J Public Health (2012) 20:425–430 DOI 10.1007/s10389-011-0475-6
ORIGINAL ARTICLE
Cooling Towers contribute to the high seroprevalence of Legionella pneumophila antibody among hotel workers Hong Sun & Lian Zhou & Xiuzhen Zhang & Qian Bian & Xiaodong Chen & Dongyue Wang & Fan Liu
Received: 29 June 2011 / Accepted: 1 December 2011 / Published online: 16 December 2011 # Springer-Verlag 2011
Abstract Objectives Regular disinfection work of the cooling towers (CTs) were carried out according to schedule, nevertheless 89.6% of the CTs were still found to be colonized by Legionella pneumophila in our survey in Changzhou city, China. The aim of the study was to observe the effect of L. pneumophila on workers exposed to contaminated cooling towers (CT). Methods A total of 364 subjects were randomly selected for the serum testing and the questionnaire survey. They were either from the hotel with contaminated CTs or without CTs (used as control). heir serum anti-L. pneumophila antibodies were tested using commercial IFA kits. Results The results showed that the prevalence of anti-L. pneumophila group 1 antibodies in the exposed population was significantly higher than control subjects (OR02.845, P00.019). The possible confounding factors were analyzed. Questionnaires results analysis found that the frequency of flu-like symptoms in the exposed group was also significantly higher; and the indoor air quality was related to the serum anti-L. pneumophila group 1. Conclusions The L. pneumophila contaminated CTs contribute to the high seroprevalence of anti-L. pneumophila antibodies; tap water and shower water are also possible contamination resources. H. Sun : L. Zhou : X. Zhang : Q. Bian : X. Chen Jiangsu Provincial Centre for Disease Prevention and Control, Nanjing 210009, China H. Sun (*) : D. Wang Changshu Centre for Disease Prevention and Control, Suzhou 215500, China e-mail:
[email protected] F. Liu Chinese Centre for Disease Prevention and Control, Beijing 100050, China
Keywords Legionella contamination . Cooling tower . Anti-Legionella pneumophila antibodies . Seroprevalence . Legionellosis
Introduction Legionella pneumophila is a common bacterial pathogen which is known to cause sporadic community-acquired pneumonia with high mortality. The common mode of transmission of L. pneumophila is aerosolization, but the major mode is considered to be aspiration of contaminated water. (Yu 1993). However, the infections originating from contaminated cooling towers (CTs) are acquired by inhalation of infectious aerosols, as indicated in a lot of surveillance (Bentham 2000; Isozumi et al. 2005; Sabria et al. 2006). Based on published data, we investigated the incidence of the contamination of cooling towers (CTs) with Legionella spp. in Changzhou, a city in the south of Jiangsu Province, China. A total of 48 CTs in hotels or shopping-malls were surveyed. Cooling towers are an integral part of the air-conditioning systems in hotels and malls, in which water is used for cooling. Therefore the CTs have the ideal warm and wet environment for the growth of micro-organisms such as L. pneumophila. Environmental surveillance in many countries has verified the existence of Legionella in CTs, and a number of countries have mandated that surveillance of CTs for the presence of Legionella spp. be enforced (Bentham 2000; Sabria et al. 2006; Borobio et al. 1987; Negron-Alvira et al. 1988). However there is a lack of such data on CTs in public places in China. To know the general situation of the L. pneumophila pollution in the water in CTs, a large-scale surveillance was designed by the Chinese Center for Disease Control and Prevention (China CDC). The present survey was part of this project and it covered more than 75% of the city’s buildings equipped with CTs.
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The serum antibody levels, due to exposure to L. pneumophila, could be used for retrospective gauging of the risk of infection. Such seroprevalence studies have been undertaken on people living and working in contaminated environments or in areas where clusters of legionellosis have occurred (Ozerol et al. 2006; Benin et al. 2002; Boshuizen et al. 2001; Darelid et al. 2001). However for China, there are very few documented studies on the serum antibody due the effect of L. pneumophila from contaminated CTs (Peng et al. 2000; Wang et al. 1998). The current study was designed to address this lack of information on L. pneumophila and its relationship to CTs and human health. In this study 202 subjects from buildings with contaminated CTs and 162 subjects from buildings with no CTs were recruited and investigated for related health effects. In this study, we surveyed the contamination of Legionella in CTs in 48 hotels or shopping malls, and then explored the prevalence of L. pneumophila antibodies of hotel employees in four selected hotels.
