Short-term effects of atmospheric pressure, temperature, and rainfall on notification rate of community-acquired Legionnaires' disease in four European countries.

Legionnaires' disease (LD) is caused by the inhalation of aerosols containing Legionella, a Gram-negative bacteria. Previous national- or regional-level studies have suggested an impact of climate on LD incidence. The objective of this study was to investigate the effect of temperature, rainfall, and atmospheric pressure on short-term variations in LD notification rate. EU/EEA Member States report their LD surveillance data to the European Centre for Disease Prevention and Control. Community-acquired LD cases reported by Denmark, Germany, Italy, and The Netherlands with onset date in 2007-2012 were aggregated by onset week and region of residence. Weather variables were extracted from the European Climate Assessment & Dataset project. We fitted Poisson regression models to estimate the association between meteorological variables and the weekly number of community-acquired LD cases. Temperature, rainfall and atmospheric pressure were all associated with LD risk with higher risk associated with simultaneous increase in temperature and rainfall. Temperatures >20 °C were not associated with a higher risk for LD. LD cases occurring during wintertime may be associated with sources less influenced by meteorological conditions.

Design and validation of a qPCR assay for accurate detection and initial serogrouping of Legionella pneumophila in clinical specimens by the ESCMID Study Group for Legionella Infections (ESGLI).

Prompt detection of Legionella pneumophila is essential for rapid investigation of legionellosis. Furthermore, as the majority of L. pneumophila infections are caused by serogroup 1 (sg1) strains, rapid identification of such strains can be critical in both routine and outbreak scenarios. The ESCMID Study Group for Legionella Infections (ESGLI) was established in 2012 and immediately identified as a priority the validation of a reliable, easy to perform and interpret, cost-effective qPCR assay to standardise the detection of L. pneumophila DNA amongst members. A novel L. pneumophila assay targeting the mip gene was designed and combined with previously published methodologies amplifying the sg1 marker (wzm) and the green fluorescent protein gene (gfp) internal process control. The resulting triplex assay was validated internationally on the three qPCR platforms used by the majority of European Legionella reference laboratories: ABI 7500 (Life Technologies), LightCycler 480 Instrument II (Roche) and Rotor-Gene Q (Qiagen). Clinical and EQA specimens were tested together with a large panel of strains (251 in total) to validate the assay. The assay proved to be 100% specific for L. pneumophila and sg1 DNA both in silico and in vitro. Efficiency values for mip and wzm assays ranged between 91.97 and 97.69%. Limit of detection values estimated with 95% confidence were adopted for mip and wzm assays on all three qPCR platforms. Inhibition was not observed. This study describes a robust assay that could be widely implemented to standardise the molecular detection of L. pneumophila among ESGLI laboratories and beyond.

Epidemic of Mycoplasma pneumoniae infection in Denmark, 2010 and 2011.

Denmark experienced two waves of Mycoplasma pneumoniae infection during autumn and early winter in 2010 and 2011, respectively. Both affected the whole country. The proportion of positive results was almost the same for both, indicating that the two waves were probably of equal size. High macrolide consumption during the epidemics did not seem to affect levels of macrolide resistance in M. pneumoniae, which remain low in Demark (1% to 3%).

Cluster of Legionnaires disease in a newly built block of flats, Denmark, December 2008 - January 2009.

During December 2008 to January 2009, two persons contracted Legionnaires' disease in a newly built block of flats in a suburb of Copenhagen in Denmark. Polymerase chain reaction and culture was used to diagnose Legionnaires' disease in this cluster. Isolates from both patients tested positive for Legionella pneumophila serogroup 1 subgroup Philadelphia sequence type 1 and the same strain was detected in hot water samples taken from the residential area indicating that the hot water supply system was the most likely source of infection. Legionella was not detected in the cold water. Two interventions were conducted to limit the Legionella colonisation of the piping and storage tanks and the effect was monitored by investigating water samples from various sites in the block of flats. Only the second intervention had a sufficient effect on the Legionella colonisation. The cluster described here points to several risk factors regarding growth of Legionella in hot water systems: (i) stagnancy of water from when the building is constructed and piping installed and until residents move in, (ii) stagnancy and low temperature (from room temperature to approximately 38°C) of water in shower hoses and (iii) failure in operation of and control measures for the hot water system.

