2017 AWT Analyst Technology Supplement by Association of Water Technologies

Issue 7

9707 Key West Avenue, Suite 100 • Rockville, MD 20850

Fall 2017

The Voice of the Water Treatment Industry

New CDC Vital Signs Report: Legionnaires’ Disease on the Rise Sampling Strategies and Test Methods for the Detection of Legionella in Potable Water Systems Advances in Legionella Testing: Methods and Interpretation New York State and New York City Cooling Tower Regulations: Are They Enough to Prevent Cases of Legionnaires’ Disease? NYS/NYC: Is the Juice Worth the Squeeze Isolation of Legionella From Environmental Samples: CDC vs. ISO Interpreting Legionella Test Results: Case Studies Illustrating Key Criteria Chlorine Dioxide for Control and Prevention of Biofilm and Legionella

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the Analyst Technology Supplement 2017

Cover Legionella bacteria.

Spotlight New CDC Vital Signs Report: ................................8 Legionnaires’ Disease on the Rise

NYS/NYC: Is the Juice Worth the Squeeze? .........32 Randy McDaniel, CWT, Weas Engineering, Inc.

In this study, non-potable environmental water samples (n=30) were analyzed in ANSI/ ASHRAE Standard 188-2015 was released in summer 2015 with the stated purpose “to establish minimum Legionellosis risk management requirements for building water systems.” Shortly thereafter, New York City and New York State created new requirements for owners of buildings with cooling towers and incorporated the ASHRAE 188-2015 water management program (WMP) concept as part of their regulation. As a result, New York City and New York State became the first to create legislation in the United States aimed at reducing Legionellosis risk from cooling towers.

To create and use water management programs, building owners and managers need to assemble a team that knows about the water systems, can identify control locations and limits, and can identify and take corrective actions when water quality is impacted. CDC developed a toolkit to help these teams to identify how buildings or devices are at risk for growing and spreading Legionella, and to implement their own personalized water management program.

Sampling Strategies and Test Methods ................10 for the Detection of Legionella in Potable Water Systems

Isolation of Legionella From ..................................34 Environmental Samples: CDC vs. ISO

Michael Coughlin Ph.D., Shivi Selvaratnam Ph.D., Elizabeth Sotkiewicz, and Steve Eisele, Weas Engineering, Inc.

Christopher Goulah, Ph.D., EMSL Analytical, Inc

In this study, non-potable environmental water samples (n=30) were analyzed in parallel by both the CDC and ISO methods. Of the 30 samples, half (n=15) returned results with positive detection of Legionellae by one or both methods. However, whereas the ISO method identified all 15 as positive for Legionella, the CDC method only identified two samples as positive for Legionella, as most fell below the limit of detection for this method. These results validate the use of the ISO method over the CDC method for the isolation of Legionella from environmental water samples.

The adoption of ASHRAE 188 has resulted in the need to validate Water Management Programs (WMP) by testing potable water for the presence of Legionella. Professional and government organizations such as the American Industrial Hygiene Association, the CDC, and OSHA provide some guidance with regard to test frequency and actionable concentrations of Legionella in a WMP. Data from several studies are presented that deal with key remaining issues related to validation of a WMP, including identification of appropriate sample locations, the number of samples that should be tested, and when PCR should be considered as an alternative test method to conventional culture techniques.

Interpreting Legionella Test Results: .....................38 Case Studies Illustrating Key Criteria Matthew Freije, HC Info

Advances in Legionella Testing:.............................20 Methods and Interpretation

Making domestic (i.e., potable) water management decisions based on only one of the recommended criteria for interpreting Legionella test results—concentration, positivity, or strain—could result in missed opportunities to prevent Legionnaires’ disease. When considering all three of the criteria, along with three additional factors, water management decision-making will be much easier and offer a greater chance of success in controlling Legionella bacteria in plumbing systems without overspending.

Janet E. Stout, Ph.D., Special Pathogens Laboratory; and Brian Verdi, Jana Jacobs, Ph.D., David Pierre, Xiao Ma, and Kyle Bibby, University of Pittsburgh, Swanson School of Engineering

Chlorine Dioxide for Control and ...........................46 Prevention of Biofilm and Legionella

New York State and New York City .......................28 Cooling Tower Regulations: Are They Enough to Prevent Cases of Legionnaires’ Disease?

Tom McWhorter, CDG Environmental, LLC

One of the greatest remaining water-related threats to human health in the United States is Legionella. Although Legionella is ubiquitous in our environment, it is especially problematic in hospitals, nursing homes, and other institutions where recirculating hot water systems serve large populations of immunocompromised people. Legionella is also problematic in cooling towers without adequate disinfection. Legionella proliferates in biofilm, from which pieces occasionally slough off and are aerosolized by the action of the cooling tower. The aerosol can then become airborne and infect people in the area surrounding the cooling tower.

Diane Miskowski, EMSL Analytical, Inc.

As result of the Legionnaires’ disease outbreak in the Bronx that occurred in summer 2015, both New York State and New York City passed emergency regulations for registering cooling towers. These regulations are the first of their kind in the US that require monitoring for Legionella and also include enforceable action levels for Legionella and Heterotrophic Plate Count (HPC) for remediating cooling towers. While these regulations are a good first step, some scientifically unsupportable requirements will hopefully prompt additional amendments to the regulations in the future.

the Analyst Technology Supplement 2017

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Calendar of Events Association Events 2018 Technical Training West East February 28–March 4, 2018 Green Walley Ranch Resort Las Vegas, Nevada

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2018 Annual Convention & Exposition September 26 – 29, 2018 Omni Orlando Resort at ChampionsGate Orlando, Florida

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2020 Annual Convention & Exposition September 30 – October 3, 2020 Louisville Convention Center and Omni Hotel Louisville, Kentucky

2021 Annual Convention & Exposition September 22 – 25, 2021 Providence Convention Center and Omni Hotel Providence, Rhode Island Also, please note that the following AWT committees meet on a monthly basis. All times shown are Eastern Time. To become active in one of these committees, please contact us at (301) 740-1421. Second Tuesday of each month

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the Analyst Technology Supplement 2017

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the Analyst Technology Supplement 2017

Presidentâ&#x20AC;&#x2122;s Message

By Marc Vermeulen, CWT

A lot has changed since our last Legionella Supplement in 2013. The passage of ASHRAE/ANSI 188-2015 and the widespread adoption of Water Management Plans (WMP) have had an impact on our industry. Members often find themselves having to explain the need for WMPs to their customers. New resources that may be helpful in educating your current or potential customer base on the need for effective water treatment include the fact sheets and toolkits from the CDC. Included in this Technology Supplement are seven articles about Legionella, including information on the CDC campaign on Legionella. In addition, there are articles on samples and test methods, New York State and New York City regulations, and the isolation of Legionella from environmental samples. Finally, there are case studies about Legionella test results and the use of chlorine dioxide for the control of Legionella. This is great information for water treatment professionals. The articles in this Supplement are provided to offer you tips, advice, and knowledge that will help you and your business succeed. We hope you find this information helpful. As always, I welcome your feedback and can be reached at president@awt.org.

the Analyst Technology Supplement 2017

New CDC Vital Signs Report: Legionnairesâ&#x20AC;&#x2122; Disease on the Rise

the Analyst Technology Supplement 2017

Legionnaires’ disease is a serious pneumonia that is deadly for about one out of every 10 people who get it. The CDC urges building owners and managers to implement newly published standards that help control the risk of Legionella, the bacterium that causes Legionnaires’ disease. People can get sick with Legionnaires’ disease when they breathe in small water droplets contaminated with Legionella. Legionnaires’ disease outbreaks are caused by common problems. While found naturally in freshwater environments like lakes and streams, Legionella becomes a problem when it grows and spreads through humanmade water systems like hot tubs, hot water tanks and heaters, large plumbing systems, cooling towers, and decorative fountains. Four common problems within water systems have been directly linked to Legionnaires’ disease outbreaks investigated by the CDC over the past 15 years. These problems include: • Process failures, like not having a Legionella water management program. • Human errors, such as when a hot tub filter is not cleaned or replaced as recommended by the manufacturer. • Equipment, such as a disinfection system, not working properly. • Changes in water quality that occur because of reasons external to the building itself, like nearby construction or a water main break. Furthermore, about half of the investigated outbreaks were linked to more than one of the problems outlined above. Learn more about this report at www.cdc. gov/vitalsigns/legionnaires. Many things can allow or even help Legionella to grow in a water system, including warm temperatures and lack of disinfectant. Nearby construction or water main breaks can also affect water quality and contribute to Legionella growth. CDC encourages building owners and managers to adopt the newly published standard—ASHRAE 188: Legionellosis – Risk Management for Building Water Systems—to help control these internal and external factors by creating and using a Legionella water management program. According to CDC Director Tom Frieden, M.D. M.P.H., “Better water system management is the best way to reduce illness and save lives.” To create and use these water management programs, building owners and managers need to assemble a team that knows about the water systems, can identify control locations and limits, and can identify and take corrective actions when water quality is impacted. CDC developed a toolkit to help these teams to identify how buildings or devices are at risk for growing and spreading Legionella, and to implement their own personalized water management program. Access the toolkit at www.cdc.gov/legionella/WMPtoolkit. More information on Legionella from the CDC can be obtained from its website: http://www.cdc.gov/vitalsigns/legionnaires/index.html.

the Analyst Technology Supplement 2017

Sampling Strategies and Test Methods for the Detection of Legionella in Potable Water Systems

Michael Coughlin, Ph.D.; Shivi Selvaratnam, Ph.D.; Elizabeth Sotkiewicz; and Steve Eisele, Weas Engineering, Inc.

the Analyst Technology Supplement 2017

Abstract The adoption of ASHRAE 188 has resulted in the need to validate Water Management Programs (WMP) by testing potable water for the presence of Legionella. Professional and government organizations such as the American Industrial Hygiene Association, the CDC, and OSHA provide some guidance with regard to test frequency and actionable concentrations of Legionella in a WMP. Data from several studies are presented that deal with key remaining issues related to validation of a WMP, including identification of appropriate sample locations, the number of samples that should be tested, and when PCR should be considered as an alternative test method to conventional culture techniques.

Introduction Since ASHRAE 188 was ratified in 2015, there is a need to establish guidelines and criteria for implementing a validation program because it is a necessary component of a Water Management Program. An intensive environmental surveillance of a major mid-west hospital’s potable water system in 2015 provided an opportunity to address some basic issues associated with validation programs. Specifically, the issue of which types of water samples to collect (hot, cold, first draw, and prolonged purge) and where to collect them (point of use, system mains) was addressed. The use of PCR as an alternative to culture methods was also investigated, as PCR offers the advantage of speed, sensitivity and objectivity compared to culture analysis.

Materials and Methods Sample Collection A 1-L potable water sample from each site was collected in a sterile widemouth screwcap polypropylene plastic bottle containing 150–200 mg sodium thiosulfate preservative.

Preparation of Samples for Bacteriological Examination Filtration Five hundred (500) mL of each sample was filter-concentrated using a 47-mm filter funnel assembly containing a 0.20 μm polycarbonate filter. After filtration, the filter was removed aseptically from the holder with sterile forceps, folded to the outside, and placed into a sterile, 50-mL centrifuge tube containing 5 mL of sterile Butterfield’s buffer. The centrifuge tube was then vortexed for 1 minute at maximum speed to elute bacteria from the filter.

