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Characteristics of clonal composition of Corynebacterium diphtheriae strain population in Russia using MLST, as well as evaluation of a possibility of using of this method during execu- tion of monitoring of diphtheria infection causative agent strains.C. diph- theriae strains, isolated in Russia in 1957 - 2015 and sent to Gabrichevsky MRIEM reference centre for diphtheria and pertussis, were studied. Gentyping of C. diphtheriae using MLST was carried out based on sequencing ofhousekeepinggene fragments. ST identification was carried out according to PubMLST Result. C. diphtheriae strains of 36 sequence-types (ST) were identi- fied on the territory of Russia - 27 previously known and 9 novel, detected for the first time. 2 sequence types ST25 and ST8 (22% and 18%) dominated. Inter-relation between phenotype. properties (toxigenicity. and biovar) and membership of C. diphtheriae strains in certain sequence- types was shown - toxigenic and non-toxigenic C. diphtheriae strains of various biovars were characterized by certain sequence-types. Changes of clonal composition of C. diphtheriae popula- tion in dynamics of epidemic process of diphtheria infection were.shown.Use of MLST allowed to characterized clonal composition of C. diphtheriae strains' population in Russia and has shown perspectives of use of this method to characterize population of diphtheria causative agent, detect epidemically significant strains and decipher foci of diphtheria infection.

Study the prevalence and mechanisms of resistance in circulating C. diphtheriae strains.664 C. diphtheriae strains isolated in 1987 - 2013 in various regions of Russia and sent to the reference center of Gabrichevsky Moscow Research Institute of Epidemiology and Microbiology were the object of the study. Antibiotic sensitivity of the strains was studied by disk-diffusion and E-test methods using 10 antimicrobial preparations. Nucleotide sequence analysis was carried out by using BLAST program and EMBL/GenBank database.Most of the studied strains turned out to be sensitive to all the antibacterial preparations used. 1.2% of C. diphtheriae strains turned out to be resistant to penicillin and 6.0% had intermediate level of resistance. 0.4 - 0.6% of the strains had intermediate level of resistance to macrolides, and 4.0 - 4.4% were resistant. 2.0% of the strains had multiple resistance. Erm(X)-specific PCR carried out in this study showed that all the C. diphtheriae strains resistant to macrolide antibiotics carry erm(X) gene.The results of the study indicate a fairly high level of prevalence for C. diphtheriae strains resistant to antibiotics.

Characterization of contemporary C. diphtheriae strains isolated in Russia by using multilocus DNA sequencing (MLST).28 toxigenic C. diphtheriae strains isolated in Russia in 2002-2012 and sent to diphtheria and pertussis reference center of Gabrichevsky Research Institute of Epidemiology and Microbiology were studied. C. diphtheriae strain genotyping was performed by using MLST based on atpA, dnaE, dnaK, fusA, leuA, odhA and rpoB gene fragments. Identification of alleles and ST was carried out according to EMBL/GenBank and PubMLST, eBurst approach was used for cluster analysis.By using MLST contemporary toxigenic C. diphtheriae strains isolated in Russia in 2002 - 2012 were characterized. 8 genotypes (ST41, ST5, ST8, ST28, ST25, ST44, ST-new1 and ST-new2) were identified, 3 among them were dominating--ST8, ST28 and ST-new1. Most of the toxigenic strains belong to biovar gravis and ST8. Among biovar mitis strains a higher heterogeneity by ST membership was noted, but with prevalence of ST28 strains.Use of MLST allowed to characterize contemporary circulating population of toxigenic C. diphtheriae strains isolated in Russia and showed perspective of application of this method for characterization of diphtheria causative agent population and detection of epidemically significant strains, as well as juxtaposing of them with genetic structure of foreign strains.

