[Clinical manifestations of Legionella pneumonia in hematology patients].

AIM: To detect the most common clinical manifestations of Legionella pneumonia (LP) in immunocompromized patients. SUBJECTS AND METHODS: Clinical manifestations, the results of investigation of bronchoalveolar lavage fluid (BALF) and urine, and the data of lung computed tomography (CT) were studied in patients with blood system diseases and acute respiratory failure (ARF). RESULTS: The diagnosis of LP was verified in 8 (10.5%) of 76 patients with blood system diseases and ARF. The disease manifested as fever, higher concentrations of inflammatory markers (procalcitonin, fibrinogen), ARF, hypoxemia, and infiltrative lung injury. Six of the 8 patients were switched to mechanical ventilation. Lung CT showed no pathognomonic signs. Five of the 8 patients were observed to have renal dysfunction. The diagnosis of LP was made on the basis of the results of BALF examination in 7 patients and urinary antigen detection in 1. The disease was caused by Legionella pneumophila serogroup 1 in 3 patients and by L. pneumophila of other serogroups in the other patients. Therapy with respiratory fluoroquinolones was performed in 5 patients. Three patients died from progressive ARF and hypoxemia. BALF results were obtained after their death and therapy for legionellosis was not initiated. CONCLUSION: The incidence of LP is 10.5% in hematology patients. The clinical manifestations of legionellosis are nonspecific; its diagnosis requires bacteriological and/or serological evidence. Due to the high risk of death, it is reasonable to preuse respiratory fluoroquinolones or macrolides in immunocompromized patients with progressive ARF and suspected Legionella pneumonia before diagnosis.

[Features of legionella biofilm formation in artificial and natural water systems].

AIM: Study the ability to form monospecies and associative biofilms as a characteristic oflegionella strains and features of organization of natural legionella biofilms in potentially dangerous water systems. MATERIALS AND METHODS: Comparative evaluation ofthe ability of 28 strains of Legionella spp. to form biofilms was determined in water according to previously developed procedure. Samples from biofilm of industrial enterprise coolers and systems of hot water supply of public buildings (hotels, trade centers, hospitals) were studied. Biofilms were studied by scanning and transmission electron microscopy methods. RESULTS: Legionella strains are divided into 3 groups by the ability to form biofilms. L. pneumophila BLR-05 strain that has the most pronounced ability to form monospecies biofilm and persistence in association with Pseudomonas aeruginosa was detected. Formation of massive legionella biofilm in association with bacteria of other taxonomic groups was detected on protective antibacterial filters in the system of hot water supply of a department of a therapeutic prophylaxis institution in the course of 2-3 weeks. Legionella biofilms on the surface of coolers resemble an aggregate of fungi, bacteria and blue-green algae enclosed into matrix. CONCLUSION: The ability to form artificial monospecies and associative biofilm may be a useful characteristic of legionella strains for evaluation of their adhesion and be used to evaluate epidemiological significance of the isolated strains. Prevention of formation of natural legionella biofilms in potentially dangerous water systems is necessary as an essential component of modern strategy of legionellosis prophylaxis.

[Prevalence of glucosyl transferase Lgt among Legionella pneumophila strains isolated from various sources].

AIM: Determine various members of Lgt glucosyl transferase family among microorganisms of Legionellaceae genus from museum collection and legionellae strains recently isolated in the Russian Federation and Germany. MATERIALS AND METHODS: Presence of 3 types of glucosyl transferase were determined in 73 strains of L. pneumophila and Legionella spp. Glucosyl transferase activity of 3 types (Lgt1, Lgt2 and Lgt3) was determined by western blotting and PCR method. RESULTS: Lgt1 and Lgt3 were detected only in members of L. pneumophila independently of isolation source and were absent in Legionella spp. strains. Lgt2 is absent in Legionella spp. strains and is detected in not all the L. pneumophila strains. Comparative analysis of detection frequency of Lgt2 in clinical strains and L. pneumophila strains isolated from the environment showed that the protein is detected in clinical strains more frequently (46%) compared with strains from the environment (23%). CONCLUSION: Lgt1 and Lgt3 as species specific markers could be used for practical purposes for identification of L. pneumophila strains. High frequency of Lgt2 isolation in clinical strains of L. pneumophila isolated from lung tissue in lethal cases of legionellosis compared with strains isolated from the environment requires a more detailed study of functional activity and substrate specificity of the glucosyl transferase.

[Serologic characteristic of Legionella pneumophila strains isolated from potentially dangerous water systems in Russian Federation in 2007 - 2011].