Acid Treatment Because water samples may contain high concentrations of non-Legionella bacteria, it was necessary to use a selective procedure to reduce their numbers before culture. One (1.0) mL of the vortexed suspension was placed into a sterile 1.5-mL centrifuge tube containing 1.0 mL of pH 2.0 acid buffer.[1] The suspension was then incubated for 5 minutes at room temperature before spreading it on the appropriate Petri plate.

Media for Legionella Growth and Isolation Buffered charcoal yeast extract (BCYE) agar containing 0.1% alphaketoglutarate was used as the base medium used for the recovery of Legionella.[2] Two types of selective BCYE agar were used in processing the samples. The first was designated BCYE complete with Oxoid™ GPVC selective supplement antibiotics; the second, BCYE complete without antibiotics.

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Sampling Strategies continued

was transferred to a clean micro-centrifuge tube and centrifuged at 14,000 x g for 2 minutes to pellet the cells. The pellet was re-suspended in 300 μL of TE buffer, followed by addition of 2 μL of Ready-Lyse Lysozyme Solution and 1 μL of RNaseA. The contents were mixed by vortexing and incubated at 37 °C for 30 minutes. This step was followed by adding 300 μL of Meta-Lysis Solution (2X) and 1 μL of Proteinase K to the tube and mixing by vortexing. Tubes were then incubated at 65 °C for 15 minutes and then transferred to an ice bath to cool the sample to room temperature (for 3–5 minutes), after which 350 μL of MPC Protein Precipitation Reagent was added to the tube and mixed by vortexing. The debris was pelleted by centrifugation for 10 minutes at 14,000 x g. The supernatant was transferred to a clean micro-centrifuge tube followed by the addition of 570 μL of isopropanol to the supernatant and inverted several times to mix. DNA was pelleted by centrifugation for 10 minutes at 14,000 x g. Isopropanol was decanted without dislodging the DNA pellet, followed by adding 500 μL of 70% ethanol and centrifugating for 5 minutes at 14,000 x g. The ethanol was decanted and the pellet air-dried for 8 minutes at room temperature, after which 50 μL of TE buffer was added. DNA extracts were stored at approximately -20 °C until they were used for qPCR assays.

Plating of Samples Plates (described above) were innoculated with 0.2 mL of either an acid-treated or non-acid-treated suspension and distributed over the agar surface with a plastic spreader. They were then incubated at 37 °C in a humidified incubator for 14 days.

Examination of Cultures for Legionella Plates were examined after 4–8 days of incubation for Legionella. Suspect Legionella colonies were streaked onto BCYE agar plate without L-cysteine and a positive control BCYE agar plate. The plates were incubated for 24–48 hours. Colonies that grew on BCYE agar, but not BCYE agar without L-cysteine, were considered to be presumptive Legionella species and later serotyped using a latex agglutination test (Oxoid, Dardilly, France) or direct fluorescent antibody (Pro Lab Diagnostics, Round Rock, Texas)

Preparation of Samples for Analysis by Real-Time Polymerase Chain Reaction (RT-PCR) DNA Extraction DNA was extracted from filtered water samples using the MetaG-Nome DNA Isolation Kit (Epicenter, Madison, Wisconsin). Water samples were filtered through a pre-sterilized 0.20-μm polycarbonate filter membrane (Millipore). The membrane was then removed and cut into two pieces and placed in a 50-mL sterile conical tube. One (1) mL of filter wash buffer containing 0.2% Tween 20 was added to the tube containing the filter pieces and vortexed intermittently for 2 minutes. This cell suspension

Quantitative PCR (qPCR) Analysis All qPCR assays were performed using a 7900 HT Fast Real-Time Sequence Detector (Applied Biosystems, Foster City, California). Reaction mixtures (20 μL) contained 10μL of 2x qPCR Master Mix (Applied Biosystems), TaqMan Environmental Master Mix 2.0 for qPCR with 0.08 μmol/L TaqMan probe (final concentration), 0.2 μmol/L primers, and 2 μL of template DNA. The primers and probes used in the assay were as described in Lu et al.[2] The sample was then held for 10 minutes at 95 °C to denature the template DNA. The following quantification cycling protocol was used: 40 cycles at 95 °C for 15 seconds and 55 °C for 30 seconds, with an extension at 72 °C for 30 seconds and a final hold at 72 °C for 5 minutes. In addition, the TaqMan Exogenous Internal Positive Control Reagents (a VIC-labelled probe) manufactured by ABITM (Life Technology) was also used as a secondary confirmation. The baseline cycles were set from three to 15, and the threshold fluorescence value was 10 times the standard deviation of the mean baseline emission. According to this protocol, a threshold of 0.2 ΔRn was used.

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Sampling Strategies continued

Biofilm Bypass Manifold A device called a Biofilm Bypass Manifold (BBM) was manufactured that was installed in six buildings on both the cold and hot water mains. The main flow of water was through the straight portion of the manifold, which also contained a copper insert with threaded unions so that the copper insert could be removed, swabbed, and reinserted into the BBM. The BBM permits sampling of water and biofilm without interrupting flow to point-of-use (POU) devices. The copper insert is made from the original piping in the mains water supply. The bypass component of the manifold is maintained partially open during times of normal flow to prevent stagnation from occurring throughout the BBM.

neither the free chlorine nor the temperature differences in these two systems had any apparent effect on the presence of Legionella spp. therein. Noting that the temperature and free chlorine range within the hot and cold water systems is so expansive, it was informative to determine if these parameters affected the incidence of Legionella spp. within a given system. The average free chlorine and temperature for samples testing positive for Legionella is essentially the same as for those samples testing negative for both the hot (p = 0.197 and 0.778 respectively) and cold water (p = 0.245 and 0.887 respectively) systems. Therefore, for first draw samples, the free chlorine concentrations and temperature differences within each of these two systems would not be expected to affect the occurrence of Legionella.

Figure 1: Biofilm Bypass Manifold

Table 2: First Draw vs. 2-Minute Purge of Sink Faucets

FIRST DRAW

COLD HOT

Table 1: Presence of Legionella in Hot vs. Cold Water First Draw Water Samples

Free Chlorine

n (%) Range Avg. SD

Temp Â°F

p Temp

35 (43) 0-1.11 0.23 0.33 71-112 91 13.2

Hot Negative

47 (57) 0-0.94 0.20 0.25 71-115 95 13.6

Cold Positive

25 (38) 0-1.29 0.67 0.52 70-101 78 7.7

Cold Negative

40 (62) 0-1.18 0.64 0.45

0.887

Hot Negative vs. Hot Positive

0.197

0.778

Total Cold vs. Total Hot

HOT

2 3 3 4

Percent 20 25 21 35 57 43 15 27 19 40

A PCR sample testing positive for any of the three targets was deemed positive for Legionella, and any samples that produced Legionella-like colonies on BCYE agar but failed to grow when subcultured on BCYE agar less cysteine was deemed culture positive. The data in Table 2 reveal that there is virtually no difference in the number of positive samples collected from first draw sink faucets unless the cold and hot water lines entering the faucet are delivered via a mixing valve. Among cold and hot first draw samples, PCR appears to be a more sensitive indicator of Legionella contamination than culture. The number of blended samples is low and therefore skews the apparent lower sensitivity of PCR versus the culture method. However, the higher percentage of positive Legionella samples encountered in the blended waters compared to both the cold and hot water samples may indicate that this type of valve is inherently more prone to Legionella contamination than both unblended hot and cold water.

71-85 77 3.6 0.245

COLD

66 64 87 81 7 7 13 11 16 10

Positive 13 16 18 28 4 3

p Cl2

Cold Negative vs. Cold Positive

BLENDED BY MIXING VALVE

Culture PCR Culture PCR Culture PCR Culture PCR Culture PCR

Range Avg. SD

Hot Positive

2-MINUTE PURGE

1.77 x 10-13 2.10x1-90

Results Free chlorine delivered by the municipal public water supply is the only source of disinfectant in both the hot and cold water systems. The range of free chlorine in both the hot and cold water in Table 1 is expansive and for the most part, is overlapping. Nevertheless, the difference between the average free chlorine values for both the hot and cold waters is highly significant (p=2.10x10-9). In spite of this highly significant difference, there is little difference between the percent of hot samples positive for Legionella (43%) versus the number of cold samples testing positive for Legionella (38%).

The relatively low number of 2-minute purge samples probably exaggerates the ability of PCR to detect the presence of Legionella compared to the culture method since there is only a difference of one positive sample between PCR samples and culture samples in both the hot and cold water. This bias is further skewed by having a low number of 2-minute purge samples. Overall, there appears to be little significant difference between the number of positive samples, both hot and cold, when comparing first draw to the 2-minute purge.

Like free chlorine, the temperature range of the hot and cold water samples is expansive and overlapping, and the difference between the average temperatures of the two systems is also highly significant (1.77x10-13). It is therefore interesting that 13

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Sampling Strategies continued

Table 3: Legionella pneumophila Serogroup 1 Isolated From Sinks by Swab and Water Samples Cold Water Hot Water Location

Plumbing Surfaces CFU/Swab

First Supply First Supply Aerator Draw Line Draw Line CFU/mL CFU/mL CFU/mL) CFU/mL (Temp) (Temp) (Temp) (Temp)

Table 4: System Water Biofilm and Planktonic Legionella

3/8â&#x20AC;? Feed Line

BBM Building

Material

Sample

Cold Hot ND

100 (85)

10 (105)

100

310

200,000 Plastic

1339-T6

26 (73)

23 (104)

BDL

200,000 Plastic

1339-T4

11 (108)

4 (114)

BDL

Plastic

1339-T3

10 (67)

BDL (59)

BDL

Plastic

1339-T2

100 (88)

100 (94)

1339-TR

23 (88)

32 (98)

120

1321

Culture

PCR

Culture

PCR

Hot

0/7

1/9

Cold

1/9

0/9

Hot

Cold

Hot

0/6

1/6

Cold

0/5

Hot

Cold

Hot

1/2

0/2

Cold

1/2

Hot

Cold

Hot

1/1

Cold

Hot

Cold

Hot

1/7

1/9

Cold

1/9

0/9

Hot

Cold

Hot

2/17

10/17

Cold

2/11

5/11

Hot

Cold

Water

1339-T8

100

Point of Use

Temp.

1A Swab

Water

200,000 200,000 Plastic 60

Plastic

1000 BDL 10 Plastic

1333-1

BDL (95)

20 (71)

BDL (75)

BDL (117)

BDL

Metal

1324-1

BDL (69)

BDL (66)

BDL (102)

BDL(121)

BDL

Metal

Swab

Water

ND: No Date

Unlike all other samples collected during this study, the samples in Table 3 were collected in the fall months as opposed to the summer months of 2015. Supply line samples were collected by removing the 3/8 inch feed line connecting it to the sink taps and discharging a liter of water from the supply line, and then collecting another liter of water from the supply line for microbiological analysis. Due to the small number of samples, it is not possible to accurately compare the effects of hot and cold water to the concentration of Legionella pneumophila serogroup 1 in this table. However, there does appear to be a correlation between the presence of Legionella pneumophila serogroup 1 in hot first draw water samples and hot water samples collected from the supply line. This observation is in contrast to the absence of Legionella pneumophila serogroup 1 in the only cold water supply line sample, but without other cold water samples to compare, the significance of this result is uncertain. As noted previously, there is a large variation in the temperature from first draw samples.

