A Complex of Pyrosequencing-Based Methods for Detection of Somatic Mutations in Codons 600 and 601 of the BRAF gene.

The aim of the study is to develop methods for the differentiation of mutations in the BRAF codon 600 and to increase the sensitivity of the K601E mutation detection. Materials and Methods: The nucleotide sequence of the BRAF codons 592-602 was identified using the PyroMark Q24 genetic analysis system. The mutations search in codon 600 was conducted using the 600-S primer in line with the following order of adding nucleotides: GCTGTCАTCTGCTAGCTAGAC (corresponding to nucleotides 1799-1786). The K601E mutation was detected using the 601-S primer in line with the following order of nucleotide addition: GCTACTCACTGTAG (corresponding to nucleotides 1801-1793). The analytical characteristics of the proposed methods for somatic mutations' detection were determined using dilutions of plasmid DNA samples containing the BRAF gene region without mutations or with one of the following mutations: V600E, V600R, V600K, V600M, and K601E. Validation was performed on 132 samples of biological material obtained from the thyroid nodules. Results: The developed methods allow to determine 2% of the V600E or V600M mutations, 1% of the V600K and V600R mutations, and 3% of the K601E mutations in samples with high DNA concentration; it is also possible to confidently detect at least 5% of the mutant allele for all mutations in low concentration samples (less than 500 copies/PCR). During biological material testing, 53 samples with the V600E mutation were detected; the proportion of the mutant allele was 4.9-50.0%. Conclusion: A complex of methods for determination of the nucleotide sequence of the BRAF codons 592-601 and the algorithm for testing samples and analyzing mutations in the BRAF codons 600-601 was developed. The method provides sufficient sensitivity to detect frequent mutations in codons 600 and 601 and allows them to be precisely differentiated.

Associations of Toll-like Receptor Gene Polymorphisms with NETosis Activity as Prognostic Criteria for the Severity of Pneumonia.

The aim of the study was to determine the molecular genetic prognostic criteria for the severity of the course pneumonia based on the analysis of the association of genetic polymorphism in toll-like receptors with the severity of NETosis. MATERIALS AND METHODS: The study included 38 patients with the main diagnosis of community-acquired pneumonia with a severe course. All the patients underwent standard clinical laboratory examinations, computed tomography of the thoracic organs, microbiological examination of blood and tracheobronchial aspirate. The level of neutrophilic extracellular traps (NETs) in blood smears was determined on the 1st-2nd and 5th-7th days of hospitalization. Genotyping of rs5743551 (TLR1), rs5743708 (TLR2), and rs4986790 (TLR4) polymorphic loci was performed by pyrosequencing. RESULTS: The level of NETs on the 1st day of admission was statistically significantly lower in heterozygous and homozygous carriers of rs4986790 (TLR4) polymorphism (AG and GG genotypes) compared with patients with the wild-type genotype (AA genotype) (p<0.05). When comparing the number of NETs with genotypes for rs5743708 (TLR2) and rs5743551 (TLR1) polymorphisms, no statistically significant correlation was found (p>0.05). The study of the NET level in dynamics demonstrated a decrease in the NETosis activity of neutrophils during the first week of hospitalization (p<0.05). The presence of the G allele in the patient's genotype for rs5743551 (TLR1) polymorphism increases the risk of a poor outcome of the disease (p<0.0001) (OR=20.3; 95% CI (4.3-135.0)). CONCLUSION: The obtained data suggest that level of NETs is a marker of the activity of neutrophils which are closely related to the studied genetic polymorphisms, and affects the prognosis of the pneumonia outcome.

Screening of somatic mutations in the JAK2 and CALR genes by high-resolution melting curve analysis.

