Liver cirrhosis as a predisposing condition for Legionnaires disease: a report of four laboratoryconfirmed

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1 Journal of Medical Microbiology (2012), 61, DOI /jmm Case Report Correspondence Zhujun Shao Liver cirrhosis as a predisposing condition for Legionnaires disease: a report of four laboratoryconfirmed cases from China Tian Qin, 1 3 Jie Xia, 2 3 Hongyu Ren, 1 Haijian Zhou, 1 Bo Tang 2 and Zhujun Shao 1 1 State Key Laboratory for Infectious Disease Prevention and Control and the National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 155 Changbai Road, Changping, Beijing , PR China Military Hospital, 100 Xisihuan Middle Road, Fengtai, Beijing , PR China Received 28 October 2011 Accepted 23 March 2012 Here, we describe four cases of laboratory-confirmed Legionella infection. Case 1 was a cultureconfirmed case of Legionella infection in a patient with liver cirrhosis. Following this, three other liver cirrhosis cases (cases 2 4) were diagnosed with Legionella infection as confirmed by quantitative real-time PCR. The cause of the pneumonia was determined as Legionella pneumophila by positive direct fluorescence assay and isolation of the causative agent. The infections were successfully treated by administering appropriate antibiotics. These cases highlight the importance of considering Legionella as a cause of pneumonia in patients with liver disease and lung infections. The strain of L. pneumophila isolated from Case 1 was characterized as being closely related to strain Philadelphila-1 (ATCC T ), which is the type strain of the species, belonging to serogroup 1 and sequence type 36 (ST36), and is known to be distributed worldwide. To our knowledge, this is the first report of Legionella infection on the Chinese mainland for a decade and highlights the need to raise awareness of diagnostic methods for Legionnaires disease in China and the requirement for further epidemiological surveillance strategies to monitor this disease. Introduction Legionnaires disease (LD) is a systemic infection caused by the Gram-negative bacteria Legionella pneumophila. The primary presentation is pneumonia. Members of the genus Legionella are typically found in both the natural environment, such as hot springs and soil, as well as artificially hydrated environments, such as water supply facilities. In studies undertaken between 1994 and 2003, Legionella species accounted for % of causative pathogens for all community-acquired pneumonia cases (Miyashita et al., 2006). Legionella pneumophila is responsible for.90 % of cases and the most prevalent disease-causing variant of this species is serogroup 1 (Yu et al., 2002). The clinical features of pneumonia caused by Legionella are diverse. Early symptoms include fever, headache, diarrhoea and coughing. During the course of the illness, stupor, respiratory- and even multiorgan failure may develop. Elevation of creatinine kinase has 3These authors contributed equally to this work. Abbreviations: BAL, bronchoalveolar lavage; LD, Legionnaires disease; PFGE, pulsed-field gel electrophoresis; qpcr, quantitative real-time PCR; SBT, sequence-based typing; ST, sequence type. also been reported as a disease-specific symptom of LD (Sopena et al., 1998). Transmission of bacteria from the environment to humans occurs via the inhalation or aspiration of Legionellacontaining aerosols (Blatt et al., 1993; Breiman et al., 1990). Epidemiological analyses based on the molecular typing of clinical isolates of Legionella have detected sporadic, epidemic, and endemic strains (Bernander et al., 2003). Strain differentiation is necessary to identify the source of contamination and determine the route of transmission. Risk factors associated with the occurrence of LD include old age, being male, smoking, having chronic heart and lung diseases, renal or hepatic failure, recent travel with an overnight stay outside of the home, recent changes in domestic plumbing, a history of cancer or haematological malignancies, steroid therapy or other immunosuppressive treatments, and diabetes mellitus (Che et al., 2008; Fields et al., 2002; Miyashita et al., 2006). In addition, numerous case reports suggest that there is a higher rate of Legionella infection among immunocompromised and splenectomized patients compared with immunocompetent patients (Jaeger et al., 1988; Jernigan et al., 1994; Kümpers et al., 2008; McClelland et al., 2004; Singh et al., 2002; Lück et al., 2008) G 2012 SGM Printed in Great Britain 1023