AIM: Study serologic diversity of Legionella pneumophila strains circulating in potentially dangerous water systems in Russian Federation by using an international panel of monoclonal antibodies. MATERIALS AND METHODS: Serotyping of 234 L. pneumophila strains isolated from coolers of industrial facilities and systems of hot water supply in Russian Federation in 2007 - 2011 was performed by enzyme immunoassay by using an international panel of monoclonal antibodies. RESULTS: Membership of the isolated strains in 14 L. pneumophila serogroups and in 7 subgroups of serogroup 1 was established. Among the isolated cultures serogroup 1 and 6, and Oxford and Heysham subgroup strains were predominant. L. pneumophila serogroup 1 strains were predominant in cooler water, and serogroup 6--in the hot water supply systems. 7 L. pneumophila strains of the serogroup 1 were typed by monoclone MAb 3/1 associated with LPS epitope that is characteristic for the most epidemically significant legionella strains. CONCLUSION: Typing by using international panel of monoclonal antibodies for characteristic and evaluation of epidemical significance of legionella strains being isolated form potentially dangerous water systems is the most informative and methodically accessible to a wide range of biological laboratories.

[Examination of the contamination aetiological agent of legionellosis in the water supply systems of medical treatment facilities].

This study considers the features of colonization of water distribution systems by Legionella in large multi-type health-care facilities in Moscow-city, Russia. The investigation of 16 pavilions in 5 multi-type health-care facilities had found the hith level of hot water systems contaminated by Legionella pneumophila (68%), including the risk units. Among the different species of Legionella pneumophila, circulating in hot water systems of health-care facilities, the following strains had prevailed: the serogroup 6 (44%), 5 (26%) and 1 (13%). The findings conclude that certain prevention measures should be taken for microbial control and ensuring water safety in water systems of health-care facilities to prevent the nosocomial cases of legionellosis.

[A case of legionellesis pneumonia verified by isolation of Legionella pneumophila serogroup 1 from bronchoalveolar lavage fluid treated with levofloxacine and tigecycline].

A male patient received non-chemotherapeutic drugs which induced deep neutropenia complicated with sepsis, bilateral pneumonia, acute respiratory insufficiency. Artificial pulmonary ventilation was applied. The examination of bronchoalveolar lavage showed the presence of the culture L. pneumophila (serogroup 1) in a concentration 2 x 10(3) CFU/ml. Antibacterial therapy with levofloxacin in a dose 1000 mg/day was conducted. In a week not only L.pneumophila but also Acinetobacter baumanii was isolated from bronchoalveolar lavage. Tigecyclin was added to levofloxacin treatment. Two air cavities were found in the left lung. The treatment reduced the size of these cavities, infiltrative changes in the lungs and respiratory insufficiency regressed. The patient was discharged from hospital This case is the first case in Russia of L.pneumophila isolation from bronchoalveolar lavage. The case is also characterized by use of tigecycline for treatment of combined legionella and akinetobacterial infection and cavities in the lungs in legionella pneumonia.

[Rate and level of contamination of potentially dangerous water supply facilities in Moscow region by Legionella pneumophila].

AIM: To assess rate and level of contamination by Legionella pneumophila of cooling water systems in industrial facilities as well as hot water supply systems of administrative buildings in Moscow region. MATERIALS AND METHODS: Cooling water systems of 8 industrial facilities and hot water supply systems of 12 administrative buildings or complexes located in Moscow or Moscow region were examined. Samples of water, washes and biofilms were studied by bacteriologic methods and RT-PCR. Results. Significant level of contamination of water systems by L. pneumophila was revealed in examined objects. Rate of contamination of cooling water systems in industrial facilities was 70%. The agent was detected in stagnant, end-capped, and rarely used segments of all hot water supply systems during decrease of water temperature to 36-52 degrees C. Visual detection of natural biofilms on the object correlated with high concentration of L. pneumophila in water samples. In some cases, associations of L. pneumophila with Pseudomonas aeruginosa were detected, including water samples from supply systems of 2 healthcare facilities. CONCLUSION: Obtained results confirm the importance of implementation of modem concept of legionellosis prevention in our country, based on regular quantitative monitoring for Legionella in potentially dangerous water objects and conduction of preventative measures if contamination exceeds acceptable level.

[Methodic bases for providing the population and potentially dangerous water objects with biosafety against the pathogens of Legionellosis].

The paper considers the key aspects of the ecology of Legionella determining the increasing role of the pathogen in the emergence of human infectious diseases in postinduatrial society. Formation of Legionella biofilms on potentially dangerous water objects with possible further aerosol or aspiration transmission of the pathogen may lead to large epidemic outbreaks and sporadic cases of the infection. Based on international experience and the results of their studies, the authors propose a current legionellosis prevention concept based on microbiological monitoring and control of potentially dangerous water objects.

[Modern conception of organization of epidemiological surveillance for Legionella infections].