Development of micro technologies based approach for express diagnostics of toxigenic C. diphtheriae strains.Corynebacterium diphtheriae 10648 (tox+) and C. diphtheriae NCTC 10356 (tox-) from Central Health Laboratory (London) reference strains were used as positive and negative controls respectively. Diagnostic kit was created by using fractions of antibodies with high avidity that were obtained by consecutive fractioning of positive antitoxic blood sera and then loaded onto polyacrylamide latex particles with the diameter of 0.81 microm. 20 Elek test positive C. diphtheriae strains and 20 tox gene PCR negative C. diphtheriae strains (i.e. non toxigenic) (Pasteur Research Institute of Epidemiology and Microbiology) were used as control. Indirect hemagglutination with anti-diphtheria antibody diagnostic kit was used as a quantitative control. PCR, Elek test and ICS test were used as quality control.The diagnostic kit obtained had specificity of 97%, sensitivity of 98%. Specimen preparation time is 15 - 20 minutes, reaction time - 2 - 3 minutes, and up to 93 specimens can be analyzed on a single microchip.The developed approach has high sensitivity and specificity, is easy to use, and fast in regard to preparation and reaction time. Portability of the apparatus allows the use of reagents in micro volumes.

To assess tolerability and immunological activity of Bubo-M vaccine and hepatitis B vaccine in patients with chronic obstructive pulmonary disease (COPD).Sixty-three patients with moderate and severe COPD aged 35-65 years were immunized against diphtheria, tetanus, and hepatitis B. Bubo-M vaccine as well as vaccine against hepatitis B were used for immunization. Immunologic effect of vaccination was assessed by measurement of serum antibody level to HBsAg as well as to diphtheria and tetanus toxoids. Assessment of antibody level to HBsAg was performed by ELISA, and levels of antibodies to diphtheria and tetanus toxoids--by micromethod in direct hemagglutination assay. Reactogenicity of Bubo-M vaccine was measured according to duration and intensity of local and systemic reactions.The local and systemic reactions were infrequent, serious adverse events after vaccination were not observed. Six months after vaccination, protective antibody titers to hepatitis B, diphtheria and tetanus were determined in all immunized persons--either healthy, or with COPD. During completion of vaccination schedule, significant reduction of acute respiratory infections rate and main disease exacerbations was noted in patients with COPD.Good tolerability and high immunogenicity of Bubo-M and hepatitis B vaccines were demonstrated in both groups of vacinees. These vaccines could be recommended for booster vaccination of adults with COPD.

Comparative assessment of efficacy of homologous and heterologous diphtheria antitoxins on the example of diphtheria intoxication.Homologous hyperimmune sera were obtained through immunization of rabbits and guinea-pigs with diphtheria toxoid according to schedule. Immune rabbit sera contained 70 - 100 IU/mL of antitoxin antibodies and guinea-pig sera contained 60 - 80 IU/mL. Equine diphtheria antitoxin was used as a heterologous one. Measurement of antitoxin level using experimental animals is based on quantitative assessment of ability of studied sera to neutralize specific dermonecrotic effect of diphtheria toxin.Concentration of antitoxin in blood of different groups of guinea-pigs immunized 2 days earlier with either heterologous equine antitoxin or homologous antitoxin was 0.06 - 0.125. IU/mL. Animals from both groups were completely protected after administration of 5.64 LD50 of toxin. Alongside with it, 50 - 75% of animals which received homologous antitoxin were protected from higher doses of toxin, whereas all animals which received heterologous antitoxin died after administration of higher doses. After administration of identical doses of homologous antitoxin to rabbits its maximal concentration was observed on the next day, was stable up to 5 - 7 days after injection, decreased two-fold to 12th day and did not change further to 15 - 16 days after injection with subsequent another two-fold decrease to 30th day (then was stable for another 5 - 10 days).Administration of homologous antitoxin compared to heterologous analogue allows to prolong time of circulation of specific antitoxic antibodies in 3 - 4 times.