2 T. Qin and others A variety of subtyping techniques have been used to identify and characterize Legionella strains, including monoclonal antibody (mab) analysis (Joly et al., 1986), amplified fragment length polymorphism (AFLP) analysis (Fry et al., 2002), pulsed-field gel electrophoresis (PFGE) (Casini et al., 2008; Zhou et al., 2010), and, more recently, sequence-based typing (SBT) (Gaia et al., 2005; Ratzow et al., 2007) and nested SBT (Ginevra et al., 2009). The SBT methodology is, in fact, a variation of multilocus sequence typing (MLST) which considers sequence variations not only in housekeeping genes but also in virulence-associated L. pneumophila genes. This method was established by the European Working Group for Legionella Infections and is now used internationally as the standard method for typing L. pneumophila. Here, we report a L. pneumophila strain that was isolated from bronchoalveolar lavage (BAL) fluid of a liver cirrhosis patient; subsequently L. pneumophila genomic DNA was detected from BAL fluid samples from three other patients with liver diseases. Genotypic characteristics of the first strain and the three strains, identified by quantitative realtime PCR (qpcr) using DNA from positive BAL fluid samples, were determined by PFGE and/or SBT analyses. To our knowledge, this is the first report of LD in a patient suffering from cirrhosis of the liver, and is also the first case of Legionella infection reported on the Chinese mainland for a decade. Thanks to a correct diagnosis, all the patients were successfully treated and fully recovered from LD. Case report A 42-year-old male, diagnosed with liver cirrhosis in early 2010, was admitted to an intensive care unit in May 2011 with signs of a respiratory infection. At admission, his blood pressure was 108/69 mmhg, temperature was 38.3 uc, pulse rate was 100 min 21 and respiratory rate was 20 min 21. Pulmonary inflammation was diagnosed by computed tomography (CT) (Fig. 1). A sputum sample was collected on day 2 post-admission and yielded Candida albicans growth, following inoculation onto Candida chromogenic agar and incubation at 28 uc for 2 days. Five days after admission, the patient s condition worsened dramatically and a further chest CT scan showed a bilateral spread of the infection in the lungs (Fig. 1). The patient developed shortness of breath, chest tightness, laboured breathing and a worsened cough. A BAL sample was collected for culture and molecular investigations. Genomic DNA was extracted from the BAL fluid. The BAL samples were centrifuged at 6000 g for 10 min and the pellets were resuspended in 2 ml sterile-distilled water (Invitrogen). Genomic DNA was extracted from 0.2 ml resuspended liquid using the QIAamp DNA Mini kit (Qiagen), according to the manufacturer s instructions. PCR analyses were performed with primers that targeted the Legionella 16S rrna gene (for the detection of members of the genus Legionella; Yamamoto et al., 1993) and the L. pneumophila mip gene (for the detection of L. pneumophila; Mahbubani et al., 1990), and qpcr analyses were performed 1024 Journal of Medical Microbiology 61 with primers that targeted the Legionella 5S rrna gene (for the detection of members of the genus Legionella). qpcr mixtures (20 ml) contained 2 ml genomic DNA from each sample and fluorescent probes to detect the products. Quantification was performed using the primers L5SL9 (59-ACTATAGCGATTTGGAACCA-39) and L5SR93 (59- GCGATGACCTACTTTCGCAT-39), which were designed to amplify a fragment of the Legionella 5S rrna gene (Mahbubani et al., 1990), and the molecular beacon HEX- CCGCGCCAATGATAGTGTGAGGC-BHQ (Diederen et al., 2006). qpcrs were conducted using a quantitative PCR instrument (Stratagene) with the following reaction conditions: an initial denaturing step of 95 uc for 2 min, followed by 40 cycles of 95 uc for 10 s and 60 uc for20s.allthe results of the PCRs and qpcrs were positive. Subsequently, Legionella colonies were grown by spreading 0.1 ml of appropriate dilutions of each sample onto buffered charcoal yeast extract agar (Oxoid) and incubating at 37 uc for 10 days. The colonies were identified as L. pneumophila serogroup 1 by using monoclonal antisera (Denka Seiken). On the basis of these results, we made a definitive diagnosis of LD. The patient was treated with clarithromycin and, after 18 days of antibiotic therapy, the patient s condition improved. Subsequently, signs of lung infection on subsequent CT scans disappeared (Fig. 1). PFGE was used (Zhou et al., 2010) to