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      Comment on Montagna, et al. Evaluation of Legionella air contamination in healthcare facilities by different sampling methods: An Italian multicenter study. Int. J. Environ. Res. Public Health 2017, 14, 670

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          Abstract

          Introduction In their recent article, Montagna et al. describe a multicenter study investigating the presence of Legionella in water and air samples of Italian healthcare facilities [1]. This is an interesting study that highlights some important gaps in our basic understanding of Legionella. One of the limitations of the study regarded the lack of information on the tap outlets (e.g., design of tap, flow rates, temperature of the water) and the bathrooms (ambient temperature, humidity, air movements, etc.) as these factors can influence aerosols produced and may facilitate the interpretation of aerosol data. For future studies it may be advantageous to characterise the aerosols produced by these outlets, using for example an aerodynamic particle sizer (APS) [2]. Furthermore, caution should be advised as some of the conclusions from the culture data are based on the detection of just 1 colony forming unit (CFU)/m3 of air (or 1 CFU per hour of sampling). Comparison of this data to the number of genomic units (GU) detected in the air by Coriolis® sampling is difficult, as the data is not presented as GU/m3. It would have been more appropriate to compare the number of GU in the water with the number of GU in the air to give a better ‘estimation’ of the emission factor. Nonetheless, the relative inability to isolate viable Legionella from the air is interesting and reinforces previous findings [3,4], including our own failures to culture airborne Legionella from both water sources [5] and compost (unpublished data). Similarly, the only successful method in these studies was liquid impingement using an all glass-cyclone sampler combined with quantitative polymerase chain reaction (qPCR). So, why is Legionella difficult to culture from the air? The authors rightly state that factors such as water chemistry, the stress of aerosolisation, and the method of sampling can influence the recovery of viable Legionella. Furthermore, it has been shown that a high concentration of Legionella (>300 CFU/mL) in shower water is required to detect Legionella in the air [6]. This may go some way to explaining why viable airborne Legionella was infrequently detected at low concentrations in this study. Perhaps the low recovery of viable Legionella from the air is a factor in the sporadic nature of Legionnaires’ disease? However, there are also significant gaps in our understanding of Legionella. Quite simply, for a respiratory pathogen that is predominantly transmitted through aerosols, we have a poor understanding of Legionella in the aerosol state. Several key questions remain and should be research priorities in light of a globally increasing incidence of Legionnaires’ disease and an increasingly elderly and immunocompromised population: What forms of Legionella are present in aerosols from different sources? Are they viable or infectious? How is Legionella partitioned in the aerosol state; e.g., how much is associated with respirable particles <5 µm in size? What impact does the water system/outlet have on this? What is the relationship between the amount of Legionella in the water and the amount of respirable Legionella in the air? Do some Legionella (e.g., sequence types commonly associated with human disease) survive better in the air compared to others? Do amoeba and/or amoeba cysts play a role in aerosol survival and airborne transmission of Legionella? How far can aerosols travel in the built environment? As the authors state, Legionella air sampling could inform quantitative microbial risk assessments (QMRA), improve our understanding of the risks posed by particular water systems, and inform mitigation measures. That said, there is a requirement for improved detection and characterisation of Legionella-containing aerosols and controlled and reproducible experiments on laboratory models and importantly, real-world systems. Until these advances are made, it is clear that the most appropriate method for controlling the risk from Legionella is to control its presence in the water, eliminating the key risk factors and limiting the numbers capable of being disseminated.

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          Detection of airborne Legionella while showering using liquid impingement and fluorescent in situ hybridization (FISH).

          Aerosols of water contaminated with Legionella bacteria constitute the only mode of exposure for humans. However, the prevention strategy against this pathogenic bacteria risk is managed through the survey of water contamination. No relationship linked the Legionella bacteria water concentration and their airborne abundance. Therefore, new approaches in the field of the metrological aspects of Legionella bioaerosols are required. This study was aimed at testing the main principles for bioaerosol collection (solid impaction, liquid impingement and filtration) and the in situ hybridization (FISH) method, both in laboratory and field assays, with the intention of applying such methodologies for airborne Legionella bacteria detection while showering. An aerosolization chamber was developed to generate controlled and reproducible L. pneumophila aerosols. This tool allowed the identification of the liquid impingement method as the most appropriate one for collecting airborne Legionella bacteria. The culturable fraction of airborne L. pneumophila recovered with the liquid impingement principle was 4 and 700 times higher compared to the impaction and filtration techniques, respectively. Moreover, the concentrations of airborne L. pneumophila in the impinger fluid were on average 7.0 x 10(5) FISH-cells m(-3) air with the fluorescent in situ hybridization (FISH) method versus 9.0 x 10(4) CFU m(-3) air with the culture method. These results, recorded under well-controlled conditions, were confirmed during the field experiments performed on aerosols generated by hot water showers in health institutions. This new approach may provide a more accurate characterization of aerobiocontamination by Legionella bacteria.
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            Occurrence of Legionella in UK household showers.

            Household water systems have been proposed as a source of sporadic, community acquired Legionnaires' disease. Showers represent a frequently used aerosol generating device in the domestic setting yet little is known about the occurrence of Legionella spp. in these systems. This study has investigated the prevalence of Legionella spp. by culture and qPCR in UK household showers. Ninety nine showers from 82 separate properties in the South of England were sampled. Clinically relevant Legionella spp. were isolated by culture in 8% of shower water samples representing 6% of households. Legionella pneumophila sg1 ST59 was isolated from two showers in one property and air sampling demonstrated its presence in the aerosol state. A further 31% of showers were positive by Legionella spp. qPCR. By multi-variable binomial regression modelling Legionella spp. qPCR positivity was associated with the age of the property (p=0.02), the age of the shower (p=0.01) and the frequency of use (p=0.09). The concentration of Legionella spp. detected by qPCR was shown to decrease with increased frequency of use (p=0.04) and more frequent showerhead cleaning (p=0.05). There was no association between Legionella spp. qPCR positivity and the cold water supply or the showerhead material (p=0.65 and p=0.71, respectively). Household showers may be important reservoirs of clinically significant Legionella and should be considered in source investigations. Simple public health advice may help to mitigate the risk of Legionella exposure in the domestic shower environment.
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              Evaluation of bioaerosol sampling techniques for Legionella pneumophila coupled with culture assay and quantitative PCR

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                Author and article information

                Journal
                Int J Environ Res Public Health
                Int J Environ Res Public Health
                ijerph
                International Journal of Environmental Research and Public Health
                MDPI
                1661-7827
                1660-4601
                04 August 2017
                August 2017
                : 14
                : 8
                : 876
                Affiliations
                Public Health England, Salisbury SP4 0JG, UK; jimmy.walker@ 123456phe.gov.uk
                Author notes
                [* ]Correspondence: Samuel.collins@ 123456phe.gov.uk ; Tel.: +44-123-582-5281
                Author information
                https://orcid.org/0000-0003-4721-0040
                Article
                ijerph-14-00876
                10.3390/ijerph14080876
                5580580
                28777305
                6705ff32-8d57-4669-b952-af66d6fb3a95
                © 2017 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 11 July 2017
                : 01 August 2017
                Categories
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                Public health
                legionella,legionnaires’ disease,aerosol,detection,risk assessment
                Public health
                legionella, legionnaires’ disease, aerosol, detection, risk assessment

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