Development of measures for limiting negative impacts of the «Atomic¼ lake on population and environment.

In arid climate, economic activities at the territory of the "Atomic" lake is one of the topical issues for the Semipalatinsk test site (STS). Hence, the boundaries of areas with radionuclides contamination, which correspond to the level of radioactive wastes at the territory adjacent to the "Atomic" lake of STS, are to be determined. The territory around the lake is used for cattle breeding and the water of the "Atomic" lake that is the one large water source is used for livestock watering. It is important to develop measures that will limit possible negative impact on population and environment. In results of the conducted research were developed measures consisting of remediation and access limitation to the stockpile of soils with contamination level corresponding to the level of radioactive waste (RW).

COMPARISON OF RESULTS OBTAINED BY ELISA AND NEUTRALIZATION TEST IN ASSESSING THE PROTECTION OF POPULATION FROM TICK-BORNE ENCEPHALITIS.

The enzyme-linked immunosorbent assay (ELISA) and the neutralization test (NT) are often used to determine the level of seropositive population and to evaluate the immunogenicity of vaccines. ELISA provides information on the total pool of antiviral antibodies, while NT allows the antiviral protection level of a person to be estimated. It is assumed that the 1:100 titer in ELISA and the 1:10 titer in NT are protective. Obviously, the ratio of the total pool and virus neutralizing antibodies can vary as a result of natural immunization or vaccination. In this study, two methods were used to study the blood serum samples taken in a group of inhabitants of the Sverdlovsk region aged from 1 to 60 years. The samples were collected before immunization and 30 days after two immunizations with inactivated vaccines against tick-borne encephalitis of different manufacturers. Immunizations were performed either according to a standard scheme (30-day interval between immunizations), or according to an emergency scheme (14-day interval). It was shown that the data on the presence of antiviral antibodies in protective titers obtained by ELISA and NT were consistent in more than 85% of cases. The discrepancies between the data are due, in the first place, to the difference in the sensitivities of the two methods. The proportion of seropositive people according to NT data is always greater than that according to the results of ELISA. Nevertheless, among 174 children, about 5% of recipients after a double immunization were seropositive according to ELISA, but did not have neutralizing antibodies in protective titers.

Immunogenicity and safety of the adult tbe vaccine «tick-e-vac¼.

About 3,000 cases of TBE are registered annually in the Russian Federation. Vaccination is the main way to prevent the tick-borne encephalitis disease. Comparative study of the reactogenicity and immunogenicity of a new vaccine «Tick-E-Vac¼ was held. Volunteers aged from 16 years old were twice immunized with the vaccines «Tick-E-Vac¼ or «Encevir¼ derived from strains of Far East subtype of TBE virus, according to standard and emergency schemes. The clinical study was randomized, comparative, blind, and controlled. The frequency, intensity, time of occurrence, and duration of local and general reactions had been recorded. The titers of antiviral antibodies in ELISA had been determined to assess the immunological efficacy of vaccination. According to the results of the clinical study, the severity of local and general reactions in initial seronegative recipients was weak or moderate. The symptoms were usually manifested within 1-2 days after injection and persisted for not more than 4 days, after which time the symptoms disappeared. There was no statistically significant difference in the reactogenicity of the vaccines after the first and after the second injection. The reactogenicity also did not depend on the gender of recipients. After the first immunization, the level of seroprotection was not less than 43%; the average geometric titer of antibodies (GTA), not less than 1:200. After the second injection, the level of seroprotection reached 90-100%; GTA, not less than 1:500. The data on the reactogenicity and immunogenicity to the original seropositive recipients is not significantly different from the data for the initial seronegative recipients. The data indicate weak reactogenicity of the vaccines «Tick-E-Vac¼ and «Encevir¼. Double vaccination with an interval of 14 or 30 days leads to the formation of expressed immune response. Thus, differences in the level of seroprotection and in antiviral titers in the cases of the standard and emergency vaccination schedules are not statistically significant. The correlation between the development in recipients of local and general symptoms and the immunological efficacy of the vaccines has not been identified.

[Usage of real time polymerase chain reaction for diagnostics of different tick-borne infections].