characterize this strain of L. pneumophila (named ICDC-LP001). The PFGE pattern of this strain was compared to those present in the reference database for L. pneumophila in China. At the time of comparison, the database contained 516 isolates of environmental L. pneumophila, isolated between 2009 and 2011, and 162 PFGE patterns, including 122 strains isolated from the city our patient originated from. This current strain showed the same pattern as the L. pneumophila reference strain Philadelphia-1 (ATCC T ) but was different to all other strains in the database (Fig. 2). Furthermore, we analysed the genomic DNA of this L. pneumophila strain, which had been extracted from BAL fluid, using SBT (Gaia et al., 2005; Ratzow et al., 2007). SBT indicated that the allele numbers for flaa, pile, asd, mip, momps, proa and neua were 3, 4, 1, 1, 14, 9 and 1, respectively. This strain was assigned the sequence type (ST) number ST36, which was the same ST as that of the type strain of L. pneumophila, Philadelphia-1 (ATCC T ). By querying the L. pneumophila SBT database (available at we found that ST36 was prevalent both in clinical and environmental strains distributed over 25 countries between 1982 and 2011, including one environmental strain that was isolated in Shanghai, China, in Subsequently, 10 additional BAL fluid samples, each from separate patients with a liver disease, were collected in order to investigate possible L. pneumophila infection (Table 1). All the patients had a lung infection with high fever, cough and sputum but had not been given a diagnosis. Each sample was cultured and DNA was extracted for molecular analyses. Unfortunately, no Legionella strain was isolated from these

3 Liver cirrhosis patient infected by L. pneumophila 16 th May 20 th May 8 th June 14 th June Fig. 1. Chest CT scans of patient 1. The date when the patient was examined is indicated. Similarity (%) JH JH BJ-33 ICDC-LP001 ATCC T (Philadelphia-1) WD FS FS1103B TL-12 JH-9 WD TL-11 FS1103C WD JH1102 WD WD FS1103A-2 JH FS FS-5 FS FS1103A BJ-27 BJ-14 JH-8 CD-2 BJ-23 FS FS-3 FS1103B-2 Fig. 2. PFGE patterns and clustering results of L. pneumophila clinical strain ICDC-LP001 compared with reference strain Philadelphia-1 and environmental isolates from the city of residence of the patient, obtained from the PFGE database. The PGFE patterns were analysed using the BioNumerics software package (version 5.1; Applied Maths). Clustering was created using the unweighted pair group method with arithmetic mean (UPGMA)

4 T. Qin and others Table 1. Details of the patients tested in this study and results obtained from culture and qpcr analysis Patient ID Age (y)/ gender Date of admission Comorbidity Detection of Legionella in BAL fluid Culture qpcr Patient 1 42/M 16/05/2011 Liver cirrhosis, lung infection, cholecystitis + + Patient 2 57/M 16/05/2011 Obstructive jaundice, diabetes, emphysema, lung infection + Patient 3 60/M 08/03/2011 Liver cirrhosis, pneumonia, splenectomy, cholecystectomy + Patient 4 33/M 15/06/2011 Liver cirrhosis, lung infection + Patient 5 48/M 14/07/2011 Liver cirrhosis, peritonitis, lung infection Patient 6 20/F 13/07/2011 Acute liver failure, hepatic encephalopathy, lung infection Patient 7 46/F 07/06/2011 Drug-induced liver injury, hepatitis, lung infection Patient 8 37/M 03/07/2011 Liver cirrhosis, hepatic encephalopathy, hepatitis, renal insufficiency, lung infection Patient 9 41/F 08/04/2011 Liver failure, hepatic encephalopathy, acute respiratory distress syndrome, lung infection Patient 10 49/M 08/07/2011 Liver cirrhosis, meningitis, lung infection Patient 11 27/F 05/08/2011 Drug-induced liver injury, cerebral oedema, hepatic encephalopathy, adult Still s disease, lung infection samples and urinary antigen tests were not performed. However, three samples were 5S rrna qpcr-positive for Legionella. The NESTED Sequence-Based Typing protocol for epidemiological typing of Legionella pneumophila directly from clinical samples (Version 1.0, available at ewgli.org) was used to genotype the three qpcr-positive samples. Seven loci were amplified and sequenced successfully for two of the samples, and only three loci were sequenced for the third sample (Table 2). The sample from patient 4 was assigned ST36, the same ST as the sample from patient 1. The sample from patient 3 was assigned ST346, which has a one-locus difference to ST36 [mip(1) for ST36 and mip(28) for ST346]. The three sequenced loci from the sample from patient 2 were pile(4), asd(1) and momps(14), the same loci as ST36 and