Legionnaires ’ disease – Results of a multicentre Canadian study

1Department of Medicine, University of Alberta, Edmonton, Alberta; 2Infectious Diseases Group, Pfizer Canada Inc, Kirkland, Quebec; 3University of Pittsburgh, Pittsburgh, Pennsylvania Correspondence and reprints: Dr Thomas J Marrie, 2F1.30 Walter C Mackenzie Health Sciences Centre, 18440 112 Street, Edmonton, Alberta T6H 2B7. Telephone 780-407-6234, fax 780-407-3132, e-mail tom.marrie@ualberta.ca Received for publication September 17, 2002. Accepted February 5, 2003 TJ Marrie, E De Carolis, VL Yu, J Stout and the Canadian Community-Acquired Pneumonia Investigators. Legionnaires’ disease – Results of a multicentre Canadian study. Can J Infect Dis 2003;14(3):154-158.

L egionnaires' disease is an acute infectious disease of which the predominant manifestation is pneumonia (1).The most common cause is Legionella pneumophila serogroup 1, however, just under half of the over 40 recognized species in the Legionellaceae family can cause Legionnaires' disease (2).A soluble antigen is present in the urine of patients with Legionnaires' disease due to serogroup 1 (3)(4)(5).A commercially available enzyme immunoassay is available that detects this antigen with a specificity of 100% and a sensitivity of 94.6% (3).We used this test to determine the frequency with which L pneumophila serogroup 1 was a cause of community-acquired pneumonia (CAP) in Canada, and to determine whether there was any geographic clustering of the cases of Legionnaires' disease.We tested a subset of the study ©2003 Pulsus Group Inc.All rights reserved

ORIGINAL ARTICLE
population for Legionella species infection using a serological assay.Our objectives were to determine the rate of seroconversion among patients with a positive urinary antigen assay and to determine whether infection with serogroups 2 to 6 occurred in Canada.

Patient populations and study design
This was a prospective observational study of CAP requiring initial hospitalization that began January 11, 1996 and ended October 31, 1997.It was conducted at 15 teaching hospitals in eight Canadian provinces.The study was approved by local ethics committees.Written, informed consent was obtained for each participant in accordance with national guidelines and the Declaration of Helsinki (Hong Kong amendment, 1989).All patients who were admitted to hospital with a diagnosis of pneumonia were screened by a study nurse.Patients who met the following eligibility criteria were approached for enrollment in the study: aged 16 years or older; a new pulmonary infiltrate on chest radiograph not attributable to an etiology other than pneumonia; two or more of the following symptoms or signs: chest pain, cough, rales, rhonchi and/or signs of consolidation, an oral temperature of less than 36.5°C or more than 38.5°C; and had not been hospitalized for at least 14 days.Sputum production was not a requirement for study entry, however, if it was ordered by the attending physician all attempts were made to collect a sample for Gram stain and culture.A study nurse completed the data collection forms and made daily visits (for a maximum of seven days follow-up while in hospital) to each patient to record progress in terms of evolution of the pneumonia and any complications that may have occurred during the hospital stay.All data were verified against the original chart by a study monitor during a site visit.Eight hundred fifty patients entered the study and all supplied informed consent.

Diagnostic testing
Blood and sputum specimens were collected at the discretion of the attending physician and processed for culture in accordance with the methodology in use at each centre's laboratory.
Acute phase serum samples were available on 837 patients and convalescent samples were available on 544 patients.All serum samples were tested for antibodies to Mycoplasma pneumoniae, Influenza viruses A and B, parainfluenza viruses 1, 2, and 3, adenovirus, and respiratory syncytial virus using a standard complement fixation technique in microtitre plates.The adenovirus and respiratory syncytial virus antigens were purchased from Flow Laboratories (USA); the influenza A and B, parainfluenza 1, 2, and 3, and M pneumoniae antigens were purchased from MA Bioproducts (USA).

