The prevalence of Legionella pneumophila in different water systems: A global systematic review and meta-analysis

The prevalence of Legionella pneumophila in different water systems: A global systematic review and meta-analysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research article The prevalence of Legionella pneumophila in different water systems: A global systematic review and meta-analysis Yadolah Fakhri, Mohammad Javad Nasiri, Anvar Asadi, Moayed Avazpour, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.2.13555/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The presence of Legionella pneumophila (LP) in aquatic habitat is a global concern. The current study was undertaken to estimate the prevalence of LP in water systems with the aid of a systematic review and meta-analysis. The searching was performed among some international databases, including Scopus, PubMed, and Embase to retrieve the related articles between 1/January/1983 and 25/July/ 2017. Therefore, thirty-six articles (with 43 studies) out of 1,541 articles collected, were included in the meta-analysis. The overall prevalence of LP in water systems was determined as 20% (95%CI: 15-25). Also, the lowest and highest pooled prevalence of LP was observed in Poland (4% (95%CI: 0-13%)) and Kuwait (98% (95%CI: 90-100%)), respectively. The lowest and highest prevalence of LP-based on water resources subgroups was a water reservoir (15% (95%CI: 2-37%) and well (40% (95%CI: 26-50%), respectively. The number of studies that used polymerase chain reaction (PCR) for detection of LP was 16/43 (37.3%) while the culture method was 27/43 (62.7%). Generally speaking, the relatively high prevalence of LP among the investigated water systems was demonstrated, which should be reduced by performing appropriate control actions. Health Economics & Outcomes Research Health Policy Infectious Diseases occurrence Legionella pneumophila water reservoir global overview Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Legionella pneumophila (LP) as a heterotrophic bacterium is a gram-negative, aerobic, non-sporogenous, and a mobile [ 1 , 2 ] which can be isolated from various environments such as soil and water systems, including water used in cooling towers and ventilation, surface water, tap water, and spring water [ 3–6 ]. The tolerance to high temperature is among the features, which could stimulate the presence, and proliferation of LP in water systems [ 7–9 ]. Although the range of optimal temperature for proliferation was indicated as 29–40 °C, rarely, it can survive in water at temperatures ranging from 0 to 63 °C [ 10 ]. Also, the efficiency of chemical or thermal disinfection can be reduced in the presence of biofilm or amoebae; which can be resulted in further contamination of water systems by LP 11 ]. The droplets of contaminated water withLP could convey the bacterium into the lungs and as a result of swallowing by macrophages [ 12 , 13 ]. Therefore, the approaching of efficient and appropriate methods is crucial to decreasing as well as controlling the prevalence of LP water systems. In this regard, according to the guideline of World Health Organization (WHO), the acceptable limit of LP in the drinking water and cooling tower systems were defined as 1 CFU/L (WHO 2008) and 1000 CFU/L [ 14 ], respectively. Legionnaires’ disease are among issues caused by these water-borne pathogens, commonly known as Legionella. Twenty-five out of 59 species in the Legionella genus, have been correlated with human disease [ 15 ]. According to Cazalet et al. (2008), almost 20 kb of 33-kb locus carrying the genes for the proteins in lipopolysaccharide biosynthesis in LP is highly specific for L. Pneumophila serogroup 1 ( LP-Sg1)[ 16 ]. Also, multigenome via comparative hybridization detected 3 genes, including lpp0837/wzm, lpp0831, and lpp0838/wzt strains only LP-Sg1 that used for real-time PCR method for identifying LP-Sg1 [ 16 ]. Moreover, Legionella pneumophila serogroup 1 ( LP) is responsible for the majority of reported cases of Legionnaires’ disease (about 90%) [ 17 ]. Legionnaires’ disease, for the first time, was diagnosed as pneumonic form [ 10 ]. The incidence of LD is increasing, especially in Europe (EU) United States of America (USA). In this context, only in 2010 and 2012, 6,305 and 4,486 cases of the Legionnaire’s disease were reported in Europe [ 18 ] and the USA [ 19 ], respectively. Some signs and symptoms include coughing, shortness of breath, high fever, muscle pains, and headaches, Nausea, vomiting, and diarrhea were associated with Legionnaires’ disease [ 10 ]. Due to the significant health effect of LP contamination in water systems, some investigations with different outcomes were performed [ 6 , 20–22 ]. In this regard, the prevalence of LP in the cooling tower systems in Kuwait and Saudi Arabic were determined as 98(95%CI: 89–100) and 2(95%CI: 1–4), respectively [ 23 , 24 ]. The culture medium and polymerase chain reaction (PCR) techniques are used to isolate the LP in water systems. The culture techniques based on ISO 11731 can be considered as gold-standard for detecting LP in water systems [ 14 ]. In the PCR method, the limit of detection (LOD) is 2 × 10 2 GU/100 mL for LP ( mip gene), while theLOD for culture methods is 1 CFU/100 mL [ 25 , 26 ]. Although culture techniques are used widely, these techniques have several limitations including a long time needed to obtain results, weak recovery, weak sensitivity, inability to detect viable but non-culturable cells (VBNC) [ 27 ]. In another hand, the polymerase chain reaction (PCR) techniques due to reproducibility, sensitivity, specificity, high-throughput and reducing in the required time to less than 24 hours are widely approached to detect LP in water systems [ 28–30 ] However, no systematic review and meta-analysis was performed regarding the prevalence of Legionella pneumophila in different water systems. Therefore, for the first time, the current study was undertaken to perform a meta-analysis to investigate the prevalence LP in the water systems based on geographical, quality of research as well as the type of water. Material and methods In this study, the systematic review was conducted in accordance with the Cochrane protocol and Prisma protocol [ 31 ]. 2.1. Search strategy The search strategy was performed on the Legionella pneumophila studies in water systems. Searching was performed in the main international databases, including Scopus, PubMed, and Embase. The following terms were used: (a) PubMed: Search ((((((legionella[Title/Abstract]) OR Legionella pneumophila[ Title/Abstract]) OR microbe[Title/Abstract]) OR bacteria[Title/Abstract])) AND ((((water[Title/Abstract]) OR drinking water[Title/Abstract]) OR tap water[Title/Abstract]) OR water system[Title/Abstract])) AND (“Legionella”[Mesh] AND “Legionella pneumophila”[Mesh]); (b) Scopus: ( ( title-abs-key ( legionella and pneumophila) or title-abs-key ( legionella) or title-abs-key ( pneumophila) or title-abs-key ( microbe) or title-abs-key ( bacteria))) and ( ( title-abs-key ( water) or title-abs-key ( water and system) or title-abs-key ( drinking and water) or title-abs-key ( tap and water) or title-abs-key ( groundwater))) and (c) Embase: ‘legionella’:ab,ti OR ‘legionella pneumophila’:ab,ti OR ‘microbe’:ab,ti. Also, the references articles were reviewed to obtain more articles. Seventeen years (1/January/1983 and 25/July/ 2017) was chosen as the searching period. 2.2. Inclusion and exclusion criteria Full-text articles were downloaded and then reviewed carefully to check if they meet the proposed criteria [ 32–34 ] such as (1) original study; (2) cross-sectional data; (3) published in English; (4) published online between 1/January/1983 and 25/July/ 2017; (5) full-text available articles; (6) existence of exact total sample size and positive samples; (7) the defined type of water was examined; and (8) accurate methods including culture or PCR techniques were mentioned. The articles were excluded when not meet our criteria. 2.3. Data extraction The obtained information from each study can be summarized as study characteristics; (the first author, year of study); total sample size; the number of positive samples; the geographical study (countries); study methodology (Culture or PCR); type of water systems (tap drinking water, hot water, water reservoir, cold water, well and spring). 2.4. Meta-analysis of data The ratio of the positive samples (p i ) to the total sample (n i ) defined as prevalence (P = p i /n i ), which is between 0 to 1 value [ 32 , 35–37 ]. Estimation of the prevalence of LP in water systems was performed using the binomial distribution model [ 38 ]. The heterogeneity ( I 2) statistics was used to determine the variation between the prevalence of Legionella penompophia among the included studies [ 39 ]. In the current study, when the heterogeneity was higher than 50%, the random effect model (REM) was used to estimate prevalence based on the defined subgroup. The Begg’s and Egger’s test was used to estimate publication bias [ 21 , 40 ]. A meta-analysis of data was conducted using Stata 12.2 intercooled version (Stata Corp, College Station, TX). All statistical analysis was significant at P-values< 0.05. Results A total number of 1541 of articles were screened in the initial screening through databases. Finally, 36 articles (43 studies) were included in the conducted systematic review and meta-analysis ( Table 1, Figure 1). Given the geographical analysis, the lowest and highest pooled prevalence of LP was observed in Poland (4 (95%CI: 0–13%)) and Kuwait (98 % (95%CI: 90–100%)), respectively ( Figure 2). Also, the global pooled prevalence of LP in water systems was determined as 20% (95%CI: 15–25%) ( Figure 2). While, the lowest and highest prevalence of LP-based on water resources subgroups was water reservoir (15% (95%CI: 2–37%) and well (40% (95%CI: 26–50%), respectively ( Figure 3). According to Begg’s (p-value = 0.029) and Egger’s (p-value = 0.024), significant publication bias among studies was noted ( Figure 4A-B); Hence, to remove the effect of publication bias, the metatrim test was performed. The pooled prevalence of LP in a water system based on metatrim in the random effect model (REM) was 21% (95%CI: 15–26%) ( Figure 5). Discussion The prevalence of LP in the water systems was obtained as an average of 20% (95%CI: 15–25%). The prevalence of LP in the different countries considerably was difference ( Figure 2), which can be associated with variation in the quality of disinfection of water, the average life of water facilities, the chemical quality of water and the type of exploitation in each country. Some factors such as water temperature, stagnation of water in pipes, the formation of biofilms on the interior