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Determination of the degree of phylogenetic relationship of Yersinia pestis strains iso- lated from the territories of natural foci of plague from the Caucasus using VNTR-typing by 25 loci (MLVA25).26 strains of Y pestis from Russian natural foci of the Caucasus were used in the study. 25 loci of tandem repeats in Y pestis genome by Le Fleche scheme were used for execution of multi-locus VNTR-analysis. Deciphering of nucleotide sequences was carried out in automatic sequencer ABI 3130 Genetic Analyser. Analysis of confinement of clusters to certain territories, objects and time of isolation of strains was carried out. using Arc GIS 10.1 program.Groups of MLVA25-types of various levels of discrimination were formed: clusters, groups and subgroups. Clusters were formed by strains ofvarious taxonomic membership: main and subspecies of Y pestis. Subgroups reflect membership of strains in certain foci, and MLVA25-types - the degree of genetic relationship.Geneticportraitsof plague causative agents obtained using MLVA25-types circulating in various natural-focal territories allow to solve problems ofboth theoretical and practical character: from interpretation of microevolution processes to the search of the source of infection and ways of its spread during possible epidemic complications.

Development and testing of a real-time PCR method for detection of Neisseria meningitiis serogroup A, B, C and W DNA.Reference strains and 187 samples of cerebrospinal fluid (CSF) from meningocci meningitis patients were used in the study. Multiplex PCR was carried out in an instrument with 5 channels of fluorescent detection:Analysis of specific serogroup loci of the genome and design of oligonucleotides for the detection of DNA of all the capsule meningococci and 4 serogroups in particular was carried out. PCR conditions were optimized; specificity was shown and analytical sensitivity was evaluated using reference strains. DNA of the following serogroups was detected during study of clinical CSF samples: A--in 103 samples (55%), B--in 45 (24%), C--in 30 (16%), W--in 5 (3%). Only DNA of meningococci capstle gene ctrA was found in 4 samples; presumably, they contained DNA of other serogroups. Multilocus sequence-typing and detection of antigenic determinants of PorA and FetA genes for 27 DNA samples of group A menincococci as well as DNA of 5 group W meningococci and 4 ungroupable was carried out.The method proposed allows to carry out serogrouping of no less than 95% of strains or DNA samples isolated from CSF of meningococci infection patients. Combined with other recommended non-cultural methods of genotyping, it may be useful for complex characteristics of pathogenic meningococci.

Development and application of real-time PCR (RT-PCR) procedure for determination of Streptococcus pneumoniae serotypes.S. pneumoniae cps-locus wzx, wzy, wzz, wcwV and galU genes were chosen as PCR targets to select serotype-specific oligonucleotide primers and fluorescent labeled probes. 89 samples of cerebrospinal fluid (CSF) obtained in 2007 - 2010 from patients with pneumococcal meningitis diagnosis undergoing therapy in the Infectious Clinical Hospital No. 2, Moscow, were studied with the aim of testing the possibility of practical use of RT-PCR.Primers and probes were selected for the determination of 16 vaccine and/or frequently encountered serotypes distributed among 4 reaction mixtures also including a pair of primers and a probe for cpsA gene detection that is present in all the capsule pneumococci (internal control). The procedure was tested on a collection of 108 pneumococci strains gathered in Research Institute of Antimicrobial Therapy and serotyped earlier by specific PCR with electrophoretic detection and serologically by using Pneumotest-Latex kit. The sensitivity and specificity of the RT-PCR was 100%. RT-PCR procedure allowed to determine pneumococcus serotype in 79% of CSF clinical samples containing S. pneumoniae DNA. Serotype 3 and 23F were detected most frequently (13%, each).RT-PCR application does not assume causative agent seeding stage, significantly reduces analysis execution time and increases sensitivity of the study. The developed procedure will allow to begin addressing the important problem--clarification of spectra and frequency of occurrence of pneumococci serotypes circulating on the territory of Russia.

