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Management of Legionella in Water Systems (2019)

Chapter: 3 Quantification of Legionnaires' Disease and Legionella

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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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Suggested Citation:"3 Quantification of Legionnaires' Disease and Legionella." National Academies of Sciences, Engineering, and Medicine. 2019. Management of Legionella in Water Systems. Washington, DC: The National Academies Press. doi: 10.17226/25474.
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3 Quantification of Legionnaires’ Disease and Legionella This chapter addresses what is known about the incidence of Legionnaires’ disease from surveil- lance systems and the occurrence of Legionella bacteria in water systems including the methods used to collect both clinical and environmental data. Both the tracking of disease incidence and monitoring the number of Legionella bacteria in various water systems are fraught with difficulties. These difficulties include deciding who to test, where and when to sample the environment, what methods to use, and how to interpret the data. Despite these challenges, advances have been made and are likely to continue as legionellosis becomes a higher public health priority. Most cases of Legionnaires’ disease are never linked to any specific environmental source, for many reasons. Most individuals are never diagnosed, even among those who seek medical care. Those who are diagnosed may have no associated clinical isolate to confirm the results of the urinary antigen test. Sampling for Legionella in buildings is routine in the United States for only a subset of acute care hospitals and other potential sources such as hotels. In addition, most states do not have the capacity to investigate environmental sources of Legionnaires’ disease, with few environmental microbiologists or engineering experts on staff in public health departments. It is still the case that information on Legion- naires’ disease stems mostly from investigations of recognized outbreaks, which account for only 4 per- cent of cases in the United States (Hicks et al., 2011). Not known is whether the environmental exposures found in outbreak investigations accurately represent the exposures for the majority of cases. More information is needed about environmental exposures that result in disease in order to estimate their risk. To assess the level of risk of Legionnaires’ disease, a quantitative microbial risk assessment (QMRA) framework can be designed using an estimate of the concentration of Legionella pneumophila (the pathogen most likely to cause disease) associated with a particular source (e.g., shower- head, hot tub, cooling tower) combined with dose-response information about the bacterium. As quanti- fication of viable Legionella in water samples increases, this framework can be used to better understand which environmental exposures are most likely to lead to cases of legionellosis. This chapter ends with a discussion of the role of QMRA in linking clinical and environmental data and informing subsequent actions as well as in determining risk-based numerical values for Legionella in water. 95 Prepublication Version - Subject to further editorial revision

96 Management of Legionella in Water Systems INCIDENCE OF LEGIONELLOSIS IN THE UNITED STATES To quantify Legionnaires’ disease incidence, national surveillance is undertaken that builds on local and state surveillance efforts. All states require that public health authorities be notified of those diagnosed with Legionnaires’ disease or Pontiac fever. In turn, states voluntarily report their numbers to the U.S. Centers for Disease Control and Prevention (CDC). Separately, states also report waterborne disease outbreaks to the CDC, including those caused by Legionella. Together this information serves as a basis for quantifying the incidence of Legionnaires’ disease and contributes to our knowledge of the epidemiology of the disease. Before describing the nation’s Legionella surveillance systems, the diagnostic tests used to identify cases of Legionnaires’ disease are briefly reviewed (building on the Chapter 2 dis- cussion). Diagnostic Tests for Legionellosis Used in Surveillance According to CDC, the preferred diagnostic tests for Legionnaires’ disease are culture of lower respiratory secretions on selective media and the urinary antigen test. Serological assays can be nonspe- cific and are not recommended in most situations, while polymerase chain reaction (PCR) is utilized by some academic and reference laboratories. Culture of sputum or bronchoalveolar lavage specimens from pneumonia patients is important to determine if Legionella is the causative agent, regardless of species and serogroup. L. pneumophila forms colonies on buffered charcoal yeast extract agar within three to five days. As discussed in Chapter 2, most non-pneumophila Legionella species (spp.) may require longer incubation times and different media, and some culture media do not support growth of certain non-pneumophila Legionella spp. Culturing Legionella is challenging because of the needs for a lower-respiratory specimen and technical expertise in the laboratory. Furthermore, a history of prior antibiotic use interferes with culture. Most hospitals do not routinely culture sputum for Legionella, although some academic health centers routinely culture bronchoscopy specimens in patients with pneumonia of unknown etiology. Culture methods are criti- cally important to epidemiologic investigations because molecular analysis can link clinical isolates to environmental samples to document the source of the exposure. Most patients with reported Legionnaires’ disease are diagnosed as a result of a positive Legionella urinary antigen test (UAT), which is available at commercial laboratories. Its advantages include ease of use, relatively high sensitivity, and the ability to noninvasively diagnose L. pneumophila serogroup 1. The UAT also has a rapid turn-around time (within hours), but this benefit is only available at the 25 percent of acute-care hospitals that conduct the test on site; otherwise, one to three days or more are required (Garrison et al., 2014; McClean et al., 2010) or sometimes longer, particularly for sites that send samples to outside laboratories. The UAT’s selectivity for L. pneumophila serogroup 1 means that patients with clinically important non-serogroup 1 L. pneumophila infections and non-pneumophila Legionella infections will be missed. Finally, as mentioned in Chapter 2, UAT results can be negative early in the disease course and are less likely to be positive with less severe disease (Mercante and Winchell, 2015). Serology is a valuable tool for epidemiologic studies, but it has little clinical impact because of the delay in receiving results (Reller, 2003). Blood samples taken three to six weeks apart are analyzed for rises in antibody titer to Legionella. In most cases of Legionnaires’ disease, a four-fold increase in anti- body titer is detected within three to four weeks although it may take longer. Thus, both sensitivity and specificity of serologic tests can be problematic. Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 97 Molecular testing for L. pneumophila consists of highly sensitive PCR and other nucleic acid ampli- fication tests. Most published studies utilize PCR testing that targets the macrophage infectivity potenti- ator (mip) surface protein of L. pneumophila (similar to the PCR tests done for environmental samples). As discussed in Chapter 2, PCR tends to detect more cases than UAT and culture tests, and it has the addi- tional advantage of being useful in patients who are already on antibiotic therapy. PCR methods can cur- rently detect L. pneumophila serogroup 1 and a few non-pneumophila species (Benitez and Winchell, 2013; Cross et al., 2016; Merault et al., 2011). Importantly, PCR for Legionella has been limited primarily to referral laboratories and research laboratories because of its difficulty, limited training, and the need for specialized instrumentation. Recently a multiplex PCR panel that includes L. pneumophila was approved by the U.S. Food and Drug Administration (FDA) for clinical use (Biofire® FilmArray® Pneumonia Panel) on sputum, endotracheal aspirates, bronchoalveolar (BAL), and mini-BAL lower-tract samples. The criteria for diagnosing legionellosis used by the CDC are given in Box 3-1. These are likely to undergo revision in 2019 (Richard Danila, Minnesota Department of Public Health, personal communi- cation, April 25, 2019). Surveillance Systems for Legionnaires’ Disease in the United States All surveillance data must be interpreted in the context of the “surveillance steps” that lead to diagnosis and reporting (see Figure 3-1). To be counted as a case, a person with legionellosis must seek medical care or be assessed as part of an outbreak. A clinical specimen (e.g., urine, respiratory) must be submitted for testing, and the specimen must be tested for the presence of Legionella. This in turn requires that the laboratory be able to identify Legionella. All cases must meet the surveillance case definition giv- en in Box 3-1. All 50 states, the District of Columbia, and U.S. territories (referred to collectively as the FIGURE 3-1 Disease surveillance steps. SOURCE: Adapted from https://www.cdc.gov/foodnet/surveillance.html. Prepublication Version - Subject to further editorial revision

98 Management of Legionella in Water Systems BOX 3-1 CDC Laboratory Criteria for Diagnosis of Legionellosis Confirmed Cases: • By culture: isolation of any Legionella organism from respiratory secretions, lung tissue, pleural fluid, or other normally sterile site • By detection of L. pneumophila serogroup 1 antigen in urine using validated reagents • By seroconversion: fourfold or greater rise in specific serum antibody titer to L. pneumophila serogroup 1 using validated reagents on specimens collected three to six weeks apart. Suspected Cases: • By seroconversion: fourfold or greater rise in antibody titer to specific species or serogroups of Legionella other than L. pneumophila serogroup 1 (e.g., L. micdadei, L. pneumophila se- rogroup 6) using validated reagents on specimens collected three to six weeks apart. • By seroconversion: fourfold or greater rise in antibody titer to multiple species of Legionella using pooled antigen and validated reagents on specimens collected three to six weeks apart. • By the detection of specific Legionella antigen or staining of the organism in respiratory secretions, lung tissue, or pleural fluid by direct fluorescent antibody (DFA) staining. • By the detection of specific Legionella antigen or staining of the organism in respiratory secretions, lung tissue, or pleural fluid by immunohistochemistry (IHC). • By detection of Legionella species by a validated nucleic acid assay (e.g., PCR). SOURCE: CDC (2010). states) require that cases diagnosed as Legionnaires’ disease or Pontiac fever be reported to local or state public health authorities. These cases are to be reported from the state to the CDC. If any step in this process does not occur, an individual ill with legionellosis will not be counted by the CDC. When cases reported through surveillance are clustered in time and space, an outbreak may be identified. As suggested in Figure 3-1, there are significant losses in numbers as one proceeds through the surveillance steps, such that the number of cases reported to the CDC is likely to be an underestimate of the true incidence of legionellosis by as much as eight- to ten-fold (Dooling et al., 2015; Mercante and Winchell, 2015; Phin et al., 2014; St-Martin et al., 2013; von Baum et al., 2008). Two national surveillance systems maintained at the CDC have the capacity to collect information on all diagnosed cases of legionellosis from states. These are the National Notifiable Disease Surveillance System (NNDSS) and the Supplemental Legionnaires’ Disease Surveillance System (SLDSS). Separately, CDC has regulatory authority over the cruise ship industry, which must report all cases of Legionnaires’ disease to the CDC. National Notifiable Disease Surveillance System Since the disease’s recognition in 1976, surveillance for legionellosis has been conducted by all states, the District of Columbia, and U.S. territories. Reporting is mandatory for all diagnosed cases of Legionnaires’ disease and Pontiac fever by healthcare providers and clinical laboratories to local and state health officials; cases must be reported within a short time period from diagnosis, usually within one to seven days. Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 99 All cases of notifiable diseases are then reported voluntarily to CDC from public health officials in states through the National Notifiable Disease Surveillance System (NNDSS). Historically, notifiable diseases have been reported weekly, and the CDC has published preliminary case counts weekly. How- ever, legionellosis reports are often sent to the CDC at irregular and sometimes lengthy intervals, such that the weekly counts may be low and the preliminary statistics for legionellosis often incomplete. Data shared on cases through this system are primarily demographic (e.g., place of residence) and clinical (e.g., date of onset of illness). Environmental source information, including the setting (e.g., hospital, hotel), type of water system (e.g., hot tub, decorative fountain), and type of water exposure (e.g., potable water, recreational untreated water) are not collected by the NNDSS. The NNDSS does not provide informa- tion on whether a case is travel-associated, healthcare-associated, or community-acquired. Supplemental Legionnaires’ Disease Surveillance System A Supplemental Legionnaires’ Disease Surveillance System (SLDSS) is available at the CDC to collect more comprehensive data on Legionnaires’ disease cases from all states. The SLDSS includes po- tential environmental exposures, such as whether a case is travel-associated or whether an individual had exposure to hot tubs, respiratory therapy equipment, or a healthcare or senior-living facility. However, these data are often incomplete and not timely, and they frequently do not identify the potential environ- mental source of exposure. Therefore, these data have been insufficient to track trends in community-ac- quired, travel-associated, or healthcare-acquired cases (Cynthia Whitney, CDC, verbal communication, March 21, 2018). In 2018, the CDC published the first surveillance summary focused on Legionnaires’ disease using NNDSS and SLDSS data from 2014 and 2015, analyzing for associations with healthcare facilities, se- nior- or assisted-living facilities, and travel (CDC, 2018a). Future summaries are planned with the goal of better understanding the burden, impact, and trends of Legionnaires’ disease over time. Critique of National Surveillance and Next Steps Given the loss of cases associated with each step in Figure 3-1, it is no surprise that the NNDSS and SLDSS do not account for most patients with legionellosis. In contrast to the steps leading to diagnosis, however, the reporting step itself is quite complete. In a 2011–2015 study conducted through the Active Bacterial Core Surveillance System to find all laboratory-confirmed cases of legionellosis, almost all cases found in the study had been previously reported through the NNDSS (Dooling et al., 2015). Having two separate surveillance systems has been problematic, and the CDC plans to address the issue. The CDC is currently integrating the NNDSS and SLDSS through the NNDSS Modernization Initiative (Sam Posner, CDC, personal communication, September 21, 2018), a CDC-wide initiative de- signed to enhance the system’s capabilities to provide more comprehensive, timely, and higher quality data. Case information that historically was sent through multiple routes will be consolidated into a single data stream. Surveillance has been frequently referred to as “data for action,” yet neither the NNDSS nor the SLDSS is robust for this purpose because states have not routinely investigated single cases for source(s) of exposure. Better understanding the source of environmental exposure could lead to improved pre- vention and control measures. Acknowledging that environmental investigation of every case is unlikely to occur because such investigations are resource intensive, more in-depth studies will be necessary to Prepublication Version - Subject to further editorial revision

100 Management of Legionella in Water Systems investigate a subset of cases by setting, source of water (e.g., potable water supply, cooling tower), and building water system for potential environmental exposure. For decades, legionellosis programs both in states and at the CDC have been given low priority compared to other preventable infectious diseases, including communicable respiratory conditions. Fur- thermore, because the programs were initially focused on outbreak detection and control, the CDC and other public health agencies did not build expertise and capacity in fields that are needed to understand legionellosis prevention and control (e.g., building water systems, environmental engineering, and indus- trial hygiene). Legionellosis surveillance has not had dedicated resources to ensure timely environmen- tal investigation of cases. Many state public health laboratories do not have the resources to identify, quantify, or subtype Legionella in water specimens; only three states have capacity to perform genome sequencing (Richard Danila, Minnesota Department of Health, email communication, September 29, 2018). CDC has recently devoted resources to legionellosis in some states through its Epidemiology and Laboratory Capacity cooperative agreements. These include Arizona, California, Colorado, Geor- gia, Illinois, Los Angeles County, Maryland, Michigan, Minnesota, Nebraska, Nevada, New York City and State, Ohio, Philadelphia, Tennessee, Utah, Virginia, Washington, DC, and Washington State. Some agreements have focused on getting public health laboratories, environmental health experts, and epide- miologists working together; others emphasize locating, registering, and testing cooling towers, whereas others focus on hotels; still others prioritize better cluster detection (Richard Danila, Minnesota Depart- ment of Health, personal communication, July 23, 2018). More efforts like these cooperative agreements are needed to help state and local health departments build their capacity for Legionella surveillance and response. New York City provides one of the most comprehensive legionellosis surveillance systems in the United States (see Box 3-2). With respect to travel-associated cases, the Council of State and Territorial Epidemiologists (CSTE) has stated that surveillance for legionellosis lacks the timeliness and sensitivity necessary to detect out- breaks of these cases (CSTE, 2005). CDC is uniquely positioned to identify connections between cases that occur in residents of different jurisdictions, which is most likely with travel-associated outbreaks. It is particularly important that travel-associated cases be reported by the states to the CDC in almost real time to prevent delays in investigation and control. Following the 2005 CSTE position statement, CDC instituted a dedicated email address to improve reporting of travel-associated cases. Europe has a more extensive reporting system for travel-associated cases, discussed later in this chapter. Academic centers currently play little, if any, role in either building or assessing prevention and control efforts for legionellosis. If the CDC chose to take a much more comprehensive approach to legio- nellosis, both the Integrated Food Safety Centers of Excellence and the Regional Centers of Excellence in Vector-Borne Diseases could serve as models. Under the Food Safety Modernization Act of 2011, the CDC designated six Integrated Food Safety Centers of Excellence at state health departments and affiliated university partners not only to identify and implement best practices in foodborne disease sur- veillance and outbreak response, but also to serve as a resource for other state, regional, and local public health professionals1. In 2017, five universities were established as regional Centers of Excellence to help prevent and rapidly respond to emerging vector-borne diseases across the United States. The goals of these centers include building effective collaboration between academic communities and public health organizations at federal, state, and local levels for surveillance, prevention, and response; training public health experts in the knowledge and skills required to address vector-borne disease concerns; and con- ducting applied research to develop and validate effective prevention and control tools and methods and to anticipate and respond to disease outbreaks. 1 See https://www.cdc.gov/foodsafety/centers/index.html, accessed March 9, 2019. Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 101 BOX 3-2 Legionellosis Surveillance Data Summary, New York City, 2007–2017 Surveillance Methods New York City (NYC) legionellosis surveillance data are comprised of reported positive Le- gionella clinical laboratory test results, clinical patient information, and patient exposure information obtained through patient interview. The NYC Health Code mandates that positive Legionella clinical laboratory test results be reported to the NYC Department of Health and Mental Hygiene (DOHMH). Electronic laboratory reports are sent to the NYC DOHMH Bureau of Communicable Disease through the Electronic Clinical Laboratory Reporting System. For each reported positive Legionella clinical laboratory test (urinary antigen test, culture, PCR, or paired serology) the NYC DOHMH conducts: (1) a medical record review using a standardized data abstraction tool; and (2) a standardized 11-page telephone or in-person interview of the patient or their next-of-kin. The healthcare facility’s medical records include chest x-ray and computed tomography (CT) scan results, along with the recorded history of the patient’s clinical symptoms and medical treatment. The patient interview collects infor- mation on the patient’s home, work, and other addresses, presenting symptoms, and health history, along with information on known water exposures, travel, and healthcare visits during the ten days before onset of symptoms (the typical disease incubation period). Information gathered from these sources is used to determine if the patient’s illness meets the case definition of a confirmed or pos- sible case of legionellosis, and to assess if there are possible exposure sources or locations that re- quire further investigation, based on the occurrence of legionellosis among other people who shared those possible exposures. Results of Trends in Reported Legionellosis Cases, NYC, 2007-2017 As shown in Figure 3-2-1, from 2007 to 2017 rates of legionellosis increased for both men and women, and in all age groups. Legionellosis cases occurred more frequently among men (62 per- cent) than women (38 percent). The majority (69 percent) of patients diagnosed with legionellosis were adults aged 55 years or older. Rates of legionellosis increased for all racial groups, with the highest rate of increase among the non-Hispanic Black/African American population. Thirty nine per- cent of all cases occurred in people who identified as non-Hispanic Black/African American (approx- imately 22 percent of New Yorkers are non-Hispanic Black/African American). Rates of legionellosis increased in all five NYC boroughs. The largest number of legionellosis cases (n = 472, 32 percent of all cases) occurred in the Bronx, home to about 17 percent of the NYC population. Rates of legio- nellosis increased in neighborhoods of all income levels. The highest rates and the greatest rate of increase occurred in very high poverty neighborhoods. FIGURE 3-2-1 Trends in Legionnaires’ disease rates per 100,000 people. Prepublication Version - Subject to further editorial revision

102 Management of Legionella in Water Systems Results from Medical Record and Patient Interview Data, NYC, 2013-2017 Health Conditions and Behaviors. The majority of legionellosis patients (72 percent) re- ported at least one chronic health condition. The most common conditions reported were diabetes (24 percent) and lung disease (19 percent). About half (45 percent) of patients reported a history of current or past tobacco smoking. Exposure Settings. About 8 percent of legionellosis cases were definite healthcare-associ- ated,1 while about 4 percent were possible healthcare-associated.2 About 9 percent of legionellosis patients reported traveling outside of NYC for at least one day during their ten-day disease incuba- tion period. Among people diagnosed with legionellosis, 23 percent reported working during their incubation period. Reported Water Exposures or Changes to Water Service. The following possible water ex- posures were reported by legionellosis patients as occurring during the ten-day disease incubation period: air humidifier, 1 percent; hot tub, 1 percent; swimming pool, 1 percent; decorative fountain, 2 percent; gym, 2 percent; respiratory equipment, 5 percent; shower outside home, 5 percent; grocery store, 10 percent. In 5 percent of cases, patients reported plumbing maintenance at the residence during the ten-day disease incubation period, and in 5 percent of the cases, patients reported a wa- ter service disruption. Clinical Diagnostic Testing The majority (90 percent) of legionellosis cases were diagnosed by Legionella urinary antigen test only. Ten (10) percent of legionellosis cases included an isolate from a clinical culture that could undergo molecular analysis for comparison to isolates from possible environmental sources. Conclusions From 2007 to 2017, legionellosis in NYC occurred at the highest rates among those who were aged 55 years and older, in neighborhoods with the highest poverty rates, and among those who identified as non-Hispanic Black/African American. From 2013 to 2017, the majority of people di- agnosed with legionellosis in NYC had chronic conditions or health behaviors that are reported risk factors for developing legionellosis, including diabetes, chronic lung disease, and tobacco smoking. Data from patient interviews and medical record reviews point to the challenges involved in using surveillance data to identify a source for individual cases of legionellosis that are not part of a clus- ter: nearly 90 percent of cases were community-associated, where numerous exposures to aerosols of water may occur during the ten-day disease incubation period. Only a very small proportion of people recall specific water exposures during their ten-day disease incubation periods. Conversely, many people may experience unrecognized aerosol exposures during that time, from cooling towers and other sources. These patient histories offered little guidance for testing possible environmental sources for individual cases of legionellosis. Only about 10 percent of cases included a clinical isolate that can undergo molecular analysis for comparison to isolates from possible environmental sources. Thus, NYC’s experience suggests that even if local and state health departments had budgetary and personnel capacity to test any and all possible environmental exposures for each individual case of legionellosis, source attribution would be possible, at best, for only about 10 percent of cases. These data indicate that any rigorous effort to better understand the sources of exposure that cause individual legionellosis cases will require well-funded, coordinated studies involving medical centers, laboratories, and health departments in areas with capacity for the consistent collection and cultivation of both clinical and environmental Legionella cultures for a substantial proportion of cas- es. This is resource intensive because most sporadic cases involve multiple possible environmental sources of Legionella exposure, and environmental isolates that do not match clinical isolates may still require on-going public health follow-up when they indicate possible disease risk from a potential environmental source. 1 Patient spent all of the ten-day disease incubation period in an acute-care hospital or nursing home. 2 Patient spent some portion of the ten-day disease incubation period in an acute-care hospital or nursing home. Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 103 U.S. Department of Veterans Affairs Surveillance System In addition to the national systems, the Veterans Health Administration (VHA) collects informa- tion on all cases of legionellosis within its healthcare system. The VHA operates the largest integrated healthcare system in the United States, with more than 1,200 sites of care, serving about 6 million vet- erans annually. In federal fiscal year (FY) 2016, 91 percent of veterans using VHA benefits were male, with a median age of 64 years and with higher morbidity than in the rest of the United States (Gamage et al., 2018), which as discussed in Chapter 2 are populations with an increased risk of contracting Le- gionnaires’ disease. As discussed in detail in Chapter 5, the VHA has a Legionella prevention policy for medical facilities to limit Legionella growth in building water systems, requiring the collection of both en- vironmental and clinical data. Concomitant to publication of the policy in 2014, the VHA Central Office implemented a national standardized Legionnaires’ disease reporting system. Compared to the CDC’s notifiable disease reporting system, the VHA collects more detailed information on each case, partly to assess if a person was exposed while inside a VHA facility. As more environmental data are collected throughout the VHA system, the surveillance system will become critical for evaluating the effectiveness of the VHA’s legionellosis prevention policies and also provide useful information for public health agen- cies and other healthcare facilities. Waterborne Disease Outbreak Reporting System of the National Outbreak Reporting System A third U.S. national surveillance system—the National Outbreak Reporting System or NORS—is also maintained by the CDC and collects data on waterborne and foodborne disease outbreaks in the Unit- ed States. CDC categorizes the sources of waterborne disease outbreaks as follows: (1) drinking water, (2) treated recreational water, (3) untreated recreational water, and (4) another environmental exposure or undetermined source. Legionella was added to this system in 2001. Data from this system are currently publicly available on the NORS dashboard;2 one can sort outbreaks by etiologic agent, year, state, setting (e.g., hotel, trailer park, hospital), water exposure (see above), and type of water system (e.g., hot tub, dec- orative fountain, cooling tower). NORS does not include detailed information on the setting and type of water system, which would be particularly useful for improving understanding of sources and conditions conducive to transmitting legionellosis. The waterborne disease outbreak reporting system was initiated in 1971 as a partnership between CDC, CSTE, and the U.S. Environmental Protection Agency (EPA). It is dependent on public health depart- ments in individual states to voluntarily provide complete and accurate data for waterborne disease out- breaks. The waterborne disease outbreak reporting system is important because outbreaks are most likely to be investigated for environmental sources. A limitation of the NORS program for legionellosis is that the database (and hence the categories of setting, water types, and water exposure) was developed for enteric pathogens, making it less useful for pathogens capable of growth in water systems and transmitted by aerosolized water. Also, NORS data for legionellosis are not updated frequently; until December 2018, only data through 2014 were available. 2 See www.cdc.gov/norsdashboard. Prepublication Version - Subject to further editorial revision

