[Anti-pertussis immunity in children in the cities of Northwestern Federal District with various population].

AIM: Evaluate the state of immunity against pertussis in children living in St. Petersburg and regional centers of Northwestern Federal District (NFD). MATERIALS AND METHODS: The level of anti-pertussis antibodies by EIA and agglutinin reaction (AR) was studied in 419 children living in St. Petersburg and by AR in 239 children living in regional centers of NFD. Blood sera in AR were studied by using liquid pertussis diagnosticum (Biomed, Russia). RESULTS: In St. Petersburg the frequency of detection of high level of antibodies was the highest in the 15 - 17 age group that indicates a high level of latent morbidity in grownups. The frequency of detection of high level of antibodies in the 3 - 4 and 9 - 10 age groups in regional centers was significantly lower, and the fraction of sera with undetected level of antibodies--significantly higher compared with St. Petersburg, that gives evidence on low circulation of causative agent, lack of "epidemizing" of children in small cities. CONCLUSION: The question of introduction of second revaccination against pertussis in children at the age of 6 is actual, because one vaccination is not enough for prolonged sustaining of population immunity intensity.

[Molecular-biological characteristic of Yersinia enterocolitica circulating in various regions of Russian Federation].

AIM: Complex characteristic by phenotype signs and main virulence genes of Yersinia enterocolitica strains circulating in various regions of Russian Federation. MATERIALS AND METHODS: 46 strains of Y. enterocolitica of 2 - 4 biotypes and 401 strains of Y. enterocolitica IA biotype isolated in 15 administrative territories of Russian Federation (Siberian, Far Eastern, Northwestern, Urals Federal Districts) from infected people, rodents, agricultural animals, birds, the environment were studied. Phagotyping was performed in the reference laboratory of the Pasteur Institute (Paris). All the Y. enterocolitica cultures were studied for the presence of ail, ystB and ystA genes by PCR method. Presence of virulence plasmid pYVwas determined by gel electrophoresis by T. Kieser method. RESULTS: 447 strains of Y. enterocolitica biotype 1A and 2 - 4 were studied. Most of the strains belonged to serotypes O:3; O:9; O: 5; O: 6,30; O:6,31; O:7,8. Phagotyping was performed for part of the strains. Phagotypes Xz and Xo were determined in biotype 1A strains. 2 - 4 biotype strains circulating in Siberia and the Far East were characterized by phagotype VIII, X3 that are present in other countries, and phagotype Xz that is spread only in Russia. Phagotypes IXa, IXb, II that are characteristic for strains from Canada, South Africa, Japan were not detected in Russian Federation. All the strains of 2 - 4 biotypes had ail and ystA genes. Most of the recently isolated strains had pYV. The only pathogenicity factor detected in 81.3% of biotype 1A strains including 14 strains from patients was ystB gene. These infections were accompanied by an expressed clinical symptomatology of enteritis and enterocolitis. CONCLUSION: Isolation of 1A biotype strains from patients necessitates execution of diagnostic studies of intestinal yersiniosis in patients with diagnosis "acute intestinal infection of undetermined etiology".

[Micro technologies in express diagnostics of toxigenic Corynebacterium diphtheriae strains].

AIM: Development of micro technologies based approach for express diagnostics of toxigenic C. diphtheriae strains. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Biodiversity and evolution of circulating bacteria and virus populations. Novel problems of medical microbiology].

Biodiversity and evolution of circulating bacteria and virus populations is a serious scientific problem, solving this problem is necessary for effective prophylaxis of infectious diseases. Principal trends of development in this field of science are described. Results of studies that were carried out and investigated biodiversity of principal pathogens in Russia and St. Petersburg in particular are presented. Risk of infectious security of society caused by increasing diversity of pathogenic microorganisms is described, and priority trends of research development in this field are specified.

[Features of cytokine levels in serum of patients with tick-borne borreliosis with different clinical signs].

AIM: To assess levels of several cytokines in blood of patients with tick-borne borreliosis (Lyme disease) with different clinical variants of the disease. MATERIALS AND METHODS: Study of complex of proinflammatory cytokines (IFNalpha, IL-1beta, IFNgamma, IL-2, IL-4, IL-8) during course of the disease was performed by solid-phase ELISA using domestic diagnostic kits (Scientific Manufacturing Organization "Proteinovyi Contur", "Cytokine" Ltd., Saint Petersburg). Levels of TNFalpha was determined by ELISA using commercial kits "Boehringer Manheim" (Austria). RESULTS: Performed comparative clinico-laboratory analysis demonstrated increased levels of LL-2, IL-4, and IL-8 in patients during acute phase of tick-borne borreliosis that could point to host's response on bacterial infection. It should be noted that in patients with arthritis levels of LL-4 and IL-2 remained high during recovery phase that probably determined by possible persistence of Borrelia burgdorferi. CONCLUSION: Further research of cytokines during Lyme borreliosis could have important diagnostic and prognostic value.

