Viable Legionella pneumophila bacteria in natural soil and rainwater puddles.

AIMS: For the majority of sporadic Legionnaires' disease cases the source of infection remains unknown. Infection may possible result from exposure to Legionella bacteria in sources that are not yet considered in outbreak investigations. Therefore, potential sources of pathogenic Legionella bacteria--natural soil and rainwater puddles on roads--were studied in 2012. METHODS AND RESULTS: Legionella bacteria were detected in 30% (6/20) of soils and 3·9% (3/77) of rainwater puddles by amoebal coculture. Legionella pneumophila was isolated from two out of six Legionella positive soil samples and two out of three Legionella positive rainwater samples. Several other species were found including the pathogenic Leg. gormanii and Leg. longbeachae. Sequence types (ST) could be assigned to two Leg. pneumophila strains isolated from soil, ST710 and ST477, and one strain isolated from rainwater, ST1064. These sequence types were previously associated with Legionnaires' disease patients. CONCLUSIONS: Rainwater and soil may be alternative sources for Legionella. SIGNIFICANCE AND IMPACT OF THE STUDY: The detection of clinically relevant strains indicates that rainwater and soil are potential sources of Legionella bacteria and future research should assess the public health implication of the presence of Leg. pneumophila in rainwater puddles and natural soil.

Summer increase of Legionnaires' disease 2010 in The Netherlands associated with weather conditions and implications for source finding.

During August and September 2010 an unexpected high number of domestic cases of Legionnaires' disease (LD) were reported in The Netherlands. To examine this increase, patient characteristics and results of source finding and environmental sampling during the summer peak were compared to other domestic cases in 2008-2011. This analysis did not provide an explanation for the rise in cases. A similar increase in LD cases in 2006 was shown to be associated with warm and wet weather conditions, using an extended Poisson regression model with adjustment for long-term trends. This model was optimized with the new data from 2008 to 2011. The increase in 2010 was very accurately described by a model, which included temperature in the preceding 4 weeks, and precipitation in the preceding 2 weeks. These results confirm the strong association of LD incidence with weather conditions, but it remains unclear which environmental sources contributed to the 2010 summer increase.

Legionella prevention in the Netherlands: an evaluation using genotype distribution.

Legionnaires' disease (LD) is an acute pneumonia caused by the inhalation or aspiration of aerosols contaminated with Legionella bacteria. The watery environment is considered to be the natural habitat for these bacteria. Therefore, drinking water legislation is used in the primary prevention of LD, focussing on the different water sources to which the general public is exposed. For 10 years, secondary prevention of LD in the Netherlands has been organised using an outbreak detection programme that eliminates potential sources of infection that LD patients have been exposed to during their incubation period. To evaluate the two prevention strategies, we compared the distribution of Legionella pneumophila serogroup 1 genotypes isolated from patient material (n = 179) with the distribution resulting from primary (n = 182) and secondary (n = 60) prevention actions, respectively. The sequence type profiles were generated using the HiMLST method that employs next generation sequencing. We found that genotypes collected during primary prevention differ to a large extent from those isolated from patients. Genotypes collected during secondary prevention efforts had a greater similarity to that of patient isolates, but could be further improved. Our results suggest that primary prevention is not aiming at the correct reservoir, whereas secondary prevention is only partially focussed. It seems that there is a still unknown reservoir.

Isolation of Legionella pneumophila from pluvial floods by amoebal coculture.

Viable Legionella pneumophila bacteria were isolated by amoebal coculture from pluvial floods after intense rainfall and from water collected at sewage treatment plants. Several isolated L. pneumophila strains belonged to sequence types that have been previously identified in patients.

Wellness centres: an important but overlooked source of Legionnaires disease. Eight years of source investigation in the Netherlands, 1 August 2002 to 1 August 2010.

Visiting wellness centres is considered safe and relaxing and might provide health benefits for visitors with certain cardiovascular, dermatological or respiratory diseases. On the other hand, wellness centres could pose health risks, especially with respect to Legionnaires' disease. We investigated the role of wellness centres in the occurrence of Legionnaires' disease by analysing the data of eight years (2002­2010) of source investigation in the Netherlands. There were 15 wellness centres identified as potential sources of infection for a total of 35 Legionnaires' disease patients. Twelve of these centres were positive for Legionella spp.: six for Legionella pneumophila, six for non-pneumophila Legionella spp.. Of the 65 positive environmental samples found during the wellness centre investigations, 41 were derived from shower heads. For two centres, the Legionella pneumophila strains in the collected samples had a genotype that was indistinguishable from the patient isolates. These results show that wellness centres are potential sources of Legionnaires' disease.