104 Management of Legionella in Water Systems European Surveillance In most European countries, laboratory-confirmed Legionnaires’ disease cases must be reported to the public health authorities of the country (e.g., in Germany, reporting is mandatory to national authori- ties within 24 hours of diagnosis). Most countries of the European Union report annually to the European Centers for Disease Control (ECDC) through the European Legionnaires’ Disease Surveillance Network (ELDSNet) (Lara Payne, ECDC, personal communication, October 6, 2018). In 2017, 30 countries partici- pated in ELDSNet. Members of this network review relevant technical documents and assist ECDC in or- ganizing an annual meeting. ELDSNet collaborates with partners, such as the World Health Organization (WHO), public health authorities of non-EU countries, and tour operators. The incidence of Legionnaires’ disease in Europe ranges widely among countries, which may largely reflect the variability in diagnosis and reporting. The burden of disease and trends are analyzed and reported in a detailed annual surveillance summary dedicated to Legionnaires’ disease (e.g., ECDC, 2019). Considerable focus of ELDSNet has been on travel-associated Legionnaires’ disease, which accounts for approximately 20 percent of cases. (The European definition of travel-associated is more restrictive than in the United States and requires a stay in an overnight accommodation in the ten days before symp- tom onset.) The operating procedures of the surveillance scheme for travel-associated Legionnaires’ dis- ease in the EU and European Economic Areas (EEA) were updated in December 2017 (ECDC, 2017a), such that these cases are reported in almost real-time. In 2015, the estimated median delay between onset of illness and report to ELDSNet was only 17 days. When a cluster is identified within an EU/EEA country, all participating countries are notified and the public health authorities where the accommodation site is located are expected to report on the investigations conducted on the accommodation site. If the timeline for reporting is not fulfilled or control measures are deemed unsatisfactory by the ECDC, the name of the accommodation site is published on the ECDC website and the International Federation of Tour Operators is notified. Trends in Reported Legionellosis in the United States From 2007 to 2017, the rate of reported legionellosis cases through the NNDSS increased from 0.91 cases to 2.29 cases/100,000 persons, with more than 7,400 cases reported in 2017. Although case reporting is officially for legionellosis, 98 percent of the case reports represent individuals hospitalized with pneumo- nia (Dooling et al., 2015). Therefore, the trends primarily reflect more severe cases of Legionnaires’ disease. It is likely that trends in treatment of outpatients with Legionnaires’ disease and Pontiac fever follow trends similar to the hospitalization data. Reported rates of legionellosis are lower in some areas of the United States (e.g., the West) than other areas. But for all areas of the country, the rates have increased from 2005 to 2015 (see Figure 3-2; Cooley, 2018). Weather patterns likely contribute to geographic differences, with warm, humid weather increasing Legionnaires’ disease risk. Population and building density as well as regional differences in water treat- ment could also be playing a role. In the United States, seasonal trends are evident, with cases rising in late spring, increasing in the summer, and peaking in late summer and fall. In 2016, 78 percent of cases were reported for the seven months of June through December. The lowest months are generally January through April. As with other variables, for all months from 2007 to 2016, the trend in incidence is generally upward. Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 105 A B C FIGURE 3-2 Rates of reported legionellosis cases by state for 2005 (A), 2010 (B), and 2015 (C). Values are cases per 100,000 population. SOURCE: Cooley (2018). After leveling off or decreasing from 2007 to 2010, European case rates have increased from 1.0 to 1.8 cases/100,000 persons from 2011 to 2017 (see Figure 3-3), with the majority of cases (69 per- cent) reported from France, Germany, Italy and Spain. Australia has also noted increases in cases of L. pneumophila between 2005 and 2014 but not of Legionella longbeacheae. L. longbeacheae disease is rarely reported in the Unites States. Figure 3-3 shows that European rates are slowing relative to those of the Unites States, with the U.S. rate superseding the European rate since 2011. Legionellosis cases can be subdivided into various categories. For example, cases may be recog- nized as part of an outbreak, a term used to describe two or more people with Legionnaires’ disease ex- posed to Legionella at the same place at about the same time. Cases not recognized as part of an outbreak Prepublication Version - Subject to further editorial revision

106 Management of Legionella in Water Systems FIGURE 3-3 U.S. and European trends in Legionnaires’ disease rate (number per 100,000 people). SOURCES: 2013–2017 European data from ECDC (2019); 2012 European data from ECDC (2018); 2011 Euro- pean data from ECDC (2017b); 2008–2010 European data from ECDC (2016); 2006–2007 European data from ECDC (2014); 2000–2009 U.S. data from Hicks et al. (2011); 2010–2015 U.S. data estimated by the Committee from https://www.cdc.gov/legionella/qa-media.html; 2016 U.S. data from CDC (2017b); 2017 U.S. data from CDC (2018b). are considered sporadic. In the United States, waterborne disease outbreaks in the NORS system are subdivided into whether the outbreak source was identified as potable water, recreational water (treated or untreated), or another water source. Frequently, cases are also categorized as either “healthcare-associated,” “travel-associated,” or “community-acquired.” “Definite” healthcare-associated cases are defined as patients that stayed over- night in a healthcare facility (e.g., a hospital or long-term care facility) for the entire ten days before symptom onset, while “possible” cases are defined as patients with exposure to a healthcare facility for a portion of the ten days preceding symptom onset (CDC, 2018a). Travel-associated cases must have a history of spending at least one night away from home, either domestically or abroad, in the ten days before symptom onset (CSTE, 2005). Cases are designated as community acquired when the patient did not spend at least one night away from home in the ten days before onset of illness or was not exposed to a healthcare facility in the ten days before onset of symptoms. Various categorizations are used below to parse occurrence data in the Unites States. Healthcare-Associated Cases Healthcare-associated cases of Legionnaires’ disease make up approximately 20 percent of all le- gionellosis cases reported in the United States. In 2015, among 21 jurisdictions that reported exposure information on more than 90 percent of cases through the SLDSS, 3 percent of cases were considered “definite” and 17 percent had “possible” exposure to a healthcare facility in the ten days before symptom onset (Soda et al., 2017). Of the definite cases, 80 percent were associated with long-term care facilities, Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 107 18 percent with hospitals, and 2 percent with both. In addition, 3 percent were associated with assist- ed- or senior-living facilities (CDC, 2018a). An analysis of case reports to the ECDC between 2011 and 2015 reported 7.3 percent as healthcare-related, 4.9 percent of cases as nosocomial (i.e., from a hospital specifically) and 2.4 percent as “other” healthcare-related cases (Beauté, 2017). Data from the VHA between 2014 and 2016 show that the rate of Legionnaires’ disease signifi- cantly increased among veterans receiving VHA healthcare services but with no exposure to a VHA healthcare facility during the disease incubation period (from 0.9 to 1.47/100,000 enrollees). The rate of Legionnaires’ disease among those with an overnight stay at a VHA facility during the disease incubation period significantly decreased (from 5.0 to 2.3/100,000 enrollees with an overnight stay). Most “definite” cases of healthcare-associated Legionnaires’ disease (11 of 13) were in long-term care VHA facilities (Gamage et al., 2018). Travel-Associated Cases The CDC has reported data on travel-associated Legionnaires’ disease from a limited number of jurisdictions. Benin (2002) found that 20 percent of Legionnaires’ disease cases were reported as possibly travel-associated between 1980 and 1998. From 2005 to 2006, 24 percent of cases reported through the SLDSS were possibly travel-associated (Smith et al., 2007). In Europe, 20 percent of Legionnaires’ disease cases reported between 2011 and 2015 were trav- el-associated (Beauté, 2017). ECDC’s case definition for travel-associated cases includes only lodging in a commercial establishment (e.g., hotel, resort), which is a more restrictive definition than the U.S. defini- tion, in which any night away from home during the incubation period was reported as travel-associated. Nonetheless, data on travel-associated cases in the United States are similar to European data. Box 3-3 discusses Legionnaires’ disease rates for cruise ships, which have plateaued. Hotels and other commercial accommodation sites have been clearly documented to be an important source of en- vironmental exposure to Legionella. BOX 3-3 Cruise Ship Industry: Legionnaires’ Disease Prevention and Control Efforts, 2007–2017 Despite a 21 percent increase in passengers and a marked increase in Legionnaires’ dis- ease outbreaks in the United States in the past decade, there was no significant increase in cruise ship associated outbreaks reported to the CDC in the five-year period from 2007 to 2011 (11 outbreaks) compared to 2012 to 2016 (12 outbreaks). In 2017, there were two cruise ship outbreaks (Sam Posner, CDC, email communication, April 17, 2019). These data suggest that prevention measures taken by the cruise ship industry appear to have been at least partially effective in addressing the threat of Legionnaires’ disease outbreaks associated with cruise trav- el. Many in the cruise ship industry have engaged Legionella consultants to assure safety of their water supply and have conducted routine environmental sampling for Legionella, including quantitative culturing. Of particular note, CDC has regulatory authority over vessel sanitation and has provided guidance to cruise ships for Legionnaires’ disease prevention for more than 20 years. The guidance and inspections of hot tubs and other potential environmental sources by the CDC’s Vessel Sanitation Program (VSP) and the adverse publicity and liability associated with outbreaks investigated by CDC using its regulatory authority may have contributed to the attentiveness of cruise lines to maintenance of their water operations. The VSP 2011 Operations Manual and updates are available at http://www.cdc.gov/nceh/vsp. Prepublication Version - Subject to further editorial revision

108 Management of Legionella in Water Systems Community-Acquired Cases Most Legionnaires’ disease cases in the United States are considered to be community-acquired (either sporadic or as part of an outbreak). This is consistent with what is found in Europe, where 70 percent of Legionnaires’ disease cases reported to ELDSNet between 2011 and 2015 were communi- ty-acquired (Beauté, 2017). Similarly, the Robert Koch Institute (2013, 2015) estimated that about 70 percent of reported legionellosis cases are neither related to an outbreak nor nosocomial, but rather acquired in private or professional surroundings. Unfortunately, most of the information on community-acquired cases in the United States comes from outbreak investigations or from the many publications on individual outbreaks. The most com- prehensive review of sporadic, community-acquired cases (Orkis, 2018) included 47 articles on sporadic cases (excluding healthcare- and outbreak-associated cases) in which a total of 28 environmental sources were identified. Potable water from single family homes, large building water systems, and car travel appeared to contribute to a substantial proportion of the sporadic Legionnaires’ disease cases. Cooling towers were also noted to be a potentially significant source. The difficulty in source attribution was noted, with definitive links using molecular typing between environmental sources and clinical isolates being made in only eight cases. The authors noted that understanding the risk magnitude of potential sources would make future public health investigations more efficient and enhance prevention efforts. den Boer (2015) performed source investigations on more than 75 percent of 1,991 patients with Legionnaires’ disease between 2002 and 2012 (source investigations were only done for clusters of dis- ease after 2006). The paper noted the difficulty and the resource intensity of investigations to locate with certainty the source of an infection, and it reported outcomes of investigations of sporadic cases together with outcomes of cluster investigations. Of the 1,484 source investigations performed, only 367 (24.7 percent) of the sources were positive for Legionella spp., and only 41 patients (2.3 percent) were found to have a clinical strain that matched the environmental source. The sources that matched included a healthcare setting (40 percent), residence (18 percent), industrial complex (8 percent), swimming pool (5 percent), wellness center (8 percent), hotel (5 percent), spa (5 percent), and car wash (3 percent). The study also examined 105 clusters associated with 266 patients based on location and geography: 26 percent of the clusters were associated with garden centers, 16 percent with healthcare facilities, 10 percent with a residence, 9 percent with wellness centers (e.g., spas, saunas), 7 percent with hotels, 5 percent with cooling towers, and 5 percent with holiday parks. Che and colleagues (2003) reported an increased risk of sporadic cases of community-acquired Legionnaires’ disease in industrial areas of France. They evaluated 880 cases from 1998 to 2000 that were not associated with an outbreak and in which individuals did not report an overnight hospital stay or traveling within ten days of disease onset. Seventy-nine percent of the cases were caused by L. pneumophila serogroup 1. A higher risk was reported in areas with exposure to aerosols and plumes of smoke, with the greatest risk being in areas with more than one industrial exposure. However, the results are inconclusive and the findings deserve further study. A study by the New York City (NYC) Department of Health and Mental Hygiene looked at the potential role of occupation among 335 community-acquired cases. Compared with the general popu- lation, legionellosis case-patients who were working in the two weeks before diagnosis were significant- ly more likely to work in transportation, repair, protective services; cleaning services; or construction (Farnham et al., 2014). Community-acquired cases are commonly attributed to private water systems, under the assump- tion that the small number of people exposed would not draw the attention of epidemiologists to in- vestigate. For example, Bonilla Escobar et al. (2014) demonstrated that a healthy, immunocompetent Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 109 young person with no other risk factors contracted Legionnaires’ disease from an improperly maintained household humidifier, but no conclusions were drawn about the frequency of humidifiers being sources of Legionella infections. In another case study, two unrelated individuals appeared to contract Legion- naires’ disease in their homes and both had solar water heaters with inadequately heated water (Erdogan and Arslan, 2016). Currently, it is largely unknown how often private water sources, particularly in individual homes, are the environmental exposure source for sporadic cases. Outbreak Data That Reveal Environmental Sources Most legionellosis outbreaks are detected through analysis of surveillance data compiled through the mandatory reporting systems described above. As discussed previously, and unlike the surveillance data reported through NNDSS or SSLDS, NORS data (now available from 2009 to 2017) are examined by water type, i.e., whether the outbreak is associated with drinking water, treated or untreated recre- ational water, or another water system. During 2013 to 2014, 19 states reported 42 outbreaks associated with drinking water; Legionella was implicated in 57 percent of the outbreaks (see Figure 3-4), 13 percent of the cases, 88 percent of the hospitalizations, and all 13 deaths3. From 2000 to 2014, NORS reported 363 outbreaks associated with treated recreational water that had a confirmed infectious etiology; 16 percent were caused by Legionella and legionellosis was confirmed or suspected to be responsible for all eight deaths (Hlavsa, 2018). During 2013 to 2014, 15 outbreaks were associated with “another” envi- ronmental exposure to water; Legionella was responsible for 63 percent of the outbreaks, 94 percent of hospitalizations, and all 17 deaths (McClung et al., 2017). Finally, 11 of 12 outbreaks associated with an undetermined exposure to water were caused by Legionella (McClung et al., 2017). 3 See www.cdc.gov/healthywater/surveillance/drinking-water-tables-figures/html. FIGURE 3-4 NORS reported drinking water-associated disease outbreaks, 2013–2014 (n=42). SOURCE: CDC (2017). Prepublication Version - Subject to further editorial revision

110 Management of Legionella in Water Systems Unfortunately, published analyses of NORS data generally do not reveal the setting (e.g., hotel, hospital) or water exposure (e.g., spa, decorative fountain), although some of the data are available and could be stratified for further analysis. The Committee analyzed NORS data between 2009 and 2017, during which 290 legionellosis outbreaks were reported. A substantial percentage of cases were asso- ciated with hotels and healthcare facilities. Other implicated locales included long-term care facilities, assisted-living or rehabilitation facilities, apartment buildings, indoor workplaces, factories or industrial settings, and prisons. Within those settings, cooling towers, hot tubs, and ornamental fountains were im- plicated. The goal of this cursory analysis is to raise awareness of the data available via the NORS dash- board that could be analyzed to determine environmental exposures associated with legionellosis cases. Garrison and colleagues (2016) analyzed data from 27 building-associated Legionnaires’ disease outbreaks (2000–2014) that were investigated by the CDC between 2000 and 2014. Common exposure settings were hotels (44 percent), long-term care facilities (19 percent), and hospitals (15 percent). Com- mon sources (within the settings) were found to be showers and faucets (56 percent), cooling towers (22 percent), hot tubs (7 percent), decorative fountains (4 percent), and industrial equipment (4 percent). By reviewing the peer-reviewed literature and government documents published between 2006 and 2017, Hamilton and colleagues (2018a) identified 119 legionellosis outbreaks globally for which an environmental source was associated with the event. Potable water was identified as the source in 42 outbreaks (30 percent), although this was not subdivided to better understand whether a specific water system or fixture deficiency was the culprit. Cooling towers, air conditioning, or evaporative condens- ers were identified in 41 outbreaks (30 percent). Cooling towers were associated with 50 percent of the confirmed cases of legionellosis and the greatest number of fatalities. Fifteen (15) percent of outbreaks occurred at hotels. One of the world’s largest outbreaks of Legionnaires’ disease was linked to a hot tub exhibited at a Dutch flower show (den Boer et al., 2002). Simply pausing at the hot tub was deemed the most important risk factor for infection, confirming that a contaminated hot tub, even if not used directly, can cause illness in susceptible people. Of particular importance is the potential role of municipal water systems. In Flint, Michigan the governor’s task force concluded that the management of the Flint River-sourced water supply may have contributed to the outbreaks of legionellosis in 2014 and 2015 in Genesee County (Flint Water Advisory Task Force, 2016), and scientific studies identified aspects of the water that were conducive to Legionella proliferation (Rhoads et al., 2017; Zahran et al., 2018). Outbreaks have also been attributed to wastewater treatment plants (Kusnetsov, 2010; Loenenbach et al., 2018). The investigation of a large outbreak of Legionnaires’ disease in NYC in 2015 illustrates how a multi-disciplinary approach to outbreak detection and subsequent investigation can lead to successful control (Box 3-4, Chamberlain, 2017). This investigation is unique in its scope, timeliness, and the extent to which clinical and environmental data were paired to determine the source of the Legionella. It also illustrates the resource intensity and difficulty of investigations of Legionnaires’ disease outbreaks. Why Are Rates of Legionnaires’ Disease Increasing? Although often put forward as potential explanations for the increase in Legionnaires’ disease in- cidence, neither improved reporting nor improved diagnosis are supported by available data as a major contributor to the rapid increase since 2000. Indeed, reporting of diagnosed cases was documented to be extremely high for the period 2011 to 2013 (Dooling et al., 2015). Currently there are very limited data available to assess the role of diagnostic testing in increased incidence. Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 111 BOX 3-4 2015 Legionnaires’ Disease Outbreak Investigation, Bronx, New York In July 2015, the New York City Department of Health and Mental Hygiene (DOHMH) detect- ed an increase in cases of Legionnaires’ disease in the South Bronx, using the surveillance system described in Box 3-2. The purpose of the investigation was to describe patient demographic char- acteristics and comorbidities, identify environmental exposures, and implement control measures. Reporting and Case Follow-up Physicians and clinical laboratories are required to report positive Legionella test results to DOHMH. For each case reported, epidemiologists review patient medical records, interview the patient (or the patient’s proxy) to determine if the report meets the CSTE/CDC national case defi- nition for legionellosis, and identify risk factors and potential exposures. Cluster Identification Analyses Two methods are used to identify clusters that could be community outbreaks of report- able diseases. Each week, the historical limits method compares case volume in the most recent four-week period with comparable data from the previous five years at the city, borough, and neighborhood levels. A separate daily spatiotemporal cluster detection method is based on the space-time permutation scan statistic, and computes a ‘‘recurrence interval,’’ which is the number of days of surveillance required for the expected number of clusters at least as unusual as the observed cluster to be equal to 1 by chance. Additionally, an automated daily algorithm compares the building identification number (i.e., a unique code for every structure in New York City) as- signed to the patient’s address with a list of health care and congregate living facilities to identify concerning events not already detected by epidemiologists. To guide environmental sampling, a multi-focused cluster test with the space-time permuta- tion scan statistic was used to assess clustering of cases of Legionnaires’ disease around cooling towers. Case Definitions An outbreak-associated case of Legionnaires’ disease was defined as clinically compatible illness meeting the national case definition for Legionnaires’ disease (Box 3-1), modified to include L. pneumophila serogroup 1 (Lp1) DNA detected by quantitative PCR (qPCR) in postmortem spec- imens, in either a resident of one of seven South Bronx ZIP codes (i.e., the outbreak zone) or in a person who worked in or visited the outbreak zone during the ten days before his or her symptom onset date (or collection date of the earliest confirmatory test if onset date was unknown) between July 2, 2015, and August 3, 2015. Legionella subtyping, as described hereinafter, was used to refine the case definition. Deaths from Legionnaires’ disease were defined as (1) patients meeting the case definition whose death was attributed to Legionnaires’ disease within 30 days of the diagnosis date, or (2) patients meeting the outbreak definition in which the Office of Chief Medical Examiner listed Le- gionella pneumonia as the immediate cause of death. Analyses of Patient Characteristics The patient demographic and clinical characteristics were summarized and adjusted odds ra- tios (aORs) and 95 percent confidence intervals (CIs) were calculated using multivariable logistic regression and the mid-P exact method to assess the relationship between fatality and comorbid- ities, smoking status, and number of days from onset to diagnosis. Odds ratios were adjusted for age and sex. Prepublication Version - Subject to further editorial revision