Legionella species urinary antigen
Patients had urine collected at baseline for detection of L pneumophila serogroup 1 antigen.An enzyme immunoassay was used according to the manufacturers instructions to detect this antigen (11).Testing was done in the microbiology laboratories of participating institutions.

Legionella species serology
A subset of the 850 patients had acute and convalescent serum samples tested for antibodies to L pneumophila serogroups 1 to 6 at VY's laboratory in Pittsburgh, Pennsylvania using an enzyme immunoassay (12).A fourfold rise in antibody titre to at least 1:256 or a stable titre of at least 1:512 was considered to be diagnostic of Legionella species infection.

Statistical analysis
The data were entered into SPSS 8.0 (SPSS Inc, USA).Differences between patient subpopulations were tested using the Pearson χ 2 test, in the case of categorical variables, and the Student's t-test or one-way ANOVA, for continuous variables.

RESULTS
Twenty-eight of the 850 patients (3.2%) had a diagnosis of Legionnaires' disease made.This diagnosis was made on the basis of a positive urinary antigen test in 18 patients; on the basis of a positive culture in four patients (L pneumophila serogroup 1: one patient; L pneumophila serogroup 2: two patients; and Legionella micdadei: one patient).The remainder were diagnosed serologically (L pneumophila serogroup 1: one patient; serogroups 2 to 6: five patients).
Two patients with serogroup 1 infection had a negative urinary antigen assay -one of these patients had the infection diagnosed serologically and the other had L pneumophila cultured from respiratory secretions.The sensitivity of the urinary antigen test for the detection of a serogroup 1 infection was 90%.
Table 1 shows the site-specific positivity rates for L pneumophila urinary antigen, which ranged from 0% to 7.6%.The latter occurred at the Quebec City site, where there was an outbreak of Legionnaires' disease associated with exposure to a contaminated cooling tower.The urinary antigen positivity rates were higher at the Quebec City site (P<0.04)and at the Halifax site (P<0.04)when compared with all the other sites.The St John's case was a truck driver from Nova Scotia who likely acquired his infection in Nova Scotia rather than in Newfoundland.When all 20 cases of serogroup 1 infection were considered, there was no difference in the rates of infection in Eastern versus Western Canada -14 of 573 (2.4%) versus six of 277 (2.2%) (P=nonsignifican).Overall, there were eight cases of nonserogroup 1 infection.Five of these occurred among the 277 patients (1.8%) from Western Canada versus three among the 573 (0.5%) from Eastern Canada (P=0.07).However, two of the serogroup 2 infections among patients from Eastern Canada (a husband and wife who had just returned to Toronto from a trip to Spain) were likely acquired in Spain.If these two cases are eliminated, the rates are 1.8% for patients from Western Canada versus one of 571 (0.017%) (P=0.01).If only those who had acute and convalescent serology tested for antibodies to L pneumophila serogroups 2 to 6 are considered, the rates were one of 150 (0.6%) for patients in Eastern Canada versus four of 84 (4.7%) for those in Western Canada (P=0.057).
Forty per cent of the L pneumophila serogroup 1 cases occurred during the months of July to September (P=0.01 for trend) (Table 2).
The 20 patients with L pneumophila serogroup 1 infection were compared with 224 patients who had a negative Legionella species urinary antigen test and negative serology tests for L pneumophila serogroups 2 to 6 (Table 3).Patients with Legionnaires' disease were more likely to present to hospital earlier after the onset of symptoms, to complain of myalgias and headaches, and to have a higher temperature at the time of admission than were patients with pneumonia due to other causes.They were less likely to have chronic obstructive pulmonary disease.There was no difference in mortality rate or length of stay.
Table 4 shows the results of serological testing on 10 patients with a positive urinary antigen test.Only three of the 10 had a positive serological test.
Fifteen of the 18 patients with a positive urinary antigen test had additional serological testing as described in the methods section.Seven patients (46.6%) had a copathogen identified.These included three cases of Streptococcus pneumoniae and one each of adenovirus, influenza virus A, influenza virus B, and Mycoplasma pneumoniae.Six of the 18 patients had blood cultures performed before antibiotic therapy and all were negative.