Efficacy of different treatment regimens with equine diphtheria antitoxin (EDA) was assessed on clinical samples as well as in experiments on animals.Protective properties and serum concentration kinetics of heterologous antibodies was studied on 12 rabbits and 51 guinea pigs after intramuscular injection of different doses of EDA, in serum samples from 26 patients, which received one intramuscular injection of EDA in various doses as well as in serum samples from 10 patients with diphtheria of different severity, which were treated with EDA in total course dose 100,000-1,500,000 IU. Antitoxin concentration in serum sample was measured with passive hemagglutination assay as well as Jensen toxin neutralization test on rabbits.Experiments on laboratory animals received EDA in dose 150 IU/kg showed high protective effect. For example, rabbits with antitoxin level 1.0-1.25 IU/ ml in serum 24 hours after injection of EDA were 50-250 times resistant to dermonecrotic effect of diphtheria toxin compared with rabbits not received EDA. Guinea pig with antitoxin level 0.5-2.0 IU/ ml in serum 2-48 hours after injection of EDA in dose 150 IU/kg were all protected against 35-50 LD50 of diphtheria toxin. After termination of EDA injection there was sharp decrease of antitoxin level and it was not detected in serum 7 days after. Increase of antitoxin level in serum of animals was not adequate to quantity of injected EDA. Study of serum samples from 26 patients received one intramuscular injection of different doses of EDA showed that doses of antitoxin from 20,000 to 30,000 IU resulted in its presence in serum in concentration 0.5-3.0 IU/ml whereas injection of 50,000 IU or 70,000-100,000 IU resulted in serum concentrations 1.25-10.0 IU/ ml and 2.5-20.0 IU/ml respectively.Relatively low doses of EDA provided relatively high level of protection against diphtheria toxin that should be taken into account during treatment of diphtheria patients.

To study features of specific immunity in patients with diphtheria using data from clinic as well as from animal experiments.Serum samples of 80 patients hospitalized to Infectious Diseases Clinical Hospital No. 2 in Moscow and treated with anti-diphtheria serum (manufactured by "Immunogen" Concern, Stavropol) were studied. Localized diphtheria of the oropharynx was diagnosed in 29 patients, diffused diphtheria--in 8, subtoxic--in 3, grade 1 toxic--in 19, grade 2 toxic--in 12, grade 3 toxic--in 9. Experimental part of the study was performed on outbred rabbits weighted 3-3.5 kg. Level of antitoxin in serum was measured in reaction of passive hemagglutination using commercial antigenic erythrocyte diagnostic kit (manufactured by Mechnikov Research Institute of Vaccines and Sera, Moscow).Intermittent administration of toxin to control rabbits which lack background immunity did not lead to changes in their immune status and after administration of anti-diphtheria antitoxin kinetics of its level in serum was analogous to that observed after administration of antitoxin to intact animals. Highest level of antitoxin (1.0-2.0 IU/ml) was observed 1-3 days after its administration, and to day 13-15 antitoxin was not detected in serum samples. Diphtheria antitoxin in concentration from 0.03 to 40.0 IU/ml was detected in serum samples in 59 of 80 (74%) studied patients. Only in 21 patients (26%) the antitoxin was not detected.Presence of antitoxin in serum argue for active immune response to infection and activation of immune memory mechansisms, which allow to predict less severe course of the disease. Absence of antitoxin in serum of patient admitted to hospital points that infectious process is developing on the background of no immunity that predicts the probable development of severe diphtheria.

To assess antibody levels against diphtheria, pertussis, and tetanus in pregnant women.One hundred and two virtually healthy pregnant women aged 18-35 years were studied. Antibodies to diphtheria and tetanus were measured in passive hemagglutination reaction with diphtheria and tetanus diagnostic kits. Antibodies to Bordetella pertussis antigens were determined in hemagglutination assay (HA) and in enzyme immunoassay (EIA).Protective titers of anti-diphtheria and anti-tetanus antibodies were detected in 91.2% and 94.1% of participants respectively, whereas high titers--in 24.5% and 27.4% respectively. Low levels of IgG to B. pertussis antigens measured by EIA were observed in 70.6% of participants whereas moderate and high levels--in 22.5% and 6.9% respectively. Conditionally protective levels of anti-pertussis antibodies measured by HA were detected in 10.8% of participants.Obtained results demonstrate high level of protection of pregnant women against diphtheria and tetanus and low level of anti-pertussis immunity.

