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.

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.

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.

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%).

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.

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.

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.