DISCUSSION
A Medline search from 1976 through 2001 revealed 33 papers dealing with Legionella species infection in Canada.Seventeen of these papers dealt with nosocomial Legionnaires' disease.From this literature it is apparent that L pneumophila serogroup 1 and Legionella longbeachae serogroup 2 were present in the water supplies of Nova Scotia hospitals (13) and that L pneumophila, Legionella micdadei and Legionella dumoffi had been recovered from clinical and or environmental specimens in the Quebec City area (14,15).L pneumophila serogroups 1, 3, 6 and 12, Legionella sainthelensi, and Legionella feelei have been reported from Ontario (16-21); L pneumophila serogroup 1 from Manitoba (22); and Legionella maccachernii from Saskatchewan (23).Nicolle et al (24) reported nine patients with community-acquired Legionellosis who were hospitalized at the Health Sciences Centre in Winnipeg between 1986 and 1991.Eight of these infections were due to L pneumophila serogroup 1 and one was due to L pneumophila serogroup 6.The current study is the first to try and determine the importance of Legionella species as a cause of CAP in Canada.We found that 2.1% of the patients were infected with L pneumophila serogroup 1.This infection was more common in Halifax and Quebec City than elsewhere; however, the Quebec City cases were related to an outbreak secondary to exposure to a contaminated water cooling tower.When all  28 cases of Legionnaires' disease were considered, cases were just as common in Western Canada as in Eastern Canada.It appeared that cases of L pneumophila serogroups 2 to 6 were more common in Western Canada when two of the three cases in Eastern Canada were removed from the analysis (these two cases appeared to be acquired in Spain).Our study has several limitations regarding conclusions about Legionella species infections in Canada: cultures of respiratory secretions for Legionella species were not done routinely, and only 234 patients had serological testing for L pneumophila serogroups 1 to 6.
Early in the course of study of Legionnaires' disease a summer-fall predominance of cases was noted (25).We noted the same seasonality.
In one of the better studies of the Legionella species urinary antigen test, Plouffe et al (5) noted a specificity of 99% and an overall sensitivity of 56%.Our overall sensitivity of 64% is in keeping with Plouffe's findings.Seven of 10 patients with positive urinary antigen in our study had negative acute and convalescent serology.Does this mean that the urinary antigen tests were false positives or that the serological tests were falsely negative?Failure of seroconversion of culture-positive cases of Legionnaires' disease is well known even when serum samples are obtained up to 10 or 12 weeks convalescent (26).Indeed the sensitivity of serology ranges from 60% to 75% (26), thus it is possible that some of our results represent falsepositive urinary antigen tests.We also documented two falsenegative urinary antigen tests.
Sopena et al ( 27) compared 48 patients with Legionnaires' disease with 125 patients with pneumonia due to other causes.By multivariate analysis the patients with Legionnaires disease were more likely to have no underlying disease, to complain of diarrhea, and to have an elevated creatine phosphokinase.Mulazimoglu and Yu (28) reviewed 14 studies of the etiology of CAP to try to determine the clinical features that distinguish Legionnaires' disease from other causes of pneumonia.They only included studies that used two methods to diagnose Legionella species infection and excluded studies that relied only on serology.They found that headache, diarrhea, neurological symptoms (especially confusion), fever of more than 39°C, hyponatremia, elevated creatinine phosphokinase, and abnormal liver function tests were the most consistent features in distinguishing Legionnaires' disease from other etiologies of CAP.We also found that headache was more common among patients with Legionnaires' disease than among patients with other causes of pneumonia.We noted that myalgias, a higher mean oral temperature, and the lack of chronic obstructive lung disease were more common among the patients with Legionnaires' disease.We also noted that patients with Legionnaires' disease presented to hospital sooner after onset of symptoms than did patients with other causes of pneumonia.
In our study the mortality rate for Legionella species serogroup 1 infection was 10% and for nonserogroup 1 infection it was 25%.Heath et al (29) studied 39 patients with Legionnaires' disease (36 were community-acquired) and found that the mortality rate was 26%.They noted that survivors had therapy with erythromycin started a median of six days from onset of symptoms compared with 11 days for those who died.We found that the median time from onset of symptoms until presentation to a hospital was 4.7 days for those with L pneumophila serogroup 1 infections.Most of our patients with Legionella species infection were treated with erythromycin, although four received azithromycin dihydrate.Edelstein (30) found that azithromycin dihydrate is more active against intracellular L pneumophila than erythromycin.He recommends treatment of Legionnaires' disease with a fluoroquinolone, which, in contrast to erythromycin, can kill intracellular Legionella species, or azithromycin dihydrate.
There are a couple of limitations to our study.Because of the requirements for informed consent, it is possible that enrollment was biased toward patients who were not seriously ill and, hence, less likely to have Legionnaires' disease.However, the overall mortality rate was 9.5%, which is similar to that in population-based studies (31,32).Another limitation is that the only test for Legionnaires' infection given to the entire population was the urinary antigen test.