Diagnostic testing for Legionnaires' disease in the Netherlands between 2007 and 2009: a possible cause for the decline in reported Legionnaires' disease patients.

Legionnaires' disease (LD) is an acute pneumonia caused by the inhalation or aspiration of aerosols contaminated with the Legionella bacteria. In the Netherlands, around 300 LD cases per year were reported between 2000 and 2008, but in 2009, the number dropped to 251, which was the lowest number in the previous 5 years of surveillance. We investigated if this decrease could be explained by the number of performed Legionella diagnostic tests in this year. We analyzed the number of tests performed between 2007 and 2009 in three large microbiological laboratories in different geographical regions in the Netherlands. Our data showed that there was no decrease in the number of patients for whom a diagnostic test for Legionella was performed in this period. These results are not in line with our hypothesis that the decrease in reported Legionella pneumonia patients in 2009 would be due to a decrease in patients for whom a diagnostic test was performed. We conclude that it is more likely that other factors such as the influence of weather patterns might explain the sudden drop in reported Legionella pneumonia patients in 2009 compared to the previous years, and it would be interesting to investigate this for the period described.

Changes in prevention and outbreak management of Legionnaires disease in the Netherlands between two large outbreaks in 1999 and 2006.

We describe an outbreak of Legionnaires' disease in 2006 in Amsterdam, the Netherlands. Comparisons with the outbreak that took place in 1999 are made to evaluate changes in legionella prevention and outbreak management. The 2006 outbreak was caused by a wet cooling tower. Thirty-one patients were reported. The outbreak was detected two days after the first patient was admitted to hospital, and the source was eliminated five days later. The 1999 outbreak was caused by a whirlpool at a flower show, and 188 patients were reported. This outbreak was detected 14 days after the first patient was admitted to hospital, and two days later the source was traced. Since 1999, the awareness of legionellosis among physicians, the availability of a urinary antigen tests and more efficient early warning and communication systems improved the efficiency of legionellosis outbreak management. For prevention, extensive legislation with clear responsibilities has been put in place. For wet cooling towers, however, legislation regarding responsibility and supervision of maintenance needs to be improved.

Genotypic comparison of clinical Legionella isolates and patient-related environmental isolates in The Netherlands, 2002-2006.

This study investigated the hypothesis that the genotype distribution of Legionella isolates from sporadic patients with Legionnaires' disease differs from that of Legionella strains in the environment. An amplified fragment-length polymorphism (AFLP) assay was used to genotype patient-derived and environmental Legionella isolates. The three Legionella pneumophila genotypes isolated most frequently from human respiratory secretions were AFLP types 004 Lyon, 010 London and 006 Copenhagen. These genotypes were cultured significantly less frequently from environmental samples (50% vs. 4%; p <0.001). The most frequently observed L. pneumophila serogroup 1 genotype among patient-derived isolates was 004 Lyon (32%). This genotype was cultured from only one of 6458 environmental samples. The positive sample contained 1.26 x 10(6) CFU/mL and originated from a whirlpool spa that had not been disinfected and had been maintained at 36 degrees C for several months. Overall, the distribution of genotypes differed significantly among patient and environmental isolates. A possible explanation is that virulent strains may exist in potential environmental sources at undetectable concentrations.

Use of surface water in drinking water production associated with municipal Legionnaires' disease incidence.

STUDY OBJECTIVES: Given an observed geographical variation in Legionnaires' disease incidence in The Netherlands, the aim of the study was to test the hypothesis that the type of drinking water production was an independent determinant of the incidence of Legionnaires' disease. DESIGN: For the 1987-2005 period, the incidence of Legionnaires' disease in The Netherlands and the price of water as a proxy for production type was studied at the municipal level. The data on the price of water were available at the municipal level. METHODS: For each of the 466 municipalities in The Netherlands a mean standardised incidence rate per 100,000 inhabitants over the 1987-2005 period was calculated, excluding patients with the most probable source of infection abroad or in hospital. Logistic regression was used to assess the relation of the price of water to the incidence rates. In order to control for diagnostic and inclusion bias, they were estimated using questionnaire data collected from all 62 medical microbiology laboratories in the country. MAIN RESULTS: The incidence of Legionnaires' disease varied between municipalities from 0.0 to 5.6 per 100,000 person-years. In univariate analysis high versus low water price was positively associated with a high municipal incidence rate (odds ratio (OR) 1.9; 95% CI 1.5-2.6). The association persisted (OR 5.1; 95% CI 3.2-8.0) after correction for diagnostic and inclusion bias. CONCLUSIONS: The price of water as a proxy for the type of water production was an independent risk factor for high municipal Legionnaires' disease incidence in The Netherlands. This can guide future prevention policies.