Evolution of views on legionellosis is analyzed in the review: from extremely dangerous rare infection to sufficiently widespread severe pneumonia emergence of which is determined by features of ecology and prevalence of natural water microorganism in potentially dangerous water systems created by man. Epidemiologic features of 3 main groups of legionellosis determined according to place of acquisition are discussed: sporadic (community-acquired) legionellosis; nosocomial legionellosis; legionellosis related with trip or travel. Modern principles of organization of epidemiologic surveillance for legionellosis based on the disease's incidence monitoring, observation on circulation of the agent in potentially dangerous water systems, conducting of prophylactic measures aimed at prevention of sporadic cases and epidemic outbreaks. Organization of epidemiological surveillance for Legionella infection in Russian Federation in present time is developed on the basis of international standards, recommendations of World Health Organization, home practical experience from elimination of epidemic outbreaks of legionellosis, and results of Legionella isolation from potentially dangerous water objects on territory of Russia.

[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.

[Using real-time polymerase chain reaction for detection of Legionella in objects of the environment].

AIM: To assess efficacy of using the method of quantitative detection of Legionella in objects of the environment by real-time polymerase chain reaction (RT-PCR). MATERIALS AND METHODS: For the development of the assay, genus-specific primers from gene coding 16S rRNAas well as species-specific primers for detection of Legionella pneumophila on the basis of mip gene sequence. For quantitative detection of L. pneumophila calibration samples of pGEM plasmid containing fragment of the mip gene in known concentration were used. Samples of water and biofilms obtained from cooling stacks of production plants, systems of autonomic water supply, humidification blocks of centralized systems of air conditioning were studied. RESULTS: Correlation of results obtained with RT-PCR and bacteriologic methods was shown during monitoring of potentially dangerous water objects as well as during epidemic outbreak of Legionella infection. Importance of samples preparation stage, during which considerable losses of DNA and inhibition of reaction could occur, is underlined. Disinfection measures on the studied objects significantly influenced on the results of the RT-PCR and can lead to false positive results. CONCLUSION: Obtained results confirm usefulness of testing of potentially dangerous water objects on the presence of Legionella based on the preliminary screening with RT-PCR for the 24 hours followed by bacteriologic testing of samples for 8 - 12 days.

[Biofilm formation by Legionella spp. in experiment].

Ability of biofilm formation was studied in 28 strains belonging to 12 species of Legionella. Optimal conditions for formation of biofilms were ascertained using reference strain Legionella pneumophila Philadelphia 1. Comparative assessment of the ability of Legionella spp. to form biofilms was performed by cultivation in proteosopepton broth (for 96 hours) and in water (for up to 2 weeks). Highest rates of biofilm formation were observed for strains of L. pneumophila and L. longbeachae. Between L. pneumophila strains the most prominent ability to form biofilms was observed in newly isolated strains BLR-05 and TOTAL 1. Opportunity to use different ability of Legionella species to biofilm formation as a epidemiologically significant marker and for modeling of biofilms of Legionella in association with other microorganisms was discussed.

[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.

[Legionella natural biofilms and their role in epidemiology of the infection: methods of study and modelling].

Ability of Legionella species to form biofilms in association with other microorganisms is the key factor of their spreading in potentially dangerous water systems. Ability of different strains of Legionella to form monospecies biofilms as well as biofilms in association with Pseudomonas aeruginosa in constant conditions was analyzed. It was shown that ability of Legionella strains to form monospecies biofilms correlates with their ability to persist in biofilms formed by P. aeruginosa.

[Modern views on the epidemiology of legionellosis: operations procedure during epidemic outbreaks and preventive monitoring].

Key aspects of legionellosis epidemiology determining the rising role of Legionella in human infectious diseases in conditions of postindustrial community were discussed. It was shown that formation of Legionella biofilms on potentially dangerous water objects with possible further aerosol or aspiration transmission of microorganisms leads to major epidemic outbreaks and sporadic cases of infection. Main principles of actions during preventive monitoring of Legionella in the environment and epidemic outbreaks of legionellosis were described.

[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.

[Assessment of bactericidal activity of disinfectants against Legionella on the biofilm model].

Comparative assessment of bactericidal activity of different disinfectants against Legionella biofilms was conducted. Monospecies biofilms of 3 strains of Legionella pneumophila obtained on plastic plates in stable conditions were used as models. It has been shown that for degradation of biofilms as well as for prophylactic action of disinfectants in preventing formation of biofilms on plastic surfaces, higher concentrations of preparations were needed as compared to their bactericidal concentrations for culture of Legionella determined by method of serial dilutions.

[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.

[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.

[Different species of Legionella plasmids and virulence].

Plasmids with a molecular weight of 2.5 to 80 MD were shown to be present in a significant portion of different-type Legionella strains including high-virulence isolates L. pneumophila, serogroup 1, and L. bozemanii detected both in Russia and abroad in different sources. Plasmid-free derivative were obtained from the L. pneumophila strains each carrying only one different-size plasmid DNA. The variants had the same virulence as the original cultures for chicken embryos and guinea pigs, when the latter were infected in the bile sac or through intraperitoneal routes, respectively; their virulence was also similar to that of strains resistant to the normal serum of guinea pigs. Hence, the infection models used by us failed to show any action of plasmids on the virulence of Legionella.