Materials and methods CTs survey Water sample collection During October 2008, water samples were collected in Changzhou city. The samples were taken from the 48 CTs in the hotels. A water sample of 500 ml was aseptically collected in a sterile glass bottle for each of the sampling points. Isolation and identification of Legionella spp. Processing of water samples for the isolation and identification of Legionella spp. was carried out by accredited laboratories in China. The standard operating procedure used was based on methods of ISO11731:1998. In short, 500 ml of the water sample was filtered through a 0.22-μm-polyamide-membrane filter (E-PaK) using microfilm filtration funnels (Millipore). The sample concentrate was suspended in 15 ml of sterile distilled water and mixed well by vortex mixing for 2 min. Then the samples were divided into three portions, untreated, heat-treated and acid-treated. For heat treatment, 1 ml of the prepared suspension was dispensed into a sterile screw-capped container and placed in a water bath at 50°C for 30 min. For the acid treatment sample, 1 ml of the test suspension was dispensed into a sterile screw-capped container, and an equal volume of acid buffer at pH 2.2 was added and left for 5 min. Then 0.1 ml of the heat and the untreated sample, and 0.2 ml of acid treated samples, was inoculated onto GVPC (Glycine, Vancomycin, Polymixin, Cycloheximide agar, Oxoid) selective agar and incubated at 37°C for 2–14 days with 2.5% CO2. Legionella only grows in the presence of cysteine, so
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presumptive Legionella colonies were confirmed by subculturing onto BCYE (with L-cysteine) and BCYE-CYS (without L-cysteine), and incubated at 37°C for 2–4 days with 2.5% CO2. The detection threshold was 300 cfu/L. Legionella latex agglutination kit (Oxoid) was used to confirm and identify the Legionella into L. pneumophila serogroup 1, and then all none-seroroup 1 isolates were further characterized using a specific antisera against L. pneumophila serogroup 2–10; Legionella-like organisms were identified with antisera against L. bozemanii and L. micdadei (Kemajia microorganism technology CO.LTD, Shanghai, China). Seroprevalence of hotel workers Group Recruited subjects were from 2 types of sites included in this study, two hotels with L. pneumophila contaminated CTs and four hotels without CTs as controls. The study sites were randomly selected in the city of Changzhou, Jiangsu Province. A total of 202 employees were recruited into the study in the hotels with contaminated CTs and 162 from hotels with no CTs. The subjects for the control group were randomly selected using computer generated random numbers from the health examination group. The demographic information was analyzed and the parameters observed were found to be comparable (Table 1). Questionnaire survey A detailed questionnaire was used to collect information on demographic characteristic (sex, age, civil state, education, working and incomes), personal habits (smoking, alcohol consumption), present and past diseases, and utilization of air-conditioning (central or independent, service frequency). Particular attention was paid to recording episodes of pneumonia and flu-like symptoms during the previous year. One hundred and ninety eight (198) of the 202 exposed subjects and 156 of 162 control subjects finished the questionnaires. Serological tests for L. pneumophila Blood samples were obtained from all 364 subjects selected for this study. The prevalence and titres of L. pneumophila antibodies against L. pneumophila serogroup 1–14 was Table 1 Demographic characteristics of the research population Variables
Exposed group N0198)
Control group (N0156)
Male/female Age (mean+SD, years) Working seniority (years)
59/139 33.1±7.4 8.4±2.7
51/105 34.4±6.5 7.6±3.1
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detected by means of an indirect immune-fluorescent assay (IFA) using Biochip Technology (Euroimmun, Lübeck, Germany), and used as evidence of previous or recent exposure. Detectable antibodies against any individual antigen at a dilution of 1:100 (manufacture’s referred positive cut-off) was considered positive, which was interpreted as having been infected at an undetermined time; no detectable antibodies at this dilution was considered a negative result. On fluorescence microscopy (Olympus, Japan), samples were scored according to the intensity and definition of cell-wall staining (3+ 0 brilliant yellowgreen staining of bacteria; 2+ 0 bright yellow-green staining; 1+ 0 definite but dim staining; Negative 0 absence of yellowgreen staining of the cells, yellow-brown auto fluorescence may occur). All samples scored 1+ or above were defined as positive. The screening tests and the confirmation tests with single antigen were performed at the same laboratory, and fluorescence microscopy was performed by the same technician. Statistical analysis The logistic regression analysis and Chi square test were employed to demonstrate whether there was a statistically significant association between the presence of Legionella in the CTs of the air-conditioning system and the corresponding antibody positive rates. The frequency of flu-like symptoms between groups was compared using nonparametric test. The data were analyzed using the statistical package SPSS 18.0 (SPSS Inc, Chicago, IL, USA). The significant difference was defined by p value of less than 0.05.