To study the magnitute of antitoxic immunity against diphtheria in different age groups of Rostov-on-Don city and Rostov region population.Antitoxic immunity against diphtheriawas studied in children 3 - 15 years old (661 persons) and adults 16 - 59 y.o. (893 persons), which were tested during 2003 - 2007 using direct hemagglutination assay manufactured by Mechnikov Scientific Production Association "Biomed" (Moscow).Proportion of children 3 - 15 y.o. seropositive to diphtheria was 98.6 +/- 0.5%. Children aged 3 years and 4 - 6 years were identified as a risk group for diphtheria as they had highest proportions of non-immune persons. Proportion of immune to diphtheria in adults was 94.4 +/- 0.8%. Maximal level of protection was observed in personsaged 16 - 17and 18 - 19 years, in which the proportion of seropositive persons was 99.1 - 100%. Personsor = 50 years old formed group of risk because persons with low or moderate titers of antitoxin were identified in this age group mostly.Obtained results provide evidence on enough high level of protection of Rostov-on-Don cityand Rostov region population against diphtheria. Group of increased risk comprised of persons aged 3 - 6 years andor = 50 years.

To study main biologic characteristics of C. diphtheriae strains circulating in North-West Region of Russia for the last 15 years.Six hundred and fifty strains of C. diphtheriae isolated from ill persons and carriers in Saint-Petersburg, Leningrad region and Vologda region at various periods of time were studied. Identification of an infectious agent was performed according to methodic guidelines MU 4.2698-98. IHA-chromatographic test (ICS-test) on the basis of MKA, polymerase chain reaction, determination of adhesive activity and susceptibility to antibiotics were performed.In recent years, circulation of C. diphtheriae strains with biologic characteristics similar to that observed in strains isolated during diphtheria epidemic and differed from that observed in strains isolated during the period of low incidence. Proportion of strains with "silent" gene between non-toxic in Elek-test C. diphtheriae increased. Decreasing of susceptibility to the range of antibiotics is observed in recent years.Revealed features of biologic characteristics of diphtheria agent circulating in post-epidemic period should be accounted during epidemiologic surveillance for diphtheria and choice of treatment of the infection.

Polymorphism of tox and dtxR genes responsible for diphtheria toxin synthesis was revealed. Seven point mutations in tox gene were detected; study of their combinations allowed to determine 10 allelic variants of the tox gene in C. diphtheriae strains. Majority of mutations did not lead to changes in substitutions in amino acid sequence of diphtheria toxin. In tox gene from 2 strains of mitis biovar, ribotype "Otchakov" isolated in Saint-Petersburg, mutation in position 1252 (G --C), which corresponds to change of glycine on arginine in amino acid sequence of diphtheria toxin (G393R), was identified. Mutation localizes in R domain of fragment B of diphtheria toxin. In the dtxR gene 16 point mutations were registered; study of their combinations allowed to determine 10 allelic variants of the dtxR gene. Two mutations led to amino acid substitutions in regulatory protein DtxR: in position 640 (C --A), which corresponds to change of leucine on isoleucine (L2141), and in position 440 (C --T), which corresponds to change of alanine on valine (A147V). Mutation A147V is characteristic for all strains of epidemic clonal group (strains of biovar gravis, ribotype "Sankt-Peterburg/Rossija", enzyme types of complex 8), dominated in Russia during diphtheria epidemic at 1990s. Strains of this group were characterized by high level of diphtheria toxin production.