Somatic mutations associated with oncological diseases, including Ph-myeloproliferative neoplasms (Ph-MPN), are very diverse, occur with different frequencies and different allelic burden levels. Therefore, at the initial stage of performing molecular-genetic diagnostic procedures, it is desirable to be able to conduct screening tests in the laboratory. This is especially important when analyzing rare and diverse mutations. Analysis of high resolution melting curves (HRM analysis), which has high sensitivity and is suitable for screening all types of mutations, in a number of studies is proposed for the analysis of Ph-MPN associated mutations in the JAK2 and CALR genes. For analysis of somatic mutations in the majority of literature sources that we reviewed, the authors use the LightCycler (Roche) thermocycler and much rarely the CFX96 (Bio-Rad), which is often presented in Russian scientific and practical and medical organizations. The aim of the study was to screen the somatic JAK2 and CALR mutations by HRM analysis using the CFX96 thermocycler and the Precision Melt Analysis software (Bio-Rad, USA) for patients with Ph-MPN. In the present research, HRM analysis was conducted on the DNA samples from patients with mutations in the JAK2 or in the CALR gene. The Precision Melt Analysis software identified all variants of the analyzed mutations, both a single nucleotide substitution in the JAK2 gene (with allelic burden level in the range of 5-40%), and various indel mutations in the CALR gene (with allelic burden level in the range of 40-50%) Therefore, the HRM analysis that was conducted on the CFX96 allows screening of highly specific mutation for the diagnosis of Ph-MPN in the exon 14 of the JAK2 gene and in the exon 9 of the CALR gene. The inclusion of this screening research in the laboratory testing algorithm improves the efficiency and accessibility of molecular genetic technologies in the diagnosis of Ph-MPN.

[A comparative analysis of allele frequencies of rs1801133 and rs1801131 of MTHFR in patients with stroke and healthy people from the Moscow region]. / Sravnitel'nyi analiz chastot allel'nykh variantov rs1801133 i rs1801131 gena MTHFR v vyborke patsientov, perenesshikh ishemicheskii insul't, i u zdorovogo naseleniia v Moskovskom regione.

AIM: To study genetic characteristics of the population of the Moscow region and analyze the association of rs1801133 and rs1801131 of MTHFR with the risk of ischemic stroke (IS). MATERIAL AND METHODS: A sample of 170 and 115 patients with atherothrombotic and cardioembolic subtypes of IS and 360 residents of the Moscow region without IS were examined. MTHFR alleles were determined by a multiplex real-time polymerase chain reaction. RESULTS AND CONCLUSION: No association between the frequencies of MTHFR alleles and the risk of ischemic stroke was found. The comparison of allele frequencies with those in Caucasian populations published in the dbSNP (NCBI) and 1000 Genomes Project databases revealed significant differences for rs1801133 from the EUR 1000 Genomes Project. The allele frequency data for MTHFR could increase the accuracy and reliability of the individual risk calculation for multifactorial diseases in the Russian population.

[Allele-specific polymerase chain reaction and electrophoretic detection in the detection algorithm clinically significant somatic mutations in the gene of calreticulin (calr).]

The detection of somatic mutations in the 9 exon of the calreticulin gene (CALR) is regulated by the clinical recommendations as a diagnostic criterion for chronic Ph-negative myeloproliferative neoplasms (MPN). Some methods of nucleic acids testing are used to identify CALR gene mutations with different requirements for special skills of personnel and expensive equipment. The purpose of this work is to compare the results of the detection of CALR gene mutations in venous blood samples by allele-specific RT-PCR with subsequent electrophoresis, fragment analysis and Sanger- or pyro- sequencing. We used 1284 blood samples of patients with suspected MPN and 20 blood donor samples. Mutations in the CALR gene of the I and II type were identified using PCR-RT with the original primers and TaqMan probes. Also, all samples were tested for mutations in the CALR gene by electrophoretic detection of PCR results in an agarose gel. The use of allele-specific RT-PCR followed by electrophoretic detection made it possible to determine clinically significant mutations in the CALR gene in 81 venous blood samples of JAK2- and MPL-negative patients, including 42 cases of type I mutation, 33 cases of type II mutation and 8 rare CALR mutations. Mutations in the 9 exon of the CALR gene were not detected in any of the 20 blood donor samples or in 121 blood samples of patients with polycythemia vera. In randomly selected 20 negative samples, CALR gene mutations were also not detected using Sanger sequencing. All positive samples were confirmed by fragment analysis, as well as with Sanger- sequencing and pyro- sequencing. The described combined approach to detect mutations of the CALR gene in peripheral blood samples can be used in clinical diagnostic laboratories that have a standard set of equipment for electrophoresis of nucleic acids and a PCR-RT. We also propose a confirmatory test based on the pyrosequencing of DNA using the system of genetic analysis "PyroMark Q24".