walls of pipes, the presence of protozoa, the chemical quality of water and pH could affect Legionella growth in aquatic systems [ 71 , 72 ]. However, the proposed temperature for growth of LP is 29–40 °C with an optimum of 35 °C; it could survive in aqueous systems at temperatures of 0–63 °C [ 10 ]. The increase in temperature above 55 °C can be considered as an efficient technique for the prevention of bacterial growth and consequently, lower incidence of legionellosis [ 73 ]. According to Leoni et al. (2001), no LP growth was observed at temperatures above 41 °C [ 74 ]. However, the growth of LP decreased with increase in temperature [ 75 ]. Moreover, LP quickly reproduces in hot water systems such as showerheads (45–50 °C) [ 61 ]. Based on one recent study, the highest prevalence of LP in hot spring was 17.6% in the 40–48.6 °C [ 7 ]. Findings showed that the prevalence of LP in cold water was higher than hot water ( Figure 4), which can be correlated with the inhibition effect of increase in temperature on LP growth. Additionally, based on ecological data, protozoans and amoeba can protect this bacteria from disinfectant, osmolality, and pH variations [ 61 , 76 ]. The influence of metallic elements such as iron and manganese on the growth of Legionella previously also has been demonstrated [ 77 , 78 ]. Due to organophilic properties of LP, its growth is limited in the water with low iron concentrations. Considering to findings of Portier et al. (2016), the iron pyrophosphate and ferric iron chelator did not affect the persistence of LPin the biofilms, but ferrous iron chelator showed a positive effect because of higher bioavailable ferrous ion. Hence, the growth of LP in the pipes that are made of iron could be stimulated. Also, manganese has an indirect effect on Legionella growth by enhancing the growth of biofilms and plantations [ 79 ]. Moreover, the protective role of manganese in enzymatic activity and increasing in resistance to oxidative stress has been confirmed [ 80 ]. In this regard, the positive correlation between Mn concentration (> 3µg/L) with a prevalence of LP in water was demonstrated [ 81 ]. The presence of a copper ion in water due to its antibacterial nature can resulted in further decline in the prevalence of Legionella[ 82 ]. However, biofilms in water systems protect Legionella against disinfection [ 83 ]. Therefore, in order to reduce the prevalence of Legionella, washing of pipes and tanks in addition to exhausting of water facilities with chlorinated hot water can be recommended. According to Oberdorfer et al. (2008), the prevalence of Legionella in the old water system of the hospital was higher than new ones [ 84 ]. Nowadays, the use of polyethylene (PE) and Polyvinyl chloride (PVC) materials in water facilities is continuously expanding. The growth of biofilm was increased with the releasing of volatile organic compounds in PVC and PE pipes [ 85 ]. Therefore, using PE and PVC in water facilities may increase the prevalence of LP in water systems. pH is another chemical parameter that affects the growth and survival of LP in water systems. The highest prevalence of LP was reported in weakly acid (pH 5–7, 37.5%) [ 7 ]. In a report by Ohno et al. (2003), the optimal range of pH for LP in water systems was defined between 6.0 and 8.0 [ 8 ]. However, other data showed that LP had not been explored in extremely acidic water [ 60 , 86 ]. Considering the frequency of detection techniques, PCR (37.3%) approximately was similar to the culture method (62.7%). While in culture techniques, only living bacteria can be detected [ 87 ], in the PCR in addition to living bacteria, the bacteria that damaged by the disinfection which their DNA remains also can be detected [ 88 ]. Moreover, in the culture technique, the incubation period of 3–14 days is recommended to facilitate the growth of bacterial colonies, while the bacteria can be damaged by used acid treatment in culture technique. In another hand, the observed limitation regarding detection sensitivity (50–60%) is another disadvantage of culture techniques[ 89 ]. Due to Culturable None but Viable (CNBV) properties, LP can survey in water for a long time without being identified by culture technique. Likewise, data have shown that Legionella prevalence in amoeba-water samples was 25–50% higher than other samples because of symbiosis survival with the amoeba [ 90 , 91 ]. Moreover, one of the most significant limitations of PCR techniques is bias due to the presence of inhibitors such as polysaccharide and chlorophyll in the samples [ 92 ]. Since the culture and PCR techniques have different ranges of detection limits, however, by using PCR technique, higher level of contamination in pathological samples can be detected quickly and accurately, approaching of both methods in the case of environmental issues such as LP might be recommended. Conclusion In the current study, the prevalence of LP in water systems in the defined subgroups were meta analyzed. A high prevalence of LP in water systems worldwide was demonstrated by the current study as a first systematic review in this field. Likewise, the higher prevalence of Legionella pneumophila was considerablein the water systems, particularly in cold water. In this context, approaching of control actions such as avoiding stagnation of water in water systems, use of high-quality water, and continuous purification of water using disinfectants factors can be recommended. Continuous monitoring of water quality to assess the effectiveness of measures to control and prevent the prevalence of legionellosis is crucial. Abbreviations LP: Legionella pneumophila; WHO: World Health Organization; PCR: polymerase chain reaction; LOD: limit of detection; VBNC: Non-culturable cells; PVC: Polyvinyl chloride; CNBV: Culturable None but Viable. Declarations Acknowledgements Not applicable Authors’ contributions Searching in international database was performed by Z.GH and MJ.NA; screening of papers based on title and abstract was performed by Y.FA, M.AZ and A.AS; Data extraction and data preparation was performed by AM.MK and AZ.AL. Meta-analysis of data and preparation of manuscript was performed by Y.FA and AM.MK. Conflict of Interest There is no conflict of interest. Funding The authors would like to thank student research committee, Shahid Beheshti University of Medical Sciences for the financial grants of this study (Code: 1396/54035). Availability of data and materials Not applicable Ethics approval and consent to participate Not applicable Consent for publication Not applicable Competing interests The authors declare that they have no competing interests References 1.Edelstein PH: Legionella jamestowniensis fatal pneumonia in an immunosuppressed man. Journal of Infection and Chemotherapy 2017, 23( 1):59–61. 2.Al-Matawah Q, Al-Zenki S, Al-Azmi A, Al-Waalan T, Al-Salameen F, Hejji AB: Legionella detection and subgrouping in water air-conditioning cooling tower systems in Kuwait. 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J Clin Microbiol 1982, 16( 4):697–699. 64.Stout J, Yu V, Yee Y, Vaccarello S, Diven W, Lee T: Legionella pneumophila in residential water supplies: environmental surveillance with clinical assessment for Legionnaires’ disease. Epidemiol Infect 1992, 109( 1):49. 65.Kanatani J-i, Isobe J, Norimoto S, Kimata K, Mitsui C, Amemura-Maekawa J, Kura F, Sata T, Watahiki M: Prevalence of Legionella species isolated from shower water in public bath facilities in Toyama Prefecture, Japan. Journal of Infection and Chemotherapy 2017, 23( 5):265–270. 66.Türetgen I, Sungur EI, Cotuk A: Enumeration of Legionella pneumophila in cooling tower water systems. Environ Monit Assess 2005, 100( 1–3):53. 67.Burak DM, Zeybek Z: Investigation of Legionella pneumophila and free living amoebas in the domestic hot water systems in İstanbul. Turkish Journal of Biology 2011, 35( 6):679–685. 68.Hwang I-Y, Park E-H, Park Y-K, Park S-H, Sung G-H, Park H-Y, Lee Y-C: Distribution of Legionella pneumophila serogroups isolated from water systems of public facilities in Busan, South Korea. Southeast Asian J Trop Med Public Health 2016, 47: 467–474. 69.Ramadan N: Prevalence of legionella among pneumonia patients and environmental water samples in an Egyptian University Hospital. The International Arabic Journal of Antimicrobial Agents 2016, 6( 2). 70.Lin H, Xu B, Chen Y, Wang W: Legionella pollution in cooling tower water of air‐conditioning systems in Shanghai, China. J Appl Microbiol 2009, 106( 2):606–612. 71.Mirmohammadlo A, Ghanizadeh G, Esmaeili D, Sepandi M, Avakh P: Legionelle pneumophila water contamination in three military hospitals of Tehran in 2013. Journal of Kermanshah University of Medical Sciences 2014, 18( 7):398–408. 72.Taylor M, Ross K, Bentham R: Legionella, protozoa, and biofilms: interactions within complex microbial systems. Microb Ecol 2009, 58( 3):538–547. 73.Darelid J, Löfgren S, Malmvall B-E: Control of nosocomial Legionnaires’ disease by keeping the circulating hot water temperature above 55 C: experience from a 10-year surveillance programme in a district general hospital. J Hosp Infect 2002, 50( 3):213–219. 74.Leoni E, Legnani P, Sabattini MB, Righi F: Prevalence of Legionella spp. in swimming pool environment. Water Research 2001, 35( 15):3749–3753. 75.Brooks T, Osicki RA, Springthorpe VS, Sattar SA, Filion L, Abrial D, Riffard S: Detection and identification of Legionella species from groundwaters. Journal of Toxicology and Environmental Health, Part A 2004, 67( 20–22):1845–1859. 76.Borella P, Montagna MT, Stampi S, Stancanelli G, Romano-Spica V, Triassi M, Marchesi I, Bargellini A, Tato D, Napoli C: Legionella contamination in hot water of Italian hotels. Appl Environ Microbiol 2005, 71( 10):5805–5813. 77.Bargellini A, Marchesi I, Righi E, Ferrari A, Cencetti S, Borella P, Rovesti S: Parameters predictive of Legionella contamination in hot water systems: association with trace elements and heterotrophic plate counts. Water research 2011, 45( 6):2315–2321. 78.Wang H, Masters S, Hong Y, Stallings J, Falkinham III JO, Edwards MA, Pruden A: Effect of disinfectant, water age, and pipe material on occurrence and persistence of Legionella, mycobacteria, Pseudomonas aeruginosa, and two amoebas. Environ Sci Technol 2012, 46( 21):11566–11574. 79.Granger HC, Stoddart AK, Gagnon GA: Direct biofiltration for manganese removal from surface water. Journal of Environmental Engineering 2014, 140( 4):04014006. 80.Arirachakaran P, Luengpailin S, Banas J, Mazurkiewicz J, Benjavongkulchai E: Effects of manganese on Streptococcus mutans planktonic and biofilm growth. Caries Res 2007, 41( 6):497–502. 81.Borella P, Guerrieri E, Marchesi I, Bondi M, Messi P: Water ecology of Legionella and protozoan: environmental and public health perspectives. Biotechnol Annu Rev 2005, 11: 355–380. 