The vaccination coverage against diphtheria in the adult population of Russia in 2006-2007 was analyzed in different age, professional and social groups and reasons of absent immunization. Vaccination coverage in adults was 95.3%, of them 92.5% were boosted (i.e. completely immunized) and only 7.5% were in process of vaccination. Vaccination coverage was high in the majority of regions. Mean vaccination coverage in adults aged 25-49 ages was 96.2-96.5% whereas in adultsor =50 years it was lower (91.8-94.8%). Mean coverage in groups of high risk for severe diphtheria was 97.7% in professional high-risk groups and somewhat lower in social high-risk groups (94.4%). The lowest vaccination rate was registered in group of migrants (81.7%). In unvaccinated persons from professional groups 33.1% were not vaccinated due to medical contraindications, 22.8%--because of vaccination refusal, and 38.1%--due to non-medical reasons. In social groups these figures were 21.8%, 28.4%, and 49.8% respectively. Reported mean adult vaccination coverage in Russia is confirmed by results of serological studies. Protective titers were detected in 91.3% tested adults. However in the group agedor =50 years rate of protected persons was lower (84-85%).

Results of the conducted study showed that naturally acquired antibacterial and postvaccinal antitoxic antibodies against diphtheria were found in human blood sera. Challenge of ADT-M toxoid to adults resulted in production of antitoxic as well as antibacterial antibodies in high concentrations. In response to challenge of ADT-M toxoid simultaneously with bacterial vaccine against diphtheria Codivac both antibacterial and antitoxic antibodies were synthesized in blood on optimal physiologic levels. This study revealed dynamics of some specific characteristics of humoral immune response after challenge of two different vaccines against diphtheria--ADT-M toxoid and Codivac vaccine.

The introduction of the immunomodulator polyoxidonium in an amount of 0.5 Mg/ml into adsorbed D(a)PT vaccine with the acellular pertussis component leads to the preservation of the protective activity of the pertusis component, diphtheria and tetanus toxoids, as well to the 4-time decrease of the content of adsorbent (aluminium hydroxide) from 2 to 0.5 mg/ml.

The child's sex was shown to influence the character of antibody formation only after immunization against diphtheria with live measles vaccine: girls exhibited stronger reaction to vaccination than boys. Children of different gender were found to have characteristic HLA DR markers of humoral immune response to diphtheria toxoid and measles vaccine. HLA DR7 proved to be the marker of low production of antibodies to diphtheria toxoid and measles vaccine in boys.

Antidiphtheria immunity in children aged 3-15 years was evaluated, depending on their age, the vaccinal preparation chosen for immunization and the immunization schedule. Adsorbed DPT vaccine was shown to have a higher immunological activity in comparison with adsorbed DT toxoid with reduced antigen content. The relationship between the tension of antidiphtheria antitoxic immunity in children and the possibility of the formation of the clinical forms of diphtheria with different severity in the patients was established. Children aged 4-6 years were considered to be a group of high risk in diphtheria morbidity, as it was among the children of this group that toxic and subtoxic forms of diphtheria were most often registered (44.9 +/- 7.1% of the examinees); in addition, a high proportion of children seronegative to this infection (26,3 +/- 2.2%) was observed in this group.

An analytical literature review dealing with the problems of resistance to causative agents of infections. The review includes the data of experimental research, as well as the author's own immunological studies in children at the clinic of acute infections. Mechanisms of resistance at different phases of the infectious process, the multivalued role of Th1- and Th2-dependent responses in different infections, the role of vaccinal immunity in the resistance of children to the causative agents of vaccine-preventable infections (diphtheria and parotitis) are analyzed.