Swab

Water 4E Swab

Water 5R Swab

Water 6S Swab

The highest colony counts were from the inside of the 3/8-inch flexible plastic tubing. The aerator and the soft plastic that composes the 3/8-inch feed lines appear to be highly associated with the presence of Legionella pneumophila, presumably existing as an adherent biofilm. With the exception of location 1339-T3, every 3/8-inch plastic feed line servicing the hot water taps possessed a Legionella biofilm. Three of the 3/8-inch lines had as much as 200,000 CFU/swab. This is in sharp contrast to the absence of any detectable Legionella biofilm on 3/8-inch feed lines that are made of copper (Locations 1333-1 and 1324-1). Not only were these two locations devoid of detectable Legionella, but the water servicing these locations had few (1331-1, 20 CFU/mL) or no detectable Legionella present.

Biofilm Bypass Manifolds (BBM) were installed on the hot and cold water mains in six buildings as identified in Table 4. They were installed on the hot water return lines and at a location furthest from the point of entry on the cold water main line. These locations were chosen to maximize the likelihood of obtaining a positive result, i.e., lowest hot water temperature and lowest disinfectant level in the cold water main line. The copper insert was created by cutting into the original copper pipe and using this section to form a replaceable copper insert. The copper insert was integrated into the system by affixing it to unions on both sides of the copper insert. The copper inserts were swabbed immedi-

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ately after the pipe was sectioned and before it was modified to become a replaceable insert. Water samples were also collected at various POU locations throughout these buildings and from the BBM itself.

Table 6: Correlation of CFU/ml and GU/ml Pearson Product Moment Correlation Coefficient: r Legionella Legionella species: pneumophila: 16S Gene mip Gene

The copper inserts from the BBMs were for the most part devoid of biofilm. Biofilm was identified in the hot copper insert in building 4E by culture and PCR, and the cold water copper insert in building 6S was identified by PCR. Therefore, out of 12 swabs, only three were positive for Legionella. Of the three positive swabs, the associated BBM water samples tested positive only in hot water from building 4E. Conversely, hot water from the BBM in building 3C and cold water from the BBM in building 4E tested positive for Legionella but was not associated with any biofilm from their respective BBM copper inserts.

PCR PCR PCR PCR Positive Positive Negative Negative Culture Culture Culture Culture

Cold Line 1 2 0 10 26 (14)

39 (21)

16 (8)

<1.0

52 -0.110

40 -0.087

29 -0.419

1-10

17 +0.455

11 -0.065

18 +0.674

11-100

10 +0.207

9 +0.518

8 +0.290

>100

2 ID

Hot Line 2 1 1 9 Total

n r

Table 7a: PCR and Culture Agreement for All Samples Collected

Hot Water 9 16 8 40 14

n r

bacterium that has replicated to sufficient density to be visible on an agar plate. Either metric can be used as an indicator of population density and under ideal circumstances, should at least be highly correlated. Since the culture method of enumerating Legionella is still considered the “Gold Standard”, Table 6 correlates CFU/mL to GU/mL, comparing a precise GU/mL values to a range of CFU/mL. The “r” value was calculated only for samples that had a detectable GU/mL or CFU/mL. For a correlation to be highly significant, an absolute r value greater than 0.50 is expected. There are only two occasions when r>0.50: when the Legionella pneumophila was present at 11–100 CFU/mL and when Legionella pneumophila serogroup 1 was present at 1–10 CFU/mL. However, these correlations appear randomly dispersed throughout the table.

Table 5: Detection of Legionella spp. by PCR and Culture in Water and Swab Samples

Cold Water

CFU/mL n r

ID: Insufficient Data

It is not possible to make a correlation between the presence of Legionella in the water taken from the BBM (buildings 2B, 3C, and 4E) and the presence of Legionella in POU locations associated with the BBM, as the number of POU samples was very low. However, in building 6S, 17 hot water POU samples and 11 cold water POU samples were taken. Based on PCR tests, there was a significant incidence of Legionella in the cold and hot POU samples, but none was found in the cold and hot mains.

Legionella pneumophila sg1: wzm Gene

109 (57)

Bracketed numbers refer to percentage

The data in Table 5 indicate that PCR is a more sensitive method of detecting Legionella spp. than culture methods. The total number of PCR positive samples totaled 35% (21+14) compared to 22% (14+8) for culture methods. Furthermore, the number of “PCR Positive Culture Negative” samples was 21% versus 8% for the converse situation. Of the total number of positive samples (26+39+16), PCR and culture were in agreement at a frequency of 32% (26/81), primarily because of the high proportion of “PCR Positive Culture Negative” samples. Negative samples for both PCR and culture reflect the overall propensity of the sample group to be negative for Legionella spp.

Total Culture Positive

Total PCR Positive

Matched Culture and PCR Positive

n % n % n %

Legionella spp./16S

51 100 75 10 35 67

Legionella pneumophila/mip

45 88 45 60 11 24

Legionella sg 1/wzm pneumophila

42 82 24 32 10 24

A total of 239 samples were processed by PCR and culture, and as noted in Table 7a, there are significant differences between the ability of culture and PCR to detect the presence of Legionella. PCR is better able to detect the presence of nonpneumophila species of Legionella (75 vs. 51), but the sensitivity of PCR decreases as the identity of Legionella becomes more precise. It is usual for PCR to detect a greater number of positives compared to culture techniques since PCR is able to detect Viable But Non Culturable (VBNC) organisms. However, the phenomenon of VBNC does not appear to present itself when positive samples

A genomic unit (GU) is a calculated value that is indicative of a single bacterium that has had its DNA amplified by PCR. Likewise, a colony-forming unit (CFU) is indicative of a single

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are compared at the species and serogroup level. Interestingly, the total number of positives at the species level is the same for both PCR and culture (45 and 45). However, PCR and culture agree with only 11 of these 45 samples, which is equal to a 24% match. At the serogroup level, PCR was inferior to culture in its ability to detect serogroup 1. When referenced to the total number of positives by culture, this also equaled a 24% match. The best match of PCR and culture occurred at the genus level, i.e., 67%.

that have the highest probability of affecting patient health. Therefore, water obtained from rooms accommodating patients in ICUs and transplant wards is highly desirable, and conversely, water from public washrooms has comparatively minimal value. It is commonly thought that hot water POU locations are more likely to harbor Legionella than cold water.[3] Neither cold nor hot water present ideal growth conditions for Legionella, so presumably it is the lack of disinfectant in the hot water that accounts for its prevalence in these systems. The data collected in this study indicates that although the differences in disinfectant levels between hot and cold water systems is substantial, this did not correlate with the number of POU locations testing positive for Legionella. This contrary result may be due to the time of year during which these samples were collected, i.e., the summer months. It is typical that Legionellosis reaches its highest levels in the summer months,[4] and this may be a consequence of the potable water coming closer to the optimum growth temperature of Legionella. A follow-up study is in progress to determine if a correlation exists between colonization of POU devices by Legionella and the temperature and free chlorine residual after a 2-minute purge. It is suspected that environmental conditions after a 2-minute purge might be more predictive of the microbial ecology of a POU device. Therefore, devices that are chronically deficient in disinfectant concentration and maintained at a temperature conducive to Legionella growth would be more likely to be culture positive after a 2-minute purge than first draw samples collected from the same device.

Table 7b: Amplicon Agreements

mip (n)

wzm (n)

Present Absent

Present 40 37 16S (n) Absent 9 Present 17 wzm Absent 35

The poor match between culture and PCR positive samples could be a result of the PCR primers not correctly identifying or annealing to the intended amplicon. One method of determining the veracity of a PCR result is to compare the performance of amplicons that constitute subsets of each other. By definition, a member of a subset will be present in the superset. Therefore, when detected, the wzm amplicon should also be associated with the detection of mip and 16S amplicons. In Table 7b, the wzm amplicon was associated with the 16S amplicon in 42% of occasions (23/55) and with the 16S amplicon in 49% of occasions (17/35). Theoretically, the percent association should have been the same for both. More problematic than this 7 percentage point difference is that mip was absent in 15 occasions when wzm was present. This alludes to either a negative PCR bias for detecting the presence of mip or a false positive bias for detection of wzm. In view of the total number of Legionella pneumophila sg1 detected by culture as constituting 87% of all Legionella spp., and that 93% (42/45) of these were serogroup 1, it appears that the PCR conditions used in this study underrepresent the total number of Legionella pneumophila present. In an analogous manner, wzm also appears to be underrepresenting the population of Legionella pneumophila serogroup 1.

Whether a sample is collected immediately upon opening a valve (first draw) or purged to remove stagnant water could affect the microbiology of the water. Due to stagnation, first draw samples would be expected to contain less disinfectant and therefore higher concentrations of bacteria than samples collected after a purge. Furthermore, loosely adhering biofilm would be sloughed within the first few seconds of opening a valve, and the bacteria therein would go by unnoticed if a purged sample were to be collected. Despite the apparent propensity for obtaining more positive samples from a first draw sample as compared to a purge, the purge sample provides information upstream of the valve. In this survey, we investigated and compared the microbiology of the water immediately upstream of the valve by removing the tubing that connects POU devices to the main supply line. Thus any residual affect that the valve might have on the perceived quality of the main supply water is obviated. In our study, we determined that water obtained from the first draw was very similar to that obtained directly from the main line. This result may be anomalous because many of the samples were obtained from a building that was largely unoccupied, and therefore, stagnation of even the cold and hot water mains was likely. Nevertheless,

Summary Environmental surveillance of a hospital for Legionella immediately begs the question as to where and how these samples should be taken. The data collected from a survey of a large midwestern hospital has provided some guidance on this matter. In a hospital setting, the matter of primary concern is to choose locations

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Sampling Strategies continued

culture, it was expected that all PCR samples would be matched with its corresponding culture result. This, however, was not the situation. It would appear, therefore, that the apparent greater sensitivity of PCR to detect the presence of Legionella (at the genus level in this study) is not solely due to the presence of VBNC bacteria. It may be that there are PCR inhibitors in some water samples.

the highest concentrations of Legionella were obtained from plastic 3/8-inch lines that connect the supply line to the valve. Aerators attached to the faucet were also contaminated with Legionella, but this was not a surprising result.[5] Interestingly, only the plastic 3/8-inch connector lines were heavily contaminated. Copper 3/8-inch connector lines were essentially free of Legionella. Whether it is the hydrophobic nature of the plastic substrate, its ability to adsorb organics, or a biodegradable component within the plastic lines that support bacterial growth is not known, but these types of connectors should become highly suspect in future environmental surveillance programs.

Although the infectious dose of Legionella is not known and would likely vary depending on the pre-existing health of an exposed individual and the exact strain of Legionella, the probability for infection to ensue will be dependent on the number of bacteria inhaled. Therefore, a method of quantifying the number of Legionella in a sample is an important metric that can be used in determining a course of action to be taken following an environmental survey. Real time qPCR permits the quantification of an amplified target. The 16S target can occur in multiple copies in a bacterial cell,[6] and therefore, it is inherently difficult to correlate the number of GUs derived by its amplification to a corresponding CFU count. However, the genes coding for the mip gene and the expression of serogroups are present at one copy per genome.[7,8] Therefore, there should be a correlation between CFUs and GUs when mip and wzm are compared to CFUs. Such a correlation, however, was not observed at low and high concentrations of viable Legionella. This lack of correlation is not reported in the literature and suggests that the extraction/ purification method and or the PCR parameters require further refinement.

