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.     

Experience of organization of flushing and disinfection of centralized system of hot water supply in town Verkhnyaya Pyshma after its colonization by Legionella

Causes of outbreak incidence of pneumonia due to Legionella infection in population of Verkhnyaya Pyshma as well as factors promoting colonization of town's hot water supply system by Legionella were discussed. Experience of organization of flushing and disinfection of hot water supply system was described, effectiveness of different methods of disinfection was evaluated.     

The Occurrence of Salmonella in Airline Meals

The occurrence of Salmonella in airline meals was studied in 1989-1992. Samples were collected from flight kitchens in 29 countries. The material consisted of 400 cold dishes and 1,288 hot dishes as well as salads, cheese plates and desserts. Total number of samples was 2211. Salmonella spp. were isolated from 6 samples; 1 contaminated sample was a cold dish prepared in Bangkok, 1 was a hot dish prepared in Mombasa and the remaining 4 contaminated samples were hot dishes prepared within one week in Beijing. The isolated serotypes were S. ohio, S. manchester and S. braenderup. The contaminated cold dish prepared by a flight kitchen in Bangkok was found to be connected with a Salmonella outbreak which occurred in Finland in 1990. Cold airline dishes containing food of animal origin seems to be more risky as a source of Salmonella infections among airline passengers.     

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.     

Quantitative real-time Legionella PCR for environmental water samples: data interpretation

Quantitative Legionella PCRs targeting the 16S rRNA gene (specific for the genus Legionella) and the mip gene (specific for the species Legionella pneumophila) were applied to a total of 223 hot water system samples (131 in one laboratory and 92 in another laboratory) and 37 cooling tower samples (all in the same laboratory). The PCR results were compared with those of conventional culture. 16S rRNA gene PCR results were nonquantifiable for 2.8% of cooling tower samples and up to 39.1% of hot water system samples, and this was highly predictive of Legionella CFU counts below 250/liter. PCR cutoff values for identifying hot water system samples containing >10(3) CFU/liter legionellae were determined separately in each laboratory. The cutoffs differed widely between the laboratories and had sensitivities from 87.7 to 92.9% and specificities from 77.3 to 96.5%. The best specificity was obtained with mip PCR. PCR cutoffs could not be determined for cooling tower samples, as the results were highly variable and often high for culture-negative samples. Thus, quantitative Legionella PCR appears to be applicable to samples from hot water systems, but the positivity cutoff has to be determined in each laboratory.     

Quantitative analyses of mycobacteria in water: adapting methods in clinical laboratories

An outbreak of occupational hot tub lung necessitated quantitative analysis of mycobacteria in water samples. We combined procedures for cultivation of mycobacteria in urine and quantitative analyses of dialysis water. Whirlpool spa water samples were analyzed showing promising results. In conclusion, quantitative mycobacterial culture of water is possible by adapting methods routinely used in clinical laboratories.     

Quantitative Real-Time Legionella PCR for Environmental Water Samples: Data Interpretation

Quantitative Legionella PCRs targeting the 16S rRNA gene (specific for the genus Legionella) and the mip gene (specific for the species Legionella pneumophila) were applied to a total of 223 hot water system samples (131 in one laboratory and 92 in another laboratory) and 37 cooling tower samples (all in the same laboratory). The PCR results were compared with those of conventional culture. 16S rRNA gene PCR results were nonquantifiable for 2.8% of cooling tower samples and up to 39.1% of hot water system samples, and this was highly predictive of Legionella CFU counts below 250/liter. PCR cutoff values for identifying hot water system samples containing >10³ CFU/liter legionellae were determined separately in each laboratory. The cutoffs differed widely between the laboratories and had sensitivities from 87.7 to 92.9% and specificities from 77.3 to 96.5%. The best specificity was obtained with mip PCR. PCR cutoffs could not be determined for cooling tower samples, as the results were highly variable and often high for culture-negative samples. Thus, quantitative Legionella PCR appears to be applicable to samples from hot water systems, but the positivity cutoff has to be determined in each laboratory.     