In human sera, studied with the use of the enzyme immunoassay, antidiphtheria postvaccinal antitoxic IgG and naturally acquired antibacterial IgG, IgM and IgA were detected. In the blood of children and adults aged up to 50 years antitoxic IgG were present at normal and high concentrations. In 50% of children antibacterial IgA were absent, while specific antibacterial IgM could be found at high concentrations. Changes in the content of antibacterial antibodies of different classes in sera were observed with age. More than 90% of adults had antibacterial IgA and IgG at normal and hig concentrations, while the level of IgM decreased. Under the influence of ecological, social, anthropogenic and other environmental factors the optimum levels of specific antibodies were replaced by anomalous ones, which led to an increased number of persons susceptible to diphtheria infection and in the intensity of the circulation of the infective agent. The deficiency of antidiphtheria antibacterial antibodies in the blood determined the necessity of correcting immunity by means of not only toxoid, but also bacterial antigens.

The data on diphtheria morbidity and the occurrence of carrier state for its causative agent at the period of 2001-2002 were analyzed. The rates of morbidity and detected carrier state for these years were 0.63-0.55 and 0.65-0.64 respectively. Nevertheless, in spite of the relatively low morbidity rates the presence of the toxic forms of diphtheria (400 patients for two years) and lethal cases (with lethality rate reaching 5.4%) indicated that the epidemic situation in diphtheria remained tense. The most unfavorable situation was observed in the North-Western and Central regions of Russia. In urban areas morbidity rates were still 2- to 3-fold higher in than in rural ones, but the latter showed a higher percentage of severe cases (46.6% in 2001 and 39.7% in 2002) and lethal outcomes (13.6% and 19.2%). The latter was indicative of drawbacks in the immunoprophylaxis, diagnostics and treatment of diphtheria in rural areas. In the total structure of diphtheria patients adults prevailed: 75%. The highest morbidity rates were registered among children aged 3-6 years, among adults in the age groups of 18-19 years and 50-59 years. The epidemic process developed mainly among the immunized population, which was indicated by a high proportion of vaccinated persons among those affected by this infection (62.8-66.6%) and a mild course of the disease in the majority of them. The present epidemiological situation in diphtheria was determined by patients not vaccinated against this infection. The proportion of severe cases among nonvaccinated children was 42.4-51.6% and lethal outcomes, 12.9-15.1%. Among nonimmunized adults these figures were equal to 43.1% and 9.3% respectively. The highest percentage of children, not vaccinated during the first years of their life, was registered among those in the asocial families, refugees and homeless persons. Among adults these were persons above 50 years old, as well as jobless persons of working age, pensioners and invalids, who had limited possibilities of undergoing vaccination due to their social position. It was these social and age groups that should be regarded as risk groups with respect to the severity of the course of diphtheria and lethality. To stabilize diphtheria morbidity, the full complex of prophylactic and antiepidemic measures, and primarily the immunization of the population, should be systematically carried out.

The retrospective evaluation of the intensity of postvaccinal immunity at different periods after the primary course of immunization against diphtheria and tetanus was made. In the sera taken from 130 children with systemic connective tissue diseases the content of antibodies to diphtheria and tetanus was determined. As revealed in this study, the protective levels of antibodies to diphtheria and tetanus toxoids were retained by both sick and healthy children for 5 years and longer. Significantly lower titers of antibodies to diphtheria and tetanus toxoids were registered in children undergoing therapy with glucocorticosteroids and cytostatics at the time of the study.

The analysis of a large number of diphtheria cases (1326) at the peak of diphtheria morbidity in Russia (1993-1994) revealed that the intensity of antidiphtheria antitoxic immunity was age-dependent with a sharp immunodeficiency in the population aged 35 years and older. As adaptive capacity of the organism decreases, the positive associative links between immunity to diphtheria and the AB0 blood groups become evident. Populations with phenotype B (III) had the largest diphtheria immune stratum at the age of 35 years and over as compared to 3 other phenotypes. The genotypic analysis of serological data may be of practical importance for the detection of the degree of predisposition of humans to infectious diseases.