Characteristic of West Nile fever (WNF) virus strains circulating in southern Russia.WNF RNA was amplified directly from clinical samples, mosquitoes and bird tissues by PCR, nucleotides were sequenced directly and analyzed comparatively.Related but different genovariants of WNF lineage 1a--"Volgograd" and "Astrakhan"--circulated during WNF outbreaks of 1999 and 2000-2003 in Volgograd and Astrakhan regions. In 2005 "Volgograd" WNF variant emerged in Astrakhan region and along with "Astrakhan" variant caused a new morbidity increase. In 2004 in sera of 2 WNF patients from Rostov region WNF lineage 2 RNA was detected, this was the first WNF clinical case caused by WNF lineage 2 outside of Africa. WNF outbreak in Volgograd region in 2007 was caused by this unique WNF lineage that may preliminary be called Russian. Finally, during a major WNF outbreak in 2010 in Volgograd and Rostov regions in clinical samples only russian genovariant WNF lineage 2 RNA was detected again.After emergence of a certain WNF genovariant the virus is capable of persisting in natural foci in southern Russia. A near disappearance of one of the WNF clones by substitution or displacement with another maybe possible. Determination of genetic characteristics of WNF strains circulating in Russia is an important element of WNF epidemiological surveillance and control of this disease.

Genetic characterization of 37 strains and CSF samples containing DNA of Haemophilus influenzae type b isolated in Moscow during 2007-2009.Multilocus sequence-typing method was used and also variant of method for capsule type determination was approbated.Ten sequence types, of which 7 were described in previous studies and 3 were revealed for the first time during this work, were detected in studied sample. ST-6 and ST-92 were the most frequently detected--9 strains (24%) of Hib belonging to each sequence type were revealed. All detected sequence types, except one, belong to clonal complex "ST-6" ("A1/A2"). Obtained data were compared with results of typing of Hib strains isolated in Moscow in 1999-2001. Genetic changes in studied population of Hib are characterized by decreased proportion of Hib belonging to ST-6 (from 54% to 24%) and increased number of sequence types belonging to clonal complex "ST-6" differing from ST-6 on more than one locus of allelic profile (from 2 types [2 strains, 5.4%] to 5 types [9 strains, 24%]).In 2007-2009, number of Hib strains with sequence type ST-95 (7 strains, 19%), which is typical for strains circulating in Russia, is markedly increased. Capsule type I was detected in 32 (86.5%) of studied strains, whereas capsule type II--in 5 (13.5%) of studied strains. Capsule type II was detected only in Hib strains with ST-80 sequence type.

To create and test the complex of polymerase chain reaction-based methods for detection of pathogens vectored by ticks in clinical and environmental samples.Real time PCR methods with hybridization-fluorescent detection were developed for detection of tick-borne encephalitis virus, Borrelia burgdorferi sensu lato, Anaplasma phagocytophillum, Erlichia muris/E. chaffeensis, and B. miyamotoi. First four methods were combined in one assay in multiprime format. Efficacy of the assay was assessed by testing of blood samples from patients with tickborreliosis (166 patients), tick-born encephalitis (22 patients) and mixed infection tick-borne encephalitis + borreliosis (21 patients) from Sverdlovsk region.It was shown that using PCR-based assay for testing the blood samples obtained during admission, it was possible to determine the etiology of disease in 39% of patients, whereas on the basis of serological data diagnosis, as a rule, is made not earlier than on 2nd week of therapy. False-positive results of PCR diagnostics were not observed. Infections caused by Anaplasma or Erlichia were not observed. It was shown that50% of cases of tick borreliosis without erythema were caused by B. miyamotoi, whereas B. burgdorferi sensu lato predominated as a causative agent of erythemic form of borreliosis.Proposed complex of methods is useful for rapid diagnostics of tick-borne infections including previously unknown infection caused by B. miyamotoi.