82.Lu J, Buse H, Gomez‐Alvarez V, Struewing I, Santo Domingo J, Ashbolt N: Impact of drinking water conditions and copper materials on downstream biofilm microbial communities and L egionella pneumophila colonization. J Appl Microbiol 2014, 117( 3):905–918. 83.Solimini AG, Cottarelli A, Marinelli L, De Giusti M: Factors influencing persistence of Legionella pneumophila serogroup 1 in laboratory cocultures. BMC Microbiol 2014, 14( 1):249. 84.Oberdorfer K, Müssigbrodt G, Wendt C: Genetic diversity of Legionella pneumophila in hospital water systems. Int J Hyg Environ Health 2008, 211( 1–2):172–178. 85.Lee Y: An evaluation of microbial and chemical contamination sources related to the deterioration of tap water quality in the household water supply system. Int J Environ Res Public Health 2013, 10( 9):4143–4160. 86.Sheehan KB, Henson JM, Ferris MJ: Legionella species diversity in an acidic biofilm community in Yellowstone National Park. Appl Environ Microbiol 2005, 71( 1):507–511. 87.Barigou M, Cavalie L, Daviller B, Dubois D, Mantion B, Delobel P, Debard A, Prere M-F, Marchou B, Martin-Blondel G: Isolation on chocolate agar culture of Legionella pneumophila isolates from subcutaneous abscesses in an immunocompromised patient. J Clin Microbiol 2015, 53( 11):3683–3685. 88.Dada R, Mishra S, Mohanty K, Kumar M, Vemprala K: Polymerase chain reaction. Manual of Cytogenetics in Reproductive Biology 2014:59. 89.Veenendaal HR, Brouwer-Hanzens AJ, van der Kooij D: Incubation of premise plumbing water samples on Buffered Charcoal Yeast Extract agar at elevated temperature and pH selects for Legionella pneumophila. Water research 2017, 123: 439–447. 90.Bartie C, Venter S, Nel L: Identification methods for Legionella from environmental samples. Water research 2003, 37( 6):1362–1370. 91.Ducret A, Chabalier M, Dukan S: Characterization and resuscitation of ‘non-culturable’cells of Legionella pneumophila. BMC Microbiol 2014, 14( 1):3. 92.Miotke L, Lau BT, Rumma RT, Ji HP: High sensitivity detection and quantitation of DNA copy number and single nucleotide variants with single color droplet digital PCR. Anal Chem 2014, 86( 5):2618–2624. Table 1 Table 1. Characteristics included of studies. Year Study Country Water resource Total sample size Positive sample size [1] Technique Ref 2011 Iran Tap drinking water 32 6 BCYE [2] [ 20 ] 2003 Iran Hot water tank 30 11 BCYE [ 41 ] 2008 Iran Water reservoir 33 5 BCYE [ 42 ] 1997 Iran Water reservoir 210 14 BCYE [ 43 ] 2011 Iran Tap drinking water 110 29 BCYE [ 44 ] 2012 Iran Tap drinking water 140 8 PCR [3] [ 45 ] 2005 Iran Tap drinking water 113 30 BCYE [ 46 ] 2012 Iran Tap drinking water 140 12 PCR [ 47 ] 2008 Iran Water reservoir 240 100 BCYE/GVPC [4] [ 48 ] 2010 Iran Cold water tank 77 14 PCR [ 49 ] 2015 Iran (Kerman) Cold water tank 78 18 PCR [ 50 ] 2015 Iran (Bam) Cold water tank 50 7 PCR 2012 Iran Cold water tank 140 10 PCR [ 51 ] 2011 Saudi Arabia Cold water tank 300 5 BCYE [ 24 ] 2013 Jordan Hot water tank 200 17 BCYE and BAP [5] [ 52 ] 2010 Taiwan Tap drinking water 706 148 BCYE and BAP [ 53 ] 2005 Morocco Hot water tank 128 25 BCYE [ 54 ] [1] LOD methods for PCR :2 × 10 2 GU/100 mL for LP (mip gene) and culture methods is 1 CFU/100 ml. [2] Buffered charcoal yeast extract : culture techniques [3] Polymerase chain reaction technique [4] Glycine-Vancomycin-Polymyxin-Cycloheximide technique [5] Blood agar plates Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4264","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":139705,"identity":"a62f43d7-d337-4454-b93e-1e98bf8dc7d5","order_by":1,"name":"Yadolah Fakhri","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYJCCDwwHDjCwsTcAmQYWROlgnAHWwnMApEWCBC0MEgkgDhFaDI4fYGz4ceZOPp/k86sbfhRIMPC3dyfg13ImgbGx58YzyzbpnLKbPUCHSZw5uwG/lgMJ7A94Phw2YJPOSbvBA9RiIJFLQMv5B4yNf0BaJM+k3fxDlJYbCYzNPDeAWiTYj90myhbJGw8bm2XOALXw5LDdljGQ4CHoF77zyQcb3xw7bCDffvzZzTd/bOT423vxa1EAhjGUyWMAJvEqBwH5BjiT/QFB1aNgFIyCUTAyAQDJ90+GX3MFtQAAAABJRU5ErkJggg==","orcid":"","institution":"Shaheed Beheshti University of Medical Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yadolah","middleName":"","lastName":"Fakhri","suffix":""},{"id":139706,"identity":"25ee446e-b195-4005-a17e-3278424871ff","order_by":2,"name":"Mohammad Javad Nasiri","email":"","orcid":"","institution":"Shahid Beheshti University of Medical Sciences School of Pharmacy","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Javad","lastName":"Nasiri","suffix":""},{"id":139707,"identity":"dea75d89-5bb3-4e03-bff2-09a63649b06f","order_by":3,"name":"Anvar Asadi","email":"","orcid":"","institution":"Kermanshah University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Anvar","middleName":"","lastName":"Asadi","suffix":""},{"id":139708,"identity":"952967ba-4e16-4c3d-a461-8674f5e10bac","order_by":4,"name":"Moayed Avazpour","email":"","orcid":"","institution":"Ilam University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Moayed","middleName":"","lastName":"Avazpour","suffix":""},{"id":139709,"identity":"a59a2631-161a-4e91-b6b7-2310caa26b71","order_by":5,"name":"Abdolazim Alinejad","email":"","orcid":"","institution":"Fasa University of Medical Science","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Abdolazim","middleName":"","lastName":"Alinejad","suffix":""},{"id":139710,"identity":"a26c6b8d-25be-4507-9487-7eb51caff381","order_by":6,"name":"Zeinab Gholami","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zeinab","middleName":"","lastName":"Gholami","suffix":""},{"id":139711,"identity":"a85f33a3-abb0-402c-a317-27f61bcf46a2","order_by":7,"name":"Amin Mousavi Khaneghah","email":"","orcid":"","institution":"UNINTER Educacional SA - Campinas Centro","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Amin","middleName":"Mousavi","lastName":"Khaneghah","suffix":""}],"badges":[],"createdAt":"2019-08-22 12:52:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.2.13555/v1","doiUrl":"https://doi.org/10.21203/rs.2.13555/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":135911,"identity":"fbf1f7e2-5bb5-4f6f-90de-8f5f33c76fae","added_by":"auto","created_at":"2019-10-23 11:54:26","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":68018,"visible":true,"origin":"","legend":"Flowchart describing the study design process based on PRISMA","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/df8c2c7b-7247-4901-80a7-b975b212bb4a/v1/Fig 1.jpg"},{"id":135913,"identity":"b6b1469c-1375-4381-b820-140750f71e83","added_by":"auto","created_at":"2019-10-23 11:54:26","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1037669,"visible":true,"origin":"","legend":"Forest plot of the prevalence of LP in the water resources based on geographical.","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/df8c2c7b-7247-4901-80a7-b975b212bb4a/v1/Fig 2.jpg"},{"id":135914,"identity":"52dd7344-f072-4521-afdd-55c33545235e","added_by":"auto","created_at":"2019-10-23 11:54:26","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1272797,"visible":true,"origin":"","legend":"Forest plot of the prevalence of LP in the water resources based on the type of water resources.","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/df8c2c7b-7247-4901-80a7-b975b212bb4a/v1/Fig 3.jpg"},{"id":135915,"identity":"7d34e2a0-1d8e-47fd-a41a-d745e1fbc4a0","added_by":"auto","created_at":"2019-10-23 11:54:26","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":26922,"visible":true,"origin":"","legend":"Evaluate publication bias based on Beggs (A) and Eggers (B) tests","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/df8c2c7b-7247-4901-80a7-b975b212bb4a/v1/Fig 4.jpg"},{"id":135916,"identity":"c70b76c0-fd64-47b9-af67-869853f1e673","added_by":"auto","created_at":"2019-10-23 11:54:27","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":136829,"visible":true,"origin":"","legend":"Sensitivity analysis (Metatrim analysis) of the prevalence of LP in the water systems","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/df8c2c7b-7247-4901-80a7-b975b212bb4a/v1/Fig 5.jpg"},{"id":13471191,"identity":"db391d61-f4ee-4aee-9f4a-1583ee136fbd","added_by":"auto","created_at":"2021-09-16 21:08:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":736830,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4264/v1/32edfa48-dc4c-4166-a8ca-4019059bf5d5.pdf"}],"financialInterests":"","formattedTitle":"The prevalence of Legionella pneumophila in different water systems: A global systematic review and meta-analysis","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eLegionella pneumophila\u003c/em\u003e (LP) as a heterotrophic bacterium is a gram-negative, aerobic, non-sporogenous, and a mobile [\u003ca href=\"#_ENREF_1\"\u003e1\u003c/a\u003e, \u003ca href=\"#_ENREF_2\"\u003e2\u003c/a\u003e] which can be isolated from various environments such as soil and water systems, including water used in cooling towers and ventilation, surface water, tap water, and spring water [\u003ca href=\"#_ENREF_3\"\u003e3–6\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003eThe tolerance to high temperature is among the features, which could stimulate the presence, and proliferation of LP in water systems [\u003ca href=\"#_ENREF_7\"\u003e7–9\u003c/a\u003e]. Although the range of optimal temperature for proliferation was indicated as 29–40 °C, rarely, it can survive in water at temperatures ranging from 0 to 63 °C [\u003ca href=\"#_ENREF_10\"\u003e10\u003c/a\u003e]. Also, the efficiency of chemical or thermal disinfection can be reduced in the presence of biofilm or amoebae; which can be resulted in further contamination of water systems by LP\u003ca href=\"#_ENREF_11\"\u003e11\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003eThe droplets of contaminated water withLP could convey the bacterium into the lungs and as a result of swallowing by macrophages [\u003ca href=\"#_ENREF_12\"\u003e12\u003c/a\u003e, \u003ca href=\"#_ENREF_13\"\u003e13\u003c/a\u003e]. Therefore, the approaching of efficient and appropriate methods is crucial to decreasing as well as controlling the prevalence of LP water systems. In this regard, according to the guideline of World Health Organization (WHO), the acceptable limit of LP in the drinking water and cooling tower systems were defined as 1 CFU/L (WHO 2008) and 1000 CFU/L [\u003ca href=\"#_ENREF_14\"\u003e14\u003c/a\u003e], respectively.\u003c/p\u003e\n\u003cp\u003eLegionnaires’ disease are among issues caused by these water-borne pathogens, commonly known as \u003cem\u003eLegionella.