The complex clinico-laboratory examination of 92 children aged 3-14 years with the localized form of stomatopharyngeal diphtheria and toxigenic C. diphtheriae (TCD) carrier state was carried out. The children were hospitalized in the clinic of drop infections during the period of 1996-1998. The hydrophobic properties, adhesive, lysozyme, antilysozyme and DNAase activities of 92 TCD strains, as well as the levels of the specific and nonspecific immunity of the macroorganism, were studied. The study revealed that the character of the infectious process in TCD carrier state was determined by the degree of the colonization activity of the infective agent. Direct correlation between the time of the persistence of TCD in the body and their hydrophobic properties, adhesive and antilysozyme activities (with r = 0.89-0.93) was established. The duration of TCD carrier state was found to be inversely related to the electrophysiological state of epithelial cells of the mucosa, the level of humoral specific antibacterial immunity and the content of local antidiphtheria IgA.

The enzyme immunoassay system (EIA) for differentiation of antibodies in therapeutic heterogeneous antitoxic serum and antibodies to Corynebacterium diphtheriae toxigenic strains in patients and carriers was developed. The use of EIA permitted the dynamic evaluation of the characteristics of humoral antitoxic and antibacterial immune response in 50 patients with the localized and disseminated forms of stomatopharyngeal diphtheria and 14 "healthy" carriers of toxigenic C. diphtheriae. As revealed in this study, the symptoms of the disease in patients with disseminated forms of stomatopharyngeal diphtheria developed in the presence of statistically significant low quantitative values of antitoxic and antibacterial antibodies to C. diphtheriae antigens. In the group of patients with the localized forms of the disease the initially low level of antitoxic antibodies was detected with the concentration of antibacterial antibodies remaining unchanged. During the period of convalescence the levels of antitoxic antibodies in both groups reached those of healthy persons. In case of localized forms of the disease the level of antibacterial antibodies decreased as compared with healthy persons, starting from the second week of the disease. The period of convalescence in the disseminated forms was characterized by the low concentration of antibacterial antibodies. Carrier state was formed in the presence of high levels of antitoxic antibodies and significantly low levels of antibacterial ones.

The present epidemiological situation in diphtheria in the Republic of North Ossetia (Alania) is analyzed. The data on diphtheria morbidity and lethality are given. The present stable situation resulted from mass immunization of the adult population which ensured the level of vaccination coverage reaching 87% in 1995.

Bubo-M, the first Russian combined vaccine, was found to have low reactogenicity. The difference between the number of postvaccinal reactions in the group of children immunized with Bubo-M (25.9%) and those in the group of children who had been simultaneously injected into different sites of the body with ADS-M toxoid (adsorbed DT toxoid with reduced antigen content) and hepatitis B vaccine (26.7%) was not statistically significant. Following immunization a considerable increase in the level of diphtheria and tetanus antibodies (p0.005) was observed in all children (100%), the level of HBs antibodies in the group of children immunized with Bubo-M (the geometric mean titer: 13,721 IU/l) essentially exceeding that observed in the control group injected with ADS-M toxoid and hepatitis B vaccine (the geometric mean of the titer: 2,441 IU/l). Bubo-M was duly registered and allowed for industrial production and medical use.

Clinical and immunological examination of 55 children aged 6-15 years with rheumatic diseases, immunized against diphtheria, was carried out. All children were immunized at the stage of clinical and laboratory remission and in some cases while undergoing a prolonged course of cytostatic therapy or therapy with nonsteroid anti-inflammatory remedies. This examination demonstrated that in the overwhelming majority of children with rheumatic diseases the diphtheria vaccinal process took an asymptomatic course and had no influence on the course of the main disease. Specific features, characteristic of the immune status of this group of children, were established. In the course of the vaccinal process the restoration of the initially inhibited characteristics (the production of TNF-alpha and IL-2) to normal values were shown to occur, which was indicative of the fact that the reserve capacities of immunocompetent cells were retained in these patients. This study also revealed that immunization of children with rheumatic diseases with adsorbed DT and D toxoids with reduced antigen content was not excessive antigenic stimulation for such children, as it did not lead to immunopathological shifts, but induced transient phase changes in immunological characteristics, similar to those in healthy children. Protective levels of antibodies to diphtheria were shown to retain for a long time with considerable prolongation of intervals between booster injections. The simultaneous course of immunosuppressive maintenance therapy in the average dosage used for the corresponding age group did not inhibit the production of protective antibodies.