ST346. Clarithromycin was given to the three qpcr-positive patients and the patients recovered. Discussion In the early stages of Legionella pneumonia, some patients present various symptoms, such as high fever, cough, sputum, diarrhoea and impaired level of consciousness, without having any breathing difficulties (Yagyu et al., 2003). These cases are often ignored, especially among patients with other diseases that also cause some of these symptoms. In the present case, the patient was a middleaged man with severe liver disease. Since the patient had fever, cough and a lung infection on first presentation and Candida albicans was isolated from the patient s sputum, we suspected he was suffering from a fungal infection. However, the patient s clinical symptoms did not improve after 5 days of hospitalization and a chest CT scan demonstrated significantly more pulmonary infiltration. We then decided to reconsider the cause of his symptoms and Legionella infection was confirmed. Clinical practice guidelines recommend testing for Legionella in selected patients, including seriously ill patients without an alternative diagnosis, older and immunocompromised patients and patients non-responsive to b-lactam antibiotics (British Thoracic Society Standards of Care Committee, 2001; Mandell et al., 2007). However, in the Chinese mainland, Legionnaires disease is usually ignored in the differential diagnosis of pneumonia as most physicians lack experience with this disease. Based on the cases reported here, we suggest that patients with liver cirrhosis suffering from lung disease should also be tested for possible Legionella infection. As the clinical signs of Legionnaires disease are usually not specific, a prompt and accurate laboratory diagnosis is crucial. In our lab, diagnostic methods were established to detect Legionella in both clinical and environmental samples, including culture, serology and conventional PCR and qpcr, as well as genotyping methods, such as PFGE, SBT and nested modified SBT. The urinary antigen test is useful for early diagnosis of Legionella infections; however, this method was not performed on any cases in this study. The sensitivity of this method is limited because the commercially available urinary antigen test detects only serogroup 1 L. pneumophila. Culture-confirmed diagnosis of Legionella infections is often hampered by early antibiotic treatment and demanding culture requirements. Legionella species do not grow on standard microbiology media, thus they are usually not detected from blood or sputum cultures. Culture isolation from BAL fluid is considered the gold standard for diagnosis, but Legionella requires 4 10 days to grow and this method is incapable of detecting viable but not culturable (VBNC) cells (Bej et al., 1991; Allegra et al., 2008). The shortcomings of culture have prompted the development and routine application of 1026 Journal of Medical Microbiology 61

5 Liver cirrhosis patient infected by L. pneumophila Table 2. Sequence-based typing of Legionella strains identified in this study, Not detected. Sample Origin of DNA Loci flaa pile asd mip momps proa neua SBT type 1 BAL fluid from patient ST36 2 BAL fluid from patient BAL fluid from patient ST346 4 BAL fluid from patient ST36 5 Strain from patient ST36 non-culture-based methods, such as qpcr, in the laboratory diagnosis of Legionnaires disease. qpcr appears to be a promising tool for the simultaneous, rapid and reliable detection of Legionella species. Here, in addition to the case described above, Legionella DNA from BAL samples of three other patients was also detected by qpcr analysis, suggesting that, in addition to established diagnostic methods such as culture and urinary antigen testing, PCR methods could significantly improve early detection in patients with suspected Legionnaires disease. Using genotyping methods, the strain from the initial case was characterized as being closely related to ST36 strains, which are distributed worldwide. The first ST36 strain to be characterized was the Philadelphia-1, isolated in January 1977 at the Centers for Disease Control and Prevention, Atlanta, GA, USA, as a result of the investigations that followed the first ever described outbreak of Legionnaires disease (McDade et al., 1977). Since then, ST36 has been identified in Germany, Canada, Netherlands, UK, Russian Federation, Japan, Belgium and China. The only Chinese ST36 strain present in the SBT database was isolated during a routine investigation of environmental samples in