\u003c/em\u003e Twenty-five out of 59 species in the Legionella genus, have been correlated with human disease [\u003ca href=\"#_ENREF_15\"\u003e15\u003c/a\u003e]. According to Cazalet et al. (2008), almost 20 kb of 33-kb locus carrying the genes for the proteins in lipopolysaccharide biosynthesis in \u003cem\u003e LP \u003c/em\u003eis highly specific for \u003cem\u003eL. Pneumophila serogroup 1 (\u003c/em\u003eLP-Sg1)[\u003ca href=\"#_ENREF_16\"\u003e16\u003c/a\u003e]. Also, multigenome via comparative hybridization detected 3 genes, including lpp0837/wzm, lpp0831, and lpp0838/wzt strains only LP-Sg1 that used for real-time PCR method for identifying LP-Sg1 [\u003ca href=\"#_ENREF_16\"\u003e16\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003eMoreover, \u003cem\u003eLegionella pneumophila serogroup 1 (\u003c/em\u003eLP) is responsible for the majority of reported cases of Legionnaires’ disease (about 90%) [\u003ca href=\"#_ENREF_17\"\u003e17\u003c/a\u003e]. Legionnaires’ disease, for the first time, was diagnosed as pneumonic form [\u003ca href=\"#_ENREF_10\"\u003e10\u003c/a\u003e]. The incidence of LD is increasing, especially in Europe (EU) United States of America (USA). In this context, only in 2010 and 2012, 6,305 and 4,486 cases of the Legionnaire’s disease were reported in Europe [\u003ca href=\"#_ENREF_18\"\u003e18\u003c/a\u003e] and the USA [\u003ca href=\"#_ENREF_19\"\u003e19\u003c/a\u003e], respectively. Some signs and symptoms include coughing, shortness of breath, high fever, muscle pains, and headaches, Nausea, vomiting, and diarrhea were associated with Legionnaires’ disease [\u003ca href=\"#_ENREF_10\"\u003e10\u003c/a\u003e]. Due to the significant health effect of LP contamination in water systems, some investigations with different outcomes were performed [\u003ca href=\"#_ENREF_6\"\u003e6\u003c/a\u003e, \u003ca href=\"#_ENREF_20\"\u003e20–22\u003c/a\u003e]. In this regard, the prevalence of LP in the cooling tower systems in Kuwait and Saudi Arabic were determined as 98(95%CI: 89–100) and 2(95%CI: 1–4), respectively [\u003ca href=\"#_ENREF_23\"\u003e23\u003c/a\u003e, \u003ca href=\"#_ENREF_24\"\u003e24\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003e The culture medium and polymerase chain reaction (PCR) techniques are used to isolate the LP in water systems. The culture techniques based on ISO 11731 can be considered as gold-standard for detecting LP in water systems [\u003ca href=\"#_ENREF_14\"\u003e14\u003c/a\u003e]. In the PCR method, the limit of detection (LOD) is 2 \u003cem\u003e× \u003c/em\u003e10\u003csup\u003e2\u003c/sup\u003e GU/100 mL for LP (\u003cem\u003emip gene), \u003c/em\u003ewhile theLOD for culture methods is 1 CFU/100 mL [\u003ca href=\"#_ENREF_25\"\u003e25\u003c/a\u003e, \u003ca href=\"#_ENREF_26\"\u003e26\u003c/a\u003e]. Although culture techniques are used widely, these techniques have several limitations including a long time needed to obtain results, weak recovery, weak sensitivity, inability to detect viable but non-culturable cells (VBNC) [\u003ca href=\"#_ENREF_27\"\u003e27\u003c/a\u003e]. In another hand, the polymerase chain reaction (PCR) techniques due to reproducibility, sensitivity, specificity, high-throughput and reducing in the required time to less than 24 hours are widely approached to detect LP in water systems [\u003ca href=\"#_ENREF_28\"\u003e28–30\u003c/a\u003e] \u003c/p\u003e\u003cp\u003eHowever, no systematic review and meta-analysis was performed regarding the prevalence of \u003cem\u003eLegionella pneumophila\u003c/em\u003e in different water systems. Therefore, for the first time, the current study was undertaken to perform a meta-analysis to investigate the prevalence LP in the water systems based on geographical, quality of research as well as the type of water.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003eIn this study, the systematic review was conducted in accordance with the Cochrane protocol and Prisma protocol [\u003ca href=\"#_ENREF_31\"\u003e31\u003c/a\u003e].\u003c/p\u003e\n\u003ch3 data-xsweet-outline-level=\"0\"\u003e2.1. Search strategy\u003c/h3\u003e\n\u003cp\u003eThe search strategy was performed on the \u003cem\u003eLegionella pneumophila\u003c/em\u003e studies in water systems. Searching was performed in the main international databases, including Scopus, PubMed, and Embase.\u003c/p\u003e\n\u003cp\u003eThe following terms were used: (a) PubMed: Search ((((((legionella[Title/Abstract]) OR \u003cem\u003eLegionella pneumophila[\u003c/em\u003eTitle/Abstract]) OR microbe[Title/Abstract]) OR bacteria[Title/Abstract])) AND ((((water[Title/Abstract]) OR drinking water[Title/Abstract]) OR tap water[Title/Abstract]) OR water system[Title/Abstract])) AND (“Legionella”[Mesh] AND “Legionella pneumophila”[Mesh]); (b) Scopus: ( ( title-abs-key ( legionella and pneumophila) or title-abs-key ( legionella) or title-abs-key ( pneumophila) or title-abs-key ( microbe) or title-abs-key ( bacteria))) and ( ( title-abs-key ( water) or title-abs-key ( water and system) or title-abs-key ( drinking and water) or title-abs-key ( tap and water) or title-abs-key ( groundwater))) and (c) Embase: ‘legionella’:ab,ti OR ‘legionella pneumophila’:ab,ti OR ‘microbe’:ab,ti. Also, the references articles were reviewed to obtain more articles. Seventeen years (1/January/1983 and 25/July/ 2017) was chosen as the searching period.\u003c/p\u003e\n\n\u003ch3\u003e2.2. Inclusion and exclusion criteria\u003c/h3\u003e\n\u003cp\u003eFull-text articles were downloaded and then reviewed carefully to check if they meet the proposed criteria [\u003ca href=\"#_ENREF_32\"\u003e32–34\u003c/a\u003e] such as (1) original study; (2) cross-sectional data; (3) published in English; (4) published online between 1/January/1983 and 25/July/ 2017; (5) full-text available articles; (6) existence of exact total sample size and positive samples; (7) the defined type of water was examined; and (8) accurate methods including culture or PCR techniques were mentioned. The articles were excluded when not meet our criteria.\u003c/p\u003e\n\u003ch3\u003e2.3. Data extraction\u003c/h3\u003e\n\u003cp\u003eThe obtained information from each study can be summarized as study characteristics; (the first author, year of study); total sample size; the number of positive samples; the geographical study (countries); study methodology (Culture or PCR); type of water systems (tap drinking water, hot water, water reservoir, cold water, well and spring).\u003c/p\u003e\n\n\u003ch3\u003e2.4. Meta-analysis of data\u003c/h3\u003e\n\n\u003cp\u003eThe ratio of the positive samples (p\u003csub\u003ei\u003c/sub\u003e) to the total sample (n\u003csub\u003ei\u003c/sub\u003e) defined as prevalence (P = p\u003csub\u003ei \u003c/sub\u003e/n\u003csub\u003ei\u003c/sub\u003e), which is between 0 to 1 value [\u003ca href=\"#_ENREF_32\"\u003e32\u003c/a\u003e, \u003ca href=\"#_ENREF_35\"\u003e35–37\u003c/a\u003e]. Estimation of the prevalence of LP in water systems was performed using the binomial distribution model [\u003ca href=\"#_ENREF_38\"\u003e38\u003c/a\u003e]. The heterogeneity (\u003cem\u003eI\u003csup\u003e2)\u003c/sup\u003e\u003c/em\u003e statistics was used to determine the variation between the prevalence of \u003cem\u003eLegionella penompophia\u003c/em\u003e among the included studies [\u003ca href=\"#_ENREF_39\"\u003e39\u003c/a\u003e]. In the current study, when the heterogeneity was higher than 50%, the random effect model (REM) was used to estimate prevalence based on the defined subgroup. The Begg’s and Egger’s test was used to estimate publication bias [\u003ca href=\"#_ENREF_21\"\u003e21\u003c/a\u003e, \u003ca href=\"#_ENREF_40\"\u003e40\u003c/a\u003e]. A meta-analysis of data was conducted using Stata 12.2 intercooled version (Stata Corp, College Station, TX). All statistical analysis was significant at P-values\u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA total number of 1541 of articles were screened in the initial screening through databases. Finally, 36 articles (43 studies) were included in the conducted systematic review and meta-analysis (\u003cem\u003eTable\u003c/em\u003e 1, \u003cem\u003eFigure\u003c/em\u003e 1). Given the geographical analysis, the lowest and highest pooled prevalence of LP was observed in Poland (4 (95%CI: 0–13%)) and Kuwait (98 % (95%CI: 90–100%)), respectively (\u003cem\u003eFigure\u003c/em\u003e 2). Also, the global pooled prevalence of LP in water systems was determined as 20% (95%CI: 15–25%) (\u003cem\u003eFigure\u003c/em\u003e 2). While, the lowest and highest prevalence of LP-based on water resources subgroups was water reservoir (15% (95%CI: 2–37%) and well (40% (95%CI: 26–50%), respectively (\u003cem\u003eFigure\u003c/em\u003e 3).\u003c/p\u003e\n\u003cp\u003eAccording to Begg’s (p-value = 0.029) and Egger’s (p-value = 0.024), significant publication bias among studies was noted (\u003cem\u003eFigure\u003c/em\u003e 4A-B); Hence, to remove the effect of publication bias, the metatrim test was performed. The pooled prevalence of LP in a water system based on metatrim in the random effect model (REM) was 21% (95%CI: 15–26%) (\u003cem\u003eFigure\u003c/em\u003e 5).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe prevalence of LP in the water systems was obtained as an average of 20% (95%CI: 15–25%). The prevalence of LP in the different countries considerably was difference (\u003cem\u003eFigure\u003c/em\u003e 2), which can be associated with variation in the quality of disinfection of water, the average life of water facilities, the chemical quality of water and the type of exploitation in each country.