The effectiveness and safety of the immunization of children with adsorbed DT toxoid with reduced component with disturbances in the reactivity of the body, caused by immunosuppressive therapy, are evaluated. The group under study included 53 children with glomerulonephritis, malignant tumors and acute lymphoblast leucosis, formerly receiving immunosuppressive drugs. The control group consisted of 24 patients with pyelonephritis who had not received such therapy. In the postvaccinal period no exacerbation of the main disease was registered in children. The manifestation and frequence of postvaccinal reactions did not differ from those observed in healthy children after the injection of adsorbed DT toxoid with reduced antigen content. Control serological testing demonstrated the presence of pronounced immune response in the vaccinees.

The genetic structure of C. dipthteriae toxigenic strains isolated in Russia during the period of more than 50 years was analysed. The use of the method of ribotyping made it possible to register 17 C. diphtheriae ribotypes. The study revealed that the genetic structure of C. diphtheriae population varied in the dynamics of the epidemic process: each epidemic cycle characterized by predominant spread of epidemic strains of definite biovars and ribotypes. Thus, C. diphtheriae strains of biovar gravis, ribotype M11, dominated in the 40-60 years and C. diphtheriae strains of biovar mitis, closely related ribotypes M1 and M1v, dominated in the 80 years. During the last epidemic rise of diphtheriae morbidity in the 90 s C. diphtheriae strains of biovar gravis, closely related ribotypes G1 and G4, dominated among circulating strains. The proportion of these ribotypes began to increase 3 years before the rise of morbidity. The data of microbiological monitoring are recommended for use in the prognostication of the development of the epidemic process of diphtheria infection.

The analysis of the level of antidiphtheria immunity in different age group of the adult population has shown that postvaccinal immunity resulting from immunization with adsorbed DT toxoid with reduced antigen content gradually decreases after the period of two years. The immune stratum remains sufficiently numerous only among persons aged 18-27 years (93.3%), persons over 38 years constitute a high risk group with respect to the possibility of diphtheria infection: the proportion of persons protected against this infection amounts to 71.3% in the age group of 38-47 years and 54.9% in the age group over 48 years, that is not sufficient.

The biochemical test of the reduction of nitrates to nitrites made it possible to identify 5.2% of strains belonging to biovar belfanti among 135 C. diphtheriae strains, initially classified within biovar mitis. Out of 7 identified C. diphtheriae belfanti strains, 2 toxigenic strains were isolated from multiple foci diphtheria. According to the results of the polymerase chain reaction, 1 out of 5 non-toxigenic strains had tox gene. All C. diphtheriae belfanti strains were found to have pronounced capacity for adhesion to sheep and human red blood cells. At the stage of the extinction of diphtheria epidemic the practical identification of C. diphtheriae belfanti strains is necessary, as increased adhesion in combination with toxigenic properties may probably promote for bacteria of this biovar to take the leading role at the period of sporadic morbidity.

624 Corynebacterium diphtheriae strains, newly isolated from patients and carriers, were studied with the use of the methods of gel immunodiffusion (Elek's test) and polymerase chain reaction (PCR). In the evaluation of 388 C. diptheriae strains, found to be toxigenic in PCR, the results of Elek's test coincided with those of PCR on 98% of cases. In 38 out of 143 strains (26.5%), nontoxigenic according to the results of Elek's test, the presence of the A-fragment of the tox-gene was established. Subculturing in nutrient media made it possible to determine the presence of toxin in 19 out of 38 of these strains; the remaining strains, isolated mainly from carriers, were found to have the "silent" gene. The advantage of using PCR for the detection of toxigenic and nontoxigenic C. diphtheriae strains of different origin was shown.