Isolation of Legionella pneumophila from clinical & environmental ...

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762 INDIAN J MED RES, JUNE 2010 Legionnaires’ disease remains a grossly under reported condition as it is difficult to distinguish the condition from other forms of pneumonia, unless the organism is specifically looked for. In spite of the under-reporting, many studies are available from Europe and North America, but studies regarding the isolation of Legionella either from clinical or environmental samples are rare from India 11,12 . We studied the presence of Legionella infection on the basis of culture in patients admitted to the hospital with signs and symptoms of lower respiratory tract infection acquired in the community. Sampling of the hospital water was also carried out to detect any Legionella contamination in the hospital water supply system so as to alert the hospital infection control committee about its consequences. Material & Methods Patient samples: This study was carried out at Sri Venkateswara Institute of Medical Sciences, Tirupati, a post graduate teaching hospital in south India. Clinical samples from respiratory tract received in the Department of Microbiology from July 2007 to December 2008 were included in this study. The samples were obtained from patients who were hospitalized with signs and symptoms of lower respiratory tract infection (LRTI). Apart from sputum, the specimens included bronchoalveolar lavage (BAL), endotracheal aspirate and pleural fluid. After careful selection, 470 samples which did not yield any bacterial respiratory pathogen on routine culture and which were smear negative for acid fast bacilli were processed for Legionella. The samples were decontaminated by acid treatment (0.2 KCl-HCl) followed by plating on buffered charcoal yeast extract agar (BCYE) (Oxoid, UK) and BCYE supplemented with polymyxin(80 u/ml), anisomycin (40 µg/ml) and cefamandole (4 µg/ml) to make BCYE selective for respiratory samples 13 . Plates were incubated at 37 0 C and observed daily for 7 days. Water samples: A total of 24 water samples were collected from the taps of various wards and ICUs. Six samples were obtained at 2 months’ interval from July 2007 to April 2008. The areas included the Medicine ward, General ward-Male, General ward-Female, Medicine ICU, Respiratory ICU, and the Emergency Room. Samples were obtained by introducing a sterile cotton swab into the opening of the taps and rotating it along the inner sides of the nozzles. The swab was then lightly streaked directly onto BCYE and then immersed in 2.5 ml acid buffer, shaken vigorously and neutralized with KOH. 0.1ml of the samples was spread with another swab onto duplicate plates of the same medium 14 . Identification: Iridescent, frosted glass colonies, 3-4 mm size, typical of Legionella spp. appearing in 3-4 days time were used for identification. Gram stain was done to show the thin, faintly stained filamentous Gram-negative morphology. Catalase positive and oxidase negative colonies showing the typical morphology were subcultured on plain BCYE and on blood agar (BA) to determine if L-cysteine is essential for growth 13 . Colonies growing only on BCYE and not on BA were presumptively identified as Legionella spp. The reference strain L. pneumophila Serogroup 1 Paris strain (CIP 107629T) obtained from French National Legionella Reference Centre, Lyon, France, was cultured in parallel with the clinical isolates and the morphology and biochemical characteristics of the clinical isolates were compared with this reference strain before definitive identification. Hippurate hydrolysis test was done by standard method 15 to identify L. pneumophila species. Briefly, 0.4 ml of 1 per cent sodium hippurate (Hi-Media, Mumbai) was inoculated with a loopful of organism and incubated overnight at 37 0 C. Next day, 0.2 ml of 3.5 per cent ninhydrin (Hi Media, Mumbai) solution in 1:1 acetone:butanol was added to the organism in hippurate solution, mixed well, and incubated at 37 0 C for 10 min. Blue purple colour developing within 20 min was considered positive. L. pneumophila CIP 107629T strain was used as a positive control and Enterobacter cloacae as the negative control. Results A total of 470 respiratory specimens from clinically suspected cases of LRTI did not yield any respiratory pathogenic bacterial isolate and were included in this study. Among the patient population studied, 12 out of the 470 (2.55%) were found to be culture positive for L. pneumophila as identified by positive hippurate hydrolysis test. The profile of these 12 patients, along with the risk factors and clinical presentation is shown in the Table. Majority were males, and were >50 yr of age. Among the risk factors alcohol and / or smoking habit was found in five patients and one third of the patients had diabetes mellitus. Three patients did not have any underlying risk factor. Fever (100%), dyspnoea (91.7%) and cough (66.7%) were the most common presenting features. Three quarters of the patients had some radiological abnormality. Maximum isolation was from sputum samples (11/12) and one was from endotracheal aspirate.
