Legionnaires' disease from a cooling tower in a community outbreak in Lidköping, Sweden- epidemiological, environmental and microbiological investigation supported by meteorological modelling.

BACKGROUND: An outbreak of Legionnaires' Disease took place in the Swedish town Lidköping on Lake Vänern in August 2004 and the number of pneumonia cases at the local hospital increased markedly. As soon as the first patients were diagnosed, health care providers were informed and an outbreak investigation was launched. METHODS: Classical epidemiological investigation, diagnostic tests, environmental analyses, epidemiological typing and meteorological methods. RESULTS: Thirty-two cases were found. The median age was 62 years (range 36 - 88) and 22 (69%) were males. No common indoor exposure was found. Legionella pneumophila serogroup 1 was found at two industries, each with two cooling towers. In one cooling tower exceptionally high concentrations, 1.2 × 109 cfu/L, were found. Smaller amounts were also found in the other tower of the first industry and in one tower of the second plant. Sero- and genotyping of isolated L. pneumophila serogroup 1 from three patients and epidemiologically suspected environmental strains supported the cooling tower with the high concentration as the source. In all, two L. pneumophila strains were isolated from three culture confirmed cases and both these strains were detected in the cooling tower, but one strain in another cooling tower as well. Meteorological modelling demonstrated probable spread from the most suspected cooling tower towards the town centre and the precise location of four cases that were stray visitors to Lidköping. CONCLUSIONS: Classical epidemiological, environmental and microbiological investigation of an LD outbreak can be supported by meteorological modelling methods.The broad competence and cooperation capabilities in the investigation team from different authorities were of paramount importance in stopping this outbreak.

Nosocomial Legionnaires' disease caused by Legionella pneumophila serogroup 6: implication of the sequence-based typing method (SBT).

Sequence-based typing (SBT) was used to determine the allelic profiles of 3 sporadic clinical isolates as well as 7 environmental isolates of Legionella pneumophila serogroup 6, isolated at the Karolinska Hospital during 2004. The clinical isolates were cultured from patients with nosocomial Legionnaires' disease (LD), while the environmental isolates were cultured from potable water sources of the hospital wards in the close vicinity of the 3 patients being investigated. The genes sequenced for the construction of the SBT profile included flaA, pilE, asd, mip, mompS and proA, in this pre-determined order and the allelic profile of the 10 isolates was identical (3, 13, 1, 28, 14, 9). Furthermore, 2 of the isolates, 1 clinical and 1 environmental, were analysed using the amplified fragment length polymorphism analysis (AFLP). The AFLP genotype of both isolates was congruent. Eight of 9 control L. pneumophila serogroup 6 isolates had the same SBT profile as the study isolates. We conclude that the environmental strain isolated from our hospital's drinking water is indistinguishable genotypically from the 3 clinical isolates of Legionella. However, this genotype of L. pneumophila is geographically widespread. Thus, results of genotyping must be evaluated in conjunction with the clinical and epidemiological data.

A community outbreak of Legionnaires' disease from an industrial cooling tower: assessment of clinical features and diagnostic procedures.

An outbreak of Legionnaires' disease (LD) occurred in Lidköping, Sweden, in August 2004. A cooling tower was identified as the probable source of infection. During the outbreak period an unexpected 3-6-fold increase in pneumonia patients was noted at the local hospital. During 7 weeks LD was diagnosed in 15 patients by urinary antigen and/or sputum culture. Additionally, 15 LD patients were diagnosed later by serology. Patients with LD were generally younger, more healthy, and more often smokers compared to other pneumonia patients. On admittance they had more severe symptoms with high fever and raised CRP levels, and more often hyponatraemia, gastrointestinal and CNS symptoms. A causative agent besides Legionella was found in 2 patients only. A significant titre rise for Mycoplasma and/or Chlamydophila pneumoniae was found in 13 of 29 tested patients with confirmed LD. We conclude that the clinical diagnosis of LD is difficult and that available diagnostic methods detect only a minority of patients in the acute phase. Therefore in severe pneumonia, empirically targeted therapy should be instituted on clinical grounds irrespective of the results of diagnostic tests. The observation of increased antibody levels for M. and C. pneumoniae suggests an unspecific immune reaction and merits further study.