Geographical variation of sporadic Legionnaires' disease analysed in a grid model.

The aim was to analyse variation in incidence of sporadic Legionnaires' disease in a geographical information system in three time periods (1990-2005) by the application of a grid model and to assess the model's validity by analysing variation according to grid position. Coordinates of the addresses at time of disease of 606 confirmed cases with Legionnaires' disease were obtained. The incidence was calculated in cells of 10 x 10 km in 25 different grids superimposed on a map of Denmark. A 95% and 99% threshold was applied to identify cells with excess incidence representing potential clusters. Four cells had excess incidence in all three time periods. The analysis in 25 different grid positions indicated a low risk of overlooking cells with excess incidence in a random grid. The coefficient of variation ranged from 0.08 to 0.11 independent of the threshold. By application of a random grid model we demonstrated that it was possible to detect small areas with excess incidence that were not detected in the present surveillance system.

Increased incidence of Mycoplasma pneumoniae infections detected by laboratory-based surveillance in Denmark in 2010.

In Denmark recurrent epidemics of Mycoplasma pneumoniae infections have been described since the 1950s at intervals of approximately four to six years. The latest epidemic occurred in 2004/05 followed by two years of high incidence and more than three years of low incidence. Due to a recent increase in diagnosed cases since late summer 2010, we conducted a survey of positive M. pneumoniae PCR tests performed by clinical microbiology departments in Denmark, which indicated that a new epidemic may be underway.

Comparison of the sensitivity of the Legionella urinary antigen EIA kits from Binax and Biotest with urine from patients with infections caused by less common serogroups and subgroups of Legionella.

The detection of urinary antigen is the most widely used method to diagnose Legionnaires' disease (LD), so it is important that these assays have a high sensitivity for the disease. In this study, we compare two kits for their ability to detect urinary antigen in urine samples from patients infected with Legionella species and L. pneumophila sero- and subgroups not considered as the most common causes of LD. Urine samples (n = 33) from 30 culture-proven cases of L. pneumophila serogroup (sg) 1, subgroup non-Pontiac infection, and urine samples (n = 35) from 32 cases of non-L. pneumophila species or non-sg 1 infection were examined using the Binax EIA and Biotest EIA kits. For both groups, the overall diagnostic sensitivity of the Binax kit was significantly better than the sensitivity of the Biotest kits (P < 0.0001). For the non-Pontiac group, the sensitivity was 81.8 and 42.4%, respectively, and for the non-sg1 group, it was 51.4 and 28.6%, respectively. It was concluded that the Binax kit was more suitable for the general diagnosis of LD than the Biotest kit, but we still need urinary antigen detection methods with higher sensitivity for non-sg1 LD.

Whole-genome sequencing for identification of the source in hospital-acquired Legionnaires' disease.

Acquisition of Legionnaires' disease is a serious complication of hospitalization. Rapid determination of whether or not the infection is caused by strains of Legionella pneumophila in the hospital environment is crucial to avoid further cases. This study investigated the use of whole-genome sequencing to identify the source of infection in hospital-acquired Legionnaires' disease. Phylogenetic analyses showed close relatedness between one patient isolate and a strain found in hospital water, confirming suspicion of nosocomial infection. It was found that whole-genome sequencing can be a useful tool in the investigation of hospital-acquired Legionnaires' disease.

Detection of Mycoplasma pneumoniae in simulated clinical samples by polymerase chain reaction. Brief report.

Polymerase chain reaction (PCR) was used to detect Mycoplasma (M) pneumoniae DNA in simulated clinical samples. Throat swabs were mixed with known amounts of broth-grown M. pneumoniae cells. An estimated detection limit of less than 40 colony forming units (cfu) was obtained without the need for time-consuming hybridization. The PCR is completed in one day and may be useful for the early detection of M. pneumoniae in clinical samples.

Pontiac fever at a sewage treatment plant in the food industry.

