Effect of different types of dry swabs and their storage conditions on the inoculation of Corynebacterium diphtheriae.

The results of evaluating the effectiveness of C. diphtheriae inoculation using different types of dry swabs in studies simulating various conditions of its storage at the preanalytical stage of a laboratory study for diphtheria are presented. A typical toxigenic strain of C. diphtheriae biovar gravis No. 665 was used. A commercial dry, sterile cotton swab probe (Ningbo Greetmed Medical Instruments Co., LTD, China), a commercial dry, sterile swab probe (plastic and viscose) (COPAN, Italy), tufters with a fluffy probe-tampon on a polystyrene applicator, standard (DELTALAB, SL, Spain). The tampons were pooled with a 24-hour bacterial culture of C. diphtheriae, then immediately seeded on Tellurite-containing blood agar and Corynebacagar. Storage conditions were simulated for 3 hours: at room conditions +(20-25)°C, in the refrigerator +(4-8)°C, in a thermostat +(37±1)°C. Optimal storage of C. diphtheriae on all three types of dry swabs at + (4-8) ° C; at +(20-25)° C - growth is observed when seeding from a cotton swab; in a swab with a fleecy probe-tampon, a decrease in the inoculation of C. diphtheriae was noted; when using a viscose swab - a significant loss of C. diphtheriae. At +(37±1)°C, a significant decrease in the inoculation of C. diphtheriae on all three types of tampons was noted, up to the absence of growth when using a viscose tampon. To exclude the loss of C. diphtheriae, it is necessary to observe the conditions for taking and storing biological material at the preanalytical stage of a laboratory study, which will improve the quality of laboratory microbiological studies for diphtheria infection.

Comparative tests of quality of bacteriological investigation with Corynebacterium diphtheriae isolation.

The results of comparative experimental studies of identification of nontoxigenic C. diphtheriae strain by three different commercial laboratories are presented. A typical nontoxigenic strain of C. diphtheriae biovar mitis was used. For the studies, three lines of ten-fold dilutions of bacterial culture were prepared, followed by control planting on the medium and counting CFU/ml. In the experiment, tampons were pooled with a 24-hour bacterial culture of a nontoxigenic C. diphtheriae strain. Tampons were provided from three different laboratories - ∑-Transwab® with Ames liquid medium (from the first and second laboratories) and a viscose tampon with coal medium (from the third laboratory). After pooled, tampons were delivered to commercial laboratories. And as a result of the experiment, Corynebacterium spp. was identified in first laboratory (103 CFU/tamp), S. epidermidis (102 CFU/ml) - in second laboratory and nontoxigenic C. diphtheriae biovar gravis - in third laboratory. The study indicates that there is a need to the supervision of bacteriological investigations conducted in various laboratories. This will improve the quality of investigations on diphtheria infection and identify of diphtheria carrier, which is a reservoir of the causative agent of diphtheria, and will contribute to the maintenance of sanitary and epidemiological well-being in our country.

Digital analysis and quantitative assessment of the cervical surface with dysplasia.

The investigation aims - a quantitative assessment of cervical surface changes with digital analysis and computer technologies in dysplasia. Colposcopy was made in 90 women from 21 to 52 years (avr. age 33,9±8,13 y.o.) with mild epithelial dysplasia (CIN1), moderate dysplasia (CIN2), severe dysplasia (CIN3). The algorithm detected indicators which provide the cervical dysplasia classification on pre cytological and pre molecular-genetic patients investigations. The outcome of an algorithm was the identification of the cervix surface condition severity by an objective quantification. The cervical dysplasia type (CIN) was classified as IndGV values. The mild dysplasia (CIN1) had IndGV=8,5, moderate dysplasia (CIN2) - IndGV=13, severe dysplasia (CIN3) - IndGV=15,6. The cervical affected surface area (IndInt) equalled 0,17 in CIN1, 0,19 in CIN2, 0,22 in CIN3. A change severity has a direct relation with a grey color value. It demonstrates quantify classification in digital analysis. The algorithm is used in real-time mode and no requires considerable material outlays. This makes it possible to use an algorithm after clinical examination and predict patient management.

PCR-based diagnosis of whooping cough in the Russian Federation.