112 Management of Legionella in Water Systems Environmental Monitoring Cooling tower sampling in the outbreak zone was prioritized per the location of patients with Legionnaires’ disease and the multi-focused cluster test. Although the city had no complete official registry of cooling towers at the time, cooling towers in the area were identified by examining city records of water credit and construction permit applications, in addition to publicly available satel- lite imagery. The sampled locations in cooling towers were thought to be most representative of the water aerosol generated. If the cooling tower basin was safe to access, a swab of biofilm was collected. Methods. The New York State Department of Health Wadsworth Center and the New York City Public Health Laboratory tested cooling tower water samples for the presence of Legionella us- ing PCR and culture methods. Use of PCR allowed for the rapid screening of samples to prioritize culture and cooling tower remediation. Samples in which L. pneumophila DNA was detected were processed and cultured at the Public Health Laboratory with standard microbiological methods. Isolates were identified as Lp1 through direct fluorescent antibody staining. Pulsed-field gel elec- trophoresis subtyping was performed at the Public Health Laboratory and Wadsworth Center with identical methods. Epidemiologic Results In total, 138 patients met the outbreak case definition of outbreak-associated Legionnaires’ disease, and 128 (93 percent) were hospitalized. Illness onset peaked on July 26, 2015, and the last patient linked to the outbreak became ill on August 3, 2015. Sixteen (12 percent) patients died, five in their homes. A total of 108 patients (78 percent) resided in the outbreak zone. Of the remain- ing 30 patients, 16 resided in other Bronx ZIP codes, nine in other New York City counties, two in other New York State counties, and three in other states. Several events led the investigation to one potential cooling tower source. On July 28, 2015, DOHMH received a physician inquiry about a cluster of respiratory illnesses among residents of a supportive housing residence for people with medical needs, including HIV infection, and the build- ing identification number analysis identified two reports of Legionnaires’ disease from this building. On July 29, 2015, the CDC notified DOHMH of a traveler who had been diagnosed with Legion- naires’ disease and had spent part of the incubation period at a hotel in the South Bronx (Building A). Building A was located less than a block away from the supportive housing residence, and the cooling tower, which was not previously known to city agencies, was detected through satellite imagery. The multi-focused cluster test identified unusual case clustering of Legionnaires’ disease cases around Building A, with a recurrence interval of 1.36 million years. Environmental Results The environmental investigation began on July 28, 2015. During the next three weeks, 55 cooling towers from 46 buildings in the outbreak zone were identified, inspected, and sampled. PCR results were available within 24 to 36 hours. Lp1 DNA was detected by qPCR in water samples from 21 cooling towers and successfully cultured from 14. An order to immediately remediate was issued to owners of cooling towers that tested positive for Lp1 by qPCR. Whole-genome sequencing of the 14 Lp1 cooling tower isolates revealed the Building A strain to be indistinguishable from the 26 outbreak-associated clinical isolates. No strain from any other cooling tower matched to the Lp1 culture obtained from any patient during the investigation, as judged by whole-genome sequencing. An order to disinfect all cooling towers within 14 days was issued to all NYC building owners on August 6, 2015. Tracking compliance with the citywide order presented its own difficulties, including the need to review more than 10,000 documents submitted to the city to demonstrate compliance. Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 113 Conclusions A large outbreak of Legionnaires’ disease resulted in severe illness and death in a NYC neighborhood. Epidemiologic, environmental, and laboratory investigations implicated a hotel cooling tower as the likely source of the outbreak. The outbreak response was expedited by a screening of water samples collected from cooling towers using a qPCR-based assay for Lp1 DNA followed by culture of PCR-positive cooling towers. Previous outbreak investigations relied on culture, which, if successful, can take several weeks to identify and subtype. Using qPCR allowed rapid screening, prioritization, and focusing of control efforts on potential outbreak sources. Both host factors and environmental factors are likely to contribute to the increased number of cases of legionellosis since 2000. As discussed in Chapter 2, increasing numbers of persons are at higher risk of acquiring Legionnaires’ disease because of aging of the population, increased use of immunosup- pressant drugs, and higher prevalence of comorbid conditions, including diabetes and chronic obstruc- tive pulmonary disease. There is a growing dependence on heating, ventilation, and cooling systems, as well as increased complexity of indoor plumbing systems in large buildings, which have a labyrinth of water lines and features ranging from hundreds of showerheads along lengthy hospital corridors to hot tubs and indoor decorative fountains. Changes in plumbing materials could play a factor. In addition, increased efforts to conserve water with attendant slower flow in plumbing systems likely enhances bio- film formation and therefore increases risk of Legionella growth in premise plumbing (see Chapter 4). In- adequate maintenance of public water supplies (e.g., water main breaks, corrosion of pipes) may increase risk for contamination of building water systems and other water devices or equipment. Contaminated environmental sources, from dental hygiene equipment to street cleaning machines, continue to be newly identified (Ricci et al., 2012; Schönning et al., 2017; Valero et al., 2017). Changing environmental conditions are also facilitating human exposure to aerosolized water containing Legionella. Multiple hydrologic factors including humidity and rainfall may influence legio- nellosis risk, and climate change, including global warming, is likely contributing to the increase in cases (see Chapter 2). Despite the increase in reported rates, most cases of legionellosis are not diagnosed, even among those who seek medical care, and there is little evidence that diagnostic testing has improved for legionel- losis between 2007 and 2016. Diagnostic testing for pneumonia in the Unites States has been generally discouraged for many reasons. Reimbursement practices deter use of microbiologic diagnostic tests for pneumonia. Professional guidelines of the American Thoracic Society and the Infectious Disease Society of America have also discouraged routine testing of hospitalized patients for community-acquired pneu- monia (Bartlett, 2011; Mandell et al., 2007). Although these guidelines are currently being updated, it is not expected that the guidelines’ approach to legionellosis will change. At one academic medical center, adherence to these guidelines for testing of patients for Legionella would have resulted in an underesti- mate of the burden of Legionnaires’ disease of at least 41 percent (Hollenbeck and Mermel, 2011). In this study, even with more robust testing than recommended by the guidelines, only 35 percent of patients discharged with a diagnosis of pneumonia had been tested. Microbiologic analysis standards in most laboratories have declined. The belief that a deep re- spiratory secretion is needed for Legionella culture has discouraged testing, although this assumption is incorrect; sputum specimens that may be inadequate for culture of other pathogens may be sufficient for culture of Legionella (Bartlett, 2011; Ingram and Plouffe, 1994). In 2011, Bartlett reviewed reasons why testing has declined for diagnosis of community-acquired pneumonia. In particular, the Clinical Prepublication Version - Subject to further editorial revision

114 Management of Legionella in Water Systems Laboratory Improvement Amendments regulations led to the demise of the “house staff laboratory” and the distancing of microbiological analysis from the site of care, which may delay diagnoses. Obviously, there are fewer options at most community and rural hospitals, many of which have only basic laborato- ries. Legionnaires’ disease diagnostics, particularly use of culture, may have declined as a result of many of these factors. It is not known whether the use of PCR has had any impact on legionellosis diagnoses, although this may change as more molecular assays gain FDA approval. There has been little, if any, federal research funding for applied research on legionellosis, which, in turn, may depress training on legionellosis in academic healthcare centers. As a result, academic health- care centers in the Unites States have limited expertise on Legionnaires’ disease. The National Institute of Allergy and Infectious Diseases has focused its Legionella funding on basic science related to Legionella and the pathogenesis of the organism (Heilman, 2015). True Incidence of Legionellosis It is difficult to determine from available data the true incidence of legionellosis in the United States, although reported cases are certainly an underestimate. Some studies have attempted to deter- mine the incidence of Legionnaires’ disease in hospitalized patients with pneumonia. A population-based study in two counties in Ohio in 1991 estimated 8,000 to 18,000 individuals were hospitalized with com- munity-acquired Legionnaires’ disease per year in the Unites States (Marston et al., 1997). From 2013 to 2015, 98 percent of patients with pneumonia in a Pittsburgh VHA hospital were tested for Legionnaires’ disease with at least one diagnostic test, documenting that at least 1.7 percent of community-acquired pneumonia and 0.6 percent of healthcare-acquired pneumonia was caused by Legionella (Decker et al., 2016). The incidence of Legionnaires’ disease among hospitalized patients was reported as 8/100,000 veterans, with an incidence of 6/100,000 for community-acquired Legionnaires’ disease. More recently, Gamage et al. (2018) reported an incidence of Legionnaires’ disease in the nationwide VHA system of 1.9/100,000 for the years 2014 to 2016. Since both VHA studies lacked data on veterans admitted to hospitals outside the VHA system, the incidence of pneumonia among veterans was underestimated. The CDC is currently working on better estimates of morbidity and mortality related to waterborne patho- gens, including Legionnaires’ disease, but these reports will not be available until late 2019. To develop its own estimate of the incidence of Legionnaires’ disease, the Committee relied on the estimate from the population-based Etiology of Pneumonia in the Community (EPIC) study of commu- nity-acquired pneumonia that required hospitalization ( Jain et al., 2015). This CDC-led study is the more recent of only two such studies conducted in the United States that determined the incidence of Legion- naires’ disease (the other being Marston et al., 1997). The EPIC study was conducted from 2010 to 2012 in Nashville, Tennessee, and Chicago, Illinois, and considered 2,488 patients. Using mainly UAT, Jain et al. estimated an incidence of community-acquired pneumonia caused by L. pneumophila of 4/100,000. Starting with this value, the Committee increased this rate to 4.44/100,000 after assuming a 90 percent sensitivity of the UAT for detection of L. pneumophila serogroup 1. This estimate is conservative; other have found that the UAT only detects of 80 percent of L. pneumophila serogroup 1 cases (Mercante and Winchell, 2015; Yzerman, 2001). Another adjustment to the estimated incidence was made to account for the fact that the EPIC study was not designed to estimate Legionnaires’ disease, and methods of enrollment and exclusion cri- teria (e.g., excluding immunosuppressed patients) as well as limited testing likely resulted in significant underestimates of the burden of community-acquired Legionnaires’ disease. The Committee assumed that the enrollment and exclusion criteria removed at least 10 percent of actual cases, leading to a rate of 4.88/100,000 people. This adjustment is conservative given other, higher estimates of hospitalized Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 115 patients with community-acquired pneumonia. For example, Rameriz and colleagues (2017) studied adults hospitalized with pneumonia in Kentucky and reported rates of community-acquired pneumonia more than double those in the EPIC study and similar to rates found by Griffin et al. (2013), a study based on national Agency for Healthcare Research and Quality hospitalization data. Ramirez et al. (2017) at- tributed the higher rates in their study compared to those in EPIC to the stringent exclusion criteria used by EPIC. Next, the Committee incorporated evidence (supported by Mercante and Winchell, 2015) that at least 20 percent of patients hospitalized with Legionnaires’ disease have non-L. pneumophila serogroup1 disease, which was not captured in the EPIC study.4 This consideration increased the rate to 6.17/100,000. The Committee then assumed that 10 percent of all legionellosis cases are healthcare-associated (see previous sections of this chapter), numbers which also would not have been captured in the EPIC study, leading to an adjusted rate of 6.85/100,000. The EPIC study gathered and analyzed data from 2010 to 2012, such that the incidence cited in that study would reflect those years. According to Figure 3-3, there has been a doubling of the number of reported cases from 2011 to 2018, and this increase should be reflected in any current rate. There is little information available on the frequency of testing or whether diagnostic testing has improved (which could account for the observed doubling), has remained stable, or declined since 2011. The Committee assumed a range from as little as 50 percent of the doubling of reported cases being real (such that the other 50 percent is attributable to improved testing) to 100 percent of the doubling being real, which leads to a rate of 10.25 to 13.7/100,000. Although plausible, the Committee did not consider the possibility that diagnostic testing had decreased, a situation that would further increase its estimate of disease cases. The U.S. Census Bureau on July 1, 2018, estimated there are 327.2 million people in the United States, of which 253.2 million are 18 years of age and older (children are excluded because there are limited data on estimates of Legionnaires’ disease rates in children).5 Thus, the Committee arrived at an estimate of 26,000 to 35,000 hospitalized cases of Legionnaires’ disease per year. The EPIC study considered only cases of community-acquired pneumonia that required hospital- ization. To determine the incidence of outpatient Legionnaires’ disease, the Committee consulted von Baum et al. (2008) who analyzed data from CAPNETZ, which is a medical competence network for com- munity-acquired pneumonia funded by the German Ministry for Education and Research. von Baum et al. (2008) documented that the fraction of individuals with community-acquired pneumonia who were treated as outpatients was similar to that of persons with community-acquired pneumonia who were hospitalized. To be conservative, the Committee made a similar assumption, although there is evidence that, in the United States, the number of outpatients diagnosed with community-acquired pneumonia substantially exceeds the number of inpatients diagnosed with community acquired pneumonia.6 Thus, the Committee arrived at an estimate of 52,000 to 70,000 cases of Legionnaires’ disease per year in the United States (or a rate of 20.5 to 27.4/100,000). This estimate of the rate is approximately ten times higher than the reported rate for 2017 and is felt to be very conservative, as it considers only those cases of Legionnaires’ disease for which treatment was sought (either inpatient or outpatient). It is a coarse analysis that does not reflect all of the uncertainties. An analysis using different methods to estimate Legionnaires’ disease in hospitalized patients with pneumonia provides further evidence that Legionnaires’ disease may be substantially underdiagnosed in the United States. Cassell et al. (2019) reviewed hospitalization data for all non-federal hospitals in Con- necticut from 2000 to 2014; using the International Classification of Diseases, they compiled time series for pneumonia and influenza, and estimated (with a mixed-effects model) the percentage of cases due to 4 31 of 32 EPIC cases were detected by UAT, with a single case detected by PCR. Cultures were not performed. 5 See https://www.census.gov/quickfacts/fact/table/US/PST045218. 6 See https://www.ahrq.gov/professionals/quality-patient-safety/hais/tools/ambulatory-care/cap-toolkit.html, accessed June 22, 2019. Prepublication Version - Subject to further editorial revision

116 Management of Legionella in Water Systems Legionella, influenza, and respiratory syncytial virus. The annual incidence rate of Legionnaires’ disease among hospitalized patients was predicted to be 11.7/100,000; this rate was also approximately ten times higher than the average reported rate during the 14-year study period. The estimates of the burden of Legionnaires’ disease put forward by both the Committee and by Cassell et al. (2019) suggest that the U.S. rate of Legionnaires’ disease may be far higher than that indicated by notifiable disease statistics. ENVIRONMENTAL MONITORING Monitoring of Legionella bacteria in water systems has been done for several reasons. Water sam- pling has often been undertaken to locate the source of the bacteria after an outbreak of Legionnaires’ disease was documented or after cases began to accumulate. Routine monitoring is done to verify that a water management plan is working and to determine background levels of Legionella. For example, mon- itoring of cooling towers or hospitals, in the absence of cases of disease, has largely focused on whether or not to implement water treatment. Presence/absence approaches, where positive results initiate action, have frequently been used rather than quantitative measures. Assessment monitoring has often been done in conjunction with water treatment to determine treatment efficacy. Monitoring is also often carried out for research purposes, which is a valuable means of providing generalizable information to the scientific and practitioner communities about conditions in water systems that are conductive to Legionella growth and the means to control it. Table 3-1 provides a general overview of various methods currently available for environmental monitoring and how each may be applied toward these four goals. Of note, there is presently a great deal of variability in how the methods are actually applied to various systems and scenarios. This is likely because choosing the most appropriate methods, which systems and locations to target for testing and how often, and what medium to sample, are dependent on specific aspects of the water system and building being sampled. These are important considerations for a build- ing’s water management plan (discussed in Chapter 5). This section describes the individual methods and compares their strengths and weaknesses for various purposes. Finally, it summarizes what decades of data collection have revealed about Legionella presence and concentrations in various engineered, envi- ronmental niches. Methods Many of the methods used to analyze environmental samples for Legionella are the same as those discussed previously for clinical studies of Legionnaires’ disease. Historically, culture-based methods have been applied as the standard method for monitoring and to obtain isolates for further characterization. However, new methods have been developed that shorten the delay inherent to culture methods and allow for more real-time information gathering. The methods for environmental monitoring still do not fully account for Legionella’s complex ecology (see Chapter 2). For example, swabbing has been used as a sampling method because Legionella are known to be associated with biofilms that form in pipes and fixtures, yet quantitative data (e.g., area swabbed, method, other measures of total biomass obtained) have not been consistently reported. Few studies address the relationship of Legionella with amoeba and instead measure mostly planktonic bacteria. Recent knowl- edge of the ecology of Legionella spp. has been slow to impact the development of new methods, even in the research arena. Prepublication Version - Subject to further editorial revision

TABLE 3-1 Sampling for Legionella in Water Systems: Purpose, Methods, and Other Considerations Purpose of Which Method(s)? Which Water Systems? Spatial/Temporal Which Medium/ Testing Considerations? Volumea to Sample? Outbreak • qPCR/PCR- Rapidly identify suspect sites for further Suspect sources? Cooling As soon as possible when Water Investigation testing towers, hot and cold taps, an outbreak is suspected. Culture needed for • Culture- Confirm viable Legionella showerheads, hot tubs, Numbers would be comparison to • Serogroup, sequence typing, whole-genome decorative fountains, etc. expected to be high in case patient isolates sequencing- Compare to patient isolates of outbreak Routine • qPCR- Monitor baseline • qPCR positives should Where there is patient risk, Continuous- Develop First draw water samples Monitoring (viable + non-viable + be followed up by e.g., point-of-use devices in feasible plan and Select one, apply VBNC) culture intensive care units, neonatal frequency (May be Biofilms are sampled consistently • Culture- Monitor • Culture negatives- Be care units stipulated for some routinely, but the value of baseline (viable and aware of VBNC entities, locales, guidance, these data over sampling culturable) • Either can be used Where there is system standards). of the water column to flag concerns and vulnerability, e.g., stagnant unclear. changes in system zones, distal taps, substandard plumbing material Mitigation • qPCR- Do numbers increase or decrease following The system subject to Before and after mitiga- Water Assessment mitigation? Note DNA from dead Legionella could still mitigation. Check upstream tion, ideally long-term. Select one or more, be detectable after disinfection. and downstream of target Assess the overall effect Biofilm- Can assess if apply consistently • Culture- Provides information on viability. system and a comparable or changes in baseline. mitigation is reaching • Amoebae co-culture- Evidence for VBNC forms? control. Sample relevant inlets sources in biofilms Changes in virulence? and outlets to point of mitigation. Research In addition to all of the methods above, consider: Water systems in place in Depends on research Water Varies according to • Amplicon sequencing to address the responses of the field. These are more question. Longer-term research question broader microbial community real-world, but where there studies are valuable Biofilm • Metagenomics—broader context of functional is a weaker understanding of but lacking. Water genes, viruses, other factors factors at play. chemistry fluctuates with Aerosols- Need to Prepublication Version - Subject to further editorial revision • Viability qPCR or flow cytometry—indicator of the time. Three or more understand transfer of viable fraction of Legionella Simulated water systems. This years may be required to Legionella from biofilms allows for controlled variables achieve stable biofilm, to respirable, infectious and statistical replication, but which short-term studies aerosols less real-world significance. overlook a Volume to be determined based on application and desired detection limit. Larger volumes provide lower detection limits, but also may dilute the Legionella present in first-flush samples 117

118 Management of Legionella in Water Systems Table 3-2 compares several methods in use for detection, isolation, characterization and quantifica- tion of Legionella from building water systems. The table includes whether the method (1) elicits a presence/ absence or quantitative result; (2) allows the bacteria to be isolated; (3) can be used routinely; (4) identifies species, serogroups or genotypes; and (5) detects bacteria that are potentially viable, culturable, or those which are inactivated (killed). Each method has advantages and disadvantages. While culture methods have remained the gold standard, they may need to be adapted or supplemented with other methods to assist in developing risk estimates and informing outbreak investigations. Depending on the application, it is likely that combinations of methods will be used in the future. Culture Methods Culture methods capture cells that grow and produce colonies on solid agar, generating quantitative data in the form of colony forming units (CFU), or in some cases in liquid media. In many early studies using these methods, no quantification was undertaken because the goal was to isolate colonies and identify serogroups using antibodies. Thus, the methods initially focused on cultivation and isolation of the bac- teria only. One major shortcoming that still exists today is the length of time it takes to culture Legionella, as results may not be available for eight or more days. This can result in precious time lost for outbreak investigation, but this delay is not typically problematic for routine monitoring. By the late 1970s and early 1980s, media formulations were focused on growth of L. pneumophila, which led to the predominance of buffered charcoal yeast extract (BCYE) agar and the use of antibiotics as well as acid or heat pretreatment. The BCYE media used for culture tests is insufficient to recover all Legionella spp., although it does not exclusively detect L. pneumophila (Lee et al., 1993). Protocols that used filtration to sample larger volumes of water as well as swab samples became more prevalent (Cordes et al., 1981; Witherell et al., 1988). By 1990, improvements had been made, yet full assessment of a standard method was not forthcoming. There was concern regarding the standardization of the methods towards improved recovery and identification. After examining methods recommended by the VHA, CDC, and a group in Germany, Ta et al. (1995) made recommendations to enhance recovery of culturable species and identification of strains. Finally, in 1998 International Organization of Standardization (ISO) cul- ture methods were updated and published (ISO, 1998). A variety of standardized and consensus-based methods are now available including Standard Methods for the Examination of Water and Wastewater (APHA, 2007); Procedures for the Recovery of Legionella from the Environment (CDC, 2005); and ISO methods ISO 11731-2 (100-ml membrane filtration) (ISO, 2004, 2017). Procedures were directed toward the isolation of culturable colonies, in part to facilitate comparison of environmental and clinical isolates during out- break investigations. A new, easier culture method specifically for L. pneumophila has been developed that uses a liq- uid-based most-probable-number (MPN) approach (Legiolert™/Quanti-Tray™, IDEXX). The compar- ative data from four studies (see Box 3-5) suggest that the method is equivalent to other methods but generally trends higher in concentration estimations, which could elicit more violations and trigger re- mediation more often. One limitation of the reported evaluations of the MPN method was the lack of confirmation tests on positive wells in the tray. None of the studies mentioned in Box 3-5 evaluated the positives with genetic confirmation, but tested only via culture. The method also does not differentiate among serogroups of L. pneumophila nor is its specificity for all 61 species of Legionella available, making further testing necessary if this information is needed. Another drawback of this MPN method is that cultures are not readily available for molecular discrimination assays. As new methods develop, there is a need for greater systematic study and reporting of information, including a full description of the types Prepublication Version - Subject to further editorial revision

TABLE 3-2 Comparison of Methods for Environmental Legionella Monitoring Potential for Potential for Discerns Serogroups/ Form of Bacteria Method Level of Use* Pros Cons Quantification Isolation Sequence Types? Measured Culture Methods ISO Yes Yes Routine Yes Culturable Standardized Time to results; may Historical data underestimate VBNC, other serogroups and species, risks CDC Yes Yes Routine Yes Culturable Standardized Time to results; may Historical data underestimate VBNC, other serogroups and species, risks AHPA Yes Yes Routine Yes Culturable Standardized Time to results; may Historical data underestimate VBNC, other serogroups and species, risks Molecular Methods^ PCR No No Research, used with No Inactivated, Can support Need to process gels cultivation VBNC+, sequencing Culturable qPCR Yes No Research, potential No Inactivated Rapid results Measures inactivated cells, less for diagnostics and VBNC+ Greater sensitivity and historical use surveillance Culturable specificity ddPCR Yes No Research, potential No Inactivated Rapid results Measures inactivated cells; few for diagnostics and VBNC+ Greater sensitivity and studies using and comparing the surveillance Cculturable specificity method Emerging Methods Next No No Research No Inactivated Provides info on how Takes special expertise, Generation VBNC+ bacteria relate to instrumentation. More cost and Sequencing Culturable microbial community time to obtain results Amoeba Co- No Yes Research Yes Culturable Improves isolation of Adds at least 3 days to cultivation culture difficult-to-culture strains Liquid-based Yes Yes Research, potential No Culturable Simple set up, may be 8 days for results Prepublication Version - Subject to further editorial revision MPN for routine use specific to Lp More difficult to confirm EMA-PCR Yes No Research No Viable Can be used with Not proven to work with molecular tools disinfection PMA-PCR Yes No Research No Viable Can be used with Not proven to work with molecular tools disinfection Flow Yes Yes Research, potential Yes Inactivated Simple set up, specific Early commercial release, limited Cytometry for routine use VBNC+ to Lp serogroups based validation, higher detection limit Culturable on antibodies 119 *Categories include Routine, Research, Potential for Routine, or Potential for Diagnostics and Surveillance; ^Molecular tools require special instruments, training, and expertise; VBNC+: Viable-but-Non-Culturable.