Genotyping of Hib strains isolated in regions of Russia as well as characterization of genetic relations of typed strains with strains isolated in other areas.Genetic characterization of 31 strains of Hib isolated in Russian regions during 2005-2008 was performed by multilocus sequence typing.Studied strains belonged to 11 variants of sequence types, 6 of which were described in previous studies, whereas other 5 were isolated for the first time during this study. The most common isolated strains were ST-92 (13 strains or 42%) and ST-6 (6 strains or 19%). Typed strains were distributed to two clonal complexes. Clonal complex "A1/A2" ("ST-6") incorporates all typed strains except ST-93 strain belonging to clonal complex "B1b" ("ST-93"). The majority of studied strains (19 or 61%) had difference from "central" sequence type of clonal complex, A1/A2 ("ST-6") on not more than one allele.Clonal structure of isolated strains is analogous to the one observed in Moscow and foreign strains.

To perform advanced antigenic characterization of meningococci belonging to serogroups A and B and circulating in Moscow according to modern nomenclature of Neisseria meningitidis strains.Method of typing of "VR" fragment of FetA protein together with methods of genetic subtyping and multilocus sequence typing was used.Detailed information about studied strains was inputed in Internet database--http://pubmlst.org/neisseria/. Typing of serogroup B strains did not allow to define dominating variant of "VR, fragment of FetA protein which is in accordance with subtyping data obtained previously. Serogroup A strains were notable for less variability of "VR" fragment variants: 6 variants were detected. For the majority of serogroup A strains, it was possible to trace connection between belonging of the strain to particular genetic subgroup and its revealed antigenic profile. For strains from genetic subgroup VI, antigenic profile P1.5-2, 10; F1-5 detected in 14(18%) strains was typical, whereas antigenic profile P1.5-2, 10; F3-5 was typical for genetic subgroup X and was detected in 50 (63%) strains. Antigenic profile P1.5-2, 10-67; F3-5 was detected in 5 (6%) strains, and other 10 antigenic profiles were revealed in one strain each.Prevalence of strains with antigenic profile P1.5-2, 10; F3-5 is explained by change of predominant genetic subgroup from subgroup VI to subgroup X in Moscow population serogroup A meningococci observed after 2003.

The aim of the study was to perform molecular genetic analysis based on multi-locus sequence typing in order to identify source of Legionnaires' disease outbreak in town Verkhnyaya Pyshma in July 2007 and genetic profile of the causative agent. Sequence-based typing protocol recommended by European Working Group on Legionella infection (EWGLI) was used. It was not possible to obtain satisfactory results of Fla gene sequencing for all samples. Obtained allelic profiles of other genes were typical for L. pneumophila. Allelic profiles of L. pneumophila isolated from patients were identical and matched with L. pneumophila DNA detected in water from hot water supply of domestic building, but differed from cooling tower's isolates and isolates from showerhead in apartment of one patient. Identity of 5 genes of L. pneumophila isolated from autopsy samples and from hot water of central hot water supply of domestic building confirms aspiration route of infection through hot water contaminated by the microorganism. L. pneumophila detected in water from cooling tower, showerhead in apartment of one patient, and from drainage canal of hot water supply station belonged to other allelic variants and, therefore, are not related with the outbreak.

Results of microbiological monitoring for serogroup A Neisseria meningitidis circulated in Moscow from 2002 to 2006 are presented. Using multilocus sequence-typing, molecular and epidemiologic characteristics of 32 cultures isolated from cerebro-spinal fluid of patients with generalized forms of meningococcal infection. Typed isolates belonged to 4 sequence types: CT-3349 (detected in 24 cultures), CT-2 (detected in 5 cultures), CT-75 (detected in 2 cultures), and CT-5803 (detected in 1 culture). All sequence types (except CT-5803) were detected in Moscow in previous years. Using Internet database (http://pubmlst.org/neisseria) they were genetically characterized and compared with data on serogroup A meningococci circulated in Moscow before 2002., meningococci belonging to epidemically dangerous genetic subgroup III were not detected between characterized strains. Typed isolates were distributed between subgroups VI and X, which are typical for the area under surveillance. Genetic changes in Moscow population of Neisseria meningitidis serogroup A, which manifested by shift of dominating genetic subgroup after 2002-2003, were analyzed.