AIM: To create and test the complex of polymerase chain reaction-based methods for detection of pathogens vectored by ticks in clinical and environmental samples. MATERIALS AND METHODS: Real time PCR methods with hybridization-fluorescent detection were developed for detection of tick-borne encephalitis virus, Borrelia burgdorferi sensu lato, Anaplasma phagocytophillum, Erlichia muris/E. chaffeensis, and B. miyamotoi. First four methods were combined in one assay in multiprime format. Efficacy of the assay was assessed by testing of blood samples from patients with tickborreliosis (166 patients), tick-born encephalitis (22 patients) and mixed infection tick-borne encephalitis + borreliosis (21 patients) from Sverdlovsk region. RESULTS: It was shown that using PCR-based assay for testing the blood samples obtained during admission, it was possible to determine the etiology of disease in 39% of patients, whereas on the basis of serological data diagnosis, as a rule, is made not earlier than on 2nd week of therapy. False-positive results of PCR diagnostics were not observed. Infections caused by Anaplasma or Erlichia were not observed. It was shown that > 50% of cases of tick borreliosis without erythema were caused by B. miyamotoi, whereas B. burgdorferi sensu lato predominated as a causative agent of erythemic form of borreliosis. CONCLUSION: Proposed complex of methods is useful for rapid diagnostics of tick-borne infections including previously unknown infection caused by B. miyamotoi.

Origin and distribution of tick-borne encephalitis virus strains of the Siberian subtype in the Middle Urals, the north-west of Russia and the Baltic countries.

Tick-borne encephalitis virus (TBEV) plays an important role in infectious human morbidity, particularly in Russia and the Middle Urals. The Siberian subtype of TBEV (S-TBEV) is dominant in the Middle Urals. Determining the origin of S-TBEV strains in this territory and also in the European part of Russia and the Baltic countries is very important for understanding the cause of its distribution. The surface glycoprotein E gene was partially sequenced in 165 S-TBEV isolates collected in the Middle Urals between 1966 and 2008. Nucleotide and amino acid sequence identity of the studied isolates is 94 and 97.4 %, respectively. Eighty per cent of them are represented by six clusters with identical amino acid sequences in the glycoprotein E fragment analysed. We have determined four types of isolate distribution in the explored territory: local, split, corridor and diffuse. The average rate of nucleotide substitutions per site year(-1) is estimated to be 1.56 x 10(-4). The age of the S-TBEV population was evaluated to be slightly less than 400 years. Phylogenetic analysis of the data and comparison with historical events indicate that the distribution of S-TBEV strains in the Middle Urals and the European part of Russia originated twice from different foci in western Siberia. This is related to the first land road into Siberia and the Trans-Siberian Way, which functioned at different times. The main reason for such rapid distribution of S-TBEV strains is the anthropogenic factor, i.e. human economic activity during the colonization of new territories in Siberia in the recent past.

[Analysis of prevalence and variability of Legionella pneumophila and Legionella spp. strains on the basis of study of allelic profiles].

AIM: To analyze prevalence and variability of Legionella strains isolated in town Verkhnaya Pyshma located in Sverdlovsk region during prophylactic surveillance of potentially dangerous water objects in 2007 - 2008. MATERIALS AND METHODS: Sequencing of mip gene was conducted for identification of species of Legionella. Multi-locus sequence typing was used for describing of allelic profiles of Legionella pneumophila strains. RESULTS: Five firstly identified on Russian territory strains of Legionella species were deposited in institute's collection. Sixty-three strains of L. pneumophila belonging to 28 sequence types were characterized. Relation between strains isolated in industrial building and from water supply system was demonstrated. CONCLUSION: Observations made on the basis of study of L. pneumophila strains isolated from cooling stacks of industrial plants confirmed potential danger of these objects as a source of dissemination of Legionella infection.

[Molecular and epidemiological characteristics of tick-borne encephalitis virus in the Sverdlovsk Region on the basis of genotype-specific RT-PCR].

Studies and complete awareness of the regional and epidemiological properties of tick-borne encephalitis virus (TBEV) allow one to improve methods for preventing, diagnosing, and treating its severe neurological infection. The authors have developed reverse-transcriptase polymerase chain reaction (RT-PCR) systems for the detection of RNA of TBEV and for the determination of its genotype in the ticks and clinical materials. RT-PTC was shown to have a higher sensitivity and specificity than the practically used enzyme immunoassay system. Despite significant variations in the spread of infected ticks in some districts of the Sverdlovsk Region (5-12%), the average regional value was 8% over the study period. The authors have studied more than a thousand of ticks collected from the nature and humans in the epidemic season of 2005-2006. There was a virtually complete predominance (more than 95%) of the Ural-Siberian genotype, with rare cases of the European genotype (slightly more than 4%) being detected. The Far-Eastern genotype was not detected.