The aim was to determine how often the PCR method is used in different laboratories in Russia. In 2018, we conducted a questionnaire survey in diagnostic laboratories of medical organizations and the Centers of Hygiene and Epidemiology that performed PCR studies to identify microorganisms of the genus Bordetella in all 85 Russian regions. We found that in 2013 the PCR was used in 33 (38.8%) regions, but in 2017 the number of regions increased to 64 (75.3%). During 2013-2017 the study has not been applied in 21 regions. The number of PCR tests performed in the laboratories of medical organizations was significantly different. There has been an increase in the number of tests for the diagnosis of pertussis among people with clinical signs of infection and among contact persons in foci of infection. Compared to the Centers of Hygiene and Epidemiology, in medical organizations the rate of introduction of the PCR was higher. Between 2013 and 2017 the proportion of samples containing DNA B.pertussis decreased, but the proportion of samples containing DNA of other representatives of the genus Bordetella increased. Moreover, in the case of isolation DNA Bordetella spp. clinicians diagnose «Whooping cough, other unspecified organism¼, since there is no information on the species of the pathogen. Thus, in order to improve the diagnosis of pertussis, it is necessary to optimize PCR tests by including target genes that allow to identify of currently relevant DNAs of different representatives of the genus Bordetella.

Status and problems of bacteriological diagnosis of diphtheria infection in the Russian Federation.

The purpose of the work was to assess the state of bacteriological diagnosis of diphtheria infection in Russia in order to establish possible reasons for the decrease in the release of C. diphtheriae. The Reference Center for Monitoring the Pathogens of Measles, Rubella, Mumps, Pertussis and Diphtheria in 2018 in 85 subjects of Russia conducted a questionnaire of laboratories of medical organizations and the Centers for Hygiene and Epidemiology of Rospotrebnadzor, carrying out bacteriological studies for diphtheria infection. It was found that the number of studies conducted over the five-year period decreased by 1.2 times. The tendency to decrease the number of bacteriological studies for diphtheria is observed in the territories of almost all federal districts. In 99% and 29% of cases, the institutions of the FBUZ Centers for Hygiene and Epidemiology and medical organizations (MO) and use in their work documents regulating bacteriological studies for diphtheria infection. In a number of territories, the list of documents used includes documents that are invalid or do not define such studies. Most organizations use dry tampons when examining for diphtheria, however, 13.1% and 53.4% of FBUZ Centers for Hygiene and Epidemiology and medical organizations (respectively) use commercial transport environments, which does not comply with regulatory documentation. Analysis of the quality of work of bacteriological laboratories showed shortcomings at the stage of preparation of media (use of donor blood, or absence of addition of blood and potassium tellurite), Elek tests (addition of horse serum or absence of serum to the medium), setting of incomplete biochemical series (absence of tests for urease and nitrate reductase), absence of standard control strains, incomplete volume of internal laboratory quality control. Given the continuing circulation of the pathogen in various countries of the world and in our country, as well as the possibility of imported cases of infection from endemic regions, the analysis was aimed at drawing the attention of specialists to the problem of improving the quality of laboratory diagnosis of diphtheria in Russia.

Development of accelerated genodiagnosis method of pertussis and pertussis-like diseases on the basis of mPCR-RT.

The aim of the work was to develop an accelerated genodiagnosis method based on mPCR-RT for the detection DNA of B. pertussis, B. parapertussis, B. holmesii. MATERIALS AND METHODS: The study used 104 strains of microorganisms, of which: 50 strains of B. pertussis, 37 - B. parapertussis, 17 - heterologous species of microorganisms. Assessment of analytical specificity was carried out using DNA strains of various microorganisms with a concentration at least 109 GE / ml. To check the analytical sensitivity we studied a series of serial dilutions of bacterial cultures of the control strains B. pertussis № 143, B. parapertussis № 38b, B. holmesii DSM 13416 with a concentration of 5x109 - 5 µm/ml. RESULTS: Insertion sequences were chosen as diagnostic targets: for B. parapertussis - a specific fragment IS1001, for B. holmesii - a specific fragment hlIS1001, for B.pertussis - a fragment IS481. To develop a genodiagnosis method specific primers were designed and combined into a single multi-primer mixture, the composition of the reaction mixture and the amplification conditions were selected. The analytical sensitivity of the developed method for detecting pertussis and pertussis-like pathogens was 5×101 GE / ml. Verification of the developed methodology of gene diagnostics showed 100% analytical specificity. CONCLUSION: An accelerated genodiagnosis method based on mPCR-RT has been developed, it allows you to identify DNA of B. pertussis, B. parapertussis, B. holmesii, which expands the possibilities of examining patients with suspected pertussis and pertussis-like diseases in order to increase laboratory confirmation of the diagnosis.