Incidence and completeness of notification of Legionnaires' disease in The Netherlands: covariate capture-recapture analysis acknowledging regional differences.

To estimate incidence and completeness of notification of Legionnaires' disease (LD) in The Netherlands in 2000 and 2001, we performed a capture-recapture analysis using three registers: Notifications, Laboratory results and Hospital admissions. After record-linkage, 373 of the 780 LD patients identified were notified. Ascertained under-notification was 52.2%. Because of expected and observed regional differences in the incidence rate of LD, alternatively to conventional log-linear capture-recapture models, a covariate (region) capture-recapture model, not previously used for estimating infectious disease incidence, was specified and estimated 886 LD patients (95% confidence interval 827-1022). Estimated under-notification was 57.9%. Notified, ascertained and estimated average annual incidence rates of LD were 1.15, 2.42 and 2.77/100 000 inhabitants respectively, with the highest incidence in the southern region of The Netherlands. Covariate capture-recapture analysis acknowledging regional differences of LD incidence appears to reduce bias in the estimated national incidence rate.

Legionnaires' disease and gardening.

Legionella longbeachae was cultured from the sputum of a patient suffering from Legionnaires' disease. Source identification efforts included analysis of samples of potting soil from the patient's garden, and a genotypically indistinguishable strain of L. longbeachae was cultured from this material. Following examination of a national collection of Legionella isolates, two more patients with indistinguishable genotypes were identified. One of these patients had visited a garden centre in the same municipality in which the index patient had acquired his potting soil. The study demonstrated the value of systematic collection of identification data and patient isolates over a prolonged period.

Risk factors for sporadic community-acquired Legionnaires' disease. A 3-year national case-control study.

OBJECTIVE: Risk factors for sporadic community-acquired Legionnaires' disease (LD) have been studied in the past, well before the widespread introduction of the urinary antigen test. Our objective was to evaluate the impact of the concomitant decrease in underdiagnosis on established and unknown risk factors for LD. STUDY DESIGN: Prospective case-control study. METHODS: From 1 July 1998 to 30 June 2001, 228 LD cases and 293 controls were included for a national case-control study. Patients were included upon notification provided that they fulfilled international criteria for confirmed LD. RESULTS: A history of diabetes mellitus, current tobacco smoking, travelling abroad, spending one or more nights away from home not leaving the country and being a driver by profession were independent risk factors. LD patients who had travelled abroad during their incubation period differed from LD patients who had not. They appeared healthier than non- or domestic travellers with respect to a history of coronary disease, pulmonary disease, current use of corticosteroids or immunosuppressives and any medication. Also the environmental risk factors differed significantly for the two groups. CONCLUSIONS: The finding of two distinct populations of LD patients calls for a differentiated preventive strategy.

Estimation of minimum infection rates with Legionella pneumophila in an exposed population.

The distribution of antibody levels to Legionella (L.) pneumophila (serotypes 1-7) was compared between subjects who worked near the source of a large outbreak of Legionnaires' disease (n=668) and a population sample of comparable age (n=480). In a previous analysis of these data, it was estimated that 80% of those working near the source were infected with L. pneumophila. However, the estimation procedure implicitly assumes that the probability of infection does not depend on the antibody level of a person before exposure. This is questionable, as antibodies could protect against infection. We have now estimated the minimum value consistent with the data on the number of infected persons. We observed that a minimum of 40% [95% confidence interval (CI) 32-48] of those working near the source and 13% (95% CI 8-18) of those working further away were infected with L. pneumophila. Implications of these findings for design options in future research are discussed.

[Three patients with pneumonia due to Legionella associated with a sauna, a cooling tower and a caravan in The Netherlands]. / Drie patiënten met Legionella-pneumonie, in verband gebracht met een sauna, een koeltoren en een caravan in Nederland.