[Bactericidal activity of colloidal silver against grampositive and gramnegative bacteria].

It was shown that colloidal silver solution prepared in cooperation with the A. F. Ioffe Physical Technical Institute of the Russian Academy of Sciences, had significant bactericidal activity. Stable bactericidal effect on gramnegative microorganisms was observed after their 2-hour exposition in the solution of colloidal silver at a concentration of 10 ppm. Grampositive capsule-forming microorganisms were less susceptible to the colloidal silver solution: their death was observed after the 4-hour exposition in the solution.

[To the differential diagnosis of arthritis of pseudotuberculous and chlamydial etiology].

AIM: to define main differential diagnostic criteria for arthritides of chlamydial and pseudotuberculous etiology and to improve patient examination tactics. SUBJECTS AND METHODS: Forty-six patients with pseudotuberculosis and 41 patients with chronic urogenital chlamydial infection with articular involvement were examined. A bacteriological method of polymerase chain reaction (PCR), agglutination test, enzyme immunoassay (EIA) (IgA, IgG, IgM), indirect hemagglutination (IHA) test were used to diagnose pseudotuberculosis. Diagnostic techniques for chlamydiasis involved cultural, direct immunofluorescence (DIF), real-time PCR, and EIA (IgM, IgG, IgA). RESULTS: Patients with pseudotuberculosis developed polyarthritis and oligoarthritis in 56 and 39%, respectively. The development of arthritides was accompanied by fever in 89%, exanthema in 57%, gastrointestinal lesion in 56%, hepatomegalia in 78%. The pseudotuberculous etiology of the disease was confirmed by the agglutination test in 71% of the patients and by IHA in 7%. EIA revealed IgG in 78% of the patients, IgA in 11%, and IgM in 29%. PCR of synovial fluid (SF), synovial shell, and other biological substrates revealed Yersinia pseudotuberculosis DNA in 43%. Chlamydiasis and polyarthritis developed in 71 and 19%, respectively. The diagnosis of chlamydiasis was verified by EIA detection of IgG and IgA in 76 and 27% of cases, respectively. DIF, PCR, and culture studies of urethral scrapes found Chlamydia in 9, 32, and 29% of cases, respectively. Examination of SF and synovial shells revealed Chlamydia trachomatis in 24% of the patients and culture studies detected the pathogen in 21%. CONCLUSION: Asymmetrical polyarthritides mainly involving the knee joints are the most common arthritides of pseudotuberculous etiology. EIA detection of serum IgG and IgA and PCR study of SF are optimal diagnostic tools. Artritides of chlamydial etiology are asymmetrical oligoarthritides predominantly involving the knee and ankle joints. Examination of urethral and cervical canal scrapes, SF by culture and PCR studies and that of serum IgA and IgG by EIA are optimal diagnostic tests.

[Features of biologic characteristics of Corynebacterium diphtheriae circulating in postepidemic period].

AIM: To study main biologic characteristics of C. diphtheriae strains circulating in North-West Region of Russia for the last 15 years. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Etiologic role of Corynebacterium non diphtheriae in patients with different pathology].

Bacteriologic examination of 1589 patients showed that, aside from C. diphtheriae, 11% of acute upper respiratory tract infections were caused by other Corynebacterium species. Such bacteria can cause infections of various localizations (bronchitis, pyelonephritis, urethritis, colpitis, dermatitis, arthritis, etc.). C. pseudodiphtheriticum and C. xerosis were isolated from clinical specimens most frequently. Corynebacterium spp. have adhesive, hemolytic, hemagglutinating, and neuraminidase activity; some of them are highly pathogenic. The most virulent, were following species: C. diphtheriae, C. pseudotuberculosis, C. urealyticum, and C. ulcerans. Corynebacterium non diphtheriae were frequently isolated from clinical specimens in association with staphylococci and streptococci. In such cases, factors of pathogenicity and resistance to antibiotics were more pronounced. Strains isolated with association with other bacteria have lost susceptibility to tetracycline, oleandomycin, penicillin, and erythromycin. It is important to be vigilant about bacteria from Corynebacterium genus in clinical settings, and thoroughly study their biologic characteristics, especially in immunocompromised patients.

[Molecular typing of the strains of Bordetella pertussis circulating in St. Petersburg during an outbreak of the disease].