[Discriminant analysis in establishing the relationship of pathogenetic mechanisms of gestational complications in urogenital infection in pregnant women.] / Дискриминантный анализ в установлении взаимозависимости Ð¿Ð°Ñ‚Ð¾Ð³ÐµÐ½ÐµÑ‚Ð¸Ñ‡ÐµÑÐºÐ¸Ñ Ð¼ÐµÑ Ð°Ð½Ð¸Ð·Ð¼Ð¾Ð² развития Ð³ÐµÑÑ‚Ð°Ñ†Ð¸Ð¾Ð½Ð½Ñ‹Ñ Ð¾ÑÐ»Ð¾Ð¶Ð½ÐµÐ½Ð¸Ð¹ при урогенитальной инфекции Ð±ÐµÑ€ÐµÐ¼ÐµÐ½Ð½Ñ‹Ñ .

The aim of the work - to establish the interconnection and interdependence of toll-like mediated pathogenetic mechanisms of urogenital infection in pregnant women from the position of epigenomics. Using discriminant analysis in 89 patients with urogenital infection in pregnant women for the first time was established a reliable evidence-based relationship and interdependence between mucosal immunity, the severity of the infectious process, clinical manifestations, symptoms of miscarriage in the background of simultaneous development of the infectious process and pregnancy. For urgent delivery (infection), urgent childbirth (infection and clinical manifestation) and premature birth, mucosal immunity determines the severity of anti-infective resistance (with increasing mucosal immunity oppression of infectious process and clinical manifestations is logged , and its decrease increases the severity of infection process and clinical manifestations); the inhibition of mucosal immunity prevails over its hyperreaction (inhibition of mucosal immunity is determined by the physiological immunodepression in response to the development of pregnancy, as well as in response to herpes virus infection when activated); the severity of the infectious process depends on the severity of clinical manifestations and symptoms of miscarriage. During miscarriage mucosal immunity provides the pathophysiological course of infectious process and the clinical manifestations and development of symptoms of misacrriage; increasing levels of mucosal immunity to hyperreaction contributes to the development of symptoms of abortion and miscarriage; not registered mutual influence of oppression, mucosal immunity and its hyperreaction; the severity of the infectious process does not depend on the severity of clinical signs and symptoms of miscarriage. In urgent childbirth (infection), the oppression of mucosal immunity does not affect the severity of clinical manifestations, symptoms of abortion and the infectious process. In urgent or premature birth, and termination of pregnancy, the oppression of mucosal immunity affects the severity of clinical manifestations, the severity of the infectious process and the symptoms of abortion; the severity of clinical manifestations and the severity of the symptoms of abortion are interrelated. In urgent birth (infection) mucosal immunity overreaction affects the severity of clinical manifestations, symptoms of miscarriage and infection; in case of term and preterm labour overreaction mucosal immunity on the severity of infection and symptoms of abortion and does not affect the severity of clinical manifestations and at the termination of a pregnancy mucosal immunity on the severity of the infectious process and does not affect the severity of clinical signs and symptoms of abortion. The levels of mucosal immunity inhibition, its hyperreaction, clinical manifestations, symptoms of pregnancy termination and the severity of the infectious process do not depend on the type of herpes simplex virus. In the absence of infection with herpes simplex virus in patients with urogenital infections of pregnant women, there is no mutual influence and the relationship between the oppression of mucosal immunity and hyperreaction of mucosal immunity, the oppression of mucosal immunity prevails over its hyperreaction. With increasing mucosal immunity oppression, increased anti-infectious resistance of the body (the decreased activity of the infectious process), and with its decrease decreased (increased activity of the infectious process). Hyperreaction of mucosal immunity influenced the severity of pregnancy termination symptoms, clinical manifestations and infectious process, and also determined the severity of pregnancy termination symptoms. The severity of the infectious process and clinical manifestations influenced the symptoms of abortion. The severity of the infectious process did not affect the clinical manifestations. During infection with herpes simplex virus type I or type I and II on the background prevalence of oppression mucosal immunity over hyperreaction mucosal immunity, the presence of relationships between them, and the impact of mucosal immunity on the severity of the infectious process and the clinical manifestations increase mucosal immunity has been shown to decrease the severity of infection and clinical manifestations (reduction of anti-infective resistance), while reducing mucosal immunity the severity of infection and clinical manifestations increased. Hyperreaction of mucosal immunity influenced the severity of pregnancy termination symptoms and determined the severity of pregnancy termination symptoms. The severity of the infectious process and clinical manifestations influenced the symptoms of abortion. The severity of clinical manifestations reflects the severity of the infectious process. In type I and type II of pregnancy, the level of mucosal immunity determines the anti-infectious resistance of the body in urogenital infection of pregnant women. Inhibition of mucosal immunity and its hyperreactions are interrelated, have an impact on each other, as a result of their integral interaction, increasing the levels of mucosal immunity leads to a decrease in the severity of clinical manifestations and the infectious process, reducing the levels of mucosal immunity contributes to the manifestation of clinical manifestations, as well as increasing the severity of the infectious process. Hyperreaction of mucosal immunity affects the severity of symptoms of abortion, infection and clinical manifestations. The infectious process and clinical manifestations determine the severity of the symptoms of abortion. In type III and type IV of pregnancy course, there is no mutual influence of mucosal immunity oppression and its hyperreaction. The levels of indicators of mucosal immunity oppression and its hyperreaction are interrelated; the increase in the severity of mucosal immunity oppression is accompanied by a decrease in clinical manifestations and severity of the infectious process and vice versa. Hyperreaction of mucosal immunity affects the severity of symptoms of abortion, infection and clinical manifestations. The infectious process determines the severity of the symptoms of abortion and clinical manifestations, acting as a leading component of gestational complications in urogenital infection of pregnant women. In the III type of pregnancy course oppression of mucosal immunity does not affect the severity of symptoms of miscarriage. In the IV type of pregnancy course, the levels of mucosal immunity oppression prevail over the indicators of mucosal immunity hyperreaction, which is due to the integral interaction of physiological inhibition of immunological reactivity of the organism in response to pregnancy and inhibition of immunological reactivity of the organism, accompanying the activation of infectious process of viral genesis. Hyperreaction of mucosal immunity determines the symptoms of abortion.