One of the desired outcomes from installing a BBM is that these devices might reduce or eliminate the need to obtain multiple POU samples. This would be the case if the mains, both hot and cold, are the primary source of Legionella and not simply acting to seed POU devices with low levels of Legionella. The samples obtained from the BBMs reveal that the hot and cold water mains, the water therein, and the pipe surfaces are seldom contaminated with Legionella. It would appear that the hot and cold water mains are probably carriers of low levels of Legionella that are transported to terminal POU devices where they can achieve higher concentration by growing biofilms on materials more able to support bacterial growth, i.e., soft plastic components. Once a POU device becomes contaminated with a Legionella biofilm, it is possible that the biofilm could grow and extend upstream of the POU device and, in the case of a recirculating hot water system, seed many other POU devices. The use of PCR presents an opportunity to reduce the time line for obtaining surveillance data from weeks to hours. PCR can also eliminate the subjectivity of interpreting colony morphology and serological reactions. PCR is not new, and there are accepted protocols for using it as an alternative to culture methods. Largely because PCR can overestimate the number of Legionella in a sample because of its ability to amplify dead and VBNC organisms, the gold standard for Legionella continues to be the culture method. But because the advantages of PCR are so important, samples collected during this survey were subjected to both PCR and culture analysis to determine if PCR can provide an equitable alternative to culture. As was expected, the total number of positive samples was greater when PCR was used and opposed to culture. This was the case when all Legionella species were tested by using a primer/probe specific to the 16S DNA. The mip gene was able to detect the presence of Legionella pneumophila at the same frequency as the culture method. However, this frequency was not associated with a perfect match of test results for a given sample. In fact a mismatch was more likely to occur with all targets, except at the genus level. Given that PCR was able to detect Legionella more often than by

References 1. http://www.cdc.gov/Legionella/health-depts/inv-tools-cluster/ lab-inv-tools/procedure-mamual.htm/#appendix1 2. J.Lu, I. Struewing, S. Yelton, and N. Ashbolt. Molecular survey of occurrence and quantity of Legionella spp., Mycobacterium spp., Pseudomonas aeruginosa and amoeba hosts in municipal drinking water storage tank sediments. Journal of Applied Microbiology; 119: 278 - 288. (2015). 3. Janet Stout, Victor L. Yu and Michele G. Best. Ecology of Legionella pneumophila within Water Distribution Systems. Applied and Environmental Microbiology; 49: 221-228. (1985). 4. European Centre for Disease Prevention and Control. Legionnaires disease in Europe. Stockholm, Sweden. http://dx.doi.org/10.2900/78974. (2013). 5. Carol A. Ciesielski, Martin J. Blaser and Wen-Lan Wang. Role of Stagnation and Obstruction of Water Flow in Isolation of Legionella pneumophila from Hospital Plumbing. Applied and Environmental Microbiology; 48: 984-987. (1984). 6. StĂ¸lhaug, Anne and KĂĽre Bergh. Identification and Differ-

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Sampling Strategies continued

entiation of Legionella pneumophila and Legionella spp. with Real-Time PCR Targeting the 16S rRNA Gene and Species Identification by mip Sequencing. Applied and Environmental Microbiology; 72: 6394–6398. (2006).

Acknowledgement The authors would like to acknowledge the US EPA in Cincinnati and in particular, Jill Hoelle for her assistance in completing qPCR analysis of samples collected during this study and for the training she provided to the microbiologists at Weas Engineering in DNA extraction, purification and qPCR analysis.

7. Specific real-time PCR for simultaneous detection and identification of Legionella pneumophila serogroup 1 in water and clinical samples. Mérault, N, Rusniok, C, Jarraud, S, Gomez-Valero, L, Cazalet, C, Marin, M, Brachet, E, Aegerter, P, Gaillard, JL, Etienne, J, Herrmann, JL; DELPH-I Study Group, Lawrence C, Buchrieser C. Applied and Environmental Microbiology; 77:1708-1717. (2010).

Dr. Michael Coughlin is director of inovation and research at Weas Engineering, Inc. He has over 30 years’ of experience in the control of microorganisms. Dr. Coughlin has authored and presented papers at several international symposia on the subjects of bioremediation, food safety, and Legionella control. He can be reached at (317) 867-4477 or at Michael.coughlin@weasengineering.com

8. Direct Detection of Legionella Species from Bronchoalveolar Lavage and Open Lung Biopsy Specimens: Comparison of LightCycler PCR, In Situ Hybridization, Direct Fluorescence Antigen Detection, and Culture. Hayden ,R. T., J. R. Uhl, X. Qian, M. K. Hopkins, M. C. Aubury, A. H. Limper, R. V. Lloyd, AND F. R. Cockerill; Journal of Clinical Microbiology; 39: 2618-2626 (2001).

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Advances in Legionella Testing: Methods and Interpretation Janet E. Stout, Ph.D., Special Pathogens Laboratory, and Brian Verdi, Jana Jacobs, Ph.D., David Pierre, Xiao Ma, and Kyle Bibby, University of Pittsburgh, Swanson School of Engineering

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While standard Legionella culture remains the gold standard for Legionella detection, advances in molecular and metagenomic test methods provide new and different information. We used these molecular methods, along with standard culture, to assess building water systems and cooling towers for Legionella colonization. Molecular methods that were used were quantitative polymerase chain reaction (qPCR) and metagenomic analysis. Metagenomics is the culture-independent study of microbial DNA to investigate and survey microorganisms present in water systems. A novel combination of genetic probes yields results by qPCR in hours for both Legionella pneumophila (all serogroups) and for L. pneumophila, serogroup 1. Our objectives were to evaluate this new qPCR method and to learn more about the microbiome of building water systems. Genetic material was recovered directly from water collected from the building water systems and analyzed by using Illumina sequencing technology.

Disease Control is not a traditional proficiency testing program and some certified laboratories have produced erratic results. The method of sample collection can also influence the result. We previously demonstrated that collection of first draw hot water resulted in a more accurate assessment of positivity at water outlets (faucets and showers) compared to lower counts, and there were negative results if the outlets were flushed for 2 minutes prior to collection [Mietzner, 2013]. For the present study, all samples were first draw hot water except for cold water entry samples. These samples were flushed for 2 minutes prior to collection to represent the incoming cold water supply.

Quantitative Polymerase Chain Reaction (qPCR) for Detection of L. pneumophila and L. pneumophila, serogroup 1 This procedure is used as a rapid method to detect and quantitate a target gene sequence of Legionella pneumophila and L. pneumophila, serogroup 1 in samples by Quantitative Polymerase Chain Reaction (qPCR). Testing can be performed in hours, compared to 7–14 days for culture.

Comparison of Culture vs. Molecular Methods Legionella Culture Culture methods for the detection of Legionella from environmental (building) water samples involve several steps including:

Water samples are concentrated by filtration prior to DNA extraction using Mo-Bio RapidWater kit. Taqman qPCR is performed on the extracted DNA with two separate primer/ probe sets: 1) Legionella pneumophila specific for a sequence within the 16S ribosomal RNA (rRNA) gene; 2) Legionella pneumophila serogroup 1 (Lp1) specific for a sequence within the LPS gene. For the Lp1 assay, a 75 bp sequence is targeted within the lipopolysaccharide (LPS) gene cluster that is specific to serogroup 1 [Merault, 2011]. Primers/probes target the gene wzm, which encodes the transmembrane component of the ABC transporter of the O-antigenic polysaccharide of LPS. Concentrations for L. pneumophila are extrapolated using a standard curve. The copies (genomic units) of DNA (x-axis) is plotted against the Ct value (y-axis) generated for each concentration in a standard curve. A linear curve is then fitted to the plot for use in determining the concentration of unknown samples.

1) P lating of the sample directly and/or after concentration by filtration. 2) Pretreatment by heat or acid treatment to inhibit competing bacteria. 3) Culture on multiple media with and without additives to inhibit competing bacteria (CCVC, DGVP, GVPC, and BCYE). 4) Incubation at 35–37 °C for 7–10 days. 5) V isual inspection every 2–3 days using a stereo microscope followed by sub-culture of suspicious colonies. 6) Presumptive confirmation of Legionella species and serogroup (1 and 2–14) using agglutinating antibodies. Definitive identification of Legionella species and individual serogroup by direct fluorescent antibody staining (DFA)

Next-Generation Sequencing for Environmental Metagenomics

Reporting of Results

Environmental metagenomics is the study of organisms in a microbial community based on analyzing the DNA within an environmental sample. Next-generation sequencing (NGS) provides the capability to profile entire microbial communities from complex samples and explore microbial populations under changing conditions. This is a culture-independent method that can identify and compare bacteria from complex microbiomes or environments and can identify strains that may not be found using other methods.

The ability to successfully isolate Legionella species and the quantification limits varies depending on the capabilities, experience, and methods (concentration by filtration, pretreatment, and processing) used by the laboratory. While there are published standard testing methods, there is no requirement for laboratories to follow the same method. Many laboratories are not accredited for Legionella testing as a specific field of testing. Proficiency panels to test the ability of the laboratory to isolate Legionella are limited. The ELITE program by the Centers for

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Methods and Interpretation continued

Potable Water Samples Testing by culture and qPCR was performed on 270 water samples from 28 buildings. Results comparing culture vs. qPCR for the L. pneumophila probe (Table 3) and for the L. pneumophila, serogroup 1 probe (Table 4) are below.

One objective of this study was to examine the microbial ecology of building water distribution systems using NGS DNA sequencing. The testing was performed in the laboratories of Special Pathogens Laboratory and the University of Pittsburgh, Civil and Environmental Engineering, and processed as previously described. We have used metagenomic analysis in previous work assessing microbial communities in water systems [Ma, 2015; Baron, 2015]. One-liter water samples were collected and represented the hot and cold water distribution systems.

Table 3: L. pneumophila Culture vs. qPCR (n=270 Potable) Culture (L. pneumophila) PCR (L. pneumophila)

Metagenomic studies are often performed by analyzing the gene coding for the prokaryotic (bacterial) 16S ribosomal RNA (16S rRNA). Variable regions of 16S rRNA can be used in phylogenetic classifications such as genus or species in diverse microbial populations. In the current study, NGS sequencing was performed by targeting the bacterial 16S rRNA gene, which we used for phylogenetic classification of the microbial populations in water systems. DNA was extracted from the sample after concentration by filtration, a bacterial 16SÂ rRNA gene amplicon library was created, and sequencing was performed using an Illumina Miseq sequencer, followed by analysis as relative abundance of taxa from phylum level to genus level.

Positive Negative 25% 75%

Positive Negative 60% 40%

Table 2: L. pneumophila serogroup 1 Culture vs. qPCR PCR Lp1

Positive

74 (27%)

71 (26%)

Negative

203 (73%)

206 (74%)

Total

277 277

PCR Lp1

Positive

38 (14%)

24 (9%)

Negative

232 (86%)

246 (91%)

Table 5: Comparison of Estimated Legionella Concentration by qPCR and Culture: qPCR Was Negative When Culture Was Negative for 96% of Samples Tested

Culture (L. pneumophila) PCR (L. pneumophila)

Culture Lp1

The quantitative aspect of the qPCR test was also evaluated. qPCR estimates of the concentration of Legionella present in the sample compared to culture are presented in Table 5. When Legionella was not detected by culture, it was also not detected by qPCR in 96% of the total samples tested. Genomic units (GU) in qPCR do not equal CFU; however, good overall correlation was observed.

Table 1: L. pneumophila Culture vs. qPCR (N=277 Cooling Towers)

Cooling Tower Samples

Potable

Total 270 270

Cooling Tower Samples Testing by culture and qPCR was performed on 277 water samples from cooling towers. Results comparing culture vs. qPCR for the L. pneumophila probe (TableÂ 1) and for the L. pneumophila, serogroup 1 probe (Table 2) are below.