\u003c/p\u003e\n\u003cp\u003eSome factors such as water temperature, stagnation of water in pipes, the formation of biofilms on the interior walls of pipes, the presence of protozoa, the chemical quality of water and pH could affect \u003cem\u003eLegionella\u003c/em\u003e growth in aquatic systems [\u003ca href=\"#_ENREF_71\"\u003e71\u003c/a\u003e, \u003ca href=\"#_ENREF_72\"\u003e72\u003c/a\u003e]. However, the proposed temperature for growth of LP is 29–40 °C with an optimum of 35 °C; it could survive in aqueous systems at temperatures of 0–63 °C [\u003ca href=\"#_ENREF_10\"\u003e10\u003c/a\u003e]. The increase in temperature above 55 °C can be considered as an efficient technique for the prevention of bacterial growth and consequently, lower incidence of legionellosis [\u003ca href=\"#_ENREF_73\"\u003e73\u003c/a\u003e]. According to Leoni et al. (2001), no LP growth was observed at temperatures above 41 °C [\u003ca href=\"#_ENREF_74\"\u003e74\u003c/a\u003e]. However, the growth of LP decreased with increase in temperature [\u003ca href=\"#_ENREF_75\"\u003e75\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003eMoreover, LP quickly reproduces in hot water systems such as showerheads (45–50 °C) [\u003ca href=\"#_ENREF_61\"\u003e61\u003c/a\u003e]. Based on one recent study, the highest prevalence of LP in hot spring was 17.6% in the 40–48.6 °C [\u003ca href=\"#_ENREF_7\"\u003e7\u003c/a\u003e]. Findings showed that the prevalence of LP in cold water was higher than hot water (\u003cem\u003eFigure\u003c/em\u003e 4), which can be correlated with the inhibition effect of increase in temperature on LP growth. Additionally, based on ecological data, protozoans and amoeba can protect this bacteria from disinfectant, osmolality, and pH variations [\u003ca href=\"#_ENREF_61\"\u003e61\u003c/a\u003e, \u003ca href=\"#_ENREF_76\"\u003e76\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003eThe influence of metallic elements such as iron and manganese on the growth of Legionella previously also has been demonstrated [\u003ca href=\"#_ENREF_77\"\u003e77\u003c/a\u003e, \u003ca href=\"#_ENREF_78\"\u003e78\u003c/a\u003e]. Due to organophilic properties of LP, its growth is limited in the water with low iron concentrations. Considering to findings of Portier et al. (2016), the iron pyrophosphate and ferric iron chelator did not affect the persistence of LPin the biofilms, but ferrous iron chelator showed a positive effect because of higher bioavailable ferrous ion. Hence, the growth of LP in the pipes that are made of iron could be stimulated. Also, manganese has an indirect effect on Legionella growth by enhancing the growth of biofilms and plantations [\u003ca href=\"#_ENREF_79\"\u003e79\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003eMoreover, the protective role of manganese in enzymatic activity and increasing in resistance to oxidative stress has been confirmed [\u003ca href=\"#_ENREF_80\"\u003e80\u003c/a\u003e]. In this regard, the positive correlation between Mn concentration (\u0026gt; 3µg/L) with a prevalence of LP in water was demonstrated [\u003ca href=\"#_ENREF_81\"\u003e81\u003c/a\u003e]. The presence of a copper ion in water due to its antibacterial nature can resulted in further decline in the prevalence of Legionella[\u003ca href=\"#_ENREF_82\"\u003e82\u003c/a\u003e]. However, biofilms in water systems protect Legionella against disinfection [\u003ca href=\"#_ENREF_83\"\u003e83\u003c/a\u003e]. Therefore, in order to reduce the prevalence of Legionella, washing of pipes and tanks in addition to exhausting of water facilities with chlorinated hot water can be recommended. According to Oberdorfer et al. (2008), the prevalence of Legionella in the old water system of the hospital was higher than new ones [\u003ca href=\"#_ENREF_84\"\u003e84\u003c/a\u003e]. Nowadays, the use of polyethylene (PE) and \u003ca href=\"https://en.wikipedia.org/wiki/Polyvinyl_chloride\"\u003ePolyvinyl chloride\u003c/a\u003e (PVC) materials in water facilities is continuously expanding. The growth of biofilm was increased with the releasing of volatile organic compounds in PVC and PE pipes [\u003ca href=\"#_ENREF_85\"\u003e85\u003c/a\u003e]. Therefore, using PE and PVC in water facilities may increase the prevalence of LP in water systems.\u003c/p\u003e\n\u003cp\u003epH is another chemical parameter that affects the growth and survival of LP in water systems. The highest prevalence of LP was reported in weakly acid (pH 5–7, 37.5%) [\u003ca href=\"#_ENREF_7\"\u003e7\u003c/a\u003e]. In a report by Ohno et al. (2003), the optimal range of pH for LP in water systems was defined between 6.0 and 8.0 [\u003ca href=\"#_ENREF_8\"\u003e8\u003c/a\u003e]. However, other data showed that LP had not been explored in extremely acidic water [\u003ca href=\"#_ENREF_60\"\u003e60\u003c/a\u003e, \u003ca href=\"#_ENREF_86\"\u003e86\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003eConsidering the frequency of detection techniques, PCR (37.3%) approximately was similar to the culture method (62.7%). While in culture techniques, only living bacteria can be detected [\u003ca href=\"#_ENREF_87\"\u003e87\u003c/a\u003e], in the PCR in addition to living bacteria, the bacteria that damaged by the disinfection which their DNA remains also can be detected [\u003ca href=\"#_ENREF_88\"\u003e88\u003c/a\u003e]. Moreover, in the culture technique, the incubation period of 3–14 days is recommended to facilitate the growth of bacterial colonies, while the bacteria can be damaged by used acid treatment in culture technique. In another hand, the observed limitation regarding detection sensitivity (50–60%) is another disadvantage of culture techniques[\u003ca href=\"#_ENREF_89\"\u003e89\u003c/a\u003e]. Due to Culturable None but Viable (CNBV) properties, LP can survey in water for a long time without being identified by culture technique. Likewise, data have shown that Legionella prevalence in amoeba-water samples was 25–50% higher than other samples because of symbiosis survival with the amoeba [\u003ca href=\"#_ENREF_90\"\u003e90\u003c/a\u003e, \u003ca href=\"#_ENREF_91\"\u003e91\u003c/a\u003e]. Moreover, one of the most significant limitations of PCR techniques is bias due to the presence of inhibitors such as polysaccharide and chlorophyll in the samples [\u003ca href=\"#_ENREF_92\"\u003e92\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003e Since the culture and PCR techniques have different ranges of detection limits, however, by using PCR technique, higher level of contamination in pathological samples can be detected quickly and accurately, approaching of both methods in the case of environmental issues such as LP might be recommended.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn the current study, the prevalence of LP in water systems in the defined subgroups were meta analyzed. A high prevalence of LP in water systems worldwide was demonstrated by the current study as a first systematic review in this field. Likewise, the higher prevalence of\u003cem\u003e Legionella pneumophila \u003c/em\u003ewas considerablein the water systems, particularly in cold water. In this context, approaching of control actions such as avoiding stagnation of water in water systems, use of high-quality water, and continuous purification of water using disinfectants factors can be recommended. Continuous monitoring of water quality to assess the effectiveness of measures to control and prevent the prevalence of legionellosis is crucial.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eLP: Legionella pneumophila; WHO: World Health Organization; PCR: polymerase chain reaction; LOD: limit of detection; VBNC: Non-culturable cells; PVC: Polyvinyl chloride; CNBV: Culturable None but Viable.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch3\u003eAcknowledgements\u003c/h3\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch3\u003eAuthors’ contributions\u003c/h3\u003e\n\u003cp\u003eSearching in international database was performed by Z.GH and MJ.NA; screening of papers based on title and abstract was performed by Y.FA, M.AZ and A.AS; Data extraction and data preparation was performed by AM.MK and AZ.AL. Meta-analysis of data and preparation of manuscript was performed by Y.FA and AM.MK.\u003c/p\u003e\n\u003ch3\u003eConflict of Interest\u003c/h3\u003e\n\u003cp\u003eThere is no conflict of interest.\u003c/p\u003e\n\u003ch3\u003eFunding\u003c/h3\u003e\n\u003cp\u003eThe authors would like to thank student research committee, Shahid Beheshti University of Medical Sciences for the financial grants of this study (Code: 1396/54035).\u003c/p\u003e\n\u003ch3\u003eAvailability of data and materials\u003c/h3\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch3\u003eEthics approval and consent to participate\u003c/h3\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch3\u003eConsent for publication\u003c/h3\u003e\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch3\u003eCompeting interests\u003c/h3\u003e\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003e1.Edelstein PH: \u003cem\u003eLegionella jamestowniensis fatal pneumonia in an immunosuppressed man.\u003c/em\u003e \u003cem\u003eJournal of Infection and Chemotherapy \u003c/em\u003e2017, \u003cem\u003e23(\u003c/em\u003e1):59–61.\u003c/p\u003e\n\u003cp\u003e2.Al-Matawah Q, Al-Zenki S, Al-Azmi A, Al-Waalan T, Al-Salameen F, Hejji AB: \u003cem\u003eLegionella detection and subgrouping in water air-conditioning cooling tower systems in Kuwait.\u003c/em\u003e \u003cem\u003eEnvironmental Science and Pollution Research \u003c/em\u003e2015, \u003cem\u003e22(\u003c/em\u003e13):10235–10241.\u003c/p\u003e\n\u003cp\u003e3.Leoni E, Legnani P: \u003cem\u003eComparison of selective procedures for isolation and enumeration of Legionella species from hot water systems.\u003c/em\u003e \u003cem\u003eJ Appl Microbiol \u003c/em\u003e2001, \u003cem\u003e90(\u003c/em\u003e1):27–33.\u003c/p\u003e\n\u003cp\u003e4.Qin T, Zhou H, Ren H, Guan H, Li M, Zhu B, Shao Z: \u003cem\u003eDistribution of sequence-based types of Legionella pneumophila serogroup 1 strains isolated from cooling towers, hot spring, and potable water systems in China.\u003c/em\u003e \u003cem\u003eAppl Environ Microbiol \u003c/em\u003e2014:AEM. 03844–03813.\u003c/p\u003e\n\u003cp\u003e5.Shen S-M, Chou M-Y, Hsu B-M, Ji W-T, Hsu T-K, Tsai H-F, Huang Y-L, Chiu Y-C, Kao E-S, Kao P-M: \u003cem\u003eAssessment of Legionella pneumophila in recreational spring water with quantitative PCR (Taqman) assay.\u003c/em\u003e \u003cem\u003ePathogens and global health \u003c/em\u003e2015, \u003cem\u003e109(\u003c/em\u003e5):236–241.\u003c/p\u003e\n\u003cp\u003e6.Özen NS, Ataman ŞT, Emek M: \u003cem\u003eExploring the Legionella pneumophila positivity rate in hotel water samples from Antalya, Turkey.\u003c/em\u003e \u003cem\u003eEnvironmental Science and Pollution Research \u003c/em\u003e2017, \u003cem\u003e24(\u003c/em\u003e13):12238–12242.\u003c/p\u003e\n\u003cp\u003e7.Huang S-W, Hsu B-M, Wu S-F, Fan C-W, Shih F-C, Lin Y-C, Ji D-D: \u003cem\u003eWater quality parameters 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other potential opportunistic pathogens in a bathroom.\u003c/em\u003e \u003cem\u003eEnvironmental Science and Pollution Research \u003c/em\u003e2017, \u003cem\u003e24(\u003c/em\u003e3):2326–2336.\u003c/p\u003e\n\u003cp\u003e14.WHO: \u003cem\u003eWorld Health Organization.Legionella and the prevention of legionellosis.\u003c/em\u003e 2007.\u003c/p\u003e\n\u003cp\u003e15.Sedlata EJ, Sedlackova H, Janska J, Holy O, Lalova I, Matouskova I: \u003cem\u003eLegionella spp. in dental unit waterlines.\u003c/em\u003e \u003cem\u003eBratisl Lek Listy \u003c/em\u003e2017, \u003cem\u003e118(\u003c/em\u003e5):310–314.\u003c/p\u003e\n\u003cp\u003e16.Merault N, Rusniok C, Jarraud S, Gomez-Valero L, Cazalet C, Marin M, Brachet E, Aegerter P, Gaillard J, Etienne J: \u003cem\u003eSpecific real-time PCR for simultaneous detection and identification of Legionella pneumophila serogroup 1 in water and clinical samples.\u003c/em\u003e \u003cem\u003eAppl Environ Microbiol \u003c/em\u003e2011, \u003cem\u003e77(\u003c/em\u003e5):1708–1717.\u003c/p\u003e\n\u003cp\u003e17.Diederen B: \u003cem\u003eLegionella spp. and Legionnaires’ disease.