pathogenetic characteristic changes of infection caused by hepatitis C virus (HCV) in individuals that belong to different groups with high risk of parenteral infection (GHRPI). Materials and methods. 3219 blood sera obtained from individuals of 5 various GHRPI and 1541 unpaid blood donors were studied for the presence of anti-HCV. Anti-HCV-positive sera were studied by using polymerase chain reaction to detect HCV RNA in qualitative and quantitative variants. Results. By using molecular-genetic methods the presence of HCV RNA and its concentration in HCV antibody-containing blood sera obtained from individuals of 5 various GHRPI and a group of unpaid donors infected early by HCV was determined. Distinctive features that characterize the process of HCV infection natural evolution in individuals from various GHRPI were established to be lower frequency of spontaneous virus elimination and higher frequency of acute infection chronization, as well as a relatively higher viral load. Conclusion. The regularities detected indicate a higher level of epidemiologic danger for individuals from GHRPI.

Aim. Conduction of a comparative proteomic mass-spectrometric (MS) analysis using a personal database of V. cholerae protein mass-spectra and genetic VNTR-typing of cholera causative agent strains. Materials and methods. V. eholerae О1 El Tor strains - 7, V. eholerae non O1/non O139 - 2. Protein profiling and VNTR-genotyping of strains was carried out on MALDI TOF-MS Autoflex (Bruker Daltonics) mass-spectrometer and SIS Cholera-strains-VNTR. Results. The established community of a proteomic profile of epidemic cholera vibrio strains isolated in 2010 - 2012 in Moscow allowed to determine Indian origin of a toxigenic strain isolated in Taganrog in 2011. M/z proteins distinguishing V. cholerae О1 and non O1/non O139 strains were identified. Proteomic analysis confirms the results ofVNTR-genotyping. Conclusion. Study and typing of V. cholerae members with determination of their origin and phylogenetic relationship is possible using a collection of V. cholerae mass-spectra.

Main scientific-research studies regarding protein bacterial toxins of the most widespread bacteria that belong to Staphylococcus spp. genus and in particular the most pathogenic species for humans - Staphylococcus aureus, are analyzed. Structural and biological properties of protein toxins that have received the name of staphylococcus pyrogenic toxins (PTSAg) are presented. Data regarding genetic regulation of secretion and synthesis of these toxins and 3 main regulatory genetic systems (agr - accessory gene regulator, xpr - extracellular protein regulator, sar - staphylococcal accessory regulator) that coordinate synthesis of the most important protein toxins and enzymes for virulence of S. aureus, are presented.