Experience of microbiologic diagnostics of acute pneumonia cases arose in town Verkhnyaya Pyshma in July-August 2007

Bacteriologic, serologic, and PCR tests of blood, sputum, serum and autopsy samples from 91 patients were performed during outbreak of pneumonia in town Verkhnyaya Pyshma in July-August 2007. Streptococcus pneumoniae was isolated from 20% of diagnostically meaningful samples of sputum and from 50% of autopsy samples. Diagnosis of Legionnaires' disease was confirmed in 48% of cases. It was shown that association of Streptococcus pneumoniae and Legionella pneumophila determined the affect during such pneumonia. Need for establishment of diagnostic procedure for patients with pneumonia including those caused by atypical agents was revealed.     

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.     

实验室内树鼩爆发间质性肺炎的报告

在动物室内饲养的树鼩于2003年4月至8月爆发了一次间质性肺炎。疫情特征为患病动物呼吸窘迫,发病急,死亡率高,主要累及新生或幼年动物,红霉素类抗生素有显著的防治效果。病理检查见光镜下肺组织呈间质性肺炎改变,肺泡腔内无渗出。推测此次疫情的病原为肺炎支原体。     

Detection of Toxoplasma gondii Oocysts in Drinking Water

The world’s largest outbreak of waterborne toxoplasmosis occurred in a municipality in the western Canadian province of British Columbia. When drinking water emerged as a possible source of infection during the outbreak investigation, a laboratory method was needed to attempt detection of the parasite, Toxoplasma gondii. The method developed was based on the current U.S. Environmental Protection Agency method for detection of Cryptosporidium oocysts. Collection of large-volume drinking water samples and cartridge filter processing were unchanged, although identification of Toxoplasma oocysts in the filter retentate was carried out by using a previously described rodent model. Validation of the method developed was tested by using oocysts from a well-characterized Toxoplasma strain.     

Proteinoid microspheres more stable in hot than in cold water

The occurrence of organisms of primitive appearance near submarine hydrothermal vents has indicated sea-floor conditions that are like those under which proteinoid microspheres are produced in the laboratory. Experimental examination of the question of whether some proteinoid microspheres might be stable in hot water has revealed proteinoids that are soluble in cold water but precipitate on heating. Unanswered questions are discussed.     

Occurrence of moulds in drinking water

AIMS: In order to determine the occurrence of filamentous fungi in public drinking water systems in Norway, water from 14 water supply networks from all over the country was sampled and analysed. Networks with both ground and surface water sources were included in this study. METHODS AND RESULTS: During a one-year period, 273 water samples were collected. Frequencies of fungi in samples from raw water, treated water and from home and hospital installations were determined on the basis of incubation of 100 ml membrane-filtered samples on dichloran 18% glycerol agar media. Filamentous fungi were recovered from 62% of all samples. In ground water 42.3% of the samples were positive for mould growth, while surface waters yielded 69.7% positive samples. CONCLUSIONS: The risk to recover moulds from surface water is three times higher compared with ground water. It is more likely to detect moulds in cold waters and showers than in hot waters. SIGNIFICANCE AND IMPACT OF THE STUDY: By analysing the water reaching the consumers, the results reported in present study indicate that filamentous fungi in drinking water is not negligible, and that moulds should be considered as part of the microbiological analysis parameters in drinking water.     

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.     

Legionnaires' disease caused by Legionella dumoffii in distilled water.

Five cases of Legionnaires'' disease caused by Legionella dumoffii were identified within an 11-month period in a hospital in the Quebec City area. In four cases bacterial isolates were obtained from clinical specimens, and in one case seroconversion was demonstrated. All the patients had been admitted to hospital within 10 days before diagnosis. Two of the patients were immunosuppressed children. Only 1 of the 40 hot water samples from the hospital yielded L. dumoffii; however, 6 of 11 distilled water samples contained the bacterium. All the patients had been exposed to distilled water, four through respiratory therapy equipment and one through a room humidifier. Following the use of sterile distilled water in the apparatus, no further cases were identified. This is the first reported outbreak of Legionnaires'' disease caused by L. dumoffii, and it is the first time that nosocomial legionellosis has been linked to contaminated distilled water in Canada.     