In three male patients with lower respiratory disease, aged 51, 32 and 63 years, Legionnaires' disease was diagnosed by urinary antigen test and culture of the respiratory-tract fluid. In the second patient, the bronchoalveolar fluid also contained Streptococcus pneumoniae and Haemophilus influenzae. All three patients recovered after treatment with azithromycin in the first, cefotaxime, vancomycin and levofloxacin in the second, and erythromycin and ciprofloxacin in the third, respectively. Legionella pneumophila pneumonia is clinically not clearly distinct from other pneumonias and has a high mortality rate when not treated with the proper antibiotics. For that reason, adequate and swift diagnosis is of great importance. The urinary antigen test meets both of these criteria. Still, it is advisable to use culture and serology as well if Legionnaires' disease is suspected in a patient, since the urinary antigen test has limitations. In addition, patient isolates are ofepidemiological importance for public health. By comparing available patient isolates with Legionella strains from water sources, it is possible to identify sources of infection. In 2002, based on this principle, a project was started in The Netherlands aimed at identifying sources of infection, thereby preventing outbreaks of Legionnaires' disease by swift elimination of the source. Since the start of the project, 29 sources have been identified. In the cases described above these were a sauna, a cooling tower and a caravan, respectively. In suspected cases, respiratory-tract fluid must be collected to make possible such a source investigation.

Diagnosis of Legionella infection in Legionnaires' disease.

Since 1977, the diagnostic tools for Legionnaires' disease have been culture and serological investigation. Both methods require considerable time to produce results and have low to reasonable sensitivity. Since the introduction of urinary antigen tests in the mid 1990s, underdiagnosis has diminished and mortality has declined, thanks to early diagnosis. To obtain the most accurate diagnosis, culture, serological investigation, and urinary antigen testing should all be performed. In the last decade, much effort has been directed toward the development of assays detecting Legionella nucleic acid. Thus far, only widely varying results with small patient series have been reported. Furthermore, these assays are labour intensive and complicated. As a result, these assays are not yet suitable for the average medical microbiological laboratory.

[Reported cases of Legionella pneumonia in the Netherlands, 1987-2000]. / Gemelde Legionella-pneumonie in Nederland, 1987-2000.

OBJECTIVE: To describe the incidence and mortality of legionnaires' disease (LD) in the Netherlands on the basis of registration, with a focus on possible sources of infection, and geographic and seasonal variations. DESIGN: Retrospective, descriptive. METHOD: The incidence and mortality rate of LD from July 1987 until December 2000 was described using registration data from the Dutch Health Inspectorate. Possible sources of infection were also listed (patients involved in the Bovenkarspel outbreak (1999) were excluded). Geographic variations in occurrence of LD were investigated by incidences computed per province. To gain insight into the possible existence of seasonal variations the mean numbers of patients per month were compared. RESULTS: During the period of interest a total of 806 persons with LD were reported, 36 of whom were excluded. The mean incidence rose from 0.27 cases per 100,000 inhabitants in the 1987-1998 period to 0.91 cases per 100,000 inhabitants in the 1999-2000 period. The male:female ratio was 2.6:1. A total of 96 persons (12.5%) died as a result of LD. There were more deaths in the group of patients with confirmed LD compared to the group of patients with probable LD. After 1998 the case fatality rate was lower than in the previous period (Fisher's exact test: p = 0.005). The potential source of infection was situated in the open population in 92.9% of cases. In 7.1% of cases the potential source was situated in a hospital. The incidence per province varied from 0.13 per 100,000 (Drente) to 0.66 per 100,000 (Limburg). During the summer months more patients were reported, most of whom had contracted the infection abroad. In contrast to the 1987-1998 period, after 1998 the incidence in the Netherlands was higher than the European mean and the incidence of confirmed LD was higher than in the United States.

Subclinical Legionella infection in workers near the source of a large outbreak of Legionnaires disease.

A survey was conducted of exhibitors at a 1999 floral trade show, where a whirlpool spa on display caused a large outbreak of legionnaires disease (LD). In total, 742 exhibitors without LD returned a questionnaire on their whereabouts during the fair and their health afterward and supplied blood samples for the detection of IgM and IgG antibodies against Legionella pneumophila. The exhibitors had higher average antibody levels than did the general population. The closer to the whirlpool that the exhibitors worked, the higher their antibody levels. Both high-normal and high titer levels were found more frequently among workers with more exposure, suggesting that serosurveys among potentially exposed subjects are a valuable tool for outbreak investigation. Some differences in health complaints were observed between the more and less exposed groups, as estimated by the workplace location, but few differences were found between groups with different antibody levels.