[The development and approbation of methodology on the basis of multiplex polymerase chain reaction in real-time to determine clinically significant micro-deletion in Y-chromosome.]

One of the prevalent genetic causes of idiopathic male sterility is related to micro-deletions in AZF locus located in Y-chromosome. In total population, rate of such micro-deletions makes up to 1:4000. however, in infertile males their rate varies from 2% to 10%. In AZF locus three subregions are distinguished: AZFa, AZFb and AZFc. The loss of one or several subregions can result in disorder of spermatogenesis of various degree - from decreasing of its activity to Sertoli-cell syndrome manifested by azoospermia or oligospermia of severe degree. Therefore, implementation of genetic testing for presence of micro-deletions in AZF locus is a necessary test in case of prognosis of male sterility and its treatment. The purpose of study is to develop and test a diagnostic system of detection of micro-deletions in subregions of AZF locus using multiplex polymerase chain reaction in real-time. As a reference method a technique was implemented described in guidelines of the European Academy of Andrology conjointly with European Molecular Genetics Quality Network. The technique testing specified analysis of 33 samples of DNA separated from blood of males with azoospermia and oligospermia of severe degree. No discordant results were received as compared with reference method. In 27 DNA samples the deletions were detected in AZF locus: 4 AZFa deletions (15%), 2 AZFb deletions (7%), 17 AZFc deletions (63%) and 6 combined deletions of AZFb+candи AZFa+b+с (22%). The proposed technique permits detect micro-deletions of subregions of AZF locus.

[The development and comparative approbation of methods of increasing sensitivity of detection of mutation V617F in gene JAK2 by pyro-sequencing].

The possibilities of early detection of chronic myelo-proliferative tumors (MPT) are determined by sensitivity of techniques implemented for finding somatic mutation V617F in gene JAK2. The mutation V617F can also be found in individuals without unfolded picture of hematological diseases. The detection of mutation even in low concentrations is associated with increasing of risk of cerebral stroke and thrombosis of arterial and venous vessels. The study was carried out to develop techniques based on COLD polymerase chain reaction and allele-specific polymerase chain reaction targeted to increasing of sensitivity of finding mutation V617F detected using pyro-sequencing. The analytical sensitivity of techniques was evaluated by control samples with different ratio of alleles. For allele-specific polymerase chain reaction analytical sensitivity amounted to 0.25% of mutant allele at concentration of analyzed control sample 10 copies of DNA per mkl. For COLD polymerase chain reaction sensitivity amounted to 0.5% at concentration 10 copies of DNA per mkl. The comparative approbation of techniques was implemented using clinical material obtained from 106 patients with suspicion on MPT. The analysis of clinical samples using COLD polymerase chain reaction revealed 13 (14%) and using technique of allele-specific polymerase chain reaction - 15 (16%) positive samples. In all 15 cases of detection of mutation clinical confirmations of diagnosis of MPT was received. The proposed techniques permit increasing efficiency of amplification of mutant DNA in analyzed samples and hence to increase sensitivity of subsequent analysis of products of amplification using technique of pyro-sequencing. Therefore, the mentioned techniques can be recommended to be applied for confirmation of diagnosis of MPT and early identification of individuals with increased risk of development of venous and arterial thromboses.

[Complex assessment of the contribution of genetic factors to the risk of ischemic stroke]. / Kompleksnaia otsenka vklada geneticheskikh faktorov v razvitie ishemicheskogo insul'ta.

AIM: To develop a method of the complex assessment of genetic risk for ischemic stroke (IS) and evaluate its effectiveness. MATERIAL AND METHODS: Genotyping of 182 patients with atherothrombotic and cardioembolic subtypes of IS and 360 healthy individuals of 48 single nucleotide polymorphic loci (SNP) associated with the risk of II and its subtypes was performed. RESULTS AND CONCLUSION: In each group of SNPs, composite indicators of genetic risk of IS in groups of patients and healthy controls were identified. Differences between the calculated values of the genetic risk in these groups were significant (p <0,05). The quality of the binary classification validated by ROC-analysis confirmed the predictive potential of the proposed method of risk calculation for determining the genetic predisposition to the development of IS.