Specific clinical, epidemiological patterns and laboratory diagnostics of enterovirus infection in the Republic of Belarus

The clinical and epidemiological patterns as well as the results of the laboratory verification of the outbreak of enterovirus infection (EVI) in Minsk during the period of summer-autumn, 2000, are presented. During this outbreak a variety of clinical forms were observed, the serous meningitis being prevalent (57.5%). Practically simultaneous occurrence of infection on the territory of all administrative districts of the city, the predominant involvement of children aged up to 14 years into the outbreak, a high proportion of simultaneous casualities in the multiple foci. A number of circulating enteroviruses (EV)--ECHO 30, ECHO 6 of three serotypes and Coxsackie B5--were simultaneously isolated from clinical material. EV of the same serotypes were isolated from tap drinking water, and neutralizing antibodies to these serotypes were often detected in the patients blood sera. Infectious EV were also present in samples of bottled water and in water reservoirs used for bathing. The routes of EV transmission and the improvement of EVI control are discussed.     

Inducing sporulation in Alternaria solani I. Effect of water treatment

The sporulation was induced when fully grown cultures were given dip or spray treatment with distilled water (cold or hot) and thereafter, kept partially covered at different temperatures. Cultures dipped in cold water (4° C) for 4 minutes or sprayed with cold water (4° C) or hot water (58° C) and thereafter incubated at room temperature (13–26° C) in diffused sunlight, produced maximum number of spores within 60 hours. Incubating water treated cultures in diffused sunlight or complete darkness and age and scraping of the cultures had a considerable effect upon intensity of sporulation. The cultures yield a number of subsequent crops of spores when scraped and given dip treatment with cold or hot water, after obtaining each crop of spores.     

The Application of New Molecular Methods in the Investigation of a Waterborne Outbreak of Norovirus in Denmark, 2012

In December 2012, an outbreak of acute gastrointestinal illness occurred in a geographical distinct area in Denmark covering 368 households. A combined microbiological, epidemiological and environmental investigation was initiated to understand the outbreak magnitude, pathogen(s) and vehicle in order to control the outbreak. Norovirus GII.4 New Orleans 2009 variant was detected in 15 of 17 individual stool samples from 14 households. Norovirus genomic material from water samples was detected and quantified and sequencing of longer parts of the viral capsid region (>1000 nt) were applied to patient and water samples. All five purposely selected water samples tested positive for norovirus GII in levels up to 1.8×10⁴ genomic units per 200 ml. Identical norovirus sequences were found in all 5 sequenced stool samples and 1 sequenced water sample, a second sequenced water sample showed 1 nt (<0.1%) difference. In a cohort study, including 256 participants, cases were defined as residents of the area experiencing diarrhoea or vomiting onset on 12–14 December 2012. We found an attack rate of 51%. Being a case was associated with drinking tap-water on 12–13 December (relative risk = 6.0, 95%CI: 1.6–22) and a dose-response relation for the mean glasses of tap-water consumed was observed. Environmental investigations suggested contamination from a sewage pipe to the drinking water due to fall in pressure during water supply system renovations. The combined microbiological, epidemiological and environmental investigations strongly indicates the outbreak was caused by norovirus contamination of the water supply system.     

Lethal pneumonia in a captive juvenile chimpanzee (Pan troglodytes) due to human-transmitted human respiratory syncytial virus (HRSV) and infection with Streptococcus pneumoniae

Background  During an outbreak of respiratory disease in captive chimpanzees ( Pan troglodytes ), gorillas ( Gorilla gorilla ), Bornean orangutans ( Pongo pygmaeus ), and red-capped mangabeys ( Cercocebus torquatus ) also staff members showed non-specific upper respiratory signs. One infant female chimpanzee with severe respiratory symptoms died despite immediate medical treatment and was submitted for necropsy.
Methods  Routine post mortem, histological and bacteriological examinations were conducted. Additionally lung tissue samples form the chimpanzee and swab samples from the staff members and the other primates were examined by PCR.
Results  A severe catarrhal to purulent bronchopneumonia and an interstitial pneumonia were found and human respiratory syncytial virus (HRSV) as well as Streptococcus pneumoniae was detected in lung samples by PCR. Swab samples from one animal keeper revealed the same HRSV sequence as of the chimpanzee.
Conclusions  Therefore, it is suggested that the outbreak of respiratory disease within a zoological institution was due to transmission of HRSV between both human and primates.