[PCR-diagnostics in investigation of Legionnaires' disease outbreak].

Results of studies of biological samples from patients with community-acquired pneumonia and samples from environment during Legionnaires' disease outbreak in town Verkhnyaya Pyshma (Sverdlovsk region) in July-August 2007 performed by PCR analysis are presented.

[Organization of work with nursing staff in Central City Hospital of Verkhnyaya Pyshma town during mass hospitalization of patients with pneumonia caused by Legionella].

Data on organization and carrying out of preventive and antiepidemic measures in hospital during hospitalization of more than 197 patients with Pontiac fever and pneumonia caused by Legionella were presented. Directions and formats of work with medical personnel, including those invited from other medical institutions, as well as their information support were determined. Optimal choice of organizational, and educational technologies, as well as ways to control of compliance with implemented measures by the staff allowed to prevent cases of nosocomial infections in patients and medical personnel.

[Work with public in foci of infection during outbreaks of pneumonia caused by Legionella in town Verkhnyaya Pyshma in July-August 2007].

One of the key elements in epidemiologic control for infectious diseases is timely and active identification of contacts, which results in in-time hospitalization of patients that are needed it and allows to conduct educational work with the public. Measures for outpatient medical and preventive help in Legionella infection which were developed on the experience of termination of outbreak of pneumonia caused by Legionella in town Verkhnyaya Pyshma in July-August, 2007 are presented.

[Epidemiologic examination of focuses of community-acquired pneumonia caused by Legionella].

Main directions of epidemiologic examination of community-acquired pneumonia focus caused by Legionella accounting for features of epidemic process of this infection are discussed. Typical questionnaires, suggested list of documents, characteristics of laboratory tests are presented. Importance of epidemiologic examination of disease focuses as a basis for adequate sanitary and antiepidemic (prophylactic) measures for suppression of outbreaks of Legionella infection are underlined.

[Standards for laboratory diagnostics of legionellosis and their application during epidemic outbreak of pneumonia in town Verkhnyaya Pyshma].

High effectiveness of application of international standards for legionellosis laboratory diagnostics was confirmed during investigation of pneumonia outbreak in town Verkhnyaya Pyshma. Use of immunochromatographic method and enzyme immunoassay for detection of Legionella antigen in urine of patients allows to confirm diagnosis of Legionella infection during several hours, promptly begin etiologic antibacterial treatment and reveal possible sources of infection in potentially dangerous environmental objects.

[Application of molecular genetic methods during Legionnaires' disease outbreak in town Verkhnyaya Pyshma].

The aim of the study was to perform molecular genetic analysis based on multi-locus sequence typing in order to identify source of Legionnaires' disease outbreak in town Verkhnyaya Pyshma in July 2007 and genetic profile of the causative agent. Sequence-based typing protocol recommended by European Working Group on Legionella infection (EWGLI) was used. It was not possible to obtain satisfactory results of Fla gene sequencing for all samples. Obtained allelic profiles of other genes were typical for L. pneumophila. Allelic profiles of L. pneumophila isolated from patients were identical and matched with L. pneumophila DNA detected in water from hot water supply of domestic building, but differed from cooling tower's isolates and isolates from showerhead in apartment of one patient. Identity of 5 genes of L. pneumophila isolated from autopsy samples and from hot water of central hot water supply of domestic building confirms aspiration route of infection through hot water contaminated by the microorganism. L. pneumophila detected in water from cooling tower, showerhead in apartment of one patient, and from drainage canal of hot water supply station belonged to other allelic variants and, therefore, are not related with the outbreak.

[Laboratory diagnostics in community-acquired pneumonia outbreak investigation in town Verkhnyaya Pyshma].

Data on laboratory study of 367 clinical samples from patients with community-acquired pneumonia diagnosis, 70 samples of cold and hot water from the systems of cold and hot water supply, 15 samples of technical water, 4 samples from open basins, 35 lavage samples from environmental objects, which came to laboratory of control for biological factors in Center of Hygiene and Epidemiology in Sverdlovsk region during outbreak pneumonia caused by Legionella in town Verkhnyaya Pyshma in July-August 2007, were presented. Procedure for laboratory diagnostics was developed, complex of laboratory methods considering the pressure of time in outbreak situation was determined.