120 Management of Legionella in Water Systems of samples compared, characterization of the genera and species eliciting false positives, and genetic characterization of the Legionella spp. and serogroups that are detected. Although culture methods have been standardized, inter-laboratory precision and accuracy are still uncertain. In a methods comparison (Ta et al., 1995), filtration, use of BCYE agar, and acid buffer treatment gave the highest recoveries. One inter-laboratory study using seeded samples for proficiency testing examined how well various laboratories performed in detecting and quantifying Legionella (Lucas et al., 2011). Ten in-house protocols (which were not described in the paper) were used, based on Ameri- can Society of Microbiology, ISO, or CDC methods. CDC and nine other laboratories including county, state, hospital, and private entities participated, with CDC as the reference laboratory. The key findings included the following: • The detection limit of the methods and laboratories were similar; samples were negative 93.1 per- cent of the time with less than 10 CFU/mL and positive 85.3 percent of the time with samples with greater than 10 CFU/mL. • Quantification errors averaged about 1 log and underestimated the expected concentrations. However, this conclusion was tenuous, as formal assessment of the quantification results were not clearly articulated in the publication. • Statistics on accuracy and precision with only ten laboratories was similar to European studies. While the details were not provided, the study concluded that sampling protocol, treatment reg- imen, culture procedure, and laboratory experience did not significantly affect the accuracy of reported concentrations. The advantages of culture include (1) its ability to compare with historical samples, (2) it is an ac- cepted measure of viability, and (3) it can be used to isolate bacteria for epidemiologic investigations. The disadvantages are that final results are not available for eight to 14 days depending on the chosen labo- ratory, making rapid decisions impossible, and the cost and expertise needed to run the method limits its widespread use. Furthermore, the method cannot capture Legionella cells in the VBNC-like state, and it favors L. pneumophila and a few other Legionella spp., such that not all Legionella spp. associated with dis- ease are identified (Lee et al., 1993). Approaches to recover the bacteria from the VBNC-like state have been reported (Oliver, 2005), including co-culture with Acanthamoeba polyphaga (Dusserre et al., 2008) as discussed below. Newer MPN methods may be easier to implement and, once fully vetted, could facilitate more widespread use by utilities, building owners, and public health laboratories. Use of Amoeba Amoeba co-culture for the recovery of legionellae from clinical and environmental samples was first described by Rowbottom (1980, 1983). While there are many bacterial pathogens that resist the digestive processes of predatory amoeba (so-called amoeba-resisting bacterial pathogens, Thomas et al., 2010), L. pneumophila is the most recognized in water systems (Corsaro et al., 2010; Tosetti et al., 2014). Amoeba of the genus Acanthamoeba are generally used for co-culture (Pagnier et al., 2008) because of the ease with which they are grown in cell culture, but different amoebal hosts and incubation temperatures may influence which specific L. pneumophila strains are recovered (Buse and Ashbolt, 2011). Use of amoe- ba from the local environment has also recovered L. pneumophila when other American Type Culture Collection (ATCC) Acanthamoeba polyphaga failed to recover any isolate (Dey et al., 2019). Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 121 BOX 3-5 Comparative Studies on Legiolert™ Four studies have evaluated Legionella occurrence and concentrations in side-by-side com- parisons of Legiolert™, an MPN method in which the sample is distributed in a tray to generate a colorometric result after eight days of incubation, to other methods used more routinely. The first comparison (Satory et al., 2017) was against the ISO 11731-2 membrane filtration method with 290 paired samples. The second comparison (Petrisek and Hall, 2018) was against the standard culture method of APHA (2007) with 491 potable water samples and 846 nonpotable water sam- ples. A third study (Rech et al., 2018) compared Legiolert™ to the CDC method (CDC, 2005) and examined 288 non-potable water samples. The fourth study (Spies et al., 2018) involved six laboratories comparing Legiolert™ (using 448 samples of 100 ml volumes) to ISO 11731-2 (100-ml membrane filtration) and ISO 11731 (1 ml direct plating). Table 3-5-1 provides the results. Con- firmation is not a part of the MPN test as described, although cultured cells could be recovered for further testing/isolation. TABLE 3-5-1 Comparative Studies on Legiolert™ Comparison Sample Sample Types Results Method Numbers ISO 11731-2 Cold and hot taps show- Overall, Legiolert™ provided a greater Membrane Fil- 290 ers circulation lines, mean concentration. There were 3.3 per- tration boiler outlets cent false positives. There was no statistical difference between the methods; Legiolert™ < 0.5 percent Standard Culture 491 Potable Water and <0.9 percent false positivity rate for Methods (APHA, potable and non-potable samples, respec- 2007) 846 Non-potable water tively. Did not mention a confirmation to L. pneumophila specifically** No differences were found between the CDC Method Non-potable water, 288 methods. Non-pneumophila found in ten (CDC, 2005) mostly cooling towers samples but only by the CDC method For the 100-mL method, four of six labo- ISO 11731-2 ratories had higher Legionella counts with (100-mL mem- Cold and hot taps, the MPN method and the other two showed brane filtration) 448 showers, building circu- no difference. With the 1-mL method, five ISO 11731 (1 mL lation systems of six labs showed no difference. The plating) specificity was found to be 97.9 percent. ** They confirmed 25 percent of the positive cells by recovering the liquid from the cells in the tray and re-isolating the bacteria on standard agar media. Prepublication Version - Subject to further editorial revision

122 Management of Legionella in Water Systems Methods to recover amoebae from environmental samples are based on those developed over the past several decades. An environmental sample is applied to a lawn of viable E. coli prey on non-nutrient agar plates (e.g., 2% Neff’s saline) and incubated at 25°C for up to two weeks, identifying any clearing zones with observable trophozoites moving away from the originally applied zone, and then re-streaking onto fresh plates (e.g., Amaro and Shuman, 2019; Lorenzo-Morales et al., 2005). The use of different prey and temperatures can recover a greater diversity of isolates, but is generally not undertaken. To isolate legionellae using the amoeba co-culture method, an environmental water sample is in- cubated with amoeba obtained from a fresh, exponential culture using several dilutions to optimize the prey-to-host ratio, and then incubating the co-culture at 30°C for 12 hours. Co-cultures are observed by phase microscopy to identify trophozoites exhibiting lysis or growth of intracellular bacteria. Finally, the Legionella is isolated on BCYE agar. Amoebae co-culture methods have not been standardized and have primarily been used in the research arena and in reference laboratories in Europe for water and clinical samples. This culture tech- nique takes at least an additional three days, whereby the sample is first co-cultured, then the resulting amoebae-resisting bacteria are grown as usual on BCYE agar or are rapidly identified by qPCR/sequenc- ing (e.g., Corsaro et al., 2009; Lienard et al., 2011). Advantages of co-culture are improved isolation and detection of viable microbes and recovery of isolates to compare to clinical isolates. Amoebae co-culture is also presumably biased toward Legionella that readily infect amoebae, thus serving as a proxy for vir- ulence within human macrophages. The disadvantages of co-culture are lack of quantification, the time to obtain results, lack of standardization, and minimal information on its utility in routine monitoring. PCR, qPCR, and dPCR There has been significant growth in the use of molecular techniques either in combination or independently for detection and characterization of Legionella in environmental samples (Borges et al., 2012). PCR was first introduced in 1985 and initially provided presence/absence data. Today PCR kits that include appropriate standards and quality controls and instruments to run the test are widely avail- able. PCR can be much less expensive than culturing Legionella and entails less time per sample, produc- ing results in hours instead of days. Because it relies on DNA sequence recognition, PCR can provide very high specificity and confidence in detecting the intended target. PCR works by cycling between high and low temperatures to separate and then anneal the DNA in a water sample. Specific, small pieces of DNA called primers direct the polymerase enzyme to copy a specific gene sequence. Finally, the genetic sequence of the DNA fragment that has been amplified is determined. The amount of target DNA produced each cycle increases exponentially, enabling easy visualization of the final PCR product by staining and verifying the correct molecular weight by size sep- aration methods, such as electrophoresis. In practice, the water sample is initially filtered, the captured bacteria are removed from the filter and lysed, and their DNA is extracted for use as the template in the PCR amplification reaction. The method detects all cells in the sample, including culturable, inactivated, and VBNC-like cells, and potentially any DNA from dead organisms. PCR approaches are available for all species in the genus of Legionella (by analyzing the 16S or 23S rRNA gene), for L. pneumophilia (mip gene), and for L. pneumophila serogroup 1 (a region of the wzm gene, spanning nucleotides 99 to 392). Primer sets have also been published for L. anisa, L. bozemanii, L. longbeachae (Saint and Ho, 1999), and L. micdadei (Cross et al., 2016). The use of L. pneumophila serogroup 1-specific primers is relatively new, but appears to be gaining momentum since it was first introduced (Mérault et al., 2011). More recently, quantitative PCR (qPCR) and droplet digital PCR (ddPCR) methods have been de- veloped, which are a great improvement over traditional PCR in that they provide quantitative infor- mation. The quantitative units of qPCR and ddPCR are gene copies (GC) per unit volume (e.g., GC/L). qPCR works the same as traditional PCR, but it incorporates a dye or probe in the reaction and uses a Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 123 specialized instrument that can detect and quantify the signal as product is formed. Comparison of the exponential product amplification curves of samples to those generated by a standard curve of positive control DNA templates of known concentration allows quantification of gene copies per reaction. Units can then be converted to gene copies per volume of sample collected and subject to DNA extraction. ddP- CR is a newer alternative to qPCR that provides rapid absolute quantification, without need for a stan- dard curve, and is less sensitive to PCR inhibitors. Consequently, ddPCR can be applied to more than one genetic marker at a time, a procedure called multiplexing. The method works by dividing the sample into about 60,000 droplets wherein the PCR reaction occurs; the numbers of positive and negative droplets then provide a most probable number of the concentration. Figure 3-5 provides the results from a seeded water sample using the primers and gene sequence for the genus Legionella (23S rRNA gene) and the L. pneumophila-specific mip gene. Because qPCR and ddPCR capture all DNA, even from dead cells, more evaluation is needed be- fore one could apply these methods during routine monitoring, particularly in environments containing high levels of disinfectants (e.g., cooling towers, hot tubs) where there is likely to be more DNA derived from dead cells. Culture and qPCR have been compared and contrasted for drinking water and cooling towers for detection of L. pneumophila and L. pneumophila serogroup 1 (Toplitsch et al., 2018). Twenty (20) drinking-water samples were examined, and the agreement was very good for L. pneumophila (90 percent positive by qPCR, 95 percent positive by culture, and 85 percent positive for both). In contrast, samples from cooling towers (n = 52) were scored as 60 percent positive using qPCR, 23 percent positive by cul- ture methods, and 19 percent positive by both methods. For L. pneumophila serogroup 1, the agreement was poor for drinking water (10 percent, 5 percent, and 0 percent positive by qPCR, culture, or both, respectively), although slightly better for cooling towers (21 percent, 13 percent, and 4 percent positive by qPCR, culture, or both, respectively). When both tests were positive, generally qPCR reported 10- to 100-fold higher concentrations, although there was a positive correlation between the two tests. Another FIGURE 3-5 Results of a seeded water sample tested for two targets measured by ddPCR. The four quadrants show the number of droplets positive for the mip gene and the 23S gene (top right quadrant) or only the mip or 23S gene (left top and bottom right quadrants, respectively). The left bottom quadrant shows the number of droplets negative for both genes. Taken together, these results produce a most probable number for gene copies for both genes. SOURCE: Courtesy of Joan Rose. Prepublication Version - Subject to further editorial revision

124 Management of Legionella in Water Systems study similarly found that quantification of L. pneumophila by qPCR trends with that by culture in both hot water and cooling tower samples, but with consistently higher estimates (Yaradou et al., 2007). Lee et al. (2011) attempted to translate CFU/L into gene copies/L by comparing international results for both metrics from 232 cooling tower samples and 506 hot- and cold-water samples. There was a 2-log dif- ference between qPCR (gene copies/L being higher) and culture (CFU/L) in cooling towers for Legionella species, but only a 0.71-log difference for L. pneumophila. For drinking water taps, there was a 1.05-log and 0.62-log difference between gene copies/L and CFU/L, respectively, for Legionella and L. pneumophila. PCR and culture-based tests can produce distinct results for several reasons. In addition to the capture of both VBNC-like and dead cells by PCR, variability in the distribution of the bacteria in any given wa- ter sample (e.g., one sample may have a clump of cells), differences in detection limits, efficiencies of the methods, and multiple gene or genome copies within a cell can result in different outcomes. The advantages of qPCR and ddPCR include rapid results, the ability to design primers that have high specificity, and low cost, which allows for large numbers of samples to be tested. The disadvantag- es are that qPCR detects cells regardless of their viability. The use of PCR methods is becoming more widespread for clinical surveillance and outbreak detection and, if applied appropriately, could also be used for routine monitoring of water systems. Cooling towers are rarely monitored routinely by qPCR, in part because of the high concentrations of disinfectant and corresponding high levels of DNA from dead cells. However, even an increase in total Legionella DNA means that growth conditions are not being controlled somewhere in the system and is worthy of further investigation. When applied consistently, qPCR can be very useful for estimating baseline numbers of Legionella, even in disinfected systems, with increases and decreases indicative of growth and death in the system. Yaradou et al. (2007) noted good correspondence between qPCR and culture-based methods targeting L. pneumophila in cooling towers and suggested that qPCR could be adapted for more wide-scale cooling-tower monitoring in the future. It is not unprecedented to move from a culture-based method to qPCR, as was done for recreational waters (i.e., beaches) for E.coli and enterococci monitoring (Gonzalez and Noble, 2014). Now that there is an ISO method for qPCR detection of Legionella (ISO, 2019), it would be appropriate to compare the two methods (qPCR and a culture method) for a variety of buildings and water systems in order to help interpret qPCR-generated data. It is likely that greater application of qPCR will occur in the future given the speed with which qPCR can provide information. Viability Analyses. To alleviate concerns that qPCR also detects non-viable bacteria, several methods have been developed that favor DNA (or RNA) detection and quantification of viable Legionella. One such method uses ethidium monoazide (EMA) or propodium monoazide (PMA) in combination with qPCR (Nocker et al., 2006; Nogva et al., 2003), referred to as viability qPCR. The first working principle is that on light exposure, both PMA and EMA bind to DNA and, as a result, this bound DNA can no lon- ger be amplified by qPCR because the qPCR primers cannot bind to EMA/PMA-bound DNA (see Figure 3-6). Second, theoretically EMA and PMA cannot enter a cell when the cell membrane is intact, which is one of the viability parameters of a microbial cell (Hammes et al., 2011). As a result, free DNA and DNA from cells with a compromised membrane are bound with EMA or PMA, and that DNA will not be amplified during qPCR. In a similar way, cell integrity vital staining can be used in combination with flow cytometry.7 Viability qPCR has been used to quantify membrane-intact legionellae cells (e.g., Chen and Chang, 2010; Lizana et al., 2017). In general, these studies showed that when disinfected water samples were exposed to PMA or EMA, the gene copy numbers of Legionella calculated were between the number of Legionella colony forming units obtained by culture and the number of gene copies obtained with qPCR 7 E.g., https://www.rqmicro.com/products/l-pneumophila-kit. Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 125 without PMA or EMA exposure. Accordingly, PMA or EMA seem to bind some of the Legionella DNA from membrane-intact cells that might still be viable after disinfection. However, serious precautions have been raised about the use of EMA and PMA to quantify viable Legionella, especially for environmen- tal samples (Kirschner, 2016). These methods are not appropriate for studies involving a disinfectant whose mode of action does not affect membrane integrity, such as UV. Furthermore, there has been a lack of consistency among viability qPCR studies. For instance, the optimal EMA or PMA concentration for the viability assay reported in one study was shown to be cytotoxic to Legionella in another (Chang et al., 2010; Reyneke et al., 2017; Scaturro et al., 2016). In addition, the PMA method can overestimate viable Legionella cells (Scaturro et al., 2016). Moreover, Taylor et al. (2014) concluded that PMA is not an appropriate method for discriminating between live and dead Legionella cultivated under environmental conditions. Similar results have been obtained with EMA and PMA treatment of Legionella cells directly harvested from drinking water biofilms or cooling tower water, although the assay worked well with laboratory grown Legionella cells (Ditommaso et al., 2014; Wullings et al., 2016). When compared to live/dead stain flow cytometry, viability qPCR for L. pneumophila overestimat- ed membrane-intact cells when a large portion of the cells were membrane-compromised but underes- timated membrane-intact cells when a large portion of the cells were membrane-intact. Thus, viability qPCR appears to be qualitative rather than quantitative. Furthermore, the performance of EMA and PMA treatment is much lower with shorter amplicon lengths (less than 200 base pairs or bp) than with larger amplicon lengths (greater than 400 bp) (Ditommaso et al., 2015; Wullings et al., 2016). According- ly, larger qPCR gene targets of Legionella may be optimal. However, most companies providing molecular tools for qPCR recommend that amplicon lengths not exceed 200 bp for optimal qPCR. Kontchou and Nocker (2019) have recently optimized the PMA assay for L. pneumophila, which includes a longer ampli- con (633 bp), higher incubation temperature, and addition of EDTA and deoxycholate. They determined FIGURE 3-6 Principles of live/dead quantification with PMA and qPCR. SOURCE: https://biotium.com/product/viability-pcr-starter-kits. Image by Biotium® Inc. Prepublication Version - Subject to further editorial revision

126 Management of Legionella in Water Systems that the membrane-intact L. pneumophila cell numbers obtained with PMA-qPCR were in agreement with membrane-intact cell numbers obtained with flow cytometry, demonstrating potential for this optimized assay, with the caveat that L. pneumophila strains were cultivated under optimal conditions. Overall it can be concluded that, although PMA or EMA treatment in combination with qPCR might have merit to distinguish between membrane-intact and membrane-compromised Legionella, additional studies on the reliability of the method, standardization of the method, and its application to environmental samples need to be performed before qPCR assays can be applied routinely to detect viable Legionella. Another promising molecular method that distinguishes between viable and nonviable Legionella detects precursor RNA, which is only produced by viable cells on exposure to fresh nutrients (Cangelosi and Meschke, 2014). To detect L. pneumophila by assaying for precursor RNA, samples are exposed to fresh nutrients for three hours, RNA is extracted, and then RNA from the precursor region of the 16S rRNA gene of L. pneumophila is specifically amplified with reverse transcriptase (such that the method is called RT-qPCR) (Boss et al., 2018). In one study, L. pneumophila in drinking water samples taken from public sport facilities was analyzed by RT-qPCR, cultivation, and qPCR. For 86 percent of the samples, the results with RT-qPCR and cultivation were consistent. In 7 percent of the samples the culture meth- od was positive but RT-qPCR was negative, whereas in the other 7 percent of the samples RT-qPCR was positive but culture was negative. In addition, 17 percent of the samples that were negative with RT-qPCR were positive with qPCR, indicating the presence of DNA from dead L. pneumophila. Others have also used RT-qPCR to detect RNA of specific genes (including virulence genes) of L. pneumophila after exposure to synthetic grey water (Buse et al., 2015) or copper (Lu et al., 2013). The specific analysis of virulence genes in these assays might not only provide information on viable L. pneumophila cells but also on their virulence potential. Although RT-qPCR seems promising, additional studies are needed in which RT-qPCR results are compared with cultivation, qPCR, and viability qPCR for detection and quantification of Legionella in different environmental samples. Next Generation DNA Sequencing A handful of studies have used next-generation DNA sequencing approaches to examine Legio- nella or other relevant members of the microbial community in drinking water systems. Amplicon se- quencing is one application that is applied to amplified PCR products obtained from DNA extracted from mixed microbial communities. Most often amplicon sequencing uses universal primers for bacterial 16S rRNA genes to profile which organisms are in a particular drinking water or biofilm sample. Organisms are identified based on the similarity of the 16S rRNA gene sequence to entries in online databases, and the term operational taxonomic unit (OTU) defines the bacteria identified. Because at best the resolution is at the genus level, the presence of pathogens cannot be ascertained. Nevertheless, amplicon sequencing has proved to be a powerful tool to reveal the surprising di- versity of microorganisms inhabiting drinking water (Pinto et al., 2012) as well as estimate their relative abundance. In one laboratory study of domestic hot water, qPCR and amplicon-sequencing-based meth- ods estimated Legionella spp. to be around 3 percent of the total community ( Ji et al., 2018). Next-gen- eration DNA sequencing can be applied directly to the DNA extract, without first PCR-amplifying a gene of interest, an approach referred to as shotgun metagenomic sequencing. The advantage of shotgun metagenomic sequencing is its potential to sequence all genes in a sample, including markers of function (e.g., nitrification, iron oxidation, virulence), and thus provide much richer functional information and taxonomic resolution (Gomez-Alvarez et al., 2012). However, currently metagenomic sequencing is very costly; consequently, researchers tend to employ less thorough sequencing, which results in false nega- Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 127 tives because of high detection limits and lack of coverage. Both amplicon sequencing and metagenomic sequencing also provide rich information about non-Legionella species in water systems and could poten- tially provide new insight into the role of microbial ecology in Legionella propagation (Dai et al., 2018). However, for potential application to Legionella monitoring, these tools are still in their infancy (Borthong et al., 2018). In the future, next-generation sequencing of both environmental and human isolates could potentially provide insight into the relationship between environmentally abundant Legionella and dis- ease and perhaps help to identify previously unidentified clusters of disease. The third application of next-generation sequencing is whole genome sequencing of individual Legionella isolates (Reuter et al., 2013). Whole genome sequencing makes possible high-resolution phy- logenetic comparisons of isolates associated with outbreaks, and it can also be adapted to determine the sequence type (Raphael et al., 2016). Raphael et al. (2019) have used whole genome sequencing on cultures of clinical specimens to reveal a highly diverse population of strains causing legionellosis in Arizona. Sampling Strategy A Legionella monitoring plan for water systems should include (1) the purpose of the monitoring, (2) what medium to sample, (3) the method to be used, and (4) where and when to sample. As discussed in Chapter 5, the precise sampling strategy should be developed and adapted to the system of interest as part of a comprehensive water management plan (see the example in Box 3-6). Monitoring for Legionella in building water systems can have many purposes including to investigate outbreaks, to support reme- diation or mitigation, to demonstrate compliance with a guideline or regulation, as part of diagnostic surveys, and for research (see Table 3-1). Once the purpose is determined, the methods should be linked to the desired information. The priority may be confirmation or quantification, determining viability, or distinguishing serogroups or sequence type. For example, culture and viability are of interest when dis- infection is being used for remediation. For compliance monitoring, the methods are usually prescribed. Surveys generally attempt to use standardized methods to facilitate comparison. Nonetheless, newer methods such as qPCR have great potential to quantitatively examine more samples at a lower cost and much more rapidly. Legiolert™ may enable greater ease in sampling at a lower cost than current culture methods, although the time to receive results remains a week. First, the water system to be sampled must be identified, such as cooling towers, residences, public buildings like hotels, resorts, hospitals, drinking water, and wastewater. In particular, points thought to be most vulnerable to Legionella growth and where potential for human exposure is high should be prior- itized. For example, within buildings, premise plumbing monitoring should include distal sites that have potential both for Legionella growth and human exposure; these include showers and taps, decorative fountains, and storage tanks. Although Legionella growth is less likely in the hotter water of recirculation lines and water heaters, sampling these locations is also important for confirmation and to provide a baseline. The various media that can be targeted for sampling include the bulk water, biofilm, or the aero- sols generated. Most sampling strategies and methods have focused on the bulk water because it is easy to collect, various volumes can be readily targeted, and it can be concentrated via filtration. In addition, first-flush samples are thought to capture water that has been stagnating (thus more likely allowing for bacterial growth), potentially better representing what has sloughed or diffused off of the biofilm. (It should be noted that most studies lack any quantitative assessment of stagnation. For example, a study of 807 drinking water samples from nine buildings found occurrences to be significantly correlated with stagnation, but this was described only qualitatively as “low withdrawals” [Völker et al., 2016)]). Prepublication Version - Subject to further editorial revision