BACKGROUND AND OBJECTIVES: During a hot and humid summer period workers became ill with fever and flu-like symptoms after repairing a decanter for sludge concentration at a sewage treatment plant. The work took place over a period of 10 days in a small closed room, while another decanter was in operation and was consequently emitting aerosol to the environment, to which the workers were exposed. The aim of this study was to determine the cause of this outbreak of febrile illness so that additional cases could be prevented. METHODS: All 5 patients were seen and examined in the Department of Occupational Medicine. Furthermore 2 of the workers had recurrent illness and were examined during hospitalization. As Pontiac fever (nonpneumonic legionellosis) was suspected, antibodies to legionellae were measured in blood samples. After positive antibody titers to Legionella pneumophila were found, samples of the sludge were collected for legionellae culture. RESULTS AND CONCLUSIONS: The clinical picture agreed with that described for Pontiac fever, and positive antibody titers to L. pneumophila serogroup 1 were found in blood from all 5 patients. L. pneumophila serogroup 1 was cultured in high amounts from sludge from the decanter. It was concluded that the fever was caused by L. pneumophila emitted to the environment by the uncovered decanter. Procedures for preventing new cases were established.

[Nosocomial Legionella pneumophila infection in a nephrology department]. / Nosokomiel infektion med Legionella pneumophila på en nefrologisk afdeling.

During the autumn and winter of 1993-94 four cases of legionellosis were diagnosed in a Department of Nephrology. Three of the patients were kidney transplanted patients. Two of the patients died. The diagnosis was based on positive culture in two patients and by positive urinary antigen test in the other two patients. Serology was negative for all four patients. Legionella pneumophila was initially found in the cold and hot shower water, in ice-water from the ice machine, from the hot water tank and in the cold water inlet to the building. The isolate from patient no. one and isolates of serogroup 5 from the ice machine and the shower water had identical REA profiles, different from the profiles of the isolate from patient no. four. We concluded that at least one of the four patients was likely to have been infected from the water in the department, either by inhalation of contaminated aerosols from the shower or by aspiration of contaminated ice-water. Precautions were taken to reduce the number of Legionella in the shower- and ice-water. In addition, restrictions in the use of showers and ice-water from the ice machine were introduced.

[An outbreak of Pontiac fever among children and adults following a whirlpool bath]. / Et udbrud af Pontiac-feber blandt børn og voksne efter brug af et spabad.

We investigated an outbreak of fever most likely due to a contaminated whirlpool among nine adults and six children visiting a holiday home. The outbreak was characterized by a high attack rate, short incubation periods, influenza-like symptoms and rapid recoveries typical of Pontiac fever. The children, however, experienced less characteristic symptoms and no sequelae compared to the adults. Evidence and presumptive evidence of Legionella (L) infection was found in eleven cases; in one case by isolation of L. pneumophila serogroup 1, in two cases by positive test for Legionella by PCR and in eleven cases with seroconversion. In contrast, two adult non-users of the whirlpool had no symptoms and no serological evidence of infection. This investigation demonstrates differences between adults and children in the clinical picture of Pontiac fever, furthermore it shows that culture and PCR of tracheal aspirate for legionellae can be used in a hospital setting for rapid diagnosis although their sensitivities are low.

Dual infections with different Legionella strains.

In 2010 a case of a dual infection with Legionella pneumophila serogroup (sg) 1 and sg 3 was identified by culture of a blood sample collected from a female Austrian patient with septic pneumonia. Subsequently all 35 European National Legionella Reference Laboratories were interviewed regarding the frequency of dual infections in legionellosis. The Reference Laboratories in Denmark, the UK and Germany reported the detection of another 14 cases of dual infections with different Legionella strains between 2002 and 2012. Among the 15 cases, there were four cases with different Legionella species, six cases with different L. pneumophila serogroups, and five cases of dual infections with L. pneumophila sg 1 with different MAb-types. The median age of the 15 cases was 56 years and the male to female ratio 1:1.14. Six of the 15 patients were receiving immunosuppressive treatment following organ transplantation (n = 3) or for underlying haematological and solid malignancies (n = 3). Five of the 15 cases died within 30 days following diagnosis. Efforts to detect dual infections with different Legionella strains will improve our ability to correctly elucidate the causative sources of infection and enhance our understanding of the epidemiology of Legionella infections.

Two years' performance of an in-house ELISA for diagnosis of Legionnaires' disease: detection of specific IgM and IgG antibodies against Legionella pneumophila serogroup 1, 3 and 6 in human serum.