Results Isolation of Legionella from CTs water samples The study showed 89.6% (43 of the 48 investigated CTs, 43/ 48) of the test CTs to be colonized by L. pneumophila. Of these, Fig. 1 The results of Legionella spp. detected from the cooling tower water. *The serum samples were collected from the staffs working in the hotel with CTs contaminated only by LP1. Lp: Legionella pneumophila
81.3% (39/48) of the positive samples had L. pneumophila serogroup 1, and the rest belonged to L. pneumophila (Fig. 1). Isolation of Legionella from other water samples The analysis of tapped water from hotels with CTs showed 30% (6/20) to be of L. pneumophila Serogroup 1 type. However 30% (3/10) of the samples from the control group were also found to be positive. In shower water, the rates were 5% (1/20) in hotels with CTs and 2.5% (1/40) for hotels without CTs. Based on these results it can be said that the exposed groups were in hotels with contaminated CTs and the control groups were in hotels without CTs; the source of exposure to L. pneumophila other than from contaminated CTs is equal. Serological tests for Legionella spp. The results of the serological tests for the 202 exposed subjects from the hotels with CTs showed that only anti-L. pneumophila serogroup 13 antibodies were not detected from the blood samples (Fig. 2). However 60.9% of the samples were positive for anti-L. pneumophila serogroup 12 antibodies, followed by anti-L. pneumophila serogroup 4 antibodies (46.0%). Anti-L. pneumophila serogroup 1 antibodies were detected in 23 samples, and most of these (96%) also showed the presence of other serogroups. For the control group, the presence of different serogroups of antibodies was similar to the exposure group, anti-L. pneumophila serogroup 12 (47.1%) > serogroup 4 (44.8%) > serogroup 14 (15.1%) > serogroup 5 (12.8%). Anti-L. pneumophila serogroup 1 antibodies were detected in only 7 samples from control subjects, and these also had anti-L. pneumophila serogroup 12 antibodies (Fig. 2). The exposed subjects were more likely to be anti-L. pneumophila serogroup 1 antibody positive (OR02.84, 1.18-6.81, P00.019), and there was also significant difference 48 Cooling Towers
43 Lp positive
5 negative
(89.6%)
(10.4%)
Non-Lp1
Lp1
4 (8.3%)
39 (81.3%)
Only Lp5
Only Lp6
*Only Lp1
Lp1+Lp3
Lp1+Lp5
Lp1+Lp6
3 (6.3%)
1 (2.1%)
35 (72.9%)
1 (2.1%)
2 (4.2%)
1 (2.1%)
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Fig. 2 Seroprevalence of L. pneumophila serogroup 1–14 in Changzhou hotel workers. A total of 202 subjects of the exposed group and 162 subjects of the control group were recruited and tested with serum anti-L. pneumophila antibodies. Exposed and control subjects showed high positive rate of L. pneumophila. Contaminated cooling towers increased the exposure risk to L. pneumophila group 1. *P