[Epidemiologic situation on pneumonia incidence in population of Sverdlovsk region in 2006-2007 and goals for improving of system for epidemiologic surveillance].

Personalized system for registration of community-acquired pneumonia cases instituted in Sverdlovsk region in 2002 is able to provide completeness of identification, accounting, and registration of pneumonia cases as well as to organize regular medical follow-up for survived persons. In addition, the automated system allows to conduct operative and retrospective analysis of morbidity according to area, age and social groups with identification of casual and risk factors as well as to timely organize the complex of preventive measures in an area.

[Development and use of the assay for Legionella pneumophila detection based on fluorescent real-time/endpoint polymerase-chain reaction].

The aim of the work was to develop a PCR-based assay for detection of L. pneumophila and L. micdadei in environmental samples as well as in clinical samples from low respiratory tract and to assess its analytic characteristics. The assay was used during investigation of the outbreak developed in July 2007 in town Verkhnyaya Pyshma (Sverdlovsk region). Polymerase-chain reaction (PCR)with fluorescent detection,sequencing and cloning of DNA fragments were used. Developed assay based on the PCR with fluorescent real-time/ endpointdetection is able to detect L. pneumophila in clinical and environmental samples and to quantify amount of bacterial DNA in water. Specificity of analysis (100%) was assessed using the panel of bacterial strains and samples from healthy individuals. Analytic sensitivity of assay and quantitation limit was 1000 GU in 1 ml. Sensitivity of the assay of artificially contaminated biological samples was 1000 bacteria in 1 ml. During outbreak investigation L. pneumophila DNAwas detected in 4 lung samples from 4 fatal cases, from 1 of 2 sputum samples, 1 of 2 bronchoalveolar lavage samples with X-ray confirmed pneumonia. Legionella's DNA was found in samples from cooling towers, central hot water supply as well as from showerheads in apartments of 3 patients. Fountain and drinking water samples were PCR-negative. Specificity of PCR-positive results was confirmed by sequencing. Use of the assay during outbreak in- vestigation allowed to confirm the diagnosis in fatal cases and quickly identify the possible source of infection.

[Epidemiologic versions in investigation of Legionnaires' disease outbreak in town Verkhnyaya Pyshma].

Materials of investigation of Legionnaires' disease outbreak in one of the town of Sverdlovsk region in summer 2007 are presented. Outbreak arose in situation of epidemiologic welfare on majority of registered infectious diseases. Epidemiologic diagnostics of the outbreak has been performed in maximal shorttime interval, during which it was necessary to investigate variety of miscellaneous versions. Active search of the source of the infection and its transmission factors resulted in desired result and allowed to terminate the outbreak in 14 days.

[Epidemiologic situation and measures for prevention of infectious diseases in Sverdlovsk region in 2006-2007].

Materials characterizing the epidemiologic situation on most significant infectious diseases in Sverdlovsk region are presented. Experience on providing the epidemiologic welfare based on systematic surveillance for activity of various factors influencing the epidemic process is described.

[Organizational support of investigation of Legionnaires' disease outbreak in town Verkhnyaya Pyshma].

Materials on organizational support of preventive and antiepidemic measures during outbreak of Legionnaires' disease in town Verkhnyaya Pyshma (Sverdlovsk region) in July-August 2007 are presented. Thanks to collaborative efforts of Federal Service for Surveillance for Protection of Consumers Rights and Human Welfare, Ministry of Health of Sverdlovsk region, health-care services in Verkhnyaya Pyshma, near located municipalities, and others it was able to establish in short term the hospital for treatment, identify source of the infection and factor of its transmission, and terminate the outbreak. Operative implementation of the mentioned measures both allowed to provide good quality medical care to all patients and to prevent occurrence of such situations on other towns of Sverdlovsk region.

[Organization of preventive and antiepidemic measures in conditions of massive outbreak of Legionnaires' disease in population of town Verkhnyaya Pyshma. Epidemiologist's work experience in ensuring biosafety].

Data on organization and carrying out of preventive and antiepidemic measures in hospital during mass hospitalization (> 190 cases) of patients with community-acquired pneumonia caused by Legionella pneumophila are presented. Experience of physicians-epidemiologists in determination of directions of work in hospital, procedures of interaction, extent and description of biosafety measures in order to prevent nosocomial spread of infection to patients and staff are described.