128 Management of Legionella in Water Systems Legionella bacteria are known to associate with biofilms and their amoeba. However, swab sam- ples have had limited value in decision making for remediation of premise plumbing. Swabs are not analyzed routinely because it is impossible to collect a representative biofilm sample from the miles of premise plumbing in a building, there is no standard method available, and there is no consistent way to report the concentrations found. Developing better methods for sampling premise plumbing biofilms is clearly a research need. Because aerosol sampling is much more complicated than sampling the bulk water and still under development, aerosols are generally not included in a sampling strategy. Nonetheless, aerosols can be collected as they are generated using various types of impingers or impactors. A research program to un- derstand the difference between measured Legionella concentrations in bulk water and in aerosols would be useful (Prussin et al., 2017). The detection methods applied should include more than one technology (likely a culture method and a molecular method, e.g., qPCR) and be quantitative. Laboratories will continue to use culture but may use more than one medium; this may be unnecessary if, for example, qPCR or dPCR was used first to examine more rapidly the concentrations of specific species or L. pneumophila serogroup 1. The detection limit should be carefully documented, addressing both the volume collected and concentrated. More experience is needed where both types of results (culture and molecular methods) are available, thus providing knowledge on their comparability. Sivaganesan et al. (2019) has compared qPCR methods to culture for fecal indicator bacteria on beaches over many years during the swim season. These data are now being analyzed in several states to address the comparable level of gene copies per 100 ml that would lead to beach closure on the same day rather than waiting 24 hours to obtain culture data. A similar ap- proach could be used for Legionella. The frequency of environmental sampling for Legionella is highly variable and ranges from once per week to once per year, depending on many factors such as the size and use of the building. Box 3-6 describes the Legionella sampling strategy applicable to large buildings with complex premise plumbing systems such as hospitals, while Box 3-7 describes the sampling strategy for cooling towers; both box- es prescribe sampling frequencies. In general, however, the numbers of samples taken and how often they are collected have been based on resources and logistics rather than on an understanding of the ecological niche of the bacteria. Temporal studies with recommendations on how often to monitor and over what time frame have yet to be undertaken. Nor has there been a clear statistical assessment of the frequency of sampling needed to capture Legionella growth, blooming, and sloughing events. To evaluate temporal changes such as seasonality, several years of monitoring would be needed. More widespread and improved national laboratory certification is needed for current approach- es and for new methods, which includes standardized protocols, quantitative assessment, training and proficiency testing. The Environmental Legionella Isolation Techniques Evaluation (ELITE) Program has oversight from the CDC, but since November 2016, the Wisconsin State Laboratory of Hygiene has managed the production and distribution of testing samples as well as analysis of laboratory results. Twice per year, participating laboratories receive cultures for verification tests.8 The program issues certificates to laboratories that successfully isolate legionellae from simulated environmental samples by culture, but it is not a laboratory certification process. New York State certifies laboratories9 as do the Quebec and Alberta provincial governments in Canada. 8 See https://wwwn.cdc.gov/elite/Public/FAQ.aspx. 9 See https://www.wadsworth.org/regulatory/elap. Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 129 BOX 3-6 Legionella Sampling Strategy in Large Buildings Large buildings, including hospitals, which house vulnerable populations, tend to formalize their management of Legionella in building water management plans (see Chapter 5 for more de- scription of these plans). Although not universal, these plans often require some form of Legionel- la sampling to gauge the effectiveness of the building’s water treatment system and to determine if the treatment needs to be modified to maintain plan effectiveness. Sampling strategies are unique to any given building, and both the water management plan and the sampling strategy are subject to change as surrounding and contributing environmental conditions change. A frequently used guidance for developing a water management plan is the American Soci- ety of Heating, Refrigeration and Air-Conditioning Engineers (ASHRAE) standard 188 (ASHRAE, 2015). This standard does not provide a sampling strategy nor address biological testing for Le- gionella, but it does reference a companion document ASHRAE 12-2000 (ASHRAE, 2000) which states that “culturing for Legionella may be appropriate if carried out for a specific purpose, such as verifying the effectiveness of a water treatment protocol.” The CDC toolkit (CDC, 2017a), which aims to make ASHRAE 188 more practical, also makes reference to environmental testing for Le- gionella to validate the effectiveness of control measures. Once water temperature, disinfectant residual, and distal point flushing programs have been considered to aid in identifying sampling locations and potential Legionella growth risks, Legionel- la sampling should be the basis for validating any water management plan, regardless of building size, configuration, or even building population composition, which are risk factors secondary to plan development. Initial samples will define the extent or even if Legionella is present and the extent to which the plan should be developed. In very general terms, the initial sampling would include bulk water samples from the water source entering the building, from storage tanks if used, and from both hot and cold water distal sites at multiple points in the building. Initial sample draws should be evaluated by competent third party entities rather than contractors or vendors who are responsible for mitigation modalities. Samples should be tested by culture (e.g., ISO, 2017) or by qPCR (e.g., ISO, 2019). When sampling, it is highly recommended to record specific sample location, temperature, disinfectant residual, pH, and plumbing zone flushing and usage. These additional data points will minimize resampling time and define the conditions contributing to any given water management issue. Both culture-based and qPCR-based monitoring must be taken into context and compared to a baseline, not interpreted in isolation. Following initial testing, water management plan development will move forward based on the test results and other associated risk factors unique to the building. At this point, Legionella sampling will evaluate changes to the plan and any required mitigation to maintain water safety for water uses within the building. For large buildings, the building manager will need to identify the potential locations where Legionella may be present and propagate, based on the number of potable water systems and the number of distribution components. Examples are as follows: • Potable sources: Some building configurations have multiple water mains. A sample should be taken from each source. • Potable tanks: If used, potable tanks should be tested. Water tanks will extend the age of water. • Potable zones: Larger buildings, particularly high-rise buildings, may have multiple building zones as a result of building height and pressures. A sample should be taken from each zone. • Distribution risers: Samples should be drawn from enough risers to provide a good evalua- tion of all risers. If sampling and testing is done frequently enough, i.e., monthly, a random selection of risers would be possible. Selection should always include risers in which water use is minimal. • Horizontal distribution: Samples should be taken from enough of the horizontal distribution to be representative of the entire length. At a minimum, the end point of the horizontal dis- tribution should be sampled to determine whether mitigation is reaching the farthest point of the system. Prepublication Version - Subject to further editorial revision

130 Management of Legionella in Water Systems • Potable hot-water: All system points listed above should be tested for both hot- and cold-water systems. Legionella is more likely to exist and propagate in hot-water systems where tem- peratures range from 29.4°C to 40.6°C (85°F to 105°F). More hot-water points should be sampled than cold. • Potable hot-water heat exchangers: Where used, they should be evaluated and sampled. • Potable hot-water return piping: Where used, it should be sampled, as conditions are often suitable for Legionella growth. Once the locations that will provide a good indication of system performance are identified, the interval for sampling can be determined. In cases where initial testing indicated there was no pres- ence of Legionella anywhere in a facility, and the building use composition indicated no risk of ex- posure to building occupants, sampling may be once every six months or even once per year. The sampling interval is also driven by the building’s risk tolerance. A hospital with a large immunocom- promised patient population and zero tolerance for Legionella may opt for more frequent sampling. In either case, the sampling strategy is dictated by risk and water management plan parameters. BOX 3-7 Legionella Sampling Strategy for Cooling Towers For routine maintenance of cooling towers, a Legionella sampling program is key in ensur- ing that the operation and maintenance activities, as well as the water treatment, are effective. In the event of a Legionnaires’ disease outbreak associated with a cooling tower, a well-planned sampling strategy can help to isolate specific components of the cooling tower system that are responsible. This sampling strategy should identify or rule out suspected sources and their trans- mission pathways. A thorough visual assessment of the cooling tower should be conducted prior to any sam- pling, to determine the condition of the various components of the cooling tower and their potential to amplify and transmit bacteria. Table 1.3 in HSG274 Part 1 (HSE, 2013) denotes the various parts of a cooling tower and Figures 1.5 and 1.6 are photographs of the cooling tower fill condi- tions (see www.hse.gov.uk/pubns/priced/hsg274part1.pdf). Operation and maintenance records, as well as water treatment records and any past sampling data, should also be examined for gaps or unusual results as well as follow-up actions and validation results. Once the equipment and its components have been visually inspected, a sampling plan should be devised to take into account any potential problems. Sampling locations for cooling towers should include the locations listed below, either in routine sampling or during an outbreak. However, these locations will vary depending on the cooling tower’s components. The CDC (2015) sampling procedure for outbreaks of disease, shown in Table 3-7-1 below, indicates the number and type of samples, and the targeted process for each location. Table 3-7-1 Legionella Sampling for Cooling Towers SOURCE: CDC (2015) Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 131 Various documents such as AIHA (2015) and PWGSC (2013) provide additional guidance re- garding the type of samples to be collected, the sampling locations, the frequency, and the proper handling and analytical methods for both routine and outbreak monitoring of cooling towers. The recommended frequency for routine Legionella monitoring may vary from weekly (PWGSC, 2013) to quarterly (HSG274, 2013) depending on the outcomes of the visual inspection, past issues, and targeted outcomes. Other physical parameters to monitor include temperature, pH, residual (free) chlorine, other disinfectant levels, and water flow rates. When preparing a routine sampling strategy for cooling towers, it should be noted that: 1. Cooling towers tend to operate fully only during the summer months, although some will be operated sporadically throughout the winter depending on cooling needs. 2. Cooling towers do not continuously circulate water even when they are in operation, which provides conditions ideal for Legionella growth. 3. Cooling towers are frequently not accessible for inspection or sampling. 4. The plumbing configuration for a group of cooling towers can be very confusing. Therefore, it is necessary to properly trace the piping for each tower to pinpoint the sampling locations that will reflect the conditions for any given cooling tower and its associated equipment. 5. Because cooling towers are essential to the operation of modern buildings, it is difficult to take a cooling tower offline to allow for inspection, sampling, disinfection, or repair. A heat transfer plan should be part of the sampling strategy. 6. Surveillance monitoring is completed on a regularly scheduled basis. However, it is recom- mended to vary the testing time for comparison purposes (e.g., after a long weekend, morn- ings, afternoons). The same applies to locations within a system when possible, for example, by sampling different heights of fill of the cooling tower. 7. Personal protection equipment including eye and respiratory protection and anti-slip footwear should always be used when working around cooling towers. Occurrence of Legionella in Water Systems Much of the emphasis for environmental sampling of Legionella has been to understand its occur- rence and (in some cases) concentrations in different locations. Sampling has focused on sites where aerosols that might contain the bacteria are formed, including cooling towers, showers, hot tubs, foun- tains, and buildings with vulnerable populations (e.g., hospitals). Over the years, better methods and lower detection limits have increased the percentage of samples that test positive for Legionella, yet con- centrations have remained variable. Despite this variability, a general picture regarding the occurrence of the genus, its various species, and serogroups is emerging. The sections below present occurrence and (when available) concentration data on cooling tow- ers, residences, hotels and resorts, recreational venues, hospitals, cruise ships, and drinking water and wastewater treatment plants. The data were generated using either culture methods that quantify colony forming units (CFU) and include cells that grow and produce colonies on solid agar, or qPCR for which the data are referred to as gene copies (GC) and that include live, VBNC-like, and dead cells with intact DNA. Data presented below represent both outbreak investigations as well as routine sampling. Prepublication Version - Subject to further editorial revision

132 Management of Legionella in Water Systems Cooling Towers Legionella data from cooling towers were collected from general surveys conducted in the absence of outbreaks as well as from outbreak investigations. One of the first studies to collect environmental data on Legionella in cooling towers was conducted in 1983 (Howland and Pope, 1983). Nine cooling towers were routinely sampled over an 18-month period (162 samples). The culture methods used only identified presumptive L. pneumophila, which was found in all samples and all systems (100 percent posi- tive). The levels were noted to be higher in systems that were used seasonally (i.e., shutdown in the winter and drained); however, the data were not presented in detail. In 1983, a 12-city study took place to investigate Legionella in potable water and cooling towers in Canada (Tobin et al., 1986). Calgary, Edmonton, Fredericton, Halifax, Mississauga, Montreal, Ottawa, Poplar River, Quebec City, Regina, Winnipeg, and Vancouver were part of the survey. Sampling oc- curred from July to September, using a 1- to 2-liter sample that was filtered and plated on BYCE agar. Of the cooling towers that were specifically examined, 28.9 percent of the samples were positive. Legionella concentrations in cooling towers were a maximum of 3.3 x 104 CFU/L with a geometric mean of 4 x103 CFU/L. Almost all isolates were L. pneumophila species including serogroups 1, 3, 4, and 6. One isolate was L. dumoffii. A 2016 study collected 196 cooling tower samples across various regions of the United States (Llewellyn et al., 2017). In this study, 62 percent were positive by qPCR for Legionella spp., 32 percent were positive for L. pneumophila, and 20 percent were positive for L. pneumophila serogroup 1. The au- thors cultured only PCR-positive samples and found that 47 percent were positive for Legionella spp., 32 percent were positive for L. pneumophila, and 24 percent were positive for L. pneumophila serogroup 1. No concentrations were reported and no geographic differences were found. A study of cooling towers in Singapore was one of the few conducted in a tropical environment (Lam et al., 2011). Over an eight-year period (2000–2008), 18,164 samples were analyzed by culture methods and 15.6 percent were positive for Legionella. However, a greater prevalence of positivity was found in the first three years, ranging from 48 to 68 percent, which then dropped to between 12 and 15 percent from 2004 to 2008. Although it was speculated that this decline was because of the switch to chloramines, the drop occurred prior to implementing the change in disinfectants (which was in 2005). Again, concentrations were not reported. Investigations into 255 industrial cooling towers in China revealed a positivity rate of 37 percent using culture techniques (Li et al., 2015). 121 isolates were characterized and all were L. pneumophila, mostly serogroup 1 (56.2 percent), although serogroups 6, 5, 8, 3, and 9 (at 20.7, 9.9, 6.6, 5.0, and 1.6 per- cent, respectively) were also identified. Concentrations between 100 CFU/L and 88,000 CFU/L were reported, with an average of 9,100 CFU/L. Widespread monitoring of cooling towers in New York City was undertaken during an outbreak of Legionnaires’ disease from November 2014 to January 2015. This included power plant cooling tow- ers, in which 29 of 30 samples were positive by PCR (although primers or genes examined were not mentioned), as well as shopping mall cooling towers, in which eight of ten were positive by PCR for L. pneumophila. Those that were positive were cultured, and 90 percent (27/30) and 12 percent (1/8) from the power plants and shopping mall cooling towers, respectively, were positive for L. pneumophila sero- group1 using serology (Benowitz et al., 2018). Concentrations were not reported in these studies. The methods used are poorly described, with no indication of the detection limit for the sampling. Walser et al. (2014) summarized 19 outbreaks associated with cooling towers from around the world, nine of which had environmental sampling data. Interestingly, the Legionella concentrations were greater than 5 x 105 CFU/L and as high as 1 x 108 CFU/L with an average 1.4 x 107 CFU/L, with the excep- tion of one outbreak from Norway (2 x 103 CFU/L). These concentrations are above the average found in the Chinese studies of 9.1 x 103 CFU/L. Attack rates were not calculated because it was unknown how Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 133 many people were exposed to the cooling towers. The concentrations were not related to the number of cases or cases/day, although there was a positive relationship between duration of the outbreak and concentrations. Residences and Public Buildings Surveillance of Legionella in residential premise plumbing taps and showers has been undertaken in many parts of the world because of the concerns associated with sporadic cases of Legionnaires’ disease in a community that cannot be linked to hospitals, hotels, or cooling towers. Some studies have linked an individual with Legionnaires’ disease to a source within their residence, such as Chen et al. (2002). L. pneumophila serogroup 6 was isolated from both the patient and his home potable water system as con- firmed by pulsed-field gel electrophoresis (a method used to fingerprint DNA from bacteria). Other stud- ies implemented over the past 30 years have tried to broadly survey environmental data from residences in China, Germany, Italy, Spain, the United Kingdom (UK), and the United States. In some cases, there was an attempt to examine levels of Legionella in taps in homes or areas of a city where Legionnaires’ disease cases had occurred (Stout et al., 1992). The data from 11 studies are shown in Figure 3-7. Taken together, these data show that the per- centages of samples positive for L. pneumophila (using culture methods for Legionella followed by colony confirmation test specific to L. pneumophila) ranged from 5 percent to as high as 33 percent. When culture methods for Legionella (without colony confirmation testing) were used, positives ranged from 8 percent to 23 percent. As expected, qPCR reported higher numbers of positive samples for Legionella spp. (28 percent to 100 percent) but not notably higher for L. pneumophila (3 percent to 64 percent). Average con- centrations for L. pneumophila reported in the various studies were 1.1 x 103 CFU/L (in the UK), 3-5 x 103 CFU/L (Spain) and 1 x 104 to 6 x105 CFU /L (Pittsburgh). Using qPCR approaches, concentrations were reported at 4.0 x 103 GC/L for L. pneumophila (UK) and 104 GC/L (China). For other Legionella species, the concentrations were 1.2 x 104 GC/L (UK) and 7.7 x 104 to 8.4 x 106 GC/L (China). Levels were found at 105 GC/L for Legionella spp. in rain barrels (where no L. pneumophila was detected). Insights are provided by the studies in Figure 3-7. Stout et al. (1992) found L. pneumophila was associated with lower water temperatures in water heaters (at or below 41oC), with no prevalence in any particular kind of tap. While many suggest warm-water taps should be sampled, the data suggest that all taps can be positive. In China, L. pneumophila was more frequently found in public buildings than in res- idential buildings, perhaps because of higher water age (Liet al., 2018). In public buildings in China, neg- ative correlations were noted between Legionella numbers and total chlorine residuals and between total 16S rRNA gene copy numbers and total chlorine in both the first draw and post flushing (Li et al., 2018). Storage appeared to increase Legionella numbers, which were slightly higher in underground systems (average 1.95 x 106 ± 2.49 106 GC /L) compared to rooftop storage (7.8 x 105 ± 1.40x 106 GC/L, P < 0.05). A German study (Dilger et al., 2018) involved 76,200 samples taken from 13,397 warm-water systems. Ninety-four (94) percent were private homes, with the rest being schools, town halls, sports facilities, hotels, hospitals, and retirement homes. While the average Legionella concentration was not re- ported, 14 percent had less than 103 CFU/L (reported per 100 mL in the paper, i.e., 100 CFU/100mL) and 0.19 percent had 104 to 105CFU/L (which according to German standards is a level at which showering would be restricted). 20.7 percent of samples were positive for Legionella spp., of which L. pneumophila was the prominent species (83.9 percent) followed by L. anisa, and 12 other species. The differences in abun- dance of the various species detected was partly explained by temperatures, as L. pneumophila was present at all temperatures from 10oC to 60oC, while L. anisa was more abundant at low temperatures and other species were limited to narrower temperature ranges. Prepublication Version - Subject to further editorial revision

134 Management of Legionella in Water Systems FIGURE 3-7 Percentage of samples positive for L. pneumophila and other species using culture and PCR from pub- lication dates of 1988 to 2018 in large surveillance studies of households. SOURCES: Pittsburgh 1988 (Lee et al., 1988); Quebec City (Alary and Joly, 1991); Pittsburgh 1992 (Stout et al., 1992); Spain (Codony et al., 2002); U.S. 2010 (Donohue et al., 2014); Philadelphia (Hamilton et al., 2018b); Italy (Totaro et al., 2017); UK (Collins et al., 2017); both China columns (Li et al., 2018); Germany (Dilger et al., 2018). Higher Acanthamoeba concentrations in taps fed by tanks compared to those fed by mains were re- ported in the studies in Hong Kong, Korea, and the UK (Boost et al., 2008; Jeong and Yu, 2005; Seal et al., 1992). L. pneumophila, Acanthamoeba, and V. vermiformis were also detected in tank and tap water in the Chi- nese study (Li et al., 2018). Donohue et al. (2014) surveyed 68 public and private cold-water taps from 2009 to 2010. Low con- centrations of L. pneumophila serogroup 1 were found, between 40 and 620 GC/L, in around 50 percent of the positive samples; yet on occasion, a high level was found up to 105 GC/L, creating an average of 1.97 x103 GC/L with a median of 62 GC/L. This study found that 47 percent of sampled drinking fountains were contaminated with L. pneumophila serogroup 1, with 18 percent of the fountains (3/17) consistently positive. The prevalence of Legionella in hot and cold water was investigated in 141 homes equipped with var- ious types of domestic water heaters (38 percent gas, 38 percent electric, 18 percent oil, and 7 percent solar) in four regions of France (Wallet et al., 2016). Samples by culture exceeded 1,000 CFU/L in 5 percent of hot water and 5.6 percent of cold water from mixing valves and taps. Results using solid phase cytometry for Legionella were strikingly higher, with a prevalence of 41 percent in hot water, 52 percent for cold water, and 53 percent for mixed water. Verhoef et al. (2004) showed that Legionella was present more often in homes that had not been in- habited for ten days than those that had been occupied. Although the results were not significant, the study suggested that some Legionnaires’ disease attributed to temporary accommodation sites (e.g., hotels) might be due to domestic exposure. Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 135 A study in Australia examined the occurrence and concentrations of Legionella in home showers using qPCR (Hayes-Phillips et al., 2019). Legionella spp. and L. pneumophila were positive in 74.6 percent (50/68) and 64.2 percent (43/68) of the showers, respectively. The researchers also demonstrated that qPCR had the potential to demonstrate increased growth potential of the bacteria and exposures at temperatures between 40oC and 60oC. Hotels and Resorts Legionella is frequently found in hotels and resorts. Papadakis et al. (2018) collected 518 samples from 119 hotels in Crete and assayed them by culture; of these, 36 percent (n = 43/119) of the hotels and 13 per- cent of the samples (n = 67/518) tested positive. The majority of positive samples were from swimming pool showers (see Figure 3-8). Like many studies, few samples (n = 5) tested positive for L. pneumophila serogroup 1. Figure 3-9 and Table 3-3 show the distribution of species, serogroups, and concentrations, respectively. The concentrations of L. pneumophila serogroup 1 ranged from 3.5 x 102 to 1.15 x 103 CFU/L. This study is similar to many surveys where a range of isolates is found, with concentrations similar to those previously reported. In Flint, Michigan, 16 samples from hotels and schools were collected from 2015 (during the Legion- naires’ disease outbreak) to 2016 (after the outbreak). No L. pneumophila was detected, but about 50 percent of the samples were positive for Legionella spp. by qPCR at 2.3 x 103 GC/L (Rhoads et al., 2017). FIGURE 3-8 Note no detection in hotel showers, Jacuzzis, or soil; however, only 2, 15, and 2 samples were collect- ed from these locations, respectively. SOURCE: Papadakis et al. (2018). Prepublication Version - Subject to further editorial revision

136 Management of Legionella in Water Systems FIGURE 3-9 Distribution of Legionella species and serogroups detected in hotel swimming pool showers by culture. SOURCE: Papadakis et al. (2018). TABLE 3-3 Concentration Ranges of Legionella Species and Serogroups Detected in Hotel Swimming Pool Showers by Culture Pool Shower Pool Shower Species/Serogroup # of Positive Samples Low CFU/L High CG L.p. sg 1 5 350 1150 L.p. sg 2 4 100 2050 L.p. sg 3 0 L.p. sg 6 1 150 L.p. sg 7 5 200 3350 L.p. sg 8 1 50 L.p. sg 13 0 L.p. sg 14 3 150 100,000 L.p. sg 15 0 L.p. sg 2-15 8 50 100,000 L. anisa 9 250 300,000 L. erythra 3 400 13,000 L. taurinensis 2 650 8,250 L. birminghamensis 1 50 L. rubrilucens 3 50 6,500 L. species 4 50 1,000 SOURCE: Papadakis et al. (2018) Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 137 In a study of 51 hotels in Greece and Corfu that had been linked to travel-associated Legionnaires’ disease (via epidemiological methods although no outbreaks were identified), Kyritsi et al. (2018) reported that 74.5 percent of the hotels were colonized with Legionella spp. The study took place between October 2011 and December 2012, and hygienic inspections and physiochemical data were also collected. Samples were primarily collected from showers (n = 496), with a few others from swimming pools (n = 36), taps (n = 8), coolers (n = 2), boilers (n = 3), cold-water tanks (n = 3), hot tubs (n = 4), cooling towers (n = 3) and one fountain, for a total of 556 samples. For each sample, 500 mL were filtered and assayed by culture methods with a detection limit of 100 CFU/L. In hot- and cold-water taps, L. pneumophila was found in 76.8 percent of the samples (with L. pneumophila serogroup 1 and L. pneumophila serogroups 2-15 at positive rates of 35.8 percent and 41.4 percent, respectively). Non-pneumophila Legionella was detected in 10.9 percent of the samples. Detection was greater in hot water (41 percent positive) and hot tubs (75 percent) compared to cold-water samples (21.4 percent). Those systems with copper piping had samples that were 12.1 percent positive versus 30.4 percent positive in systems without copper. Free chlorine levels of greater than 0.375 mg/L were negatively associated with Legionella. The following parameters were positively associated with Legionella in the cold-water systems (pH > 7.45, heterotrophic bacteria > 2.5 x 104 CFU/mL, conductivity > 1,775 uS/cm (at 25oC), hardness > 321 mg CaCO3/L, and calcium concentrations > 150 mg CaCO3/L) (Ky- ritsi et al., 2018). The regulations in Greece set a limit of 103 CFU/L for Legionella. Some of the hotels in this study that were deemed unsatisfactory using parameters such as hygiene and chlorine were also above this limit for Legionella. Recreational Venues. Recreational sources such as hot tubs and hot-spring baths have long been associated with outbreaks of Legionnaires’ disease and Pontiac fever, primarily caused by L. pneumophila serogroup 1. Table 3-4 shows the concentration data collected from recreational waters by Leoni et al. (2018) during outbreak investigations that included environmental monitoring using culture techniques. The Legionella concentrations were generally greater than 105 CFU/L in these outbreaks, with little associ- ation among cases, attack rates, and concentrations. Pontiac fever outbreaks showed much higher attack rates than Legionnaires’ disease. TABLE 3-4 Attack Rates, Case Numbers, and Legionella Concentrations of Selected Outbreaks of Recreational Waters Venue Attack Rate (%) Cases Concentrations (CFU/L) Pontiac Fever indoor whirlpool 38 13 1.00E+06 hotel whirl spa 66-72 45 9.00E+04 resort spa 86 6 100 Legionnaires’ Disease public bathhouse 0.13 23 8.80E+05 public bathhouse 0.2 34 8.42E+04 hot-spring bath 1.5 295 1.60E+06 public bathhouse 0.13 9 1.30E+06 public whirlpool spa ? 3 1.50E+05 SOURCE: Leoni et al. (2018). Prepublication Version - Subject to further editorial revision