\u003c/em\u003e \u003cem\u003eJ Infect \u003c/em\u003e2008, \u003cem\u003e56(\u003c/em\u003e1):1–12.\u003c/p\u003e\n\u003cp\u003e18.Beauté J, Zucs P, de Jong B: \u003cem\u003eon behalf of the European Legionnaires’ Disease Surveillance Network.\u003c/em\u003e \u003cem\u003eLegionnaires’ disease in Europe \u003c/em\u003e2009, \u003cem\u003e2010.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e19.CDCP: Centers for Disease Control Prevention. 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\u003cem\u003e41(\u003c/em\u003e6):497–502.\u003c/p\u003e\n\u003cp\u003e81.Borella P, Guerrieri E, Marchesi I, Bondi M, Messi P: \u003cem\u003eWater ecology of Legionella and protozoan: environmental and public health perspectives.\u003c/em\u003e \u003cem\u003eBiotechnol Annu Rev \u003c/em\u003e2005, \u003cem\u003e11:\u003c/em\u003e355–380.\u003c/p\u003e\n\u003cp\u003e82.Lu J, Buse H, Gomez‐Alvarez V, Struewing I, Santo Domingo J, Ashbolt N: \u003cem\u003eImpact of drinking water conditions and copper materials on downstream biofilm microbial communities and L egionella pneumophila colonization.\u003c/em\u003e \u003cem\u003eJ Appl Microbiol \u003c/em\u003e2014, \u003cem\u003e117(\u003c/em\u003e3):905–918.\u003c/p\u003e\n\u003cp\u003e83.Solimini AG, Cottarelli A, Marinelli L, De Giusti M: \u003cem\u003eFactors influencing persistence of Legionella pneumophila serogroup 1 in laboratory cocultures.\u003c/em\u003e \u003cem\u003eBMC Microbiol \u003c/em\u003e2014, \u003cem\u003e14(\u003c/em\u003e1):249.\u003c/p\u003e\n\u003cp\u003e84.Oberdorfer K, Müssigbrodt G, Wendt C: \u003cem\u003eGenetic diversity of Legionella pneumophila in hospital water systems.\u003c/em\u003e \u003cem\u003eInt J Hyg Environ Health \u003c/em\u003e2008, \u003cem\u003e211(\u003c/em\u003e1–2):172–178.\u003c/p\u003e\n\u003cp\u003e85.Lee Y: \u003cem\u003eAn evaluation of microbial and chemical contamination sources related to the deterioration of tap water quality in the household water supply system.\u003c/em\u003e \u003cem\u003eInt J Environ Res Public Health \u003c/em\u003e2013, \u003cem\u003e10(\u003c/em\u003e9):4143–4160.\u003c/p\u003e\n\u003cp\u003e86.Sheehan KB, Henson JM, Ferris MJ: \u003cem\u003eLegionella species diversity in an acidic biofilm community in Yellowstone National Park.\u003c/em\u003e \u003cem\u003eAppl Environ Microbiol \u003c/em\u003e2005, \u003cem\u003e71(\u003c/em\u003e1):507–511.\u003c/p\u003e\n\u003cp\u003e87.Barigou M, Cavalie L, Daviller B, Dubois D, Mantion B, Delobel P, Debard A, Prere M-F, Marchou B, Martin-Blondel G: \u003cem\u003eIsolation on chocolate agar culture of Legionella pneumophila isolates from subcutaneous abscesses in an immunocompromised patient.\u003c/em\u003e \u003cem\u003eJ Clin Microbiol \u003c/em\u003e2015, \u003cem\u003e53(\u003c/em\u003e11):3683–3685.\u003c/p\u003e\n\u003cp\u003e88.Dada R, Mishra S, Mohanty K, Kumar M, Vemprala K: \u003cem\u003ePolymerase chain reaction.\u003c/em\u003e \u003cem\u003eManual of Cytogenetics in Reproductive Biology \u003c/em\u003e2014:59.\u003c/p\u003e\n\u003cp\u003e89.Veenendaal HR, Brouwer-Hanzens AJ, van der Kooij D: \u003cem\u003eIncubation of premise plumbing water samples on Buffered Charcoal Yeast Extract agar at elevated temperature and pH selects for Legionella pneumophila.\u003c/em\u003e \u003cem\u003eWater research \u003c/em\u003e2017, \u003cem\u003e123:\u003c/em\u003e439–447.\u003c/p\u003e\n\u003cp\u003e90.Bartie C, Venter S, Nel L: \u003cem\u003eIdentification methods for Legionella from environmental samples.\u003c/em\u003e \u003cem\u003eWater research \u003c/em\u003e2003, \u003cem\u003e37(\u003c/em\u003e6):1362–1370.\u003c/p\u003e\n\u003cp\u003e91.Ducret A, Chabalier M, Dukan S: \u003cem\u003eCharacterization and resuscitation of ‘non-culturable’cells of Legionella pneumophila.\u003c/em\u003e \u003cem\u003eBMC Microbiol \u003c/em\u003e2014, \u003cem\u003e14(\u003c/em\u003e1):3.\u003c/p\u003e\n\u003cp\u003e92.Miotke L, Lau BT, Rumma RT, Ji HP: \u003cem\u003eHigh sensitivity detection and quantitation of DNA copy number and single nucleotide variants with single color droplet digital PCR.\u003c/em\u003e \u003cem\u003eAnal Chem \u003c/em\u003e2014, \u003cem\u003e86(\u003c/em\u003e5):2618–2624.\u003c/p\u003e"},{"header":"Table 1","content":"\u003cp style=\"margin-bottom: .0001pt; text-align: left; line-height: 200%; text-autospace: ideograph-numeric; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; line-height: 200%; font-family: verdana, geneva; color: #000000;\"\u003eTable 1.\u0026nbsp; Characteristics included of studies.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"text-align: right; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; line-height: 106%; font-family: verdana, geneva; color: #000000;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse: collapse; border: none;\" width=\"912\"\u003e\n\u003ctbody\u003e\n\u003ctr style=\"height: 13.5pt;\"\u003e\n\u003ctd style=\"width: 69.85pt; border: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 13.5pt;\" width=\"93\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"color: #000000; font-family: verdana, geneva; font-size: 10pt;\"\u003e\u003cstrong\u003eYear Study\u003c/strong\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 105.15pt; border: solid windowtext 1.0pt; border-left: none; padding: 0in 5.4pt 0in 5.4pt; height: 13.5pt;\" width=\"140\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"color: #000000; font-family: verdana, geneva; font-size: 10pt;\"\u003e\u003cstrong\u003eCountry\u003c/strong\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 94.2pt; border: solid windowtext 1.0pt; border-left: none; padding: 0in 5.4pt 0in 5.4pt; height: 13.5pt;\" width=\"126\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"color: #000000; font-family: verdana, geneva; font-size: 10pt;\"\u003e\u003cstrong\u003eWater resource\u003c/strong\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.95pt; border: solid windowtext 1.0pt; border-left: none; padding: 0in 5.4pt 0in 5.4pt; height: 13.5pt;\" width=\"107\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"color: #000000; font-family: verdana, geneva; font-size: 10pt;\"\u003e\u003cstrong\u003eTotal sample size\u003c/strong\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.7pt; border: solid windowtext 1.0pt; border-left: none; padding: 0in 5.4pt 0in 5.4pt; height: 13.5pt;\" width=\"116\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"color: #000000; font-family: verdana, geneva; font-size: 10pt;\"\u003e\u003cstrong\u003ePositive sample size\u003ca style=\"color: #000000;\" href=\"#_ftn1\" name=\"_ftnref1\"\u003e\u003cspan style=\"line-height: 106%;\"\u003e[1]\u003c/span\u003e\u003c/a\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 1.95in; border: solid windowtext 1.0pt; border-left: none; padding: 0in 5.4pt 0in 5.4pt; height: 13.5pt;\" width=\"187\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"color: #000000; font-family: verdana, geneva; font-size: 10pt;\"\u003e\u003cstrong\u003eTechnique\u003c/strong\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 107.95pt; border: solid windowtext 1.0pt; border-left: none; padding: 0in 5.4pt 0in 5.4pt; height: 13.5pt;\" width=\"144\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"color: #000000; font-family: verdana, geneva; font-size: 10pt;\"\u003e\u003cstrong\u003eRef\u003c/strong\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 17.7pt;\"\u003e\n\u003ctd style=\"width: 69.85pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"93\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e2011\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 105.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"140\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eIran\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 94.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"126\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eTap drinking water\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"107\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e32\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.7pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"116\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e6\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 1.95in; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"187\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eBCYE\u003ca style=\"color: #000000;\" href=\"#_ftn2\" name=\"_ftnref2\"\u003e\u003cspan style=\"line-height: 106%;\"\u003e[2]\u003c/span\u003e\u003c/a\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 107.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"144\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e[\u003ca style=\"color: #000000;\" href=\"#_ENREF_20\"\u003e20\u003c/a\u003e]\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 17.7pt;\"\u003e\n\u003ctd style=\"width: 69.85pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"93\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e2003\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 105.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"140\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eIran\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 94.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"126\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eHot water tank\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"107\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e30\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.7pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"116\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e11\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 1.95in; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"187\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eBCYE\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 107.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"144\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e[\u003ca style=\"color: #000000;\" href=\"#_ENREF_41\"\u003e41\u003c/a\u003e]\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 17.7pt;\"\u003e\n\u003ctd style=\"width: 69.85pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"93\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e2008\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 105.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"140\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eIran\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 94.