Positive Negative

23% 77%

Table 4: L. pneumophila serogroup 1 Culture vs. qPCR

Results: Detection of Legionella in Building Water Systems

37% 63%

Positive Negative

Estimated qPCR Total Concentration Quantity (GU/well)

Culture Total Match (CFU/mL) Tested

No Percent Match Matched

Not Detected

<23.8

<20

232

223

96%

Low

23.8 - 145

<100

88%

Moderate

145 - 2055

100-1,000

66%

High

>2055 >1,000 24 16 8 67%

Next-Generation Sequencing for Environmental Metagenomics

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The microbial community structural (microbiome) was analyzed using next generation DNA sequencing (NGS) techniques. By this method, Legionella spp. positivity for the water samples collected from the five buildings tested was 54.5% (6/11), 50.0% (5/10), 83.3% (5/6), 33.3% (2/6) and 100.0% (6/6).

Conclusions

Taxonomic assignments of sequences were assigned to 40 different phyla. The top seven phyla with highest relative abundance were Proteobacteria, Bacteroidetes, Acidobacteria, Gemmatimonadetes, Cyanobacteria, Planctomycetes, and Thermi. Figure 1 depicts the ability of this method to show differences in the phyla of the microbial community by sample location tested in the building (hot vs. cold; source vs. outlet).

Our data shows that the qPCR results for Legionella pneumophila and for L. pneumophila serogroup 1 were often comparable to culture for potable water samples.

Analytical methods in microbiology provide different information and have advantages and disadvantages associated with each method. Determining which method to use depends on the circumstance and the sensitivity and specificity of the test and cost.

The ability to probe specifically for L. pneumophila serogroup 1 is a significant advance in the utility of qPCR for Legionella environmental surveillance. Given that L. pneumophila serogroup 1 is responsible for the majority of disease and is the basis for the primary tool used to make the diagnosis of Legionnairesâ&#x20AC;&#x2122; disease, having a rapid and specific test can be invaluable. The rapid turnaround time for qPCR (hours vs. days for culture) would assist water treatment professionals in evaluating the performance of the water treatment program or remediation in controlling Legionella. This is particularly important in an outbreak or case investigation when program adjustments/corrective actions need to be made quickly. Cooling tower samples tested with the L. pneumophila qPCR showed higher positivity compared to culture, 60% vs. 37%, respectively (Table 1). The higher qPCR positivity in cooling tower samples may represent dead bacteria or cross reactivity with other non-Legionella bacteria. However, we saw comparable positive results with the L. pneumophila serogroup 1 qPCR vs. culture, 26% vs. 27%, respectively (Table 2).

Figure 1: Average relative abundance of the top 11 bacterial phyla in cold water, hot water return line, and water outlets in a commercial highrise building. Although Legionella was detected by NGS, it is not shown because its relative abundance was <0.01%.

Potable water samples tested with the L. pneumophila and L. pneumophila serogroup 1 qPCR showed comparable results between culture and qPCR; 23% qPCR positive vs. 25% culture positive for L. pneumophila, and 9% qPCR vs 14% culture positive for L. pneumophila serogroup 1, respectively (Tables 3 and 4).

Table 6: Dataset from Building 1 Demonstrates Differences in Detection Methods Sample Legionella Legionella NGS Legionella Type qPCR Culture genus_16s

(+/-) CFU/mL Spp. (+/-) CFU/mL Spp.

Tank_CW _ _ _ _ _ _

Distal Site

HWR

_ _ + 10 Lp1

Distal Site

<100

Lp1

Distal Site

<100

Lp1

Distal Site _ _ _ _ _ _

The strong correlation between negative culture results and negative qPCR results supports the use of qPCR as a negative screen. If qPCR is positive, culture should be performed to confirm the presence of Legionella and to recover the organism for future investigation. The availability of qPCR results in approximately two days is a significant advantage, especially during outbreak investigations.

(+/-)

It has been estimated that >99% of the microbial numbers in nature are nonculturable by available techniques. Hence, new culture-independent methods are needed to study the function and diversity of microorganisms in the environment. Metagenomics is an expanding field within microbial ecology that provides a glimpse into the total environmental microbial community in the manmade environment of building water systems (built environment). Next generation sequencing is a relatively new tool and is available primarily for research applications. However, the

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instrumentation is becoming more affordable and may offer new insights into the microbiome of the built environment and for Legionella building risk assessments.

References Mietzner, S. Schaeffer, A, Yassin M, Wagener M, Stout JE. Testing for Legionella in healthcare facilities: evaluation of the reproducibility of Legionella test results and the impact of time on viability and variability. American Journal of Infection Control 2013: 41; S25-S145.

Table 7 summarizes the advantages and disadvantages for the Legionella test methods of culture, qPCR, and metagenomic sequencing. Table 7: Comparison of Legionella Testing Methods: Advantages and Disadvantages Method Advantages

Disadvantages

Comment

Culture • Considered the • Some species may be “gold standard” inhibited and not • Standardized ISO recovered methods; isolation and • Time from processing to identification techniques; final result 7–14 days • Least expensive (preliminary in 4–5 days)

• Laboratories differ in competency and proficiency despite being ELITE certified. • Culture should be performed by accredited laboratories.

qPCR • Rapid and automated – • Live and dead results in 2 days. Legionella detected – • Comparable sensitivity and potential to specificity compared to overestimate risk culture with some probes. • Quantification in genomic units (GU/L) can’t compare to CFU

• Good correlation between negative culture results and negative qPCR result • Can be used to quickly screen for process control

Metagenomics • Metagenomics provides • Expensive, mostly a more information about the research tool. diversity of organisms, their • Identifying low-abundance relative abundance and community members is the community structure. currently limited

• Powerful tool to study the microbiome of the water systems in the built environment

Merault, N.; Rusniok, C.; Jarraud, S.; Gomez-Valero, L.; Cazalet, C.; Marin, M.; Brachet, E.; Aegerter, P.; Gaillard, J. L.; Etienne, J.; Herrmann, J. L.; Lawrence, C.; Buchrieser, C. Specific real-time PCR for simultaneous detection and identification of Legionella pneumophila serogroup 1 in water and clinical samples. Appl. Environ. Microbiol. 2011, 77 (5), 1708−1717. Ma X, Baron JL, Vikram A, Stout JE, and Bibby K. Fungal diversity and presence of potentially pathogenic fungi in a hospital hot water system treated with on-site monochloramine. Water Research. 2015:71C;197-206. Baron JL, Harris JK, Holinger EP, Duda S, Stevens MJ, Robertson CE, Ross KA, Pace NR, and Stout JE. Effect of monochloramine treatment on the microbial ecology of Legionella and associated bacterial populations in a hospital hot water system. Systematic and Applied Microbiology. 2015:38;198205. Dr. Janet Stout is a senior consultant at Special Pathogens Laboratory at the University of Pittsburgh, Swanson School of Engineering. Dr. Stout has been a significant contributor to AWT’s Analyst and its conventions. Dr. Stout is a world-renowned expert on Legionella. She can be reached at (877) 775-7284 or jstout@specialpathogenslab.com.

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New York State and New York City Cooling Tower Regulations: Are They Enough to Prevent Cases of Legionnairesâ&#x20AC;&#x2122; Disease? Diane Miskowski, EMSL Analytical, Inc.

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The laboratory tests used in this outbreak included Whole Genome DNA Sequencing (WGS), the European Working Society of Legionella Infectionsâ&#x20AC;&#x2122; method of Sequence Based Typing (SBT) for Legionella pneumophila serotype 1 (Lp1), and Pulsed Field Gel Electrophoresis (PFGE). Since the SBT and PFGE comparisons of environmental and clinical isolates were identical and were not able to discriminate between the isolates, WGS was used. That test revealed one nucleotide difference (out of approximately 3.4 million nucleotides) that ultimately linked the cooling tower of the Opera House Hotel to the outbreak. Despite the compelling evidence, potable water samples from nearby buildings would have been needed to see if they were also associated with the outbreak.

As result of the Legionnairesâ&#x20AC;&#x2122; disease (LD) outbreak in the Bronx that occurred in summer 2015, both New York State and New York City passed emergency regulations for registering cooling towers. The New York City regulations became final in May 2016. New York State regulations became final in July 2016 These regulations are the first of their kind in the U.S. that require monitoring for Legionella and also include enforceable action levels for Legionella and Heterotrophic Plate Count (HPC) for remediating cooling towers. While these regulations are a good first step, there are some scientifically unsupportable requirements that hopefully will prompt additional amendments to the regulations in the future. There were actually three clusters of LD outbreaks that occurred last year in the Bronx, New York. The first disease cluster occurred from December 2014 through January 2015 in the northeast Bronx. Twelve people were diagnosed with LD, with eight people being residents of the Co-op City. (Co-op City has 14,000 apartment units, 35 highrise buildings, seven clusters of townhouses, eight parking garages, three shopping centers, a high school, two middle schools, and three grade schools.) Testing of selected cooling towers in Co-op City revealed that some were contaminated with Legionella. While no determination was made that the cooling towers were the cause of any of the reported cases of LD in this cluster, the cooling towers of Co-op City were physically cleaned and disinfected with chlorine anyway. It is unknown whether building potable water samples were taken in the residences where the infected people lived.

The third cluster of 15 cases was identified in the East Bronx on September 25, 2015, and lasted until October 7, 2015. One patient died. Because several of the facilities involved were hospitals, potable water samples from these locations were taken, in addition to cooling tower samples, to rule out hospital-acquired cases. A NYCDHMH report released on November 2015 indicated that the Legionella identified in the cooling tower of the Bronx Psychiatric Center using PFGE matched the clinical samples taken from four patients. It is unknown whether SBT or WGS were used to further analyze the Lp1 isolates from this outbreak, even though NYCDHMH determined this third outbreak to be separate and distinct than the big outbreak that occurred in July and August.

The second cluster of LD cases occurred from July 2, 2015, until August 3, 2015, and was located in the South Bronx. This cluster received much media attention because it caused 138 cases of LD with 16 deaths. On the basis of epidemiologic, environmental, and laboratory evidence obtained, New York Department of Health and Mental Hygiene (NYDHMH) determined that the Opera House Hotel cooling tower was the likely source. The use of whole genome DNA sequencing determined that the isolate obtained from cooling tower at the Opera House Hotel matched the clinical isolates obtained from a total of 26 patients linked to the outbreak. While the epidemiological pattern and laboratory analysis of the outbreak certainly suggested this outbreak was linked to the cooling tower at the hotel, it is unclear from information obtained from NYDHMH via a Freedom of Information Act request whether building potable water samples from the Opera House Hotel (or other suspect locations) were taken to rule out separate infections or co-infections from potable water.

While New York State and New York City reacted promptly, and this article is not meant to be critical of their action, it does reveal additional information that would have been needed to make their regulations more soundly science-based. Perhaps the largest piece of scientifically unsupportable information driving the promulgation of these laws pertains to the number of Legionella cases for potable water versus cooling tower. It has been documented worldwide that most of the LD cases result from exposure to contaminated building potable water rather than cooling tower water. To their credit, the New York State regulations partially address this, as they also mandate testing and remediation of building water systems in healthcare facilities (New York City does not). It is also widely known that many of the LD cases from potable water are associated with traveling and staying in hotels. Yet, despite the number of hotels in both the state and city, those building water systems are not included in the regulations. In addition, New York City is home to a very large number of rooftop potable water storage tanks (many are wooden and very old) that must tested for E.coli, yet no testing is required for Legionella. These rooftop potable water storage tanks are not included in these new regulations.