Nosocomial transmission of Legionella pneumophila to a child from a hospital's cold-water supply.

Human Legionella infections mainly consist of community-acquired and nosocomial pneumonia and rarely affect children. We describe a nosocomial infection with Legionella pneumophila, serogroup 1, subgroup OLDA, in an immunocompromized 2-y-old girl at a paediatric clinic. L. pneumophila identical to that of the patient was found in the hospital's cold-water but not in the hot-water distribution system. Transmission of Legionella to the girl most probably occurred by Legionella-contaminated cold water mixed and heated by water from the hot-water system. Mixing of hot and cold water probably occurred through thermostatic water mixing valves connected to showers regulated by a handle at the shower head. Nosocomial Legionella infection might thus have occurred, although circulating hot water temperatures never dropped below 53 degrees C and cultures for surveillance of Legionella from central parts of the hot-water system have been consistently negative. Legionellae were successfully eliminated from the hospital's cold-water distribution system by hot water flushing at 73 degrees C for 1h.

In vitro activities of three carbapenems against recent bacterial isolates from severely ill patients at Swedish hospitals.

To study the in vitro activity of imipenem, meropenem and ertapenem against common pathogens isolated from patients in intensive care, haematology and dialysis/nephrology units at 7 Swedish university hospitals, a total of 788 isolates were collected during 2002-2003. The distribution of the isolates was as follows: Escherichia coli (n = 140), Klebsiella spp. (n = 132), Proteus spp. (n = 97), Enterobacter spp. (n = 113), Pseudomonas aeruginosa (n = 126), Acinetobacter spp. (n = 53) and Enterococcus faecalis (n = 127). The susceptibility to the 3 carbapenems was determined by E-test, and the MICs were interpreted according to SRGA criteria. All 3 carbapenems were highly active against Enterobacteriaceae. The overall susceptibility to imipenem, meropenem and ertapenem was 90%, 98% and 93%, respectively. Against Enterobacteriaceae, Enterobacter spp. excluded, ertapenem had an equal or lower MIC(90) than meropenem. Apart from being the most active carbapenem against Enterobacteriaceae, meropenem was also the most active carbapenem against P. aeruginosa, whereas imipenem was the most active drug against Acinetobacter spp. The carbapenems are still potent antibiotics. With the introduction of ertapenem, and an expected increase in the carbapenem consumption due to an increased prevalence of strains with extended-spectrum beta-lactamases, continuous surveillance of carbapenem resistance appears to be warranted, with special attention to P. aeruginosa, Enterobacter and Proteus spp.

The presence of a specific genotype of Legionella pneumophila serogroup 1 in a hospital and municipal water distribution system over a 12-year period.

The genotypic distribution of Legionella pneumophila serogroup 1 was investigated in the water distribution system of a 450-bed Swedish hospital and the surrounding community. A single genotype identified by amplified fragment length polymorphism (AFLP) analysis, was found in all 34 hospital isolates and in 18 out of 20 community isolates over a 12-y surveillance period. All isolates were either monoclonal antibody subtypes Benidorm or Bellingham. In a geographical comparison, the hospital genotype was also identified in 2 out of 6 Swedish hospitals, both located within 100 km of the studied community. In all, 70 isolates originating from 7 Swedish communities clustered in 4 groups, each also containing 1 AFLP type as defined by the European Working Group on Legionella Infections (EWGLI). It was concluded that a single Legionella pneumophila serogroup 1 genotype may colonize a large water distribution system over a long period of time, and that certain clones seem to be widely spread in the environment. Results from molecular typing of isolates originating from a limited geographical area must, therefore, be interpreted cautiously in epidemiological investigations of Legionnaires' disease.

A hospital-associated outbreak of Legionnaires' disease caused by Legionella pneumophila serogroups 4 and 10 with a common genetic fingerprinting pattern.

