[THE PATHOGENIC POTENTIAL OF MORAXELLA CATARRHALIS AND STAPHYLOCOCCUS EPIDERMIDIS UNDER INFLAMMATORY PROCESSES OF UPPER RESPIRATORY TRACTS].

The frequent isolation from biological material of Moraxella catarrhalis under bronchitis and pneumonia and Staphilococcus epidermidis under rhinitis and sinusitis requires profound investigation offactors ofpathogenicity ofthe mentioned microorganisms. The genetic and phenotypic markers of virulence of strains M. catarrhalis and S. epidermidis are examined. Their etiologic role in development of infection processes of respiratory tract and middle ear is determined The most of M catarrhalis strains isolated under bronchitis and pneumonia have gene mcaP responsiblefor production ofprotein McaP that provides adhesion to epithelium cell of host and lipolitic activity of bacteria. The strains isolated from patients with pneumonia had the most adhesive activity. The cluster of genes ICA with leading role of gene icaA is responsible for for availability offactors of intercellular adhesion in Staphilococci strains. In the clinical samples from patients with sinusitis this gene is detected 5 times more frequently than from healthy individuals. In phenotypic tests, expression of gene icaA in S. epidermidis isolated from patients is three times higher than in strains isolated from healthy individuals. To establish etiologic role of M. catarrhalis and S. epidermidis and to develop tactic of therapy of patients with bronchitis, pneumonia and sinusitis complex approach is needed, including detection of genetic and phenotypic markers of virulence in isolated microorganisms.

Typing and clustering of Yersinia pseudotuberculosis isolates by restriction fragment length polymorphism analysis using insertion sequences.

Yersinia pseudotuberculosis is an enteropathogen that has an animal reservoir and causes human infections, mostly in temperate and cold countries. Most of the methods previously used to subdivide Y. pseudotuberculosis were performed on small numbers of isolates from a specific geographical area. One aim of this study was to evaluate the typing efficiency of restriction fragment length polymorphism of insertion sequence hybridization patterns (IS-RFLP) compared to other typing methods, such as serotyping, ribotyping, and multilocus sequence typing (MLST), on the same set of 80 strains of Y. pseudotuberculosis of global origin. We found that IS100 was not adequate for IS-RFLP but that both IS285 and IS1541 efficiently subtyped Y. pseudotuberculosis. The discriminatory index (DI) of IS1541-RFLP (0.980) was superior to those of IS285-RFLP (0.939), ribotyping (0.944), MLST (0.861), and serotyping (0.857). The combination of the two IS (2IS-RFLP) further increased the DI to 0.998. Thus, IS-RFLP is a powerful tool for the molecular typing of Y. pseudotuberculosis and has the advantage of exhibiting well-resolved banding patterns that allow for a reliable comparison of strains of worldwide origin. The other aim of this study was to assess the clustering power of IS-RFLP. We found that 2IS-RFLP had a remarkable capacity to group strains with similar genotypic and phenotypic markers, thus identifying robust populations within Y. pseudotuberculosis. Our study thus demonstrates that 2IS- and even IS1541-RFLP alone might be valuable tools for the molecular typing of global isolates of Y. pseudotuberculosis and for the analysis of the population structure of this species.

[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".

[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.

[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.

[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.

Immunochromatographic strip test for rapid detection of diphtheria toxin: description and multicenter evaluation in areas of low and high prevalence of diphtheria.

An immunochromatographic strip (ICS) test was developed for the detection of diphtheria toxin by using an equine polyclonal antibody as the capture antibody and colloidal gold-labeled monoclonal antibodies specific for fragment A of the diphtheria toxin molecule as the detection antibody. The ICS test has been fully optimized for the detection of toxin from bacterial cultures; the limit of detection was approximately 0.5 ng of diphtheria toxin per ml within 10 min. In a comparative study with 915 pure clinical isolates of Corynebacterium spp., the results of the ICS test were in complete agreement with those of the conventional Elek test. The ICS test was also evaluated for its ability to detect toxigenicity from clinical specimens (throat swabs) in two field studies conducted within areas of the former USSR where diphtheria is epidemic. Eight hundred fifty throat swabs were examined by conventional culture and by use of directly inoculated broth cultures for the ICS test. The results showed 99% concordance (848 of 850 specimens), and the sensitivity and specificity of the ICS test were 98% (95% confidence interval, 91 to 99%) and 99% (95% confidence interval, 99 to 100%), respectively.

[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 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 immunological indices in pathology due to yersiniosis]. / Immunologicheskie pokazateli pri patologii, obuslovlennoi iersiniozami.

Immunological characteristics in the time course of infectious processes in pseudotuberculosis and yersiniosis were studied. In diseases caused by Y. pseudotuberculosis highly virulent strains, serovar I, the suppression of T-cell-mediated immunity and a decrease in the characteristics of nonspecific resistance, especially at the peak of the process, as well as the tardy production of specific antibodies, were observed. For the relapsing course of infection, less pronounced decrease of immunological characteristics, whose values remained low also at the period of expected convalescence, was established. The study of the immune status in yersiniosis showed differences in immunological characteristics, depending on the serovar of the infective agent. In comparison with yersiniosis caused by Y. enterocolitica, serovar O9, yersiniosis caused by Y. enterocolitica, serovar O3, was found to produce more profound disturbances in the immune system and to have the potential possibility for the development of autoimmune complications.