Growth of the whooping-cough morbidity during the last years in Russia and other countries with 40-year-long history of immunization gave rise to significant interest of researchers to variability of the Bordetella pertussis population. Comparative assay of the genomes of the B. pertussis strains circulating in St. Petersburg in 1998-2000 and strains used to produce domestic vaccines AKDS was performed using the pulse-field electrophoresis and sequencing. It was found that most strains of B. pertussis circulating during this period were distinguished from the vaccine strains by the DNA-profile and structure of genes involved in encoding of biosynthesis of the S1 subunit of the whooping-cough toxin (ptxS1) and pertactin (prn). It was shown that 62% of wild-type strains had electrophoretic profiles IV alpha and IV beta, whereas vaccine strains had electrophoretic profiles II and III. Circulation of strains with profiles IV alpha and IV beta was found to correlate with the whooping-cough morbidity rate in vaccinated children. Our results and data of other researchers were compared and analyzed.

[Yersinia lipopolysaccharide and its biological activity].

The data on the structure and biological activity of the lipopolysaccharide (LPS) of Yersinia as an important virulence factor are analyzed. The biological effects of LPS are characterized by dose dependence: small doses stimulate the intensity of phagocytosis, while large doses decrease phagocytic activity and produce cytotoxic effect. Yersinia LPS plays an important role in the development of such consequences of yersiniosis as reactive arthritis, erythema nodosum, Reiter's syndrome. Yersinia LPS is a widespread component for the diagnostics of yersiniosis and pseudotuberculosis.

[Detection of virulence markers in clinical strains of Yersinia]. / Vyiavlenie markerov virulentnosti u klinicheskikh shtammov iersinii.

The dispersion of plasmid pYV associated virulence markers in 474 Yersinia strains isolated from people has been studied. The ability to autoagglutination, calcium dependence of growth and the specific antigens were identified in 157 strains of traditionally pathogenic Yersinia enterocolitica serovars 03, 09, Yersinia pseudotuberculosis serovar I. They were not found in 223 strains of other 12 serovars of Yersinia enterocolitica, in 40 strains of Yersinia frederiksenii, Yersinia kristensenii, Yersinia intermedia. The proportion of virulent clones in the population of Yersinia is noted to depend on the conditions of its existence in vivo or in vitro. Identification of virulence markers is acknowledged to be expedient in epidemiological and ethiological estimation of the role of isolated Yersinia strains.

[Pathogenic properties of Corynebacterium diphtheriae]. / Kharakteristika osnovnykh patogennykh svoistv Corynebacterium diphtheriae.

The main pathogenic properties of 73 C. diphtheriae strains (their adhesive, invasive and cytotoxic activity) were characterized in the cultures of cells HEp-2 and Vero. The quantitative determination of the toxigenicity of 381 strains in the indirect hemagglutination test was made, and the strains were distributed by the degree of their toxigenicity. The characteristics of C. diphtheriae obtained with the use of in vitro experimental models, coincided with the severity of clinical manifestations of the diphtheria in humans, which made it possible to regard the models used in this study as adequate. On the basis of the chosen criteria the characterized strains could be subdivided into highly, moderately and low virulent and the degree of their potential epidemic danger could be determined.

[The use of polymerase chain reaction in the diagnosis of diphtheria infection]. / Primenenie polimeraznoi tsepnoi reaktsii v diagnostike difteriinoi infektsii.

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.

[The cultural properties and virulence of Yersinia]. / Kul'tural'nye svoistva i virulentnost' iersinii.

Study of the cultivation properties of 82 enterobacterial strains has revealed that the colonies of virulent Y. enterocolitica (serovars O3, O9) and Y. pseudotuberculosis (serovar I) are temperature-sensitive. This sign, closely connected with the presence and expression of the virulence plasmid with a molecular weight of 44-48 MD, is not characteristic of other strains. Virulent Yersinia grown in nutrient agar for 48 hours at 37 degrees C form colonies which are smaller in diameter than those formed during cultivation at 26 degrees C (with the significance of differences t greater than or equal to 4), their diameter at 37 degrees C not exceeding 1.0 mm. The test for the determination of the temperature-sensitive morphology of Yersinia colonies, along with the tests for other virulence markers, is probably suitable for the detection of the causative agents of yersiniosis or pseudotuberculosis.

[The role of the outer-membrane antigens of Yersinia pseudotuberculosis in the pathogenesis and diagnosis of pseudotuberculosis]. / Rol' antigenov naruzhnoi membrany Yersinia pseudotuberculosis v patogeneze i diagnostike psevdotuberkuleza.

The degree of manifestation of the virulent properties of Y. pseudotuberculosis strains was found to be directly related to the concentration of protein with a mol. wt. of 103 kD (invasin), localized in the outer membrane and necessary for the realization of the process of the penetration of the infective agent into eukaryotic cells. Antiserum to INV-2-BSA, an active synthetic fragment of invasin, capable of detecting one antigenic band with a mol. wt. of 103 kD, was used for the creation of a diagnostic enzyme immunoassay system. The new variant of the diagnostic system was shown to be highly effective in the diagnosis of the disease at an early period.