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Cooling Tower Regulations continued

The American Public Health Association of Standard Methods for HPC tests includes recommendations on agar choices for either low nutrient samples or chlorinated samples. The Standard Methods procedures for choosing which agar to use do not specifically address cooling tower water samples, which are both nutrient rich and highly halogenated or oxidized. Neither the New York State nor New York City regulations specify which agar to use. Various publications document R2A agar results that are 300% higher than Plate Count Agar results. EMSL studies of 100 different cooling tower samples and 30 potable water samples has also substantiated this higher R2A agar result. Although, in a few cases we have found cooling tower samples with Plate Count Agar results that were higher than R2A results, but not significantly.

At this time, the infectious dose for Legionella is unknown. However, it is known that it is variable, as virulence factors depend on environmental conditions, the species and serotype of Legionella that are present, and the relative health of the people being exposed. Therefore, the action levels set by these regulations may result in unnecessary and costly disinfection or will not be protective of the most susceptible population. Both the New York State and New York City regulations require HPC testing using either dip slides or laboratory analyzed culture tests. However, only the New York City regulations tie HPC levels to specific regulatory actions. Industry practitioners have known for many years that HPC results from nonpotable and potable water sources have no correlation to Legionella concentrations. Therefore, the NYC regulation having an action level tied to an HPC level may result in excessive and costly remediation attempts without reducing the number of Legionella cases. (In over 2,000 New York City samples tested, EMSL has seen about 10% where HPC results exceeded the action level for disinfection while having Legionella results that were nondetectable or less than the Legionella action level.)

The New York State proposed final regulations for monitoring Legionella in the building water of healthcare facilities includes a section requiring healthcare facilities to implement disinfection when 30% or more of the building potable water distal sites are positive for Legionella. While this 30% rule of thumb has been used in the industry for some time, it is an anecdotal rather than a scientifically proven observation. For testing of potable water, it is very important to use a sample size that is large enough to minimize false negative results. However, New York State does not specify the sample size to be used for healthcare building water samples. The CDC recommends a minimum of a 1,000-mL sample for potable water, while many laboratories and clients prefer to use a 100 to 250-mL sized sample container for potable water to save on shipping costs.

A study by EMSL Analytical, Inc. of commercially available dip slides made in the United States has revealed that the use of these dip slides has never been formally validated. There have been a few limited result comparison studies from selected laboratories. However, such studies do not meet scientifically accepted validation criteria. There is no regulatory oversight or good manufacturing process specifications for these dip slides. This results in inconsistencies of reagents and materials used between batches, lots, and manufacturers that cannot be reconciled. While is it likely that New York City made a judgment call on regulating HPC levels using dip slides, the use of an invalidated and unregulated device to determine a regulated action level is not legally defensible.

To reiterate, the New York State and New York City regulations are indeed a very important first step toward minimizing the overall number of cases of Legionellosis while offering guidelines to other states experiencing higher than normal cases of LD. It is hoped that as they obtained more data as a result the regulations, they will address the items presented here to make these regulations scientifically and legally defensible.

Regarding the differences between dip slide results and laboratory culture tests, it is not uncommon to find an order of magnitude result difference (in either direction) when comparing both tests. In addition, the agar used in dip slides does not follow the Standard Method agar formulation for HPC using either R2A agar (low nutrient samples) or Plate Count Agar (chlorinated samples) that state accredited labs must use.

Diane Miskowski, MPH, has retired from EMSL Analytical, Inc. She had 30 years of experience in the areas of microbiology, laboratory management, and industrial hygiene, with a focus on aerobiology and exposure to pathogens. Over her career, Ms. Miskowski presented and published articles on such topics as Legionella, environmental mycology, and sampling strategies for microbiological assessments.

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NYS/NYC: Is the Juice Worth the Squeeze? Randy McDaniel, CWT, Weas Engineering, Inc. ANSI/ASHRAE Standard 188-2015 was released in summer 2015 with the stated purpose â&#x20AC;&#x153;to establish minimum Legionellosis risk management requirements for building water systems.â&#x20AC;?1 Shortly thereafter, New York City and New York State created new requirements for owners of buildings with cooling towers and incorporated the ASHRAE 188-2015 water management program (WMP) concept as part of their regulation. The New York City Local Law 77 of 2015 required the implementation of the WMP, which they referred to as the maintenance program and plan, by March 1, 2016. As a result, New York City and New York State became the first to create legislation in the United States aimed at reducing Legionellosis risk from cooling towers.

Figure 1 was created from the 52-Week Year-End Report. Cases reported in 2010 were taken from the 2011 report to help flush out provisional reporting. As a result, the cases reported in 2016 may not fully reflect the final cases.3-8 Data is also collected by all 50 states, New York City, and the District of Columbia. Figure 2 shows the states with the highest percentage of cases of Legionellosis. Although not a state, New York City made this list. This can partly account for their increased attention to the public health issue of Legionellosis. About 37% of all cases of Legionellosis are reported in Ohio, New York (Upstate), Pennsylvania, California, and New York City. Population may attribute to higher reported cases. However, when comparing the average cases of Legionellosis to the 2013 Census, Ohio still leads the United States with 35.35 cases of Legionellosis per 1 million people. Figure 3 lists the top 10 states.

These laws created a database for existing and new cooling towers. They require Legionella testing every 90 days. Positive results are reported to the state with corrective actions. Results in excess of 1,000 CFU/mL must be reported in NYC. Cleaning must be conducted at least twice annually, while cleaning, disinfection, and inspection must be conducted annually. Regular testing of the cooling tower must be frequent and documented to include temperature, conductivity, pH, and biocide concentration. Microbial monitoring and visual inspections are to be conducted weekly. Quarterly inspections are to be documented every 90 days of operation and prior to seasonal startup.2 These regulations have increased the time and costs associated with operating a cooling tower.

Figure 2: Legionellosis Cases Per 1 Million Residents Legionellosis by State Percent of Cases 9.00% 6.75% 4.50% 2.25%

The CDC (Centers for Disease Control and Prevention) publishes the Morbidity and Mortality Weekly Report (MMWR), which is available online. Because Legionellosis is a reportable disease, data is collected on this disease. It has been well published that cases of Legionellosis have been trending upward in the United States. A review was collected on data from the CDC MMWR.

0.00%

Upstate

Figure 3: Legionellosis in 2016 and 2017 in New York and Ohio Legionellosis Cases per 1,000,000 Residents

Figure 1: Reported U.S. Cases of Legionellosis Reported US Cases of Legionellosis

36.00

7,000

27.00

5,250

18.00

3,500

9.00

1,750 0

OH NY PA CA NYC FL IL MI NJ TX MA MD NC VA TN IN

0.00 2010

2011

2012

2013

2014

2015

2016

NYC

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Upstate

Table 1: Legionellosis in 2016 and 2017 in New York and Ohio

Legionella bacteria in cooling towers have resulted in fewer cases of Legionellosis in New York City. Further improvement would be anticipated should efforts be focused on potable water systems as set forth in the AHSRAE Standard. The June 2, 2017, release of the CMS Memorandum Requirements to Reduce Legionella Risk in Healthcare Facility Water Systems to Prevent Cases and Outbreaks of Legionnaires’ Disease (LD),12 will likely result in greater efforts to develop and follow WMPs by Medicare certified facilities. Lawmakers in states like Ohio should consider regulation that compels compliance with ASHRAE 188-2015 or the CDC Toolkit. In the end, the efforts taken to reduce the presence of Legionella will improve public health. The juice is definitely worth the squeeze.

NYC Ohio NYC Ohio

References

Source

2016 2016 2017 2017

1. ANSI/ASHRAE Standard 188-2015, 1. PURPOSE

The frequency of Legionellosis cases in New York City and Upstate New York, coupled with the publicity of the Bronx Legionnaires’ Disease outbreak of 2015, can explain why New York was the first to enact regulation. John Caloritis reported at the 2017 AWT Annual Convention and Exposition that positive tests for Legionella in NYC cooling towers were down 30–35% since the regulations went into effect.9 Granted, this reduction reflects data collected by that company and may not fully reflect the entire market. Regardless, the data is encouraging. According to the CDC, “keeping Legionella out of water is the key to preventing infection.”10

MMWR Volume 66, Issue 8

MMWR Volume 66, Issue 21

102

116

MMWR Volume 66, Issue 37

151

307

302

408

2. NYC Health, (2016, May 20). Cooling Tower Requirements: What Building Owners Should Know. Retrieved https://www1. nyc.gov/assets/doh/downloads/pdf/cd/cooling-tower-FAQs.pdf 3. MMWR Weekly, Vol. 60, No. 52, Table II. (2012, January 6). Retrieved https://www.cdc.gov/mmwr/index2011.html

Table 1 compares the reported cases of Legionellosis in New York City and Ohio from the MMWR in 2017. Based on the data from 2015, New York City reported 437 cases of Legionellosis vs. Ohio’s 572 reported cases—30.9% higher. The data shows that the cases of disease were trending fewer year over year in New York City, while cases were trending greater year over year in Ohio. Data from MMWR Volume 66, Issue 37 revealed a rapid increase in reported cases from 2016 to 2017 in New York City; however, the reported cases in Ohio exceed New York City by nearly 35%. It could be surmised that the additional regulation in New York City curtailed the rate of reported cases of Legionellosis as the population of Legionella has been reduced in their cooling towers. The effort to combat Legionella with increased monitoring, cleaning, testing, and response to positive results is a promising approach to help reduce the frequency of Legionellosis.

4. MMWR Weekly, Vol. 61, No. 52, Table II. (2013, January 4). Retrieved https://www.cdc.gov/mmwr/index2012.html 5. MMWR Weekly, Vol. 62, No. 52, Table II. (2014, January 3). Retrieved https://www.cdc.gov/mmwr/index2013.html 6. MMWR Weekly, Vol. 63, No. 52, Table II. (2015, January 9). Retrieved https://www.cdc.gov/mmwr/index2014.html 7. MMWR Weekly, Vol. 64, No. 52, Table II. (2016, January 8). Retrieved https://www.cdc.gov/mmwr/index2015.html 8. MMWR Weekly, Vol. 65, No. 52, Table II. (2017, January 6). Retrieved https://www.cdc.gov/mmwr/index2016.html 9. J.D. Caloritis, Biofilm Monitoring, Legionella Update, AWT 2017 Annual Convention. (2017, Sept. 14) 10. Centers for Disease Control and Prevention. Fast Facts. Retrieved https://www.cdc.gov/legionella/fastfacts.html

The CDC released a report in June 2016 studying the details regarding outbreaks of Legionnaires’ disease in North America.8 About 56% of the outbreaks were linked to potable water, with about 22% linked to cooling towers. However, the percentage of actual number of confirmed and suspected cases was about 45% for cooling towers and 44% for potable water. The nature of cooling tower drift makes it possible to disseminate Legionella over a broader area, potentially infecting more people. The median number of infections per cooling tower outbreak was 22, with only 10 cases per potable water outbreak.11

11. Garrison LE, Kunz JM, Cooley LA, et al. Vital Signs: Deficiencies in Environmental Control Identified in Outbreaks of Legionnaires’ Disease – North America, 2000–2014. MMWR Morb Mortal Wkly Rep 2016;65:576 – 584. DOI: http://dx.doi.org/10.15585/mmwr.mm6522e1 12. CMS Memorandum (2017, June 02 Revised June 9) Requirements to Reduce Legionella Risk in Healthcare Facility Water Systems to Prevent Cases and Outbreaks of Legionnaires’ Disease (LD). Retrieved https://www.cms.gov/Medicare/ProviderEnrollment-and-Certification/SurveyCertificationGenInfo/ Downloads/Survey-and-Cert-Letter-17-30.pdf

Conclusion The voluntary development and implementation of WMPs by building owners as outlined by ASHRAE 188-2015 has been less than spectacular. However, when compelled by regulations such as the laws enacted by New York State and New York City, compliance improves. It appears as if the efforts made to reduce

Randy McDaniel, CWT, is the strategic accounts manager for Weas Engineering, Inc. He can be contacted at (317) 867-4477 or randy.mcdaniel@weasengineerng.com. 33

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Isolation of Legionella From Environmental Samples: CDC vs. ISO Christopher Goulah, Ph.D., EMSL Analytical, Inc.