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"126\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eWater reservoir\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"107\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e33\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.7pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"116\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e5\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 1.95in; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"187\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eBCYE\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 107.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"144\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e[\u003ca style=\"color: #000000;\" href=\"#_ENREF_42\"\u003e42\u003c/a\u003e]\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 17.7pt;\"\u003e\n\u003ctd style=\"width: 69.85pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"93\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e1997\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 105.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"140\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eIran\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 94.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"126\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eWater reservoir\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"107\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e210\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.7pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"116\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e14\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 1.95in; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"187\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eBCYE\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 107.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"144\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e[\u003ca style=\"color: #000000;\" href=\"#_ENREF_43\"\u003e43\u003c/a\u003e]\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 17.7pt;\"\u003e\n\u003ctd style=\"width: 69.85pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"93\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e2011\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 105.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"140\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eIran\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 94.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"126\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eTap drinking water\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"107\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e110\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.7pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"116\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e29\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 1.95in; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"187\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eBCYE\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 107.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"144\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e[\u003ca style=\"color: #000000;\" href=\"#_ENREF_44\"\u003e44\u003c/a\u003e]\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 17.7pt;\"\u003e\n\u003ctd style=\"width: 69.85pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"93\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e2012\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 105.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"140\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eIran\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 94.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"126\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eTap drinking water\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"107\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e140\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.7pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"116\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e8\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 1.95in; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"187\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003ePCR\u003ca style=\"color: #000000;\" href=\"#_ftn3\" name=\"_ftnref3\"\u003e\u003cspan style=\"line-height: 106%;\"\u003e[3]\u003c/span\u003e\u003c/a\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 107.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"144\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e[\u003ca style=\"color: #000000;\" href=\"#_ENREF_45\"\u003e45\u003c/a\u003e]\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 17.7pt;\"\u003e\n\u003ctd style=\"width: 69.85pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"93\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e2005\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 105.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"140\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eIran\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 94.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"126\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eTap drinking water\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"107\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e113\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.7pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"116\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e30\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 1.95in; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"187\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eBCYE\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 107.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"144\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e[\u003ca style=\"color: #000000;\" href=\"#_ENREF_46\"\u003e46\u003c/a\u003e]\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 17.7pt;\"\u003e\n\u003ctd style=\"width: 69.85pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"93\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e2012\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 105.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"140\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eIran\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 94.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"126\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eTap drinking water\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"107\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e140\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.7pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"116\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e12\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 1.95in; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"187\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003ePCR\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 107.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"144\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e[\u003ca style=\"color: #000000;\" href=\"#_ENREF_47\"\u003e47\u003c/a\u003e]\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 17.7pt;\"\u003e\n\u003ctd style=\"width: 69.85pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"93\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e2008\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 105.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"140\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eIran\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 94.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"126\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eWater reservoir\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"107\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e240\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.7pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"116\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e100\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 1.95in; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"187\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eBCYE/GVPC\u003ca style=\"color: #000000;\" href=\"#_ftn4\" name=\"_ftnref4\"\u003e\u003cspan style=\"line-height: 106%;\"\u003e[4]\u003c/span\u003e\u003c/a\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 107.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"144\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e[\u003ca style=\"color: #000000;\" href=\"#_ENREF_48\"\u003e48\u003c/a\u003e]\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 17.7pt;\"\u003e\n\u003ctd style=\"width: 69.85pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"93\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e2010\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 105.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"140\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eIran\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 94.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"126\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eCold water tank\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"107\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e77\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.7pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"116\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e14\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 1.95in; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"187\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003ePCR\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 107.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"144\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e[\u003ca style=\"color: #000000;\" href=\"#_ENREF_49\"\u003e49\u003c/a\u003e]\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 17.7pt;\"\u003e\n\u003ctd style=\"width: 69.85pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"93\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e2015\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 105.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"140\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eIran (Kerman)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 94.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"126\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eCold water tank\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"107\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e78\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.7pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"116\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e18\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 1.95in; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"187\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003ePCR\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 107.