The aim of this study was to evaluate the performance of an in-house ELISA for the diagnosis of Legionnaires' disease (LD) by detection of IgM and IgG antibodies against Legionella (L.) pneumophila serogroups (sg) 1, 3 and 6. The evaluation was done throughout a two-year period in a diagnostic routine laboratory. Furthermore, the sensitivity of four different methods, the in-house L. pneumophila antibody test (ELISA), the urinary antigen test (Binax® EIA), an in-house PCR and culture, both alone and in combination was evaluated. From 2008 to 2010, 12,158 serum samples from 10,503 patients were analysed. During the same period, 361 cases of laboratory-confirmed LD cases were recorded in Denmark, but of these only 113 had a serum sample examined. The positive predictive value of the in-house ELISA was calculated to be 12.8 and the negative predictive value was 99.6, using only the confirmed LD cases as true positives. The sensitivity of the in-house ELISA for the detection of IgM and IgG antibodies in the confirmed LD cases was 61% and 36%, respectively. By combining the two ELISA assays the sensitivity increased to 66%. The sensitivity of the Legionella urinary antigen test (Binax® EIA) was 63%, of the in-house PCR 87% and of culture 69%. When all the different methods were combined, a higher sensitivity was calculated--for in-house ELISA (IgM+IgG) and Binax® EIA 91%, in-house ELISA (IgM+IgG) and in-house PCR 93%, in-house ELISA (IgM+IgG) and culture 93%, Binax® EIA and in-house PCR 79%, Binax® EIA and culture 68% and in-house PCR and culture 94%. This study confirms that the detection of IgG and IgM antibodies by ELISA is an important diagnostic tool, also during the initial phase of the disease. Furthermore, we showed that LD in Denmark with or without serum samples collected exhibits the same age and sex distribution and epidemiology, as in the rest of Europe, i.e., mostly men are infected, infections are mostly community acquired, followed by infection from travelling abroad. Apart from patients with notified LD, the patients investigated by serology were evenly distributed in all age groups; there was only a slightly higher ratio of men tested for "atypical pneumonia" in the serology laboratory.

The occurrence of Legionella species other than Legionella pneumophila in clinical and environmental samples in Denmark identified by mip gene sequencing and matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

In Denmark, several laboratories use PCR as a routine diagnostic method for Legionnaires' disease, and almost all PCR-positive samples are investigated by culture. From 1993 to 2010, isolates of Legionella species other than Legionella pneumophila were obtained from respiratory samples from 33 patients, and from 1997 to 2010, 42 isolates of Legionella non-pneumophila species were obtained and saved from water samples from 39 different sites in Denmark. Macrophage infectivity potentiator gene (mip) sequencing was used as a reference method to identify the Legionella non-pneumophila species. Only one of the 75 isolates did not meet the acceptance criterion of a similarity of ≥98% to sequences in the database. The species distribution between clinical and environmental isolates varied. For the former, four species were detected, with Legionella bozemanae and Legionella micdadei predominating (both 44%). For the latter, eight species were detected, with Legionella anisa predominating (52%). The distribution among the Danish clinical isolates was different from the general distribution both in Europe and outside Europe, where L. bozemanae and Legionella longbeachae are the most commonly found clinical Legionella non-pneumophila species. The 75 isolates were also investigated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS): 64 were correctly identified, with a score of ≥2.0; eight had a score of <2.0, but only two of these were wrongly identified; and three gave no results with MALDI-TOF MS. Both mip sequencing and MALDI-TOF MS are robust methods for Legionella species identification.

Dynamics of Legionella antibody levels during 1 year in a healthy population.

A total of 522 Danish blood donors were followed during 2004-2005 to describe the seroepidemiology of Legionella infections in healthy individuals from a general population. Antibodies to Legionella spp. were measured by indirect immunofluorescence antibody test. The prevalence of Legionella antibodies (titre 1:128) was 26.8% and remained fairly constant during the year of follow-up. However, 6.9% of the blood donors developed a fourfold or greater rise in antibody titres. A history of visits to Danish summer cottages was associated with both Legionella seropositivity (OR 1.53, 95% CI 1.02-2.30) and seroconversion (OR 2.66, 95% CI 1.21-5.83). There were no consistent associations between either levels of antibody titres or seroconversion and self-reported health symptoms, absence from work due to illness, or to any risk factors. We conclude that community-acquired Legionella infections are frequent; however, they rarely result in severe illness.