Table. Characteristics of patients with legionellosis (n=12) Characteristics No. (%) Age (yr) Range Age groups (yr) 20-30 31-40 41-50 >50 Males Females RISK FACTORS Smoking status Current Ex Smoker Never Alcohol consumption Alcoholic Non alcoholic Co-morbid illnesses None Diabetes mellitus Ischaemic heart disease Asthma End stage renal disease Clinical presentation Fever Cough Absent Productive Dry Dyspnoea Haemoptysis Chest pain Vomiting Diarrhoea X- ray finding (Consolidation) ANBUMANI et al: LEGIONELLA ISOLATION FROM PATIENTS & ENVIRONMENT 763 Among 24 samples of water tested, 8 (33%) were positive for L. pneumophila by culture. Among these samples, maximum isolations were in the months of July and October (three each), and one each in January and April. The colony count was in the range of 180- 356/ plate (swab). Discussion 24-76 3 (25) 1 (8.3) 0 8 (66.7) 10 (83.3) 2 (16.7) 1 (8.3) 2 (16.7) 9 (75) 2 (16.7) 10 (83.3) 3 (25) 4 (33.3) 3 (25) 3 (25) 1 (8.3) 12 (100) 4 (33.3) 6 (50) 2 (16.7) 11 (91.7) 2 (16.7) 1 (8.3) 4 (33.3) 1 (8.3) 9 (75) Epidemics were the original presenting scenario for Legionnaires’ disease in the 1980s, but most cases are now known to be sporadic. Numerous observational studies of patients with community-acquired pneumonia (CAP) requiring hospitalization have documented that the incidence of Legionnaires’ disease ranges from 2 to 9 per cent 6,16 . A recent Australian study has found that 3.4 per cent of CAP was caused by Legionella 17 , while another study from UK reported the corresponding figure to be 3 per cent 18 . Our study showed Legionelle infection in 2.55 per cent patients. Male population and age >50 yr are established predisposing factors which have been found in the present study also. In these patients, fever was the most common finding (mean temperature at admission 39 0 C) followed by respiratory symptom like cough and dyspnoea. Studies 6,19 have found that a significant population of patients have mild to moderate disease and do not have expected co-morbidities like diabetes mellitus which are also the finding in our study. Apart from systemic and respiratory symptoms, gastrointestinal symptoms like nausea, vomiting, and diarrhoea are known to be common 4 as seen in our patients also. In our study, majority (75%) had some radiological abnormality, which is a common feature of Legionella pneumonia and hence Legionnaires’ disease cannot be distinguished from other causes of CAP on radiological grounds alone 4 . For these reasons confining laboratory testing for this organism specifically to high risk patients will overlook a notable number of cases and our study also supports the practice of placing more emphasis on ascertaining the aetiology of pneumonia 19 . Centres for Disease Control and Prevention (CDC) does not support routine environmental culture for Legionella because of the supposedly ill defined relationship between the presence of the organism in water system and risk of acquiring the infection 20 . In our study, Legionella could be isolated from the hospital water supply over a nine months period. There is an increased yield of Legionella from the swab compared to the filtration concentration technique 21 . This can be explained by the fact that swab technique results in direct sampling of the organisms present in the biofilm consortium, which gives a greater yield than sampling of water. It has been recommended that swab samples be collected as part of any environmental Legionella sampling protocol 21 . Our study has not included hospital acquired pneumonia patients and the potential sources of the infections were located outside this hospital. So no immediate conclusion can be drawn whether it is causing hospital acquired infections. However, it has been advocated that if the water supply is colonized by Legionella, then Legionnaires’ disease should be included in the differential diagnosis of hospital acquired pneumonia 22 . In a study from various hospitals, positive environmental sampling raised the suspicion, and once culture for Legionella of hospitalized patients started, cases were discovered 23 .
Page 1: Indian J Med Res 131, June 2010, pp

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