[COMPOSITION OF POPULATION OF DIPHTHERIA CAUSATIVE AGENT STRAINS IN RUSSIA].

AIM: 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. MATERIALS AND METHODS: 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 of <> gene 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. CONCLUSION: 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.

[Quantitative and qualitative composition of axilla microbiota in practically healthy individuals].

AIM: Characteristics of quantitative and qualitative composition of cultured microorganisms isolated from axilla skin of practically healthy individuals. MATERIALS AND METHODS: 77 practically healthy individuals aged 18 to 40 years were examined. Species identification of microorganisms was carried out byculture-morphologic, tinctorial and biochemical properties using time-of-flight mass spectrometer and rpoB gene amplification with subsequent direct sequencing. RESULTS: Quantitative evaluation of microbial composition of axilla skin microbiota in most of the practically healthy individuals varied in the 4-5 lg CFU/ml interval, whereas seeding of skin by this microbiota at the level of 8 lg CFU/ml was not detected. 158 strains of 24 microorganism species were identified in this biotope. Most of these strains (68.9%) belonged to Corynebacterium genus, 21.6% of strains--to Staphylococcus genus, 7.6% of strains--to Micrococcus genus and 1.9% of strains--Candida albicans. 16 species of corynebacteria were isolated with predomination of C. tuberculostearicum (40.3%), C. amycolatum (18.4%) and C. ureicelerivorans (14.8%) strains. The microbial landscape in most of the examined individuals (77.9%) was presented by microorganism association. CONCLUSION: Quantitative and qualitative species composition of cultured microorganisms isolated from axilla skin biotope of practically healthy individuals was characterized for the first time.