46 Haemophilus influenzae strains of serotype b, isolated on the territory of Moscow and compared with foreign strains, were characterized by the method of multilocus sequencing-typing. Among the strains circulating in Moscow 10 variants of sequence-types were observed; of these, only one variant (CT-6) had been described earlier. The analysis of the data revealed that the strains under study were distributed in two clonal complexes; the overwhelming majority of the strains belonged to sequence-type 6. The distribution of Moscow strains in clonal complexes repeated the clonal organization detected in foreign strains. The conclusion was made that lower morbidity rate in Hib meningitis in Moscow (in comparison with that in the countries of Europe and America before the introduction of prophylactic vaccination) was not due to the peculiar genetic features of the circulating strains.

The analysis of meningococcal strains of different serogroups, isolated from the liquor of patients in Moscow, which was carried out with the method of multilocus sequencing-typing (MLST), was presented. At the periods of epidemic morbidity rises in Moscow the prevalence of group A meningococcal strains, belonging to subgroups III with sequence-types 5 (in the 1970s) and 7 (in 1996), was noted, and at a period between epidemics strains of genetic subgroups VI and X were isolated. Meningococcal strains, groups B and C, isolated in 1995 - 2002, had, as a rule, unique sequence-types, differing both one from another and from N. meningitidis sequence-types detected in other countries. Among group B meningococci the prevalence of strains belonging to clonal complex ST-18 was noted, while for group C meningococcci strains belonging to clonal complex ST-41/44 were most typical. Such genetic variability of circulating meningococci was regarded as characteristic of the period between epidemics, observed in Moscow since the end of the 1980s.

32 Russian patients with late complement component deficiency (LCCD) were immunize with tetravalent meningococcal polysaccharide vaccine (A + C + W135 + Y). Their immune response and infectious morbidity rate were followed for 6 years and the partial protective efficacy of vaccination was demonstrated. As the antibody-mediated complement-induced bactericidal activity of plasma was completely absent in persons with LCCD, the bactericidal action of human neutrophils on meningococci of groups A, W135 and B was studied under the conditions of incubation with serum samples collected from persons with LCCD before and after vaccination. In LCCD serum alone the exponential growth of meningococci was observed, while the addition of human neutrophils resulted in the essential inhibition of the growth of meningococci (up to their complete elimination). The proportion of serum samples stimulating the elimination of group A and W 135 meningococci by neutrophils was almost 40% of the serum samples collected before vaccination and significantly increased among the serum samples collected after vaccination (up to 84%) or revaccination (up to 90%). At the same time the capacity of an individual serum sample to promote the bactericidal effect of neutrophils against meningococci correlated with its content of specific anti-polysaccharide IgG and IgM antibodies, as well as antibodies to the inner core of lipopolysaccharide. The interaction of neutrophils with meningococci was significantly inhibited after incubation in heat-inactivated serum, suggesting that this interaction was partly mediated along the following path: the binding of IgM and IgG antibodies with bacteria--the activation of complement and the deposition of C3 complement on bacteria--the binding of meningococci with CR3 receptors of neutrophils.

The samples of spinal fluid arriving to the Clinical Infectious Hospital in 1994-1996 with the clinical diagnosis "generalized form of meningococcal infection" or "purulent meningitis of unclear etiology" were studied. The etiological agent was bacteriologically identified in 35% of 487 patients (in 25% of cases Neisseria meningitidis, in 7% of cases Streptococcus pneumoniae and in 2% of cases Haemophilus influenzae, type b, were detected). The method of latex agglutination, used in this study, was highly specific (100%) and moderately sensitive (67%); this method made it possible to diagnose 25% of cases additionally (N. meningitidis in 15% of cases, S. pneumoniae in 5% of cases and H. influenzae in 3% of cases). Diagnostics with the use of PCR was characterized by high specificity (97%) and sensitivity (85%) relatively to the "golden standard" of microbiological diagnostics. There were few false positive results (3 samples), caused probably by contamination at the moment of taking the samples. For this reason the results obtained by PCR could be used for diagnostic purposes even in cases of negative results given by other methods. Tests with the use of PCR made it possible to diagnose 29% more cases additionally (in 26% of cases N. meningitidis DNA and in 3% of cases S. pneumoniae DNA were detected. Thus the complex of methods used in this study permitted the detection of the etiological agent altogether in 87% of cases.