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of the sample during transport, which could result in a false negative result. Similarly, the sample must be transported on ice to prevent continued growth of any bacteria present that could overwhelm or inhibit the slow-growing Legionella. It is recommended that samples arrive at the environmental testing laboratory within 48 hours of collection.

Abstract In this study, nonpotable environmental water samples (n=30) were analyzed in parallel by both the CDC and ISO methods. Of the 30 samples, half (n=15) returned results with positive detection of Legionellae by one or both methods. However, whereas the ISO method identified all 15 as positive for Legionella, the CDC method only identified two samples as positive for Legionella, as most fell below the limit of detection for this method. It should be noted, however, that at least one sample determined to be ‘no Legionella detected’ by the CDC method was identified as containing >10,000 CFU/mL of Legionella pneumophila serotype 1 by the ISO method. This was primarily due to the presence of Pseudomonas aeruginosa that was not selected against by either the antibiotics present in the GVPC plates or the acidification during the acid-treatment of the CDC method. Only after the heat treatment required by the ISO method was this inhibitory background flora eliminated. Both scenarios represent significant points of weakness for the CDC method, as <15% of the samples determined to contain Legionella were detected by this technique. These results validate the use of the ISO method over the CDC method for the isolation of Legionella from environmental water samples.

NYS requires all Legionella samples to be analyzed at a laboratory certified by the Environmental Laboratory Approval Program (ELAP). To obtain certification from NYS, commercial Legionella laboratories are required to isolate and enumerate Legionella bacteria according to ISO method 11731:1998 for potable and nonpotable samples. Previously, most commercial laboratories both in NYS and nationwide followed the method prescribed by the Centers for Disease Control and Prevention (CDC) and utilized the CDC’s Environmental Legionella Isolation Techniques Evaluation (CDC-ELITE) proficiency testing program. While the two methods—CDC and ISO—operate on the same fundamental principles, there are methodological differences that can result in variances in the detection of Legionella from identical environmental samples.

Materials and Methods Sampling A total of 30 water samples were collected from various cooling towers located in proximity to western New York State. Water samples (>250 mL) were collected in accordance with ISO 19458:2006 into sterile containers containing an excess of sodium thiosulfate to neutralize any residual oxidizing biocides in the water. Samples were transported to the laboratory as soon as possible and processed within 48 h of collection.

Introduction Background During summer 2015, a series of fatal legionellosis outbreaks forced the New York City (NYC) Department of Health and Mental Hygiene (DOHMH) to take emergency action to contain the rapid spread of the infection. After determining that the source of the outbreak was a centrally located cooling tower, emergency ordinances were put in place that mandated the disinfection of all cooling towers citywide. The city passed permanent regulations to maintain a set schedule of maintenance and biological monitoring of all cooling towers within the NYC limits. Requirements included daily water quality measurements, weekly biological process control indicators, and quarterly Legionella testing. The New York State (NYS) Department of Health (DOH) quickly followed suit and published “Protection Against Legionella,” which established similar regulations statewide to cover both cooling towers and healthcare facilities. The state regulations require owners of cooling towers and healthcare facilities to have a water management plan in place and to monitor their water systems for biological content at regular intervals.

Processing of water samples per CDC protocol (2005) The CDC method states that “non-potable water rarely requires concentration and can be processed directly”; therefore, 5 mL of each sample was transferred to a sterile 50-mL conical and stored at 4 °C until plating. Each sample was plated on a total of six plates: (1) BCYE, (2) PVC, (2) GVPC, and (1) GVPC –Cys, each receiving 0.1 mL of sample. The plates were then wrapped in plastic wrap and transferred to a 36 °C incubator for seven days of incubation. Processing of water samples per ISO 11731 (1998) Each 250 mL sample was mixed well by agitation and then filtered through a 0.4-μm polycarbonate filter (Pall). The filter was then aseptically removed from the filter holder and placed with 5 mL of isotonic buffer (a 1:10 dilution of ¼-strength Ringer’s buffer) in a 50-mL sterile conical and vigorously vortexed for 2 minutes. Each 5 mL concentrated sample was thoroughly mixed and then divided into three portions: one portion was acid-treated for 5 ± 0.5 minutes using HCl pH 2.0 in KCl; one portion was heat-treated at 50 °C ± 0.5 °C for 30 ± 2 minutes; and one portion was left untreated. All three methods were applied to the same concentrated sample, so results were not

NY sampling requirements Sampling for Legionella in cooling towers is required every 90 days from one or more of the following locations: tower sump, tower makeup, heat exchanger inlet/outlet, or the tower pack. It’s recommended that healthcare facilities sample 10 percent of selected outlets—both hot and cold—semiannually. In both cases, the samples must be collected in sterile vessels containing sufficient sodium thiosulfate to neutralize any residual chlorine present. This is essential to prevent continued disinfection 35

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Isolation continued

affected by variations in filtration/resuspension. Samples were stored at 4 °C until plating. 0.1 mL of each treatment of each sample was plated on a single GVPC plate, with the exception of the heat-treated portion, which was also plated on a single GVPC –Cys plate. Plates were then wrapped in plastic wrap and transferred to a 36 °C incubator for 10 days of incubation.

Table 1: Legionella Detection by ISO 11731:1998 Culture Method Lab Method Identificaton* Sample Processed Number

Analysis All plates were analyzed on day 4 of incubation to monitor the growth of any Legionella colonies. Representative colonies from any putative Legionella were transferred to BYCE and BCYE –Cys to confirm growth. Enumeration was performed on days 4, 7, and 10 (ISO only), with day 7 being the final count for the CDC method. Results Of the 30 samples, half (n=15) returned results with positive detection of Legionellae by one or both methods. The ISO method identified all 15 as positive for Legionella (Table 1). The CDC method only identified two samples as positive for Legionella, as most fell below the limit of detection for this method (Table 2). There were several other samples (see samples 12 and 16) that were detected using the ISO method at levels well above the limit of detection for the CDC method but were not observed in the CDC method processed samples. This is possible in situations where the sample plates are not overgrown in the initial plating and the acid-treatment reprocessing is not required to be performed. The most striking omission from the positive samples on Table 2 was sample 13. This sample contained >10,000 CFU/mL as determined by the ISO method but was found to be “None Detected” using the CDC method. Upon examination of the culture plates, it was found that the untreated and acid-treated plates in both methods contained multiple colonies of Pseudomonas aeruginosa that was not selected against by the antibiotics present in the GVPC plates nor by the acid-treatment. Only the heat-treatment step in the ISO method was sufficient to eliminate this potent inhibitor of Legionella as background flora.

Concentrated (acid treated)

Legionella 44.80 pneumophila (sero 1)

Concentrated (acid treated)

Legionella 1.60 pneumophila (sero 1)

Concentrated (heat treated)

Legionella 6.00 pneumophila (sero 1)

Concentrated

Concentrated (acid treated)

Legionella 8.00 pneumophila (sero 1)

Concentrated (acid treated)

Legionella 2.40 pneumophila (sero 1)

Concentrated

None Detected

Concentrated

None Detected

Concentrated

None Detected

Concentrated (heat treated)

Legionella sp. (not L. pneumophila)

0.20

Concentrated (heat treated)

Legionella sp. (not L. pneumophila)

0.20

Concentrated Legionella 89.00 (acid treated) pneumophila (sero 1)

Concentrated (heat treated)

Concentrated

None Detected

Concentrated

None Detected

Concentrated (acid treated)

Concentrated (heat treated)

None Detected

Concentrated

None Detected

Concentrated

None Detected

Concentrated

None Detected

Legionella 12000.00 pneumophila (sero 1)

Legionella 31.25 pneumophila (sero 1)

Legionella 95.00 pneumophila (sero 5)

Concentrated

None Detected

Concentrated

None Detected

24 Concentrated

Legionella 8.75 pneumophila (sero 5)

Concentrated

None Detected

Concentrated

None Detected

Concentrated Legionella 0.40 (acid treated) pneumophila (sero 1)

Concentrated (heat treated)

Concentrated

30 Concentrated

None Detected

21 Concentrated

Discussion One of the more critical differences between the two methods is the way in which nonpotable water samples are initially processed. The CDC method directs all nonpotable samples to be directly plated onto selective buffered charcoal yeast extract (BCYE) media, both with and without various antibiotics and in the presence and absence of the amino acid cysteine, which is a strict nutritional requirement of Legionella. Direct plating of water samples provides a limit of detection (LOD) of 10 CFU/mL

Final Results (CFU/mL)

Legionella 4.00 pneumophila (sero 6) None Detected

Legionella 9.00 pneumophila (sero 1)

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Isolation continued

plating can lower the LOD to 0.2 CFU/mL. This lowered LOD provides a buffer zone to the maintenance engineers so that when Legionella are detected, there is an opportunity to readjust the disinfection protocol to remain compliant without triggering a regulatory action.

Table 2: Legionella Detection by CDC Culture Method Lab Sample Method Identificaton* Number Processed 1 Direct Plating 2 Direct Plating 3 Acid Treated 4 Direct Plating 5 Acid Treated 6 Acid Treated 7 Direct Plating 8 Direct Plating 9 Direct Plating 10 Direct Plating 11 Direct Plating 12 Direct Plating 13 Acid Treated 14 Direct Plating 15 Direct Plating 16 Direct Plating 17 Acid Treated 18 Direct Plating 19 Direct Plating 20 Direct Plating 21 Direct Plating 22 Direct Plating 23 Direct Plating 24 Direct Plating 25 Direct Plating 26 Direct Plating 27 Acid Treated 28 Acid Treated 29 Direct Plating 30 Direct Plating

Final Results (CFU/mL)

Legionella 30.00 pneumophila (sero 1) None Detected ND None Detected ND None Detected ND None Detected ND None Detected ND None Detected ND None Detected ND None Detected ND None Detected ND None Detected ND None Detected ND None Detected ND None Detected ND None Detected ND None Detected ND None Detected ND None Detected ND None Detected ND None Detected ND Legionella 100.00 pneumophila (sero 5) None Detected ND None Detected ND None Detected ND None Detected ND None Detected ND None Detected ND None Detected ND None Detected ND None Detected ND

Another difference between these two methods is the secondary processing of the sample prior to plating. The CDC method directs samples with high background to be reprocessed using acid treatment, while the ISO method mandates that all samples be independently acid treated and heat treated. The heat treatment has been validated by multiple studies to be more effective at decreasing the levels of background flora that normally flourish on the highly selective BCYE media even in the presence of multiple antibiotics. This is particularly useful with regard to reducing bacterial species that are inhibitory to Legionella growth; for example, the presence of Pseudomonas aeruginosa can result in false negative results. The last methodological difference does not relate to the efficacy of the process, but rather to the overall efficiency and cost to perform the test. The CDC method utilizes four variations of the BCYE agar during the initial plating of the sample: BCYE; BCYE with Polymyxin B, Vancomycin, and Cycloheximide (PVC); PVC with glycine (GVPC); and GVPC without cysteine (GVPC-). In addition, the PVC and GVPC agar are plated in duplicate, resulting in a total of six plates. If acid treatment is required to reduce background flora, as is usually the case for nonpotable samples, this is doubled to 12 plates. The ISO method performs the secondary acid and heat treatments up front and plates each treatment on a single GVPC plate. The heat treatment is also plated on the GVPC- plate as a negative control, bringing the total number of plates utilized during the ISO method to four. The decrease in the number of plates combined with the amount of time saved by virtually eliminating the need to reprocess the samples results in a highly streamlined method.

based on the common practice of 0.1 mL being plated. Current NYC regulations set the action level for the presence of Legionella in cooling towers at 10 CFU/mL; at that level, immediate disinfection, review of the treatment program, and retesting of the waters within three to seven days is required. Therefore, any positive sample using the CDC method would require immediate action with no warning that Legionella may be starting to proliferate in the system.