Shanghai in These data suggest that L. pneumophila ST36 is present in the environment in China. Legionnaires disease is a notifiable disease in many countries worldwide, but not in China (Centers for Disease Control and Prevention, 2011; Joseph & Ricketts, 2010). In the past decade, no case of Legionnaires disease has been reported on the Chinese mainland, and no clinical strains have been identified. In the present study, we confirmed a case of L. pneumophila infection by laboratory investigations and described, for the first time, the genotypic characteristics of a novel clinical strain in China. A disease-causing endemic strain, Philadelphia-1, already exists in China and has caused disease, but the current system that monitors Legionella is focused on environmental water samples rather than clinical samples. This clinical case highlights the need to consider diagnostic methods for Legionnaires disease in China, and the requirement for further epidemiological surveillance strategies to monitor this disease. The appearance of this strain indicates the need for improved public health disease control strategies in China in the future. Acknowledgements This publication made use of the EWGLI Sequence-Based Typing Database for Legionella pneumophila (available at org) provided by the European Working Group for Legionella Infections (EWGLI) in conjunction with the Health Protection Agency and the European Center for Disease Prevention and Control. This study was supported by grants from the Science Foundation for Youth from China CDC (2011A102) and the Science Foundation for State Key Laboratory for Infectious Disease Prevention and Control from China CDC (2011SKLID202). References Allegra, S., Berger, F., Berthelot, P., Grattard, F., Pozzetto, B. & Riffard, S. (2008). Use of flow cytometry to monitor Legionella viability. Appl Environ Microbiol 74, Bej, A. K., Mahbubani, M. H. & Atlas, R. M. (1991). Detection of viable Legionella pneumophila in water by polymerase chain reaction and gene probe methods. Appl Environ Microbiol 57, Bernander, S., Jacobson, K., Helbig, J. H., Lück, P. C. & Lundholm, M. (2003). A hospital-associated outbreak of Legionnaires disease caused by Legionella pneumophila serogroup 1 is characterized by stable genetic fingerprinting but variable monoclonal antibody patterns. J Clin Microbiol 41, Blatt, S. P., Parkinson, M. D., Pace, E., Hoffman, P., Dolan, D., Lauderdale, P., Zajac, R. A. & Melcher, G. P. (1993). Nosocomial Legionnaires disease: aspiration as a primary mode of disease acquisition. Am J Med 95, Breiman, R. F., Cozen, W., Fields, B. S., Mastro, T. D., Carr, S. J., Spika, J. S. & Mascola, L. (1990). Role of air sampling in investigation of an outbreak of Legionnaires disease associated with exposure to aerosols from an evaporative condenser. J Infect Dis 161, British Thoracic Society Standards of Care Committee (2001). BTS guidelines for the management of community acquired pneumonia in adults. Thorax 56 (Suppl. 4), IV1 IV64. Casini, B., Valentini, P., Baggiani, A., Torracca, F., Lorenzini, C., Frateschi, S., Matteoli, B. & Privitera, G. (2008). Comparison of two molecular methods used for subtyping of Legionella pneumophila 1 strains isolated from a hospital water supply. Water Sci Technol 58, Centers for Disease Control and Prevention (2011). Legionellosis United States, MMWR Morb Mortal Wkly Rep 60, Che, D., Campese, C., Santa-Olalla, P., Jacquier, G., Bitar, D., Bernillon, P. & Desenclos, J. C. (2008). Sporadic communityacquired Legionnaires disease in France: a 2-year national matched case-control study. Epidemiol Infect 136,

6 T. Qin and others Diederen, B. M. W., de Jong, C. M. A., Kluytmans, J. A. J. W., van der Zee, A. & Peeters, M. F. (2006). Detection and quantification of Legionella pneumophila DNA in serum: case reports and review of the literature. J Med Microbiol 55, Fields, B. S., Benson, R. F. & Besser, R. E. (2002). Legionella and Legionnaires disease: 25 years of investigation. Clin Microbiol Rev 15, Fry, N. K., Bangsborg, J. M., Bergmans, A., Bernander, S., Etienne, J., Franzin, L., Gaia, V., Hasenberger, P., Baladrón Jiménez, B. & other authors (2002). Designation of the European Working Group on Legionella Infection (EWGLI) amplified fragment length polymorphism types of Legionella pneumophila serogroup 1 and results of intercentre proficiency testing using a standard protocol. Eur J Clin Microbiol Infect Dis 21, Gaia,V.,Fry,N.K.,Afshar,B.,Lück, P. C., Meugnier, H., Etienne, J.,Peduzzi,R.&Harrison,T.G.