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" rowspan=\"2\" width=\"144\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; layout-grid-mode: char;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e[\u003ca style=\"color: #000000;\" href=\"#_ENREF_50\"\u003e50\u003c/a\u003e]\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 17.7pt;\"\u003e\n\u003ctd style=\"width: 69.85pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"93\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e2015\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 105.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"140\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eIran (Bam)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 94.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"126\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eCold water tank\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"107\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e50\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.7pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"116\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e7\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 1.95in; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"187\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003ePCR\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 17.7pt;\"\u003e\n\u003ctd style=\"width: 69.85pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"93\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e2012\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 105.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"140\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eIran\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 94.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"126\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eCold water tank\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"107\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e140\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.7pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"116\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e10\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 1.95in; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"187\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003ePCR\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 107.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"144\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e[\u003ca style=\"color: #000000;\" href=\"#_ENREF_51\"\u003e51\u003c/a\u003e]\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 17.7pt;\"\u003e\n\u003ctd style=\"width: 69.85pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"93\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e2011\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 105.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"140\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eSaudi Arabia\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 94.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"126\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eCold water tank\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"107\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e300\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.7pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"116\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e5\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 1.95in; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"187\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eBCYE\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 107.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"144\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e[\u003ca style=\"color: #000000;\" href=\"#_ENREF_24\"\u003e24\u003c/a\u003e]\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 17.7pt;\"\u003e\n\u003ctd style=\"width: 69.85pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"93\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: 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normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eTaiwan\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 94.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"126\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eTap drinking water\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"107\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; 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embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eBCYE and BAP\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 107.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"144\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e[\u003ca style=\"color: #000000;\" href=\"#_ENREF_53\"\u003e53\u003c/a\u003e]\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 17.7pt;\"\u003e\n\u003ctd style=\"width: 69.85pt; border: solid windowtext 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"93\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e2005\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 105.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"140\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eMorocco\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 94.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"126\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eHot water tank\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 79.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"107\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e128\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 86.7pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"116\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e25\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 1.95in; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"187\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal; direction: rtl; unicode-bidi: embed;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003eBCYE\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 107.95pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 17.7pt;\" width=\"144\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"font-size: 10pt; font-family: verdana, geneva; color: #000000;\"\u003e[\u003ca style=\"color: #000000;\" href=\"#_ENREF_54\"\u003e54\u003c/a\u003e]\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cspan style=\"color: #000000; font-family: verdana, geneva; font-size: 10pt;\"\u003e\u003ca style=\"color: #000000;\" href=\"#_ftnref1\" name=\"_ftn1\"\u003e\u003cspan style=\"line-height: 106%;\"\u003e[1]\u003c/span\u003e\u003c/a\u003e LOD methods for PCR :2 \u0026times; 10\u003csup\u003e2\u003c/sup\u003e GU/100 mL for LP (mip gene) and culture methods is 1 CFU/100 ml. \u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"text-indent: 0in; margin: 0in 0in 14.0pt 0in;\"\u003e\u003cspan style=\"color: #000000; font-family: verdana, geneva; font-size: 10pt;\"\u003e\u003ca style=\"color: #000000;\" href=\"#_ftnref2\" name=\"_ftn2\"\u003e\u003cspan style=\"line-height: 106%;\"\u003e[2]\u003c/span\u003e\u003c/a\u003e Buffered charcoal yeast extract\u003c/a\u003e: culture techniques\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan style=\"color: #000000; font-family: verdana, geneva; font-size: 10pt;\"\u003e\u003ca style=\"color: #000000;\" href=\"#_ftnref3\" name=\"_ftn3\"\u003e\u003cspan style=\"line-height: 106%;\"\u003e[3]\u003c/span\u003e\u003c/a\u003e Polymerase chain reaction technique\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan style=\"color: #000000; font-family: verdana, geneva; font-size: 10pt;\"\u003e\u003ca style=\"color: #000000;\" href=\"#_ftnref4\" name=\"_ftn4\"\u003e\u003cspan style=\"line-height: 106%;\"\u003e[4]\u003c/span\u003e\u003c/a\u003e Glycine-Vancomycin-Polymyxin-Cycloheximide technique\u003c/em\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan style=\"color: #000000; font-family: verdana, geneva; font-size: 10pt;\"\u003e\u003ca style=\"color: #000000;\" href=\"#_ftnref5\" name=\"_ftn5\"\u003e\u003cspan style=\"line-height: 106%;\"\u003e[5]\u003c/span\u003e\u003c/a\u003e Blood agar \u003cem\u003e plates\u003c/em\u003e\u003c/span\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"occurrence; Legionella pneumophila; water reservoir; global overview","lastPublishedDoi":"10.21203/rs.2.13555/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.2.13555/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The presence of Legionella pneumophila (LP) in aquatic habitat is a global concern. The current study was undertaken to estimate the prevalence of LP in water systems with the aid of a systematic review and meta-analysis. The searching was performed among some international databases, including Scopus, PubMed, and Embase to retrieve the related articles between 1/January/1983 and 25/July/ 2017. Therefore, thirty-six articles (with 43 studies) out of 1,541 articles collected, were included in the meta-analysis. The overall prevalence of LP in water systems was determined as 20% (95%CI: 15-25). Also, the lowest and highest pooled prevalence of LP was observed in Poland (4% (95%CI: 0-13%)) and Kuwait (98% (95%CI: 90-100%)), respectively. The lowest and highest prevalence of LP-based on water resources subgroups was a water reservoir (15% (95%CI: 2-37%) and well (40% (95%CI: 26-50%), respectively. The number of studies that used polymerase chain reaction (PCR) for detection of LP was 16/43 (37.3%) while the culture method was 27/43 (62.7%). Generally speaking, the relatively high prevalence of LP among the investigated water systems was demonstrated, which should be reduced by performing appropriate control actions.","manuscriptTitle":"The prevalence of Legionella pneumophila in different water systems: A global systematic review and meta-analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2019-08-24 17:31:45","doi":"10.21203/rs.2.13555/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6da1b510-d734-4e43-9802-2143f3dd318d","owner":[],"postedDate":"August 24th, 2019","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":23202,"name":"Health Economics \u0026 Outcomes Research"},{"id":23203,"name":"Health Policy"},{"id":23204,"name":"Infectious Diseases"}],"tags":[],"updatedAt":"","versionOfRecord":[],"versionCreatedAt":"2019-08-24 17:31:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4264","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"identity":"rs-4264","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}