As additional states follow New Yorkâ&#x20AC;&#x2122;s lead and start to regulate testing for Legionella, the question of which method to utilize will come up more frequently. Based on the procedural differences between the CDC and ISO methods, the ISO method is a more appropriate choice for regulators, engineers, and environmental laboratories. Christopher Goulah, Ph.D., is the Legionella technical manager for EMSL Analytical, Inc. He is an accomplished microbiologist with nearly 20 years of experience. Dr. Goulah has spoken on global infectious agents at national speaking engagements and has multiple peer-reviewed articles published in a variety of scientific journals. He can be reached at (716) 651-0030 x 1407 cgoulah@emsl.com

In contrast, the ISO method directs all samples (potable and nonpotable) to be concentrated prior to plating. Typically, a 250 mL water sample submitted for analysis that is concentrated and subsequently resuspended in 5 mL of isotonic buffer prior to

the Analyst Technology Supplement 2017

Interpreting Legionella Test Results: Case Studies Illustrating Key Criteria

Matthew Freije, HC Info

the Analyst Technology Supplement 2017

Introduction

Interpreting Legionella Test Results: Case Studies Illustrating Key Criteria

Six Criteria Should Be Considered to Respond Successfully to Legionella Test Results for Samples From Plumbing Systems When reviewing Legionella test results for samples collected from plumbing systems, considering all six of the following will help in finding opportunities to prevent Legionnaires’ disease:

When considering all three of the criteria, along with three additional factors, water management decision-making will be much easier and offer a greater chance of success in controlling Legionella bacteria in plumbing systems without overspending.

• Concentration per sample (CFU/mL) • Positivity

Sampling’s Proper Place in Managing Building Water Systems and Complying with ANSI- ASHRAE Standard 188-2015

• Legionella strains

The purpose of testing building water systems for Legionella—at least with respect to ANSI- ASHRAE Standard 188-2015—is to validate a water management program by providing data that indicate the program, as implemented, is effective in controlling Legionella bacteria.

• Equipment-specific remediation

• Breakdown of findings • Occupant susceptibility Concentration per sample The Veterans Health Administration (VHA 2014) and the Centers for Disease Control and Prevention (CDC 2003) do not acknowledge Legionella concentrations in interpreting Legionella test results, but instead view results as positive or negative.

According to ASHRAE 188, whether or not to establish a water management program for a building should be based on the types of water systems in the building rather than on Legionella test results. For example, an owner of 20 hotels should not decide whether to implement a water management program for each of the properties based on Legionella test results, but rather based on the types of water systems.

Some organizations, however, have recommended interpreting Legionella test results for plumbing systems based on the concentration per sample (see Table 1), with the idea that action will be taken if the Legionella level exceeds the threshold. Likewise, if Legionella is not found, or found at a concentration below the threshold, the system will be considered “safe” and no action taken.

Decisions Must Be Based on Reliable and Thorough Data Good practices in interpreting Legionella test results will not matter much unless the results are based on adequate sampling, data recording, and laboratory analysis.

Concentrations matter. Other factors being equal (susceptibility of the person exposed and the efficiency of transmission from the water to the person’s lungs), the primary risk factor for contracting Legionnaires’ disease is the number of Legionella organisms in the water to which a person is exposed. Clearly, 1,000 CFU/mL presents a greater risk than 1 CFU/mL.

Sample at a frequency appropriate to your building type and situation and specify an adequate number of samples for each round. Plan your samples strategically—systems and devices, locations, water versus swab, hot versus cold, pre-flush vs post-flush.

However, interpreting Legionella results based solely on concentrations per sample has limitations that could result in missed opportunities to prevent disease. According to the World Health Organization, the infective dose for humans can be assumed to be low—possibly even a single organism (WHO 2002). Therefore, any Legionella finding could indicate a health risk, especially in situations in which Legionella transmission is efficient (e.g., a breathing apparatus connected to a humidifier) or the persons exposed are immunocompromised.

Execute the sampling well. Collect samples properly. Record data to maximize useful information. Select a laboratory that is CDC ELITE certified and highly proficient. Ensure that the laboratory's speciation and detection limits are consistent with your needs.

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Table 1: Concentration Thresholds Given for Plumbing Systems

Table 2: Comparison of Legionella Results Reported by Three CDC ELITE Certified Laboratories for 445 Duplicate Samples

Excerpted and adapted from the online course “Interpreting Legionella Water Test Results and Responding Appropriately,” courtesy HC Info. Used with permission.

Laboratories A and B

Laboratories A and C

Threshold CFU/mL

Percentage difference in CFU/mL reported (absolute value/average)

145%

120%

Average CFU/mL difference

33.2*

14.5

Agreement in strains reported

81.1%

95.2%

France

1 in public facilities* 0.1 to prevent HAIs* 0.05 in at-risk patient areas*

Germany 1* Netherlands 1* South Australia

Switzerland

0.1 in hospitals

0.1 if positivity > 50%* 1 in any sample*

USA CDC

any Legionella-positive

USA OSHA

USA VHA

any Legionella-positive

Samples for which one laboratory 37/172 L-positive samples (22%) reported < 10 CFU/mL and the other > 10 (total for both laboratory pairs) *Excluding four samples for which the absolute difference exceeded 1,000 CFU/mL

Positivity Interpreting domestic water Legionella results based on the percentage of samples in which Legionella is found (positivity) has been recommended based on studies indicating that 30% positivity was a key threshold associated with Legionnaires’ disease in hospitals (Best 1983; Kool 1999; Stout 2007).

* Converted from CFU/L to CFU/mL

Moreover, the concentration reported by laboratories for a sample can vary significantly, even several fold. Agreement among the three laboratories for which the summary of duplicate sample test results is shown in Table 2 is actually quite good compared to other laboratory pairs HC Info has tested, yet the percentage difference in results averaged 145% for one pair and 120% for the other. The average difference between laboratories A and B was 33.2 CFU/mL, even excluding the four samples with differences greater than 1,000 CFU/mL. Water management teams that base decisions on a threshold of 10 CFU/ml per OSHA should note that for 37 of the 172 samples (22%) for which at least one of the two laboratories found Legionella, one reported >10 CFU/mL and the other <10 CFU/mL.

As with concentrations, interpreting Legionella results based solely on positivity has limitations that could result in missed opportunities to prevent disease. A literature review (Allen 2012) has pointed out limitations of using 30% positivity as a strict risk assessment tool, concluding that the concentration per sample, Legionella strain, and building-specific factors need to be considered—along with positivity—to properly apply test results. Positivity must be applied only to samples collected from the domestic water (plumbing) system, even if other devices and water systems—cooling towers, whirlpool spas, decorative fountains— are sampled on the same day, recorded on the same log, and sent to the lab in the same shipment.

Data gathered by the CDC shows even greater variation in quantitation reported by laboratories—approximately a 10-fold difference—indicating that numerical differences less than a factor of 10 are statistically insignificant. Decisions based on relatively minor numerical differences (e.g., 8 versus 20 CFU/mL or 50 versus 110) are not scientifically defensible. The CDC cites this data as the reason for not endorsing “action trigger” tables.

And, even among samples from plumbing systems, positivity must not be applied blindly. A representative number of samples must be collected for positivity to be meaningful.

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Legionella strains Some Legionella strains have caused more cases of Legionnaires’ disease than others. Data shows that Legionella pneumophila serogroup 1 (Lp1) has caused the majority of reported cases in the United States, although the oft-cited percentage of cases caused by Lp1 could be biased by the fact that the urinary antigen test—which detects only that strain—is the one most commonly used in hospitals for detecting Legionella. Other strains that have been associated with reported cases—for example, other serogroups of Legionella pneumophila and the species bozemanii, dumoffii, and micdadei—may cause more cases than are being recognized.

Breakdown of findings In addition to evaluating whether Legionella positivity or concentrations exceed the thresholds you have set for your domestic hot and cold water systems overall, a breakdown of the findings may help in pinpointing specific problems and solutions. A breakdown can be performed thoroughly and quickly only if sample information was recorded in sufficient detail and entered into a database. Check to see if Legionella concentrations or positivity were significantly higher for: • Certain types of fixtures (e.g., electronic faucets, shower hoses, foot pedal faucets, bar sinks)

CDC guidance indicates that finding any Legionella strain is a concern. Others have recommended taking action only if the most pathogenic species and serogroups are found.

• Pre-flush versus post-flush samples • Some buildings or water loops versus others

Opportunities to prevent Legionnaires’ disease could be missed if action is taken only when certain Legionella strains are detected, especially when Legionella positivity and concentrations are high. Finding any Legionella strain indicates the water system— as presently designed, operated, and maintained—is conducive to Legionella growth, at least for the strain detected.

• Hot versus cold water The breakdown data can be especially useful over several sampling rounds. Breaking down results could save a facility a lot of time and money. Too many facilities take an all or nothing approach to domestic water remediation—doing nothing or disinfecting everything. In some cases, a rifle approach is necessary to solve problems without overspending, and reviewing a breakdown of data is the best way to hit the target.

Moreover, the reported strain may not have been identified correctly, or there could have been other strains in the sample that were not identified. Duplicate sample test results show that CDC ELITE certified laboratories are not always consistent in strains reported. One may report a pathogenic species (e.g., L. bozemanii) while another reports a species that is less pathogenic or not pathogenic (e.g., L. anisa). One may report L. pneumophila serogroup 1 for a sample in which another lab reports another serogroup.

Equipment-specific remediation If Legionella findings indicate that remedial steps are appropriate for certain types of equipment (e.g., ice machines; piped water dispensers; water heaters), then take those steps even if Legionella positivity and concentrations are below your thresholds for the domestic water system overall.

Although the laboratories that tested the 445 duplicate samples summarized in Table 2 had remarkable agreement in the strains reported, correctly identifying the Legionella strain for 81% to 95% of Legionella-positive samples is not reliable enough to base water management decisions entirely on identifying one Legionella strain versus another.

Occupant susceptibility If Legionella is found in a sample from an outlet in a room occupied by a high-risk person, take appropriate measures to protect those persons (e.g., install faucet and shower filters validated to block Legionella or restrict water use), even if Legionella positivity and concentrations are below your thresholds for the domestic water system overall.

To base decisions only on strain also assumes that scientists and public health authorities have enough data to conclude that only certain strains present a significant risk of disease.

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Case Studies Illustrating Opportunities for Good or Bad Decisions

Case Study 2: Hospital A If Hospital A takes remedial action only when domestic water Legionella positivity exceeds 30%, it will overlook the clues for reducing Legionella risk that are indicated by the results shown in Table 4.

The following case studies illustrate the importance of all six criteria.

Case Study 1: Hotel D If the water management team for Hotel D interprets Legionella results for its domestic water system based strictly on concentrations exceeding 10 CFU/mL, it would have concluded from the results shown in Table