138 Management of Legionella in Water Systems Hospitals There is great concern about Legionella infections in hospitals because of their susceptible popu- lations. As mentioned in Box 3-6, in many large hospitals Legionella monitoring has been undertaken to confirm that water treatment is suppressing bacterial growth in the premise plumbing. The goal for most hospitals is to detect no Legionella. Monitoring is undertaken to provide assurance to patients and managers of the building that controls are working. Culture methods are used most frequently, and any positive results tend to instigate investigation and remediation. Stout et al. (2007) examined Legionella culture data from 20 hospitals in 14 U.S. states between 2000 to 2002 (see Table 3-5). As few as ten and as many as 80 samples were collected per hospital. Legionella (specifically L. pneumophila serogroup 1, L. pneumophila serogroups 2-14, and L. anisa) was detected in 70 percent of the hospitals. These investigators characterized “high level colonization” as when 30 percent or more of the distal outlets were positive for L. pneumophila. A total of 668 samples were collected and 21.4 percent were positive for L. pneumophila serogroup 1, 9.4 percent for L. pneumophila serogroups 2-14, and 9.9 percent for L. anisa. At hospitals that were positive, the percentages ranged from 5 to 83 percent, 5 to 67 percent, and 4 to 28 percent for L. pneumophila serogroup 1, L. pneumophila serogroups 2-14, and L. anisa, respectively. Eleven (11) hospitals had L. pneumophila serogroup 1 but only four of these had known cases of Legionnaires’ disease. TABLE 3-5 Legionella Detection in Premise Plumbing of 20 Hospitals Cases of >30% of distal water L. pneumophila L. pneumophila Hospital L. anisa Legionellosis outlets positive for sg 1 sg 2‐14 Location %+ (#+/total) Identified L. pneumophila %+ (#+/total) %+ (#+/total) CA Yes Yes 47 (7/15) 0 (0/15) 13 (2/15) PA Yes Yes 30 (12/40) 25 (10/40) 0 (0/40) NY Yes Yes 36 (8/22) 0 (0/22) 0 (0/22) IA Yes Yes 35 (19/55) 0 (0/55)) 0 (0/55) NE No Yes 83 (58/70) 0 (0/70) 24 (17/70) OH No No 25 (11/44) 0 (0/44) 0 (0/44) AZ No No 20 (10/49) 12 (6/49) 16 (8/49) MI No No 5 (2/44) 14 (6/44) 7 (3/44) FL No No 17 (2/12) 0 (0/12) 8 (1/2) WV No No 12 (7/58) 0 (0/58) 12 (7/58) CA No No 7 (3/42) 0 (0/42) 0 (0/42) OH No No 0 (0/57) 67 (38/57) 28 (16/57) TN No No 0 (0/28) 7 (2/28) 4 (1/28) MA No No 0 (0/20) 5 (1/20) 0 (0/20) KY No No 0 (0/10) 0 (0/10) 0 (0/10) MI No No 0 (0/44) 0 (0/44) 0 (0/44) DE No No 0 (0/23) 0 (0/23) 9 (2/23) NY No No 0 (0/12) 0 (0/12) 0 (0/12) NY No No 0 (0/13) 0 (0/13) 0 (0/13) MI No No 0 (0/10) 0 (0/10) 0 (0/10) SOURCE: Stout et al. (2007) Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 139 Two hospitals in Flint, Michigan, were tested after an outbreak of Legionnaires’ disease in 2014 and 2015. The prevalence and concentrations of Legionella from October 2015 and March 2016 were measured using qPCR (see Table 3-6 and Rhoads et al., 2017). These two time points corresponded to before and after the Flint drinking water was switched from the Flint River back to Lake Huron; Octo- ber 2015 was also identified as near the end of the outbreak. The percent positives ranged from 3 to 74 percent for L. pneumophila and from 29 to 94 percent for Legionella spp. Concentrations in the positive samples were similar (103 GC/L), regardless of the percent positive. Nonetheless, both percent positives and concentrations were considerably higher in October 2015 compared to March 2016. Although dozens of hospitals are monitoring for Legionella, long-term monitoring data are not readily available. Box 3-8 describes the Legionella monitoring program and its results, as well as the en- gineering approaches used, in one hospital after a decade of testing the water in the hospital’s premise plumbing. This extensive database suggests that non-detects can be achieved and that improvements in water treatment of hospital plumbing systems assist in achieving this outcome. Monitoring has also been used to prove that remediation efforts in hospitals are successful after an outbreak. A nosocomial outbreak of Legionnaires’ disease in 2013 in Australia was followed by extensive cleaning of the water system using heat, flushing, and chlorination (Bartley et al., 2016). The environ- mental monitoring used culture methods, which attempted to match the clinical isolates to water isolates from the patients’ rooms (showers and taps were cultured). Overall 18 percent of the water samples were positive for L. pneumophila serogroup 1 ranging from 6.3 percent to 71.4 percent positive in one of the wings of the hospital. The premise plumbing was treated with 60°C water for ten minutes, yet positive samples were still detected (5/89, 5.6 percent). Disinfection was then carried out by flushing the system with a chlorinated alkaline detergent (pH = 10.0) and then superchlorinating with 10 mg/L free chlorine. Three cycles of treatment were needed to rid the hospital of Legionella. TABLE 3-6 Percentage of Samples Positive and Average Concentrations for Legionella spp. and L. pneumophila at Hospitals in Flint, Michigan, October 2015 and March 2016, by qPCR Average Average Total # of Lp % L spp. % Locations Concentration Concentration samples #+ Positive #+ Positive GC/L GC/L October 2015 Total 98 51 52 3,000 80 82 3,300 Hospital A 46 34 74 3,000 43 94 3,400 Hospital B 52 17 33 3,000 36 69 3,100 March 2016 Below Total 44 1 2 16 36 2,300 quantification Below Hospital A 35 1 3 10 29 2,500 quantification Healthcare Below 9 0 0 6 67 1,900 facility quantification Grand total 142 52 36.5 96 67.7 SOURCE: Rhoads et al. (2017). Note: GC=gene copy detected by qPCR. Prepublication Version - Subject to further editorial revision

140 Management of Legionella in Water Systems BOX 3-8 Hospital Monitoring: Reviewing an 11-year Data Set A hospital on the east side of NYC has maintained records of Legionella testing of its po- table water for more than a decade. From 2007 to 2017 there were only three positive cultures. Interestingly, the positive cultures were all found in bulk water samples of distal sites, while swab samples from the same sites gathered at the same time tested negative. This analysis describes the testing methodology and system configuration, and it reviews potential conclusions that may be drawn from the results. History. The hospital includes a high-rise tower with less than 500 registered inpatient beds. The hospital sees approximately 23,500 inpatients per year in this facility. The patient population is primarily immunocompromised and is highly susceptible to waterborne pathogens, including Legionella. In 1999, the hospital experienced what potentially was the first nosocomial case of Legionella. The patient was diagnosed with Legionella jordanis and Legionella bozemanii sero- group 2. Both Legionella types were also detected in environmental samples of potable water in the hospital. Potable Water System. The primary water source to the hospital is the NYC water sup- ply. Hospital floors at basement, ground, first, second and third levels are supplied from street pressure; all other floors (4 to 21) are supplied from two gravity roof tanks. Inpatient beds are on floors 4 to 19. Two wooden water tanks are located on the roof, each with a total capacity of 10,000 gallons, of which 4,750 gallons are held as fire reserve and 5,000 gallons are available for domestic use. The hospital’s water heaters are the instantaneous type with minimal storage capacity. Tem- perature is set at 60°C (140°F) and mixed locally at faucets and shower bodies. Circulating pumps on the hot-water returns operate in a continuous mode. Inpatient bathrooms, sinks and showers, nurse server sinks and all other potable distal sites from the fourth to the 19th floor are fed from 18 pairs of hot and cold risers. Water distribution begins in the ceiling of the 19th floor and ends in the ceiling of the third floor. Hot-water returns with balancing valves are at the base of each hot-water riser and return back to the heaters on the 20th floor. Secondary Water Treatment. Following the first diagnosed Legionnaires’ disease patient in 1999, the hospital installed secondary water treatment to prevent Legionella growth and prop- agation in the building plumbing in March 2000. Research and discussions with the hospital’s infection control group indicated that long-term mitigation should primarily address the potable hot-water system. To ensure effective treatment levels were maintained, quarterly water testing for Legionella was performed after secondary treatment was installed; these longitudinal records provided the basis and the data for this review. Water Testing Protocol. During the analysis period, potable water testing was performed quarterly. The bulk of the samples were taken twice at each distal site, once by swab and once a first draw of bulk water. All samples were drawn and sent to a third-party lab, overnight delivery, in lab-provided containers. The swabs were taken from inside the faucet/shower with the screen/ head removed. Water Testing Dataset. The review of sampling data began in March 2007 and continued through December 2017. Both water column and swab samples were collected quarterly from approximately 40 to 46 locations that were either showers or faucets. Showers represented 85 percent of the samples collected (1,445 total samples, half were swabs) and faucets represented the other 15 percent (253 samples, half were swabs). Of the 1,698 samples collected over the 11 years, only three were positive. One sample from a shower was positive for L. pneumophila serogroup 1 (140 CFU/mL) and the other two positive samples were Legionella anisa (8 CFU/mL and 10 CFU/mL from a faucet and shower, respectively). The faucet sample positive for L. anisa was taken at a sink in a newly renovated ICU prior to occupancy. It should be noted that while Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 141 the shower water tested positive, the swab samples taken at the same time from the same location tested negative. All tests were performed by culture; the detection limits were 10 CFU/swab sample and 1 CFU/mL for the water sample. It is unclear if seasonality was involved, although the posi- tives were found in spring and fall (March and September). Engineering data such as disinfectant residual, pH, temperature, and estimated water age were not measured or recorded at the time of sampling. Conclusions. Ten years of quarterly testing were performed from 2007 to 2017. During this period, 1,698 tests were performed, resulting in three positive cultures. The three positives were all obtained from the bulk water samples while the corresponding duplicate swab samples were nega- tive. Over the 11-year period of testing, and after the implementation of secondary water treatment, the level of positivity was reduced to two-tenths of one percent, substantially below the 28 percent positivity rate at system implementation measured in the year 2000. Cruise Ships and Ferries Goutziana et al. (2008) studied Legionella on cruise ships and ferries in Greece. No Legionella was found in the ten cruise ships’ water systems. However, 14 of the 21 ferries were positive when 276 sam- ples of hot and cold water were analyzed, and remediation commenced. There was greater contamina- tion in the ferries’ hot-water systems, with 38, 34, 19, 15, and 7 percent of the samples positive for Legio- nella spp., L. pneumophila, L. pneumophila serogroup 1, L. pneumophila serogroups 2-14, and L. pneumophila serogroup 1 concurrent with other serogroups, respectively. In cold water, 18, 15, 11, 4, and 2 percent of the samples were positive for Legionella spp., L. pneumophila, L. pneumophila serogroup 1, L. pneumophila serogroups 2-14, and L. pneumophila serogroup 1 concurrent with other serogroups, respectively. In an- other similar study, 12 cruise ships were found to be negative for Legionella, while 28 ferries were sampled and found to be positive 81 percent of the time (Mouchtouri and Rudge, 2015). Drinking Water and Wastewater Many fewer monitoring studies have focused on drinking water or wastewater systems compared to the other categories, with most studies undertaken as investigative special surveillance studies. A na- tional study found Legionella spp. in 12 of 18 samples (67 percent positive by qPCR) from the sediments of drinking water storage tanks of ten states (i.e., Alabama, Arizona, California, Illinois, New Jersey, North Carolina, Ohio, Pennsylvania, Tennessee) at average concentrations of 5.2 x 103 cell equivalents(CE)/ gram of wet weight of sediment (Lu et al., 2015). L. pneumophila was found in 33 percent of the samples and L. pneumophila serogroup 1 was found in 28 percent. (To facilitate comparison with other studies, dry weight rather than wet weight should have been recorded.) Developing consensus on methods and data reporting is needed for these types of investigations in order to begin to build national databases and to understand the role of drinking water in seeding of premise plumbing. Drinking water and reclaimed water were examined for Legionella species by Garner et al. (2018) using qPCR (see Table 3-7). Prevalence was higher in reclaimed water compared to potable water (89 percent versus 55 percent), and concentrations of gene copies were 10- to 100-fold higher in reclaimed water. There was no quantification of L. pneumophila, although it was annotated in samples using metag- enomic approaches. Prepublication Version - Subject to further editorial revision

142 Management of Legionella in Water Systems TABLE 3-7 Legionella spp. by qPCR in Potable and Reclaimed Waters and Biofilms Sample % Positive (n=) Gene Copy/L Potable water POE 67 (15) 5.6 x 105 Potable water POU 56 (102) 4.7 x 105 Reclaimed water POE 91 (22) 3.8 x 107 Reclaimed water POU 87 (96) 9.6 x 107 Swabs from potable water POU 52 (60) 1.9 x 105 Swabs from reclaimed water POU 92 (51) 5.6 x 106 Note: averages in the final column were determined from positive samples only. POE refers to the point of entry to the distribu- tion system while POU refers to the point of use from the distribution system. SOURCE: Garner et al. (2018). The best known example of a drinking water source playing a major role in an outbreak of Legion- naires’ disease occurred in Flint, Michigan, in 2014–2015. The outbreak coincided with a change in the source and treatment of drinking water for the City of Flint. In the absence of proper chemical corrosion control, this change in source water led to drastic increases in iron levels in the water and also risked dis- rupting biofilms coating the surfaces of pipes, releasing Legionella into the potable water supply of many buildings. Box 3-9 discusses this case in greater detail. Wastewater treatment plants have been identified as sources for Legionnaires’ disease or Pontiac fever in different countries. In 2013, a large outbreak of legionellosis (159 cases) occurred in Warstein, Germany. The source for the outbreak was a cooling tower that received river water into which a bi- ological wastewater treatment plant discharged (Maisa et al., 2015). The effluent of this wastewater treatment plant contained high numbers of L. pneumophila (approximately 107 CFU/L), and genotyping showed identical patterns in patient strains and strains from the wastewater treatment plant (Maisa et al., 2015). Investigations at the treatment plant showed that the aerobically pre-treated wastewater con- tained high numbers of cultivable legionellae (108 to 1010 CFU/L) (Noguiera et al., 2016), demonstrating that legionellae were capable of multiplying in this treatment process. BOX 3-9 2014- 2015 Legionnaires’ Disease Outbreaks in Flint, Michigan Flint is an industrial city in Genesee County, Michigan, whose economy boomed in the 1960s. Subsequent changes in the auto industry decreased factory jobs, with unemployment peaking around 17 percent in 2009 and reaching about 5 percent in 2018 (Bureau of Labor Statistics). By 2018 the Flint population had decreased by about 50 percent. Since 1954, the municipal water source was Flint River water treated at the Flint Water plant. In 1967, the city began purchasing water from Detroit Water and Sewerage Department (DWSD), which treats Lake Huron water at the Fort Gratiot plant. To reduce costs, in April 2014, the city switched back to Flint River water treated at the Flint Water plant. However, corrosion control measures were inadequate. Within a few weeks of the switch, residents complained of not only red and smelly water, but also skin rash- es, respiratory irritation, and gastrointestinal problems. By the end of 2014, the Genesee County Health Department also recognized an increase in legionellosis cases, a pattern that repeated in the summer of 2015. In October 2015, the municipal water was switched back to DWSD, with ap- propriate corrosion control. A massive flushing program was also in place through spring of 2016. At that point, 79 Legionnaires’ disease cases and 12 deaths had been reported. By the following summer, legionellosis cases had declined to historic baseline rates (Rhoads et al., 2017; Zahran et al., 2018). Table 3-9-1 gives a timeline of the Flint water crisis events. Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 143 TABLE 3-9-1 Timeline of the 2014 - 2015 Flint Water Crisis Michigan Department of Environmental Quality (DEQ) approves source water switch and a Flint River changeover ceremony is held. April 2014 On April 25th, the community begins receiving treated Flint River water. May 2014 Complaints begin of poor water quality (smell, taste, discoloration). June 2014 6 cases of legionellosis occur. August 2014 Flint water tests positive for E. coli. Two boil water advisories are issued. September 2014 32 total cases of legionellosis have occurred. November 2014 City increases hydrant flushing to address red water concerns. City receives official violation notice from DEQ for violations of the December 2014 Safe Drinking Water Act (SDWA) for total trihalomethanes (TTHMs). End of 2014 42 total cases of Legionellosis have occurred in 2014. February 2015 High levels of lead are found at a residence (up to 397 ppb). May 2015 3 cases of legionellosis occur. June 2015 9 cases of Legionellosis occur. City receives second violation notice from DEQ for violations of the June 2015 SDWA for TTHMs. Flint installs a granular activated carbon filter to control TTHMs by July 2015 removing organic matter. September 2015 44 total cases of Legionellosis have occurred in 2015. October 2015 Flint switches back to DWSD-treated water from Lake Huron. Governor Snyder appoints the Flint Water Advisory Task Force to October 2015 investigate. For corrosion control, Flint increases phosphate concentration from 1 December 2015 to 2.5 mg/L. January 2016 Federal emergency declared by President Obama. March 2016 Flint Water Advisory Task Force Report issued. Massive “Flush for Flint” campaign to ensure corrosion control is deliv- May 2016 ered throughout. Summer 2016 Legionnaires’ disease cases return to pre-2014 levels. SOURCE: Adapted from Masten et al., 2016. Epidemiology. During the period that Flint residents received treated Flint River water, their risk of Legionnaires’ disease was elevated 6.3-fold (Figure 3-9-1A). That the switch in source and treatment of the municipal water system accounted for this increased disease risk was supported by additional independent epidemiological analyses. When purported hospital-associated cases were disregarded, the risk remained elevated by a factor of 5.7. After boil-water advisories were announced, the odds of Legionnaires’ disease cases among Flint residents declined, most likely due to reduced water use among Flint residents after the boil-water advisory (Zahran et al., 2018). And, in communities bordering Flint, the probability of legionellosis cases in each census tract correlated to their number of residents who commuted into Flint (Zahran et al., 2018). Prepublication Version - Subject to further editorial revision

144 Management of Legionella in Water Systems FIGURE 3-9-1 Spike in Legionnaire’s disease cases coincident with switch in water supply and increased variation observed in the Flint water distribution system. A. Quarterly Legion- naires’ disease incidence in Genesee County, MI, 2010 through 2016. The count of Legion- naire’s disease cases in Genesee County as compiled in the Michigan Disease and Surveil- lance System at the quarterly time step. B. Free chlorine at eight monitoring locations in Flint’s water distribution system, 2013-2016. Free chlorine (mg/L as Cl2) was reported weekly during the three water regime phases defined above (vertical lines) and the periods and dates (year/week) shown at eight locations in Flint. SOURCE: Zahran et al. (2018). Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 145 Water Quality Monitoring. In addition to inadequate corrosion control of Flint River water, multiple water parameters conducive to L. pneumophila persistence or growth were reported. These included slightly elevated distribution water temperature, elevated organic matter, high iron concentrations, and elevated or depleted chlorine residual (Figure 3-9-1B; Masten et al., 2016; Rhoads et al., 2017; Zahran et al., 2018). Iron, an essential nutrient for L. pneumophila (Mengaud and Horwitz, 1993), also inactivates chlorine. Indeed, during the period Flint received corrosive river water, as the concentration of free chlorine in water in a census track declined, the probability of Legionnaires’ disease cases in that sector increased (Zahran et al., 2018). Microbiological Monitoring. Water samples collected in October 2015 from the cold-water taps of public restrooms in two Flint hospitals (Schwake et al., 2016) contained Legionella and L. pneumophila gene copy numbers considerably higher than those reported previously for U.S. drinking water systems in the absence of a legionellosis outbreak (Donohue et al., 2014) (mean concentration range of 1,170 and 2,480 versus 2 gene copies/mL). In fall of 2016, after Flint had switched back to Lake Huron water, a surveillance study of 130 residences cultured L. pneumophila from 13 Flint homes. Of the 16 L. pneumophila strains isolated from premise plumbing, one was serogroup 1, and the rest were serogroup 6 (Byrne et al., 2018). In contrast, all 33 clinical isolates submitted from 2013 to 2016 to the Michigan Depart- ment of Health and Human Services Bureau of Laboratories by hospitals in southeast Michigan were L. pneumophila serogroup 1 (Byrne et al., 2018), consistent with widespread diagnosis by the urinary antigen test. Conclusions. During the time that Flint River water was used as the city’s primary source, corrosion within the Flint municipal water system created conditions favorable for Legionella per- sistence and proliferation. L. pneumophila strains naturally present at low levels within the Flint distribution system and building premise plumbing would likely thrive with the influx of organic carbon and iron and the concomitant drop in free chlorine characteristic of the corrosive treated Flint River water. Unfortunately, the small number of clinical and environmental L. pneumophila isolates collected during the 2014-2015 outbreak limited the molecular epidemiology attempts to identify the source(s) and parameters that accounted for this outbreak. Only 11 patient isolates were collected in Genesee County between 2014 and 2015 and available for whole genome se- quencing, and only eight of these were associated with any exposure in Genesee County during the outbreak. This emphasizes the importance of collecting clinical isolates for tracking potential sources of disease. It should be noted that if the Flint hospitals where patients were likely exposed to Legionella had had effective water management plans including on-site controls of Legionella, there likely would have been very different outcomes in terms of patients contracting Legionnaires’ disease. Indeed, disease cases stemming from one hospital in Flint decreased dramatically after a biocide system was installed in the hospital. Nonetheless, while the Flint outbreak is an example of the failure of an important barrier—treatment of the building water system—it is also unique in highlighting the role of drinking water utilities in creating conditions conducive to Legionella prolif- eration in premise plumbing. Prepublication Version - Subject to further editorial revision