(2005).Consensus sequence-based scheme for epidemiological typing of clinical and environmental isolates of Legionella pneumophila. JClinMicrobiol43, Ginevra, C., Lopez, M., Forey, F., Reyrolle, M., Meugnier, H., Vandenesch, F., Etienne, J., Jarraud, S. & Molmeret, M. (2009). Evaluation of a nested-pcr-derived sequence-based typing method applied directly to respiratory samples from patients with Legionnaires disease. J Clin Microbiol 47, Jaeger, T. M., Atkinson, P. P., Adams, B. A., Wright, A. J. & Hurt, R. D. (1988). Legionella bozemanii pneumonia in an immunocompromised patient. Mayo Clin Proc 63, Jernigan, D. B., Sanders, L. I., Waites, K. B., Brookings, E. S., Benson, R. F. & Pappas, P. G. (1994). Pulmonary infection due to Legionella cincinnatiensis in renal transplant recipients: two cases and implications for laboratory diagnosis. Clin Infect Dis 18, Joly, J. R., McKinney, R. M., Tobin, J. O., Bibb, W. F., Watkins, I. D. & Ramsay, D. (1986). Development of a standardized subgrouping scheme for Legionella pneumophila serogroup 1 using monoclonal antibodies. J Clin Microbiol 23, Joseph, C. A. & Ricketts, K. D. on behalf of the European Working Group for Legionella Infections (2010). Legionnaires disease in Europe Euro Surveill 15, Kümpers, P., Tiede, A., Kirschner, P., Girke, J., Ganser, A. & Peest, D. (2008). Legionnaires disease in immunocompromised patients: a case report of Legionella longbeachae pneumonia and review of the literature. J Med Microbiol 57, Lück, P. C., Schneider, T., Wagner, J., Walther, I., Reif, U., Weber, S. & Weist, K. (2008). Community-acquired Legionnaires disease caused by Legionella pneumophila serogroup 10 linked to the private home. J Med Microbiol 57, Mahbubani, M. H., Bej, A. K., Miller, R., Haff, L., DiCesare, J. & Atlas, R. M. (1990). Detection of Legionella with polymerase chain reaction and gene probe methods. Mol Cell Probes 4, Mandell, L. A., Wunderink, R. G., Anzueto, A., Bartlett, J. G., Campbell, G. D.,Dean,N.C.,Dowell,S.F.,File,T.M.,Jr,Musher,D.M.&other authors (2007). Infectious Diseases Society of America/American Thoracic Society consensus guidelines on the management of community-acquired pneumonia in adults. Clin Infect Dis 44 (Suppl. 2), S27 S72. McClelland, M. R., Vaszar, L. T. & Kagawa, F. T. (2004). Pneumonia and osteomyelitis due to Legionella longbeachae in a woman with systemic lupus erythematosus. Clin Infect Dis 38, e102 e106. McDade, J. E., Shepard, C. C., Fraser, D. W., Tsai, T. R., Redus, M. A., Dowdle, W. R. & Laboratory Investigation Team (1977). Legionnaires disease: isolation of a bacterium and demonstration of its role in other respiratory disease. N Engl J Med 297, Miyashita, N., Matsushima, T., Oka, M. & Japanese Respiratory Society (2006). The JRS guidelines for the management of community-acquired pneumonia in adults: an update and new recommendations. Intern Med 45, Ratzow, S., Gaia, V., Helbig, J. H., Fry, N. K. & Lück, P. C. (2007). Addition of neua, the gene encoding N-acylneuraminate cytidylyl transferase, increases the discriminatory ability of the consensus sequence-based scheme for typing Legionella pneumophila serogroup 1 strains. J Clin Microbiol 45, Singh, N., Stout, J. E. & Yu, V. L. (2002). Legionnaires disease in a transplant recipient acquired from the patient s home: implications for management. Transplantation 74, Sopena, N., Sabrià-Leal, M., Pedro-Botet, M. L., Padilla, E., Dominguez, J., Morera, J. & Tudela, P. (1998). Comparative study of the clinical presentation of Legionella pneumonia and other community-acquired pneumonias. Chest 113, Yagyu, H., Nakamura, H., Tsuchida, F., Sudou, A., Kishi, K., Oh-ishi, S. & Matsuoka, T. (2003). Chest CT findings and clinical features in mild Legionella pneumonia. Intern Med 42, Yamamoto, H., Hashimoto, Y. & Ezaki, T. (1993). Comparison of detection methods for Legionella species in environmental water by colony isolation, fluorescent antibody staining, and polymerase chain reaction. Microbiol Immunol 37, Yu, V. L., Plouffe, J. F., Pastoris, M. C., Stout, J. E., Schousboe, M., Widmer, A., Summersgill, J., File, T., Heath, C. M. & other authors (2002). Distribution of Legionella species and serogroups isolated by culture in patients with sporadic community-acquired legionellosis: an international collaborative survey. J Infect Dis 186, Zhou, H., Ren, H., Zhu, B., Kan, B., Xu, J. & Shao, Z. (2010). Optimization of pulsed-field gel electrophoresis for Legionella pneumophila subtyping. Appl Environ Microbiol 76, Journal of Medical Microbiology 61