[Protective properties of recombinant IgA1 protease from meningococcus].

The study of enzymatic and protective properties of recombinant IgA1 protease in active and mutant form showed that active form of IgA1 protease exhibited species - and type-specificity for mouse and human immunoglobulins. Mutant form, which did not exhibit enzymatic activity, had protective properties against meningococcal infection, induced by meningococcus serogroup A, B and C protecting the mice from lethal infection by living virulent culture of heterologous serogroups of meningococcus. Obtained results make it possible to consider IgA1 protease as a perspective preparation at the stages of development of polyvalent vaccine for protection the people from meningococcal infection of various etiology.

[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.

[Use of several parametric characteristics for study of epidemic features of meningococcal infection and bacterial meningitis].

UNLABELLED: Continuous surveillance for main indicators of epidemic process of meningococcal infection and etiology of bacterial meningitis. Epidemiologic surveillance was electronically-based and was performed on the basis of specially developed standardized case reporting form. Main parameters of epidemiologic surveillance included 10 indexes. Information on 18,519 cases was obtained from 47 regions of Russian Federation. Organization of personalized registry of patients with meningococcal infection and bacterial meningitis allowed to reveal etiologic structure of the disease, territorial differences in serogroup landscape of clinical isolates of meningococci, shortcomings in laboratory diagnostics as well as features of meningitis caused by pneumococci and Haemophilus influenzae type b. CONCLUSION: Necessity of continuous epidemiologic surveillance for meningococcal infection and bacterial meningitis with mandatory use of modern methods of laboratory diagnostics as well as organization of official registration system for meningitis caused by pneumococci and Haemophilus influenzae type b providing decrease of morbidity and development of vaccination tactics was substantiated.

[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.

[Growth medium for Neisseria meningitidis isolation].

Growth medium for isolation of N. meningitidis, which do not require addition of serum and based on previously developed medium for cultivation of bacteria from Haemophilus genus (without growth factors V and X) was constructed. Selective properties of the medium in relation to meningococci were determined by addition of vancomycin and colistin--antibacterial supplement inhibiting growth of nonpathogenic Neisseria and outside microflora. Developed medium was successfully approved during examination of children for nasopharyngeal carriage of meningococci.

[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.

[Analysis of the Moscow population of Neisseria meningitidis strains by the method of multilocus sequencing-typing].

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.

[Detection of antibiotic contents in cerebrospinal fluid]. / Opredelenie soderzhaniia antibiotikov v spinno-mozgovoi zhidkosti.

The suggested method provides for the semi-quantitative detection of antibiotics in liquor made for the purpose of prognosticating the possibility of cultural secretion of infectious agents of purulent bacterial meningitis (PBM); it is also a basis for the need in using the non-cultural diagnostic tools. The method can be used in detecting the pharmacokinetic specificity of antibacterial drugs now in use and of their combinations with the aim of making corrections in antibiotic therapy. It is based on inhibiting the surface growth of test-culture by a liquor-dissolved antibiotic with the growth-delay zone being shaped. The preparation concentration is estimated in U/ml of the penicillin equivalent (PE). Examinations of 131 liquor samples (primary puncture) obtained from patients with meningitis showed clear-cut correlations between the antibiotics' concentrations, cultural verification of the PBM diagnosis and the efficiency of non-cultural examinations. 82.14% of cultures were derived from liquor samples with the negative antibacterial activity (range 1); 7.14% of cultures were derived from liquor samples containing the antibacterial preparation of up to 0.5 U/ml PE (range 2); culture of secondary PBM agents (S. aureus) were isolated from liquor samples containing up to 10 U/ml PE (range 3); and no microbe agents were isolated from liquor samples containing above 10 U/ml PE (range 4). The designed method, when used, demonstrated a high efficiency and significance of laboratory PBM diagnosis in presence of the antibacterial liquor efficiency.

[Multilocus sequence-typing for characterization of Moscow strains of Haemophilus influenzae type b]. / Kharakteristika moskovskikh shtammov Haemophilus influenzae tipa b metodom mul'tilokusnogo sekvenirovaniia-tipirovaniia.

Haemophilius influenzae, type b (Hib) bacteria, were genotyped by multilocus sequence typing (MLST) using 5 loci (adk, fucK, mdh, pgi, recA). 42 Moscow Hib strains (including 38 isolates form cerebrospinal fluid of children, who had purulent meningitis in 1999-2001, and 4 strains isolated from healthy carriers of Hib), as well as 2 strains from Yekaterinburg were studied. In MLST a strain is characterized, by alleles and their combinations (an allele profile) referred to also as sequence-type (ST). 9 Sts were identified within the Russian Hib bacteria: ST-1 was found in 25 strains (57%), ST-12 was found in 8 strains (18%), ST-11 was found in 4 strains (9%) and ST-15 was found in 2 strains (4.5%); all other STs strains (13, 14, 16, 17, 51) were found in isolated cases (2.3%). A comparison of allelic profiles and of nucleotide sequences showed that 93% of Russian isolates, i.e. strain with ST-1, 11, 12, 13, 15 and 17, belong to one and the same clonal complex. 2 isolates from Norway and Sweden from among 7 foreign Hib strains studied up to now can be described as belonging to the same clonal complex; 5 Hib strains were different from the Russian ones.