146 Management of Legionella in Water Systems Wastewater treatment plants that service wood-, plant- or food-processing industries in Den- mark, Finland, Sweden, and the United States have also been identified as a source of L. pneumophila (Castor et al., 2005; Gregersen et al., 1999; Kusnetsov et al., 2010). At these locations, only workers at the treatment plants became ill with Legionnaires’ disease or Pontiac fever. L. pneumophila at relatively high concentrations (107 to 109 CFU/L) was mainly observed in sludge and effluent at these plants. Two recent separate outbreaks of Legionnaires’ disease in The Netherlands were traced to bi- ological wastewater treatment plants that treat animal waste (Loenenbach et al., 2018; Alvin Bartels, Dutch National Institute for Public Health and the Environment, personal communication, July 2018). L. pneumophila was observed in high numbers (106 to 108 CFU/L) in their aeration ponds, which contain nutrient-rich water and operate at 35°C. Genotyping of the L. pneumophila strains demonstrated that the same sequence type (ST 1646) was observed in patients and in the treatment plant aeration ponds (Loenenbach et al., 2018). Box 3-10 describes the investigation of a Norwegian outbreak of Legionnaires’ disease attributed to a wastewater treatment plant. BOX 3-10 Wastewater Treatment Plant Identified as a Source for Legionnaires’ Disease A wastewater treatment plant in Norway was identified as a source for Legionnaires’ disease, leading to 56 cases and ten deaths in 2005, and five cases and two deaths in 2008 (Borgen et al., 2008; Nygård et al., 2008). This plant treated wastewater from a wood refinement factory using both an air-treatment process (air scrubber) and a biological treatment process (aeration ponds). In the air-treatment process, process air was mixed with fresh air before it entered the air scrub- bers, where it was sprayed with water. In the biological treatment process, microbial degradation of organic substances from the wastewater was achieved in two large aeration ponds (30,000 m3 of liquid), and the effluent of the plant was discharged to a river (Olsen et al., 2010). In 2005, the same genotype of L. pneumophila serogroup 1 was observed in patients and in water sampled from the air scrubbers and from a river sample downstream of the wastewater treatment plant (Nygård et al., 2008). Since the water temperature in the air scrubbers was ap- proximately 40°C and it expelled greater than 4 m3 of water per hour as aerosol and was never disinfected, initially the air scrubbers were the suspected source for the outbreak in 2005 (Nygård et al., 2008). However, despite the control measures taken for the air scrubbers, a second outbreak linked to the same plant occurred in 2008 (Borgen et al., 2008). Additional research showed that L. pneumophila serogroup 1 was present in high numbers in the aeration ponds (108 to 1010 CFU/L) and in the effluent of the plant (up to 106 CFU/L) that was discharged in the river (Olsen et al., 2010). In river water downstream of the treatment plant (up to 1.6 km downstream), L. pneumophila serogroup 1 was detected at 104 to 106 CFU/L. The L. pneumophila strains were genotyped, and the same sequence type isolated from the patients in 2005 and in 2008 was also observed in the aeration ponds and the river. Moreover, air samples taken above the aera- tion ponds were consistently positive for L. pneumophila by PCR, and cultivation detected up to 3,300 L. pneumophila CFU/m3 air (Blatny et al., 2008). Air samples taken upwind of the aeration ponds were generally negative for L. pneumophila, but downwind samples were regularly positive. Therefore, the aeration ponds (and not the air scrubbers) were identified as the primary source of the 2005 and 2008 outbreaks (Olsen et al., 2010). Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 147 Occurrence Summary The vast majority of studies reviewed for this chapter reported presence/absence data but not quantitative concentration data, making it difficult to draw meaningful conclusions about the extent of Legionella risk from built water systems. Nonetheless, the preceding section makes it clear that over the 30 years that Legionella data have been gathered, the percent positives and concentrations found have not changed significantly over time or with building or device type. Thus, whether large-scale surveys exam- ine cooling towers, residences, hotels, or hospitals, between 30 and 80 percent of the samples are positive for Legionella species and 3 to 20 percent are positive for L. pneumophila. The more limited set of studies for which concentrations were reported demonstrates that higher concentrations of Legionella are associated with higher disease risk. For example, the studies of Legionella outbreaks associated with cooling towers suggest that duration of the outbreak, but not the total number of cases, is related to Legionella concentrations averaging greater than 106 CFU/L (Walser et al., 2014). One small study in Flint, Michigan, showed positivity levels in hospital taps dropping from 55 percent to 2 percent for L. pneumophila along with concentrations dropping from 106 CFU/L to below detection limits once the outbreak subsided. Similarly, in two Flint hospitals there was a drop from 80 percent to 40 percent positivity for Legionella spp. (with no drop in concentrations) after the outbreak (Rhoads et al., 2017). Non-detectable CFU/L is possible in hospital taps as shown by data obtained from a major hos- pital’s 11-year monitoring program (see Box 3-8). A number of the studies cited in this chapter included environmental monitoring that recorded concentrations of culturable Legionella. The Walser et al. (2014) review of cooling tower outbreaks from France, Germany, Italy, New Zealand, The Netherlands, Norway, Spain, and the UK reported Legionella concentrations (for nine outbreaks) ranging from 2.0 x 103 to 1.0 x 108 CFU/L with an average of 1.39 x 107 CFU/L. Leoni et al. (2018) evaluated nine recreational outbreaks of Pontiac fever and Legionnaires’ disease associated with hot tubs and bathhouses. Their work reported Legionella concentrations ranging from 8.4 x 104 to 1.6 x 106 CFU/L with an average of 8.0 x 105 CFU/L. An outbreak associated with a wastewater treatment plant showed that Legionella concentrations from the aerators ranged from 2.0 x 106 to 2.2 x 109 CFU/L with an average of 1.1 x 109 CFU/L (Loenenbach et al., 2018). Finally, Orkis et al. (2018) reviewed data from sporadic cases of disease from several environments (e.g., apartments, homes, high rises, and associated showers and storage tanks) and reported a range of 1.0 x 104 to 2.0 x 105 CFU/L with an average of 1.0 x 105 CFU/L. These data were contrasted to routine sampling concentrations of Legionella from reclaimed water, residential properties, hotel showers, and industrial cooling towers (Codony et al., 2002; Johnson et al., 2018; Li et al., 2015; Papadakis et al., 2018). The results are graphed in Figure 3-10. The goal of this exercise was to see if there was an obvious break in the data between sporadic cases and outbreaks, similar to an analysis done for Cryptosporidium (Haas and Rose, 1995). The Committee identified the concentration of 5 x 104 CFU/L as such a break. Hence, a Legionella concen- tration of 5 x 104 CFU/L should be considered an “action level”—that is, a concentration high enough to warrant serious concern and to move remediation forward immediately. A lower action level may be necessary to protect those at higher risk for legionellosis such as hospital patients, particu- larly those in intensive care, cancer, and solid-organ transplant units. Prepublication Version - Subject to further editorial revision

148 Management of Legionella in Water Systems FIGURE 3-10 Concentrations of culturable Legionella during outbreaks and routine monitoring from various envi- ronments (ranges shown as bars, averages shown as diamonds). Red solid lines are outbreaks. Green dashed lines are routine sampling. The orange solid line is from sporadic cases. The solid black line is the 5 x 104 CFU/L action level identified by the Committee as a break between sporadic cases and outbreaks. SOURCES: Cooling towers outbreaks (Walser et al., 2014), recreational water outbreaks (Leoni et al., 2018), waste- water treatment plant outbreaks (Loenenbach et al., 2018), sporadic cases from buildings (Orkis et al., 2018), re- claimed wastewater ( Johnson et al., 2018), residences (Codony et al. 2002), showers at hotel pools (Papadakis et al., 2018), and industrial cooling towers (Li et al., 2015). QUANTITATIVE MICROBIAL RISK ASSESSMENT FRAMEWORK FOR LEGIONELLA Quantitative microbial risk assessment (QMRA) is the process whereby the risk associated with exposure to pathogens is assessed (Haas et al., 2014). It evolved from the National Academies of Sciences, Engineering, and Medicine’s framework on risk assessment (see Box 3-11), which focused on chemical and physical environmental hazards. QMRA can also be used to assess the Legionnaires’ disease risk from exposure to waters containing L. pneumophila under various scenarios (e.g., aerosols from toilets, showers, or cooling tower drift). Risk assessment has multiple applications in understanding and controlling problems from Legionella. For example, given an acceptable level of risk in a particular venue or application (e.g., hos- pital showers, cooling towers), one can use QMRA to estimate the concentration of L. pneumophila in the breathing zone (or ultimately, in the water being aerosolized) that would result in that risk. This con- centration could be used as a standard, criterion, or operational target to which one would compare the results of routine environmental sampling for Legionella to determine whether it is necessary to remediate a building water system and to what extent (i.e., the “how clean is clean” problem). This does not imply Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 149 BOX 3-11 Risk Terminology and Definitions Kaplan and Garrick (1981) set forth the concept of risk as the likelihood of a consequence from a hazard, with attendant uncertainty and variability. As delineated in a framework set forth by the National Academies of Sciences, Engineering, and Medicine (NAS, 1983), human health risk assessment involves the delineation of a hazard, assessment of exposure, determination of the dose-response relationship and aggregation in a risk characterization. These terms are formally defined as: Risk: The potential for realization of unwanted, negative consequences of an event (Committee on Foundations of Risk Analysis, 2015). Hazard identification: The process of determining whether exposure to an agent can cause an increase in the incidence of a health condition (NAS, 1983). Dose-response assessment: The process of characterizing the relation between the dose of an agent administered or received and the incidence of an adverse health effect in exposed pop- ulations and estimating the incidence of the effect as a function of human exposure to the agent (NAS, 1983). Exposure assessment: The process of measuring or estimating the intensity, frequency, and duration of human exposures to an agent currently present in the environment or of estimating hypothetical exposures that might arise from the release of new [agents] into the environment. In its most complete form, it describes the magnitude, duration, schedule, and route of exposure; the size, nature, and classes of the human populations exposed; and the uncertainties in all estimates (NAS, 1983). Risk characterization: The process of estimating the incidence of a health effect under the vari- ous conditions of human exposure described in the exposure assessment. It is performed by com- bining the exposure and dose-response assessments. The summary effects of the uncertainties in the preceding steps are described in this step (NAS, 1983). Risk management: Risk management is the process of weighing policy alternatives and selecting the most appropriate regulatory action by integrating the results of risk assessment with engineer- ing data and with social, economic, and political concerns to reach a decision (NAS, 1983). conducting QMRA for each situation, but rather developing a generic QMRA for types of buildings or exposures to develop actionable cleanup targets (e.g., cleanup such that the average of ten air samples does not exceed a certain value). Another application of QMRA is outbreak investigations. In this situation the plausibility of a particular source being the cause of an outbreak can be determined by back-calculating the Legionel- la concentrations that would have been there if in fact that site was the cause. There are many other applications of QMRA in the design or remodeling stage of a building. QMRA can inform design deci- sions and determine, for example: (1) the length of a shower hose that should not be exceeded to avoid unacceptable amplification of pathogens; (2) the setback distances from populations for large industrial cooling towers; or (3) the adequacy of building-level hydraulic design to maintain acceptable microbial quality. In all the above cases, even in the absence of precise data for all inputs, risk can be calculated by estimating the uncertainties for each input and propagating them through the calculations. Prepublication Version - Subject to further editorial revision

150 Management of Legionella in Water Systems From a mechanistic standpoint, a Legionella QMRA can be conducted by going through the se- ries of steps shown in Figure 3-11. Given a recovery-corrected concentration of infectious and viable Legionella in water, the aerosol generation rate can be computed. Some enrichment of Legionella in the aerosol may occur, since bacteria selectively accumulate at air-water interfaces (Schäfer et al., 1998). The size distribution of bacterial-laden aerosols is important with respect to transport, survival, and passage to the lungs. Once aerosols of the appropriate size are inhaled, the inhaled dose can be used to determine the risk from the exposure via application of a dose-response model. There are dose-response models for L. pneumophila that have been derived from animal experiments and validated against outbreaks (Arm- strong and Haas, 2007a, 2008). These are consistent with the beta-Poisson and exponential models (Haas, 2015), such that there is no “threshold” dose below which zero risk occurs. In other words, for any dose, no matter how small, there is a finite non-zero risk of infection thence illness, since even a single organ- ism can, in some fraction of hosts, multiply to a biologically significant level in vivo. FIGURE 3-11 Framework for Legionella QMRA. SOURCE: Revised from Hamilton and Haas (2016). Reproduced with permission of R S C Publications in the format Book via Copyright Clearance Center. Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 151 Because the QMRA approach relies on dose-response models from only a few selected strains for which animal testing has been performed, one uncertainty is the incorporation of any strain variability, or variability associated with prior history of bacterial exposure, such as the acquisition or expression of virulence factors (Buse et al., 2015). Also, the current dose-response models are only for L. pneumophila serogroup 1; the relative potency of other serogroups is unknown. The dose-response models do not ac- count for any differences in host characteristics, such as age, gender, or immune status. If actual data are available for microorganisms at one of the intermediate points in the flow chart, it is possible to start the QMRA at that point. For example, size-resolved microbial concentrations in aerosols might exist which could be used as a starting point (Step F). There have been over 18 exposure assessments and more than ten full risk assessments conducted on L. pneumophila (Hamilton and Haas, 2016). In some cases, concentrations may only be reported as presence/absence. In this situation, concen- trations can be estimated using an MPN approach. This is discussed and illustrated in Box 3-12, which indicates that non-detects can be informative if the volume examined is known. Non-detects, as well as samples that are “too numerous to count” (TNTC), can also be informative for exposure assessment as long as the volumes examined and the cut-offs for TNTC are known (Haas and Heller, 1988). Environmental measurements of Legionella are frequently made using molecular methods, with qPCR being the most prevalent technique. However, direct sequencing approaches (Timms et al., 2017) may become more common. (A discussion of these and putative viability assays is found earlier in this chapter.) Exposure estimates are necessary to produce good risk estimates, and the number of samples collected in a monitoring program and their detection limits should be sufficient to determine exceed- ance or compliance with an acceptable risk value. The number of samples can be determined using standard quality control statistics. As was made obvious earlier in this chapter, the chosen Legionella sampling method may influence the measurements of occurrence and concentration. For example, a recent study comparing the con- centrations of Legionella spp. in wastewater treated for non-potable reuse found dramatic differences be- tween the results from culture, qPCR, and EMA-qPCR methods ( Johnson et al., 2018), as shown in Figure 3-12. Culture-based methods generally reported the lowest occurrence and concentrations. Regardless of the sampling method used for exposure assessment, quantification of any microor- ganism carries with it many sources of variability. Some variability may be inherent in the time-to-time and place-to-place differences in actual microbial levels, which is irreducible by more sampling. This variability was exemplified by a detailed investigation of hot- and cold-water outlets in nine residential homes and hotels in Cologne, Germany (Völker et al., 2016). The 807 samples taken showed significant variability (up to 4 logs) in Legionella spp. concentrations in flushed samples between sampling points within a single building and, for a given point, between hours in a day or between weeks. Other variabil- ity may be due to the experimental techniques themselves, including sample collection, concentration, decontamination, processing, and detection. Only a true end-to-end comparison can assess the extent of this intrinsic variability. Such a study requires that a sample be spiked with a known number of or- ganisms and then processed through the entire protocol (i.e., concentration, decontamination, detection) to assess the recovery and its variability. An example of such a study is Bonilla et al. (2015). Sufficient numbers of samples should be taken to make the effect of this intrinsic variability small with respect to the irreducible variability. Prepublication Version - Subject to further editorial revision

Management of Legionella in Water Systems Prepublication Management of Legionella in Water Systems ionella in Water Systems Prepublication Prepublication Management of Legionella in Water Systems   Prepublication   152 Management of Legionella in Water Systems   BOX 3 BOX 3‐12  BOX 3‐12    BOX 3‐12  Using Presence/Absence Data  Using Presence/Absence Data to Estimate Concentrations  BOX 3‐12    Using Presence/Absence Data to Estimate Concentrations  Using Presence/Absence Data to Estimate Concentrations    BOX 3-12 Using Presence/Absence Data to Estimate Concentrations    Consider that a number (N) of samples, each o samples are found to be positive.  The fraction positive is then � �have been collected. Of these, P Using Presence/Absence Data to Estimate Concentrations � Consider that a number (N) of samples, each of volume (V) have been collected.  Of these, P    samples are found to be positive.  The fraction positive is then � � o be positive.  The fraction positive is then � � .  What is the estimate for the thenconcentration () of microorganisms in the system fro Consider that a number (N) of samples, each of volume (V) �.  What is the estimate for the  � Consider that a number (N) of samples, each of volume (V) have been collected.  Of these, P  � .  What is the estimate for the  at a number (N) of samples, each of volume (V) have been collected.  Of these, P  samples are found to be positive.  The fraction positive samples are found to be positive.  The fraction positive is then � � .  What is the estimate for the for � Consider that a number (N) of samples, each of volume (V) have been collected.  Of these, P  � samples are found to be positive. The fraction positive is � � concentration () of microorganisms in the system from which the samples were drawn?  This is  . What is the estimate  microorganisms in the system from which the samples were drawn?  This is  which the samples were drawn? This the concentration (μ) of microorganisms in the system from concentration () of microorganisms in the system from which the samples were drawn?  This is  analogous to the single dilution MPN analysis, which h concentration () of microorganisms in the system from which the samples were drawn?  This is  analogous to the single dilution MPN analysis, which has long been discussed (e.g., Cochran, 1950).   is analogous to the single dilution MPN analysis, which has long been discussed (e.g., Cochran, analogous to the single dilution MPN analysis, which has long been discussed (e.g., Cochran, 1950).   analogous to the single dilution MPN analysis, which has long been discussed (e.g., Cochran, 1950).   the assumption that the microorganisms in the Under the assumption that the microorganisms in the ngle dilution MPN analysis, which has long been discussed (e.g., Cochran, 1950).   system from which the samples are 1950). Under Under the assumption that the microorganisms in the system from which the samples are drawn are  Under the assumption that the microorganisms in the system from which the samples are drawn are  �� � ����� � �� � ����� � ��  � ��  on that the microorganisms in the system from which the samples are drawn are  the best (maximum likelihood) esti- drawn are randomly distributed, i.e., Poisson, the following is randomly distributed, i.e., Poisson, the following is the Under the assumption that the microorganisms in the system from which the samples are drawn are  �� � ����� randomly distributed, i.e., Poisson, the following is the best (maximum likelihood) estimate:  �� � ����� � ��  randomly distributed, i.e., Poisson, the following is the best (maximum likelihood) estimate:  �� � ����� �For many other organisms (although not for Legionella), the distribution of microorganisms in water is  ��  mate: randomly distributed, i.e., Poisson, the following is the best (maximum likelihood) estimate:  d, i.e., Poisson, the following is the best (maximum likelihood) estimate:  (1)  (1)  (1)  (1)  For many other organisms (although not for Legionella For many other organisms (although not for Legionella), the distribution of microorganisms in water is  not random but more heterogeneous than the Poisson distribution ( El‐Shaarawi et al., 1981; Gale et al.,  For many other organisms (although not for Legionella), the distribution of microorganisms in water is  anisms (although not for Legionella), the distribution of microorganisms in water is  not random but more heterogeneous than the Poisson not random but more heterogeneous than the Poisson distribution ( El‐Shaarawi et al., 1981; Gale et al.,  For many other organisms (although not for Legionella), the distribution of microorganisms in 1997; Pipes et al., 1977).  This may be because of intrinsic variability in the environment, or variability in  not random but more heterogeneous than the Poisson distribution ( distribution ( El-Shaarawi et al., re heterogeneous than the Poisson distribution ( El‐Shaarawi et al., 1981; Gale et al.,  El‐Shaarawi et al., 1981; Gale et al.,  1997; Pipes et al., 1977).  This may be because of intrin overdispersion parameter “k”; small k values indicate greater overdispersion, and the limit of � � � is  water is not random but more heterogeneous than the Poisson 1997; Pipes et al., 1977).  This may be because of intrinsic variability in the environment, or variability in  the enumeration, or both.  An alternative to the Poisson is the negative binomial distribution with an  overdispersion parameter “k”; small k values indicate greater overdispersion, and the limit of � � � is  1981; Gale et al., 1997; Pipes et al., 1977). This may be because of intrinsic variability in the en- 1997; Pipes et al., 1977).  This may be because of intrinsic variability in the environment, or variability in  977).  This may be because of intrinsic variability in the environment, or variability in  the enumeration, or both.  An alternative to the Poisso the enumeration, or both.  An alternative to the Poisson is the negative binomial distribution with an  overdispersion parameter “k”; small k values indicate greater overdispersion, and the limit of � � � is  meter “k”; small k values indicate greater overdispersion, and the limit of � �⁄� is  the Poisson distribution.  vironment, or variability in the enumeration, or both. An alternative to the Poisson is the negative �� � ���� � �� persion, and the limit of k→∞ is the Poisson distribution.�� � � ��  the enumeration, or both.  An alternative to the Poisson is the negative binomial distribution with an  r both.  An alternative to the Poisson is the negative binomial distribution with an  overdispersion parameter “k”; small k values indicate  the Poisson distribution.  binomial distribution with an overdispersion parameter “k”; small k values indicate greater overdis- �� � ���� � ����⁄ �� � ���� � ����⁄� � ��  (2)  the Poisson distribution.  �� � ���� � ����⁄� � ��  (2)  �� � ���� � ����⁄� � ��  tion.  Figure 3‐12‐1 below is a plot of the estimate for �� given the fraction of samples positive in the case of  the Poisson distribution.  (2)  the Poisson as well as negative binomial distributions with different “k” values.  Below a fraction positive  (2)  Figure 3‐12‐1 below is a plot of the estimate for �� giv Figure 3‐12‐1 below is a plot of the estimate for �� given the fraction of samples positive in the case of  of approximately 10 percent, the impact of heterogeneity is negligible.  The utility of this approach can  Figure 3-12-1 is a plot of the estimate for μV given the fraction the Poisson as well as negative binomial distributions  of samples positive in the case of as negative binomial distributions with different “k” values.  Below a fraction positive  �is negligible.� The utility of this positive of approximately 10 percent, the impact of heterogeneity ����� � ���� ����� and therefore  Figure 3‐12‐1 below is a plot of the estimate for �� given the fraction of samples positive in the case of   is a plot of the estimate for �� given the fraction of samples positive in the case of  be illustrated with a simple example.  Suppose 50 mL samples are used and less than 5 percent of them  the Poisson as well as negative binomial distributions with different “k” values.  Below a fraction positive  the Poisson as well as negative binomial distributions with different “k” values. Below a fraction � � illustrated with a simple example. Suppose 50 mL samples are used and less ����������.  the Poisson as well as negative binomial distributions with different “k” values.  Below a fraction positive  are found to be positive for Legionella.  From equation (1), �� of approximately 10 percent, the impact of heterogen of approximately 10 percent, the impact of heterogeneity is negligible.  The utility of this approach can  are found to be positive for Legionella.  From equation (1), �� � ����� � ���� � ����� and therefore  approach can be of approximately 10 percent, the impact of heterogeneity is negligible.  The utility of this approach can   percent, the impact of heterogeneity is negligible.  The utility of this approach can  be illustrated with a simple example.  Suppose 50 mL s be illustrated with a simple example.  Suppose 50 mL samples are used and less than 5 percent of them  are found to be positive for Legionella.  From equation (1), �� � ����� � ���� � ����� and therefore  itive for Legionella.  From equation (1), �� � ����� � ���� � ����� and therefore  � � ����������.  than 5 percent of them are found to be positive for Legionella. � � ����������.  be illustrated with a simple example.  Suppose 50 mL samples are used and less than 5 percent of them   simple example.  Suppose 50 mL samples are used and less than 5 percent of them are found to be positive for Legionella.  From equation � � ����������.  From equation (1), and therefore   FIGURE 3‐12‐1  Estimation of Mean Density Given Fraction of Positive Samples of Volume vs. Poisson  compared to Negative Binomial Distributions.  3‐72      FIGURE 3‐12‐1  Estimation of Mean Density Given Frac   FIGURE 3‐12‐1  Estimation of Mean Density Given Fraction of Positive Samples of Volume vs. Poisson  mation of Mean Density Given Fraction of Positive Samples of Volume vs. Poisson  compared to Negative Binomial Distributions.  FIGURE 3‐12‐1  Estimation of Mean Density Given Fraction of Positive Samples of Volume vs. Poisson  compared to Negative Binomial Distributions.  compared to Negative Binomial Distributions.  ve Binomial Distributions.  FIGURE 3-12-1 Estimation of Mean Density Given Fraction of Positive Samples of Volume vs. Poisson 3‐72 compared to Negative Binomial Distributions. 3‐72  3‐72  3‐72  Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 153 FIGURE 3-12 Seasonal occurrence (A) and concentration (B) of Legionella (in CFU/mL for culture, and genomic units per mL for qPCR and EMA-qPCR). SOURCE: Johnson et al. (2018). For L. pneumophila, there are no good published studies to assess the intrinsic variability of different sampling methods, including culture techniques, molecular techniques, or various proprietary test kits. However, one known factor (albeit with coliforms) is that the variability associated with methods that result in actual concentrations tends to be less than variability associated with MPN-type techniques, although this does depend on how many actual counts are enumerated, and the protocol (number of di- lutions and replicates per dilution) of an MPN. As an example, early work by Thomas and Woodward (1955) showed that the MPN enumeration of coliform tended to have about 2.5 times the coefficient of variation for replicates than the membrane filter colony count methods. QMRA Case Studies for Legionella As an example of a forward QMRA, the risks associated with Legionella exposure in aerosols gen- erated from toilet flushing using reclaimed wastewater were examined by Hamilton et al. (2018c). The key inputs required were:10 • The concentration of L. pneumophila; in this analysis, the monitoring results from several water re- use facilities were used ( Johnson et al., 2018) in which Legionella spp. were measured using culture techniques, qPCR, and EMA-qPCR, the latter of which is thought to be more closely related to viability (Mansi et al., 2014). • Measurements of aerosol concentrations in a respirable size range in the vicinity of the toilet after flushing; the size-resolved concentrations from Johnson et al. (2013) were used and aerosols in the range of 1 to 10 µm were considered respirable. • Respiration rate for light activity of 0.013 to 0.017 m3/min from the Exposure Factors Handbook (EPA, 2011) was used. 10 This is designated as “Model 2” in the paper. Three different models, yielding a span of results, were compared. Prepublication Version - Subject to further editorial revision