An outbreak of eight cases of pneumonia caused by Legionella pneumophila non-serogroup 1 (non-sg 1) occurred at a Swedish university hospital in 1993. Including previous and subsequent sporadic cases, the total number of culture-positive patients was 13. Twelve available non-sg1 isolates from patients were compared to 50 environmental water isolates using a monoclonal antibody test for serogrouping and amplified fragment length polymorphism analysis (AFLP). Of the 12 hospital-associated Legionella non-sg 1 patient isolates, 4 were serogrouped as sg 4, 7 as sg 10, and one as sg 6. Using AFLP fingerprinting all serogroup (sg) 4 and 10 isolates were genetically related except for minor variations. Furthermore, sg 4 isolates were identical in AFLP to sg 10 isolates. Patient isolates were also identical to isolates found in the water system of several hospital buildings, but quite unrelated to isolates obtained in a subsequent outbreak at the same hospital caused by L. pneumophila sg 1. Serogroup variations in outbreaks may occur despite a common molecular fingerprinting pattern. Evidently, the L. pneumophila sg 4 and 10 strains were closely related genetically, which raises the question whether this variation in phenotype is due to a genetic event or to a variable phenotypic expression. Genetic fingerprinting should be used in conjunction with serogrouping in epidemiological investigations.

A hospital-associated outbreak of Legionnaires' disease caused by Legionella pneumophila serogroup 1 is characterized by stable genetic fingerprinting but variable monoclonal antibody patterns.

An outbreak of 18 pneumonia cases caused by Legionella pneumophila serogroup 1 occurred at a Swedish university hospital 1996 to 1999. Eight clinical isolates obtained by culture from the respiratory tract were compared to 20 environmental isolates from the hospital and to 21 epidemiologically unrelated isolates in Sweden, mostly from patients, by using pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism analysis (AFLP), and monoclonal antibody (MAb) typing. All patients and most environmental isolates from the outbreak hospital belonged to the same genotypic cluster in both PFGE and AFLP. This genotype was distinctly different from other strains, including a cluster from a second hospital in a different part of the country. The MAb subtype of the outbreak clone was Knoxville except for three isolates that were Oxford. A variation in the MAb reactivity pattern was also found in a second genotypic cluster. These changes in the MAb reactivity pattern were due to the absence or presence of the lag-1 gene coding for an O-acetyltransferase that is responsible for expression of the lipopolysaccharide epitope recognized by MAb 3/1 of the Dresden Panel. In all MAb 3/1-positive strains, the lag-1 gene was present on a genetic element that was bordered by a direct repeat that showed a high degree of sequence homology. Due to this homology, the lag-1 gene region seemed to be an unstable element in the chromosome. MAb patterns are thus a valuable adjunct to genotyping methods in defining subgroups inside a genotypic cluster of L. pneumophila sg 1.

Clinical utility of urinary antigen detection for diagnosis of community-acquired, travel-associated, and nosocomial legionnaires' disease.

The clinical utility of Legionella urinary antigen assays for the diagnosis of Legionnaires' disease was assessed by using samples from 317 culture-proven cases. The sensitivities of the Binax enzyme immunoassay (EIA) and Biotest EIA were found to be 93.7 and 94.4% for travel-associated infection and 86.5 and 76.0% for community-acquired infection but only 44.2 and 45.7% for nosocomially acquired infection, respectively.

A multicenter evaluation of genotypic methods for the epidemiologic typing of Legionella pneumophila serogroup 1: results of a pan-European study.