Molecular epidemiology of serogroup a meningitis in Moscow, 1969 to 1997.

Molecular analysis of 103 serogroup A Neisseria meningitidis strains isolated in Moscow from 1969 to 1997 showed that four independent clonal groupings were responsible for successive waves of meningococcal disease. An epidemic from 1969 to the mid-1970s was caused by genocloud 2 of subgroup III, possibly imported from China. Subsequent endemic disease through the early 1990s was caused by subgroup X and then by subgroup VI, which has also caused endemic disease elsewhere in eastern Europe. A 1996 epidemic was part of the pandemic spread from Asia of genocloud 8 of subgroup III. Recent genocloud 8 epidemic disease in Moscow may represent an early warning for spread of these bacteria to other countries in Europe.

[The prospects for the diagnosis of bacterial meningitis]. / Perspektivy diagnostiki bakterial'nykh meningitov.

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.

[The serosubtyping of serogroup B meningococci]. / Serosubtipirovanie meningokokkov serogruppy B.

The results of the study of 116 Neisseria meningitidis strains, isolated from patients at different territories of Russia at the period of 1983-1992, by the method of the enzyme immunoassay are presented. 13.8 +/- 3.2% of the strains were found to have stereotype proteins and 59.5 +/- 4.5%, subtype proteins. In the population of circulating meningococcal strains no absolute prevalence of any single serotype or subtype was established. The comparison of the tendency in the course of morbidity rate and the state of the serosubtype composition of isolated group B N. meningitidis stains is indicative of the favorable situation with respect to meningococcal infection and the importance of further observation of the circulating strains.

[The type subtyping of meningococci by means of whole-cell immunoenzyme analysis]. / Tipo-subtipirovanie meningokokkov metodom tsel'nokletochnogo immunofermentnogo analiza.

In this work the method of the whole-cell enzyme immunoassay, used for the serotype-subtyping of meningococci by means of specific monoclonal antibodies, is described. High specificity of the method, the simplicity of the assay procedure and evaluation of its results, as well as the availability of this method for practical use, have been demonstrated. The results of this investigation confirm the importance of the evaluation of type-subtype appurtenance of reference and laboratory strains used in experiments. Study of 72 meningococcal strains obtained from patients has revealed their polyclonal character in respect of their type-subtype signs.

[The characteristics of suppurative meningitis morbidity caused by Streptococcus pneumoniae and Haemophilus influenza type B]. / Osobennosti zabolevaemosti gnoinymi meningitami, vyzvannymi Streptococcus pneumoniae i Haemophilus influenzae tipa B.

Of the 1,018 patients with purulent bacterial meningitis, hospitalized at the 2nd Clinical Infectious Hospital in Moscow during the period of 1980-1987, the diagnosis was confirmed in 54.7%; of these, meningitis of pneumococcal etiology was established in 44.8% and meningitis caused by H. influenzae, type b, in 23.8% of the patients. In meningitis of pneumococcal etiology high risk groups included mainly adults, especially those over 50 years, and children under 3 years of age. In meningitis of H. influenzae etiology high risk groups included mainly young children under 2 years of age. Meningitis of pneumococcal etiology was characterized by considerable death rate (on the average, 20%), while in meningitis of H. influenzae etiology death rate was 3 times lower. Pneumococci of serotypes 1, 3, 6, and 19 were found to be of the highest etiological importance for adults and pneumococci of serotypes 19, 6, 12, and 1, for children. In recent years greater etiological role of serotype 42 in adults was noted. The study of the spread of meningitides of different etiology is a high-priority task for this country.

[Preservation of microorganisms by deep freezing]. / Khranenie mikroorganizmov v usloviiakh glubokoi zamorozki.

Meningococci, pneumococci, and hemophilic bacilli may be stored at -70 degrees C. Only half (54.4 percent) of Meningococcal cultures were viable after 11 months storage, 41.6 percent retained their serologic properties. Conditions for stable long storage of meningococci at -70 degrees C are to be searched for. Pneumococci retained their initial parameters for 8 months, this permitting recommendation of such storage for these bacteria. No data on the possibility of hemophilic bacilli storage is available yet.

[A modified method of latex agglutination in capillaries for the detection of meningococcal antigens]. / Modifitsirovannyi metod lateks-aggliutinatsii v kapilliarakh dlia obnaruzheniia meningokokkovykh antigenov.

The suggested microvariant of the latex agglutination test in glass capillaries is not inferior to the traditional latex agglutination on the glass in sensitivity and specificity. The microvariant consists in the following: glass capillaries 0.8 mm in diameter 10 cm long are filled with the pathological material (3/5 of the tube length) and with the diagnostic latex preparation (1/5 of the tube length) so that no air hole is formed between them. The time of the reaction may be prolonged to 20-30 min without deteriorating its specificity. The results are recorded using a 4+ system, similarly as with the latex agglutination on the glass. The described modification appears to be useful for practical laboratories: it saves the latex preparation 4-fold and takes 2 times less pathologic material.