154 Management of Legionella in Water Systems • Number of flushes per day; a value of 5/d was used (DeOreo et al., 2016). • Time of exposure to aerosol per occurrence; a range of 1 to 5 minutes exposure per flush was used based on Lim et al. (2015). • Dose-response relationship for L. pneumophila developed by Armstrong and Haas (2007a) from the underlying data of Muller et al. (1983) and Fitzgeorge et al. (1983) were used. In particular, the first bullet (concentration) has uncertainty because of the issues associated with envi- ronmental measurements of viable infectious L. pneumophila discussed above. The final bullet (dose-re- sponse) has uncertainty because of the use of animal models on a particular strain of Legionella, although this has been shown to be consistent with human outbreaks (Armstrong and Haas, 2007b). Several factors not considered could be of importance. These include the difference between Legionella spp. and L. pneumophila, the possibility of accumulation of microorganisms at air–water inter- faces and thus selective enrichment in the aerosols (Blanchard, 1989), and any inactivation of microor- ganisms in the period between aerosol formation and inhalation. Based on this analysis, using the three different means of enumerating bacteria in the water (i.e., culture techniques, qPCR, and EMA-qPCR), the annual risks (median) were estimated to be: • 3.2 x 10-9 (using culture) • 1.02 x 10-7 (using qPCR) • 2.56 x 10-8 (using EMA-qPCR) When compared to a common benchmark of 1/10,000 annual risk, these estimates were substantially lower. It is also possible to perform a reverse QMRA (Soller et al., 2010), in which the starting point is the desired risk of a scenario (Step L in Figure 3-11); then, the calculations are run “backwards” to ascertain the water quality (Step A in Figure 3-11), aerosol concentration, etc. corresponding to that desired risk. An example of a reverse QMRA is the work of Schoen and Ashbolt (2011), of which a portion is sum- marized here. They considered the risk of Legionella exposure during a single showering event. Starting from a maximum inhaled L. pneumophila dose of 1-100 CFU,11 they considered what the water concen- tration in the shower might be to attain that level. Key inputs required for their reverse QMRA were: • Aerosol production rate and microbial partition coefficient (from bulk water to aerosol); values used were based on the experiments of Perkins et al. (2009). • A respiration rate of 0.012 – 0.025 m3/min was used (EPA, 2004). • Size-specific aerosol deposition fractions in the lungs from Schlesinger (1989) were used. • Duration of exposure in the shower was assumed to be 15 minutes (Perkins et al., 2009). With this analysis, they computed that a bulk air concentration of 35 to 3,500 CFU/m3, and a bulk water concentration of 3.5 x 106 – 3.5 x 108 CFU/L would be required to attain the delivered dose. In all cases, the performance of a QMRA (either in the forward or reverse directions), requires a substantial number of input parameters, each of which may have uncertainty. The resultant risk estimate (or in the case of a reverse QMRA, the exposure estimate) will also not be known with certainty. The calculation of these uncertainties is possible using a variety of techniques, with Monte Carlo methods being the most common. 11 This would produce a risk unacceptably high in the general population, but was used as an extreme example. Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 155 Acceptable Risk A key question for any QMRA is what level of risk should be regarded as acceptable. This is not (sole- ly) a scientific question, but must be informed by policy, economic, and other social factors. In developing U.S. drinking water regulations for virus and protozoa, the EPA was informed by an annual risk level of 10-4 infections/year (Regli et al., 1991). For regulation of carcinogens, a range of 10-4 to 10-6 cases/lifetime has been used as a range of acceptability (Travis and Hattmer-Frey, 1988). The World Health Organization has widely promoted the use of 10-6 DALY/person-year as being acceptable for microorganisms in drinking water (Havelaar and Melse, 2003; WHO, 2008). The broad community of stakeholders in the Legionella arena need to be engaged in a deliberative process to develop acceptability levels in different venues (Renn, 1999). It may be that different venues with different types of exposure and different exposed populations should have different acceptability levels—for example, hospitals with acute susceptible populations and relatively short stays, versus cooling towers with broad, potentially frequent exposure to the general population. The level of risk that may be regarded as acceptable is associated with the type of hazard (how well it is understood, natural versus human-derived), the consequences (e.g., death) and the ability to control the exposure. For drinking waterborne pathogens, The Netherlands has codified an annual risk of 10-4 (1 in- fection in 10,000 over a one-year time frame). In the United States, for drinking water standards primarily aimed at controlling mild to moderate gastroenteritis, a value of 10-4 infections per year is also considered acceptable. However, it is important to examine the daily risk versus an annual risk, as both are incurred every day in the context of drinking water. Annual risk is translated to a daily risk via the relationship below (Haas, 1996): Pannual = 1 — (1 — Pdaily)365 If daily risk varies day to day, then the annual risk can be computed as follows: 365 Pannual = 1 — П (1 — Pi) i=1 For an exposure that is relatively continuous to a large population, an annual risk level may be an appro- priate approach to control. This could be pertinent to exposures such as large industrial cooling towers. For exposures that may only be short-term, especially to susceptible subpopulations, the control of daily risk could be appropriate. This could be pertinent to situations such as hospitals and nursing homes. Use of a daily risk level could lead to a different monitoring and control scheme. This is illustrated by the hypothetical Figure 3-13 below. The red line indicates the uniform daily risk that would correspond to 1/10,000 infections per year. The black plot illustrates a random set of daily risks that over the course of the year would result in the same annual risks, despite a high degree of day-to-day variability. For shorter-term exposures, therefore, a population would be exposed to higher risks from time to time rather than to a uni- form risk. In the case of Legionella, there is a lack of data to know how variable day-to-day exposures, and hence the resultant risks, might be. In addition to the choice of annual or daily (or some other time period) averaging for assessing ac- ceptability of risk in particular venues, the choice of the endpoint metric needs to be addressed by risk managers. As noted above, both 10-4 annual risk of infection and 10-6 DALY per person per year have been put forth as useful endpoint metrics in the context of drinking water. These were developed with regard to the risks of gastroenteric pathogens such as enteric bacteria, viruses, and protozoans (e.g., Giardia and Cryptosporidium). Such organisms have mild to moderate health consequences, such that the 10-4 annual risk of infection and 10-6 DALY endpoints produce similar results with respect to acceptable microbial quality of water (i.e., the concentration of Cryptosporidium in water). Prepublication Version - Subject to further editorial revision

156 Management of Legionella in Water Systems FIGURE 3-13 Sketch of uniform daily risk vs. variable daily risk. However, the severity of legionellosis leads to a much higher ratio of DALYs per infection as noted in Table 3-9. Compared to cryptosporidiosis, legionellosis is more than 300-fold more consequential. Hence, endpoints of 10-4 annual risk of infection and 10-6 DALY are not equivalent in this case. TABLE 3-9 Ratio of DALYs to Infections for Various Pathogens Conveyed Via Water Illness DALYs/100 infections Cryptosporidiosis 0.3 Norovirus 0.3 Salmonellosis 0.3 Hepatitis 17 Legionellosis 97 SOURCE: Abstracted from van Lier et al. (2016). This is also illustrated in Figure 3-14, in which the water concentrations corresponding to accept- able risk based on per exposure or annually (using either infections or DALYs as the endpoint) are graphed for different types of exposures. In this case, faucet, shower, and toilet exposures using both conventional and water-efficient fixtures are tabulated. Once a risk manager has decided what endpoint metric and acceptability level and what averaging period (if any) are appropriate, then the corresponding water con- centration can be determined. For example, if an acceptable annual risk of 10-4 has been chosen, then the concentration of L. pneumophila measured at a conventional faucet, toilet, or showerhead should be no more than 105, 8.6 x 105, and 1.4 x 103 CFU/L, respectively (see Table 3-10). On the other hand, if acceptable risk is based on the 10-6 DALY, then the concentration of L. pneumophila measured at a conventional faucet, toi- let, or showerhead should be no more than 103, 8.8 x 103, and 14 CFU/L, respectively. (These numbers are revisited in Chapter 5 as thresholds to help interpret monitoring data.) Risk management decisions need to be developed for target levels of acceptability to Legionella in various settings. While U.S. practice has been to use a 1/10,000 annual infection endpoint as a measure of Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 157 FIGURE 3-14 Curves of L. pneumophila concentration versus risk from conventional (conv) and water efficient (eff) fixtures. CSI = clinically symptomatic infection (i.e., disease). SOURCE: Hamilton et al. (2019). https://pubs.acs. org/doi/10.1021/acs.est.8b03000>, and include a notice to readers that further permissions related to the material excerpted should be directed to the ACS. acceptability in drinking water (Rose et al., 1991), WHO has promoted use of a 10-6 DALY annual risk as an endpoint because of the increased severity of Legionnaires’ disease. Which risk target is more appropriate, and whether an annual or a daily (or some other time period) average is more appropriate, are specific questions that need to be addressed by risk managers. The examples above focused on exposure to aerosols in the indoor environment from plumbing fix- tures. It is also possible to conduct a QMRA for exposure to cooling towers and other aerosols in the out- door environment (see Hamilton et al., 2018c), but these circumstances require much more site-specific information. This includes (1) characteristics of the cooling tower, including aerosol generation rate and height, (2) the concentration of L. pneumophila within the water producing the aerosol, and (3) wind direc- tion (relative to exposed population), velocity, and meteorological conditions (atmospheric stability). TABLE 3-10 L. pneumophila Concentrations in Various Plumbing Fixtures that Correspond to Target Risk Levels. Critical Average Concentration Devices/Fixtures (CFU/L) Target Risk Value: 10-4 infections per person per year Conventional faucet 104,000 Conventional toilet 857,000 Conventional shower 1,410 Target Risk Value: 10-6 DALY per person per year Conventional faucet 1,060 Conventional toilet 8,830 Conventional shower 14.4 NOTE: Median estimates from a Monte Carlo simulation. SOURCE: Hamilton et al. (2019). Prepublication Version - Subject to further editorial revision

158 Management of Legionella in Water Systems How to Respond to Data and Information Generated from Sampling The role of liability in the control and prevention of Legionnaires’ disease has been mixed in the United States. Multi-million-dollar lawsuits are not uncommon for Legionnaires’ disease when the en- vironmental source is tracked to a large building or other entity where the owner and/or other persons are responsible for the safety of those served by an implicated water system. Manslaughter charges have been filed on rare occasions. To protect their clients, some lawyers have advocated that the water facil- ities considered at-risk (e.g., hotels, hospitals) test their water for Legionella as part of a water manage- ment plan, while others have advocated that it is better not to test since results could potentially be used against their client. This latter argument will probably not become entrenched as testing becomes more common, and “not knowing” may hurt rather than help the defense. The growing number of litigants and large size of settlements may result in the insurance industry pushing many clients with water systems serving the public into improving their prevention program for legionellosis. A challenge inherent in implementation of Legionella control programs by healthcare centers, as- sisted-living facilities, hotels, and other commercial buildings, as well as public water supplies, is balanc- ing professional or commercial responsibility with notification of the public when disease cases or water system contaminations occur. No guidelines currently exist, for hospitals or other building management or municipalities, on how to release information when Legionella and Legionnaires’ disease are detected. Such guidelines are critically important, given the need to provide accurate, actionable information to the public, while protecting patient confidentiality, and taking resource limitations for the entities involved in releasing information into account. CSTE’s Legionnaires Disease Surveillance Working Group plans to focus on risk communication, including notification and disclosure, with regards to Legionnaires’ disease outbreak investigations and will be coordinating with other relevant national organizations, in- cluding the CDC, in the coming months (Monica Schroeder, CSTE, personal communication, April 26, 2019). A policy framework for risk communication should be developed by a coalition of stakeholders, with representatives from infectious disease, epidemiology, microbiology, and public health; healthcare and assisted-living management; hotel and resort management; cooling tower and municipal water man- agement; insurance; liability and privacy law; and ethics. The work of this coalition could be informed by the European Legionnaires’ Disease Surveillance Network (ELDSNet); the Public Services and Procure- ment Canada’s Legionella Management Communications and Actions Protocol; the federal Sunshine Act to increase transparency in government; and the CDC Foodborne Diseases Active Surveillance Network (FoodNet), a national food safety policy and prevention effort that monitors trends, attributes illness to source, and disseminates information about current foodborne illnesses to the public. CONCLUSIONS AND RECOMMENDATIONS This chapter has demonstrated that Legionnaire’s disease rates have been rising in the United States and Europe for the past 20 years, and current reported incidence is likely a substantial underestimate of the actual disease burden. The Committee estimates 52,000 to 70,000 cases of Legionnaires’ disease in the United States each year. There are many sources of Legionella risk in engineered water systems, from cooling towers to premise plumbing to hot tubs. Most of the occurrence data gathered from these sources has not been reported as concentrations, making it difficult to discern trends over time and con- duct microbial risk assessment. In only a few outbreak investigations have clinical and environmental data been linked to definitively show that a particular water system was the etiological source of disease cluster. The following conclusions and recommendations are made to improve surveillance and diagno- Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 159 sis of legionellosis, monitoring of water systems for Legionella, and identification of sources of exposure for both sporadic and outbreak-associated Legionnaires’ disease. There is an urgent need to develop better clinical tools that will capture more Legionnaires’ disease cases and identify pathogenic Legionella beyond L. pneumophila serogroup 1. The increasing rates of legionellosis, combined with its associated morbidity and mortality, demand improved diagnos- tics. First, hospitals in both rural and urban areas should have access to on-site urinary antigen testing to facilitate more targeted antimicrobial therapy and to increase disease recognition. Second, efforts to develop standardized molecular methods for Legionella diagnoses (including non-pneumophila species and pneumophila serogroups other than serogroup 1) should be prioritized by research laboratories and federal agencies. Such methods could increase understanding of the extent of the underestimate of re- ported disease rates and should be accessible outside of research and academic institutions. Finally, the U.S. Department of Health and Human Services should fund multi-center prospective studies of clinical respiratory samples using these new assays to better understand prevalence and diversity of the Legionella species and serogroups causing clinical disease. Once the “true” diversity of human-infectious legionellae is identified, a range of environmental niches could then be explored to identify isolates representing genotypes by niche and preferred methods for their identification from environmental samples. There is also a need for education and a cultural shift from empiric treatment to use of available and future diagnostic tools for Legionella to better characterize the true incidence of legionellosis in the community. The CDC should strengthen the (soon-to-be-merged) NNDSS and SLDSS to include environ- mental exposures as feasible, including both the potential exposure setting and the type of related building water systems. Although all cases will not receive thorough environmental investigations, at a minimum it should be discerned whether a case may be associated with a healthcare facility, accom- modation site, hot tub or other well-recognized potential source, as well as some information about the building water system and any known deficiencies (e.g., water main breaks) during the incubation pe- riod. Similarly, within NORS, the CDC should consider housing Legionella outbreak data in a separate database from enteric pathogens to make NORS more useful for legionellosis prevention and control. In addition, timely analyses by setting and type of water system, with more frequent updating of publicly available data, would improve the usefulness of NORS for assessment of Legionella prevention efforts. An improved understanding of sporadic, community-acquired cases of Legionnaires’ disease is critical to reducing the rising rates observed over the last 20 years. Determining the most common sources of sporadic disease will require well-funded, population-based studies in multiple juris- dictions (e.g., cities, counties, states). Such studies would require the recruitment of multiple medical centers with an adequate number of Legionella cases each year, willingness and capacity to collect clinical samples for Legionella culture, environmental personnel with knowledge of how to sample the most likely sources of exposure for legionellosis patients, and laboratory capacity to reliably grow Legionella from clinical and environmental samples. In the United States, clinical cultures are currently available for less than 10 percent of cases; thus, an effective study would have to dramatically improve on the current capacity to obtain cultures from patients. Enhanced clinical culture capacity is also essential to accu- rately assess the contribution to disease from non-pneumophila Legionella, and L. pneumophila that is not serogroup 1 (recommended above). The CDC should work with states to gain closer to real-time reporting and investigation of travel-associated cases. Many outbreaks of travel-associated disease can be best detected at the national Prepublication Version - Subject to further editorial revision

160 Management of Legionella in Water Systems level, since many of the patients who report staying in a hotel or other accommodation during the incu- bation period have crossed state lines. Currently, reporting of travel-associated cases from many states is neither timely nor complete. Better understanding travel-associated cases is an easy target for interven- tion, as these data are often readily available from patient interviews, can help to link individual cases to larger clusters, and may help to identify opportunities to limit further exposures. Although additional Legionella program efforts are underway in some states, these efforts are not comprehensive, and most state health departments are severely lacking (both in resources and expertise) in their programs of surveillance, prevention, and control for Legionnaires’ disease. Regional Centers of Excellence for prevention and control of legionellosis could serve as a backbone to strengthen the capacity of state health departments to detect and investigate cases of Legionnaires’ disease. These centers could be modeled on the Integrated Food Safety Centers of Excellence and the Centers of Excellence for Vector Borne Diseases, with modifications to include the relevant disciplines needed for Legionella applied research and control. The Centers could undertake critical applied research (e.g., optimizing culture methods and comparing them to new methods and coordinating the in-depth, multi- ple-jurisdiction studies of environmental exposures recommended above). By building a cadre of experts in Legionella prevention and control that includes industrial hygienists and engineers, these centers could promulgate best practices for prevention and control measures (see Chapter 4). Finally, these Centers could train and assist building managers as they create water management plans, and they could initiate certification programs for those responsible for the safety of water systems in built environments (see Chapter 5). A systematic study to compare culture methods for L. pneumophila (and other pathogenic le- gionellae) with qPCR, viability-qPCR and RT-qPCR is needed to determine comparability. qPCR and its variants offer a more rapid method to quantify Legionella in the environment and could be used consistently to inform decisions on decontamination and restoration of affected systems, to investigate the bacteria’s ecology and exposure pathways, and as a quality control method. Yet, there are few com- parisons of methods, and a better sense of real world performance under “normal” and “bloom” con- ditions is needed. There are reasons why culture techniques may underestimate the true Legionella risk (e.g., VBNC cells) whereas qPCR might overestimate risk (due to response to nucleic acid in nonviable organisms). Whether use of viability qPCR or RT-qPCR could balance these issues in unknown. With side-by-side comparisons of methods in a broad range of settings, it may be that PCR-based or other simplified methods or test kits could be shown to be useful predictors of human health risk and adequacy of remediation. By reviewing dozens of Legionella studies on various building types from around the world, the Committee found the Legionella occurrence data to be highly variable and sparse, making compar- isons among studies difficult and detection of spatial and temporal trends almost impossible. The available data suggest that cooling towers, hot tubs, showers, and wastewater treatment plants can be hot spots for growth of Legionella and exposures. This data set could be improved by adopting standard- ized molecular methods that allow for greater quantitation and more rapid results. Improved environ- mental monitoring methods could facilitate a temporal and spatial assessment of changes in Legionella levels within buildings in several special studies to better understand background levels, potential expo- sure, and ultimately risk. Finally, a collaborative, widespread national survey of Legionella that included distribution systems, premise plumbing in various types of buildings, and cooling towers would be useful for further understanding the concentrations of concern and the risks of sporadic Legionnaires’ disease. Prepublication Version - Subject to further editorial revision

Quantification of Legionnaires’ Disease and Legionella 161 The Committee’s analysis of studies on Legionella occurrence that collected concentration data sug- gests that a Legionella concentration of 5 x 104 CFU/L should be considered an “action level,” that is, a concentration high enough to warrant serious concern and trigger remediation. This concentration could be used for many purposes, including to set an acceptable risk level for Legionnaires’ disease and for regulations and guidelines on Legionella management in building water systems (see Chapter 5). There is a good framework to perform QMRA for various L. pneumophila exposures. To strengthen these tools, additional knowledge is needed about the impact of virulence and strain dif- ferences, phenotypic alterations in potency and aerosol survival, and generation rate of aerosols from various devices. Data on exposures, especially for cooling towers, are lacking. Also, validation of models for predictive growth of L. pneumophila in water systems is required. QMRA has many applications from setting action levels for the occurrence of L. pneumophila in different venues or targets for remediation to informing design and permitting decisions about pipe length, setback distances for large industrial cooling towers, and building-level hydraulic design to maintain acceptable microbial quality. QMRA can be used to determine Legionella concentrations in building water systems that correspond to certain Legionnaires’ disease risk levels. REFERENCES Alary, M., and J. R. Joly. 1991. Risk factors for contamination of domestic hot water systems by Legionella. Appl. Environ. Microbiol. 57:2360-2367. Amaro, F., and H. Shuman. 2019. Selection of Legionella virulence-related traits by environmental proto- zoa. Methods Mol. Biol. 1921:55-78. American Industrial Hygiene Association (AIHA). 2015. Recognition, evaluation and control of Legionella in building water systems. Falls Church, VA: AIHA. American Public Health Association/American Water Works Association/Water Environment Federa- tion (APHA/AWWA/WEF). 2007. Detection of pathogenic bacteria. Legionella. In: Standard Methods for the Examination of Water and Wastewater, 21st edition. Washington, DC: American Public Health Association/American Water Works Association/Water Environment Federation. Armstrong, T. W., and C. N. Haas. 2007a. A quantitative microbial risk assessment model for Legion- naires’ disease: Animal model selection and dose–response modeling. Risk Anal. 27(6):1581-1596. Armstrong, T. W., and C. N. Haas. 2007b. Quantitative microbial risk assessment model for Legionnaires’ disease: Assessment of human exposures for selected spa outbreaks. J. Occup. Environ. Hyg. 4:634- 46. Armstrong, T. W., and C. N. Haas. 2008. Legionnaires’ disease: Evaluation of a quantitative microbial risk assessment model. J. Water Health 6:149-66. American Society of Heating, Refrigeration and Air-Conditioning Engineers (ASHRAE). 2000. Minimiz- ing the risk of legionellosis associated with building water systems. Atlanta, GA: ASHRAE. ASHRAE. 2015. Standard 188 legionellosis: Risk management for building water systems. Atlanta, GA: ASHRAE. Bartlett, J. G. 2011. Diagnostic tests for agents of community-acquired pneumonia. Clinical Infectious Diseases 52(S4):S296-S304. Bartley, P. B., N. L. Ben Zakour, M. Stanton-Cook, R. Muguli, L. Prado, V. Garnys, K. Taylor, T. C. Bar- nett, G. Pinna, J. Robson, D. L. Paterson, M. J. Walker, M. A. Schembri, and S. A. Beatson. 2016. Hospital-wide eradication of a nosocomial Legionella pneumophila serogroup 1 outbreak. Clinical Infectious Diseases 62(3):273-279. Prepublication Version - Subject to further editorial revision

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Legionnaires’ disease, a pneumonia caused by the Legionella bacterium, is the leading cause of reported waterborne disease outbreaks in the United States. Legionella occur naturally in water from many different environmental sources, but grow rapidly in the warm, stagnant conditions that can be found in engineered water systems such as cooling towers, building plumbing, and hot tubs. Humans are primarily exposed to Legionella through inhalation of contaminated aerosols into the respiratory system. Legionnaires’ disease can be fatal, with between 3 and 33 percent of Legionella infections leading to death, and studies show the incidence of Legionnaires’ disease in the United States increased five-fold from 2000 to 2017.

Management of Legionella in Water Systems reviews the state of science on Legionella contamination of water systems, specifically the ecology and diagnosis. This report explores the process of transmission via water systems, quantification, prevention and control, and policy and training issues that affect the incidence of Legionnaires’ disease. It also analyzes existing knowledge gaps and recommends research priorities moving forward.

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