OBJECTIVES: To compare genotypic methods for epidemiologic typing of Legionella pneumophila serogroup (sg) 1, in order to determine the best available method within Europe for implementation and standardization by members of the European Working Group on Legionella Infections. METHODS: Coded isolates (114) of L. pneumophila sg 1 comprising one epidemiologically 'unrelated' (79) and one 'related' panel of isolates (35) were sent to 12 laboratories in 11 European countries. Analysis was undertaken in each laboratory using one or more of the following methods: ribotyping, restriction fragment length polymorphism analysis, restriction endonuclease analysis, pulsed-field gel electrophoresis (PFGE), PCR using arbitrary/repeat sequence primers (AP-, AP/rep-PCR), and amplified fragment length polymorphism (AFLP) analysis. Results were analyzed visually or using gel analysis software. Each method was assessed for its: index of discrimination (D), epidemiologic concordance (E), speed of application and ease of use. In addition, phenotypic analysis was performed in two laboratories using monoclonal antibodies (mAbs). RESULTS: The D of each of the genotypic methods ranged from 0.840 for ribotyping to 0.990 for PFGE using Sfil: E ranged from 0.06 for AP- and AP/rep-PCR to 1.00 for ribotyping using Pstl/EcoRI and AFLP: in general, E was inversely related to D. Although offering only limited discrimination (D=0.838), mAb typing was both rapid and highly epidemiologically concordant (E=1.00). CONCLUSIONS: Two methods, PFGE using Sfil and AFLP, were selected for further study. AFLP is rapid and highly epidemiologically concordant (E=1.00), but is not highly discriminatory. This method will be developed as a rapid screening tool. PFGE using Sfil is highly discriminatory but, in the present study, yielded low values of E (0.12-0.71). Attempts will be made to rigorously standardize this method for use as the reference method. Primary screening of isolates by mAb subgrouping is recommended.

A multicenter evaluation of the Biotest legionella urinary antigen EIA.

OBJECTIVES: To undertake a multicenter study to evaluate the Biotest legionella urinary antigen enzyme immunoassay (EIA) performance against those EIAs already in use in 14 European laboratories. METHODS: Each laboratory examined urine specimens from appropriate patients using both their current assay and the Biotest EIA. Each examined: a standard panel of 12 coded urine samples (distributed by Biotest); a panel of 10 coded urine samples provided as part of a European external quality assurance (EQA) scheme; urine samples from patients with proven legionnaires' disease (LD); urine samples from patients with pneumonia of microbiologically proven cause other than LD; and urine samples submitted for routine examination. Thus, the performance of the Biotest assay (in comparison with current EIAs), its specificity and utility, and the inter-laboratory agreement were assessed. RESULTS: Inter-laboratory agreement was excellent, with all participants obtaining the expected results for 20 of 22 coded urine specimens. Specificity, determined using 123 specimens from patients with infections of known etiology, was 100%. The Biotest EIA gave positive results in 86% of specimens which had been positive in the laboratories' current EIAs, and in 94.6% of those specimens which were positive for Legionella pneumophila serogroup 1. CONCLUSION: The Biotest EIA is simple to use and specific and the results obtained in different laboratories show excellent agreement. The assay compares well existing EIAs, at least for L. pneumophila serogroup 1

A nested polymerase chain reaction for detection of Legionella pneumophila in clinical specimens.

OBJECTIVE: Because presently used methods for diagnosis of Legionella pneumonia lack sufficient sensitivity and sometimes specificity and rapidity, the detection of Legionella spp. by amplification of nucleic acids might be valuable. However, performing polymerase chain reaction (PCR) on clinical samples such as sputum is difficult because of the presence of extraneous DNA and inhibitors of the reaction. An attempt to circumvent these problems was made. METHODS: A nested PCR method was devised using primers from the mip gene of Legionella pneumophila. This PCR was tested on pure cultures of legionellae and clinical isolates of other bacteria. Clinical samples (bronchoalveolar lavage fluid, bronchial aspirate and sputum) from patients who suffered from legionellosis and samples from patients who suffered from other causes of pneumonia were also tested. RESULTS: The PCR was specific for L. pneumophila and no non-Legionella bacteria reacted. Ten to 50 colony forming units of Legionella in the sample could be detected. Twenty-two of 25 clinical samples were positive among patients suffering from pneumonia proven to be due to L. pneumophila serogroups 1, 3, 4, 5 and 6. Two of the three negative samples were from patients who had been treated with adequate therapy for at least 2 days and were culture negative. However, nine other culture-negative samples were PCR positive, of which seven came from patients who had been treated for 3-7 days. All pneumonia patients in the control group proved negative in PCR. A commercial kit for DNA preparation from clinical samples, based on absorption of nucleic acids to silica gel, was superior to the traditional phenol/chloroform extraction and increased the rapidity, simplicity and sensitivity of the procedure. CONCLUSIONS: A nested, simplified and rapid PCR method using mip primers proved to be more sensitive than culture and as sensitive and specific as other PCR procedures previously reported.