[The comparison of three molecular genetic techniques for identifying major mutations in gene HFE related to development of inherent hemochromatosis.]

The three main mutations in gene HFE (C282Y, H63D, S65C) are the cause of development of 97% of cases of inherent hemochromatosis. It is known that about 85% of patients with inherent hemochromatosis are either homo-zygotic agents of mutation C282Y or carry compound-heterozygote C282Y/H63D. Therefore, the molecular genetic study intended for detection of these three mutations in gene HFE takes important place in diagnostic of inherent hemochromatosis. The study was organized to develop methods for detection of mutations C282Y, H63D, S65C on the basis of two molecular genetic methods - polymerase chain reaction in real-time and pyrosequenation. As reference method was used published method by Moyses C.B. et al. (2008). These methods were applied to analyzing 129 DNA samples. There were no discordant results. Among analyzed clinical DNA samples, mutant alleles of gene HFE were detected in 42 samples (32.5%)ю The mutation C282Y is detected in heterozygotic condition in 4 samples (3.1%); mutation H63D was detected in heterozygotic condition in 31 samples (24%) and in homo-zygotic condition in 4 samples (4%). The mutation S65C encountered in heterozygotic condition in one sample (0.8%) and in one sample compound-heterozygote H63D/S65C was detected (0.8%). The comparative characteristic of these three methods was made according the following parameters: time, number of analysis stages and convenience of interpretation of results. The main merit of method based on polymerase chain reaction in real-time is time of analysis implementation. The main merit of method based on pyrosequenation is automatic identification of genotype.

[Real-time pcr procedure for determination of Streptococcus pneumoniae serotypes].

AIM: Development and application of real-time PCR (RT-PCR) procedure for determination of Streptococcus pneumoniae serotypes. MATERIALS AND METHODS: 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. RESULTS: 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). CONCLUSION: 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.

[A method for determination of Neisseria meningitidis serogroup A, B, C and W by real-time PCR].

AIM: Development and testing of a real-time PCR method for detection of Neisseria meningitiis serogroup A, B, C and W DNA. MATERIALS AND METHODS: 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: RESULTS: 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. CONCLUSION: 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.

[The quantitative testing of V617F mutation in gen JAK2 using pyrosequencing technique].

The somatic mutation V617F in gen JAK2 is a frequent cause of chronic myeloprolific diseases not conditioned by BCR/ABL mutation. The quantitative testing of relative percentage of mutant allele can be used in establishing severity of disease and its prognosis and in prescription of remedy inhibiting activity of JAK2. To quantitatively test mutation the pyrosequencing technique was applied. The developed technique permits detecting and quantitatively, testing percentage of mutation fraction since 7%. The "gray zone" is presented by samples with percentage of mutant allele from 4% to 7%. The dependence of expected percentage of mutant fraction in analyzed sample from observed value of signal is described by equation of line with regression coefficients y = - 0.97, x = -1.32 and at that measurement uncertainty consists ± 0.7. The developed technique is approved officially on clinical material from 192 patients with main forms of myeloprolific diseases not conditioned by BCR/ABL mutation. It was detected 64 samples with mautant fraction percentage from 13% to 91%. The developed technique permits implementing monitoring of therapy of myeloprolific diseases and facilitates to optimize tactics of treatment.

[The detection of activating somatic mutations in gene KRAS using pyrosequencing technique].

The technique to detect all possible variants of mutations in 12, 13 and 15 codons of gene KRAS was developed on the basis of the pyrosequencing technology. The analytical characteristics of the developed technique were identified. The limit of detection for mutations G34T, G35A and G38A detected on the cloned control samples consisted 3%. The limit of blank for various mutations consisted from 0.3% to 4.1%. The system was tested on clinical samples. The 7 different types of mutations were identified and detected in quantitative format. No discrepancy of pyrosequencing data with results of sequencing according Sanger was established.