CONCLUSIONS
Legionella species infections constitute a small percentage of cases of CAP requiring admission to hospital in Canada.These cases occur throughout Canada, and a comprehensive diagnostic approach is necessary for their diagnosis.It is not practical to carry out comprehensive testing for Legionella species infection on all patients who are admitted to hospital so this is best reserved for those in whom there is a high clinical suspicion of Legionnaires' disease, and those with rapidly progressive pneumonia.L pneumophila serogroup 1 infections may be more common in Halifax than in the rest of Canada.The higher rate of this infection in Quebec City was related to an outbreak.Infection with L pneumophila serogroups 2 to 6 is just as common as infection with serogroup 1.

ACKNOWLEDGEMENTS:
We would like to thank Dr JA Inverso (Pfizer Inc) for her assistance in this study.We are grateful to S Ioannou and NG Moghaddam (Pfizer Canada Inc), and the fol- lowing clinical study coordinators: M Dambreville RN, H Stalts RN, J Clark-DiPrata RN, B Peters RN, K Heney RN, J Graham RN, F Brisebois RN, H Patil RN, G Patrick RN, T Muir RN, F Habel RN, E Condon RN, S Roberts RN, A Lindemulder RN, D Paget-Dellio RN, M Jones RN, C Hammerberg RN, and H Duckworth RN.Legionnaires' Disease -Results of a Canadian Study Can J Infect Dis Vol 14 No 3 May/June 2003 157

TABLE 4 Results of serological testing (ELISA) of patients with a positive urinary antigen for Legionella pneumophila serogroup 1
The following investigators participated in this study: Dr M Gribble, University of British Columbia, Vancouver, British Columbia; Dr S Field and Dr T Louie, University of Calgary, Calgary, Alberta; Dr S Houston, University of Alberta, Edmonton, Alberta; Dr K Williams, University of Saskatchewan, Saskatoon, Saskatchewan; Dr L Nicolle, University of Manitoba, Winnipeg, Manitoba; Dr R Grossman and Dr I Salit, University of Toronto, Toronto, Ontario; Dr R Saginur, University of Ottawa, Ottawa, Ontario; Dr D Gregson, University of Western Ontario, London, Ontario; Dr R Duperval, Université de Sherbrooke, Sherbrooke, Quebec; Dr M Laverdière; Université de Montréal, Montreal, Quebec; Dr M Rouleau; Université Laval, Ste-Foy, Quebec; and Dr J Hutchinson, Memorial University of Newfoundland, St John's, Newfoundland.This research study was funded by Pfizer Canada Inc, Anti-Infectives Group, Kirkland, Quebec.