[The protective activity of Yersinia pseudotuberculosis antigens]. / Protektivnaia aktivnost' antigenov Yersinia pseudotuberculosis.

Samples of Y.pseudotuberculosis (serovar I) antigens, represent a high-molecular lipopolysaccharide (LPS) fraction with a mol. wt. of 22.5 kD and fractions of outer membrane proteins isolated by the method of M. Osborn and R. Munson (1974), were tested in comparison with the activity with live cells of Y. pseudotuberculosis I attenuated mutant KV 9/2, having lost its Cad plasmid of virulence with a mol. wt. of 47 MD and carrying 2 attenuating markers: resistance to crystal violet and nalidixic acid. In experiments on guinea pigs pathomorphological studies demonstrated high protective activity of Y.pseudotuberculosis I attenuated mutant KV 9/2 and a pronounced protective effect achieved after the immunization of the animals with complex biopolymers, including a high-molecular LPS fraction and outer membrane proteins.

[The incidence of finding and characteristics of Klebsiella pneumoniae strains isolated in neonatal infections]. / Chastota vstrechaemosti i kharakteristika shtammov Klebsiella pneumoniae, vydelennykh pri infektsiiakh novorozhdennykh.

The clinical and laboratory study of 190 hospitalized children revealed that in 122 cases the course of the underlying disease was complicated by Klebsiella infection. From different pathological material obtained from 122 patients 158 K. pneumoniae cultures were isolated. These cultures were mostly isolated from the respiratory organs (69%), from patients with the generalized infection (21.6%), and less frequently from operative wounds and burn surfaces (5.8%), the gastrointestinal tract (1.4%) and the urinary system (1.4%). The isolated clinical strains were multiresistant to antibiotics and contained plasmid DNA with molecular weights of 70 and 105 MD (60.4%). K. pneumoniae were shown to produce an aggravating effect on the course and outcome of the underlying disease, mainly in preterm babies or those born asphyxiated or with congenital defects of development, as well as in patients with immunodeficient states. The probability of infection was directly related to the character and number of medical manipulations on an infant, mainly in connection with artificial ventilation of the lungs and transfusion therapy.

[Invasiveness and cytotoxicity as criteria in assessing Yersinia attenuation]. / Invazivnost' i tsitotoksichnost' kak kriterii otsenki attenuatsii iersinii.

The electron microscopic study of cells HEp-2 and the complex microbiological and morphological study of tissues and organs of guinea pigs and mice infected with the isogenic pairs of Yersinia strains (Y. pseudotuberculosis I and Y. enterocolitica 09) differing in the presence of the calcium-dependence plasmid, as well as Y. pseudotuberculosis mutants resistant to rifampicin, nalidixic acid or crystal violet without this plasmid, have revealed that the invasiveness and cytotoxicity of the infective agents are not directly related to the presence of the above-mentioned plasmid in these bacteria. The use of the quantitative characteristics of virulence, such as penetration ability, intracellular multiplication, dissemination and the formation of degenerative changes, has made it possible to find out that the mutants of Y. pseudotuberculosis I, yielding the negative result in the keratoconjunctivitis test and resistant to the above-mentioned antimicrobial substances, can be arranged in the following order according to the degree of attenuation: rifr mutants--nalr mutants--kvlr mutants. In contrast to Y. pseudotuberculosis I, the loss of the calcium-dependence plasmid by Y. enterocolitica 09 is accompanied by an essential decrease in their invasive and cytotoxic properties, but this relationship is indirect and unstable. The proposed criteria intended for use in the evaluation of the degree of the manifestation of the invasive and cytotoxic properties of bacteria can be useful for the selection of optimally attenuated Yersinia strains showing promise as vaccine strains.

[Immunoelectrophoretic analysis of the antigenic composition of Yersinia]. / Immunoélektroforeticheskii analiz antigennogo sostava iersinii.

The antigenic composition of 24. Y. pseudotuberculosis newly isolated and reference strains, 7 Y. enterocolitica strains, as well as Y. pestis vaccine strain EV, has been studied by the method of immunoelectrophoresis in agar. The antigenic composition of these bacteria has been found to be complicated and to comprise not less than 8-11 antigens, and among them nonspecific protein antigens common for enterobacteria, the common generic antigen, the antigen common with Y. pestis, as well as O-antigens specific for each serovar are identified. Immunoelectrophoretic study has shown the possibility of Y. pseudotuberculosis O-antigen, serovar I, with Salmonella sera, serogroup A, and Y. enterocolitica 09 with brucellar and cholera sera.