[Polymorphism of ABCB1 and ABCG2 genes encoding drug transporters and their investigation by pyrosequencing].

Published data on the effect of transporter propeins of the ABC family in the features of drug pharmacokinetics in the human organism are reviewed. Clinical significance of mononucleotide polymorphisms in ABCB1 and ABCG2 genes is analyzed. A set of genetic tests based on pyrosequencing for determing the allele state in rs1128503 (1236 T > C), rs2032582 (2677T > A/G), rs1045642 (3435 T > A/C), rs2231142 (421C > A), and rs72552713 (376 C > T) polymorphisms in ABCB1 and ABCG2 genes has been developed and verified. These tests can be used in both research work and clinical practice for calculating recommended doses of various drugs, predicting their therapeutic efficacy, and determining groups of risk with respect to the developent of undesired secondary reactions.

[Genetic characteristics of Neisseria meningitidis strains obtained from healthy carriers during meningococcal infection outbreaks].

AIM: Genetic and antigenic characterization of Neisseria meningitidis strains isolated during meningococcal infection outbreaks from individuals in contact with patients with generalized form of meningococcal infection. MATERIALS AND METHODS: Strains obtained in 2007 - 2009 in Moscow during examination of individuals that were in contact with patients during meningococcal infection outbreaks were analyzed. Multilocus sequence typing, genetic subtyping and typing of VR fragment (FetA) techniques were used. RESULTS: Data regarding investigated strains were submitted to the database at http://pubmlst.org/neisseria/. Previously undescribed sequence types were found in 12 strains, sequence-type could not be determined in 2 strains, 2 strains lacked VR fragment (FetA). Serogroup A meningococci had "P1.5-2,10: F3-5" antigenic profile and belonged to ST-75 and ST-3349 sequence-type, these data does not support the emergence of epidemically significant strains in the territory under surveillance. All typed serogroup C strains and 1 serogroup B strain are of "ST-41/44 complex/Lineage 3" clonal complex. Subtypes of serogroup C meningococci strains match subtypes of strains that cause generalized forms of infection, while serogroup B strains isolated from the carriers and strains isolated from the patients had different antigenic profiles. Ungrouppable strains had notably higher level of genetic and antigenic diversity: only 6 of 16 strains (37.5%) could be sequence-typed using earlier data, all these strains are of clonal complex "ST-53 complex" that consists mostly of strains isolated from the carriers. CONCLUSION. Ratio of meningococci population circulating in Moscow and subpopulation capable of causing generalized form of meningococcal infection (GFMI) is different for meningococci of various serogroups. Ungrouppable strains isolated from the carriers are highly different from strains causing GFMI.

[Genetic characteristics of Haemophilus influenzae type b isolated from children with bacterial meningitis in Moscow in 2007-2009].

AIM: Genetic characterization of 37 strains and CSF samples containing DNA of Haemophilus influenzae type b isolated in Moscow during 2007-2009. MATERIALS AND METHODS: Multilocus sequence-typing method was used and also variant of method for capsule type determination was approbated. RESULTS: 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%]). CONCLUSION: 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.

[Genetic characteristic of Haemophilus influenzae type B strains isolated in Russian regions].

AIM: 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. MATERIALS AND METHODS: Genetic characterization of 31 strains of Hib isolated in Russian regions during 2005-2008 was performed by multilocus sequence typing. RESULTS: 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. CONCLUSION: Clonal structure of isolated strains is analogous to the one observed in Moscow and foreign strains.

[Monitoring for Neisseria meningitidis species using sequencing of variable fragments of surface proteins FetA and PorA genes].

AIM: 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. MATERIALS AND METHODS: Method of typing of "VR" fragment of FetA protein together with methods of genetic subtyping and multilocus sequence typing was used. RESULTS: 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. CONCLUSION: 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.

[Genetic subgroups of Neisseria meningitidis serogroup A isolated from patients with disseminated forms of meningococcal infection in Moscow, 1969-2006].

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.

[Genetic relationships of Moscow and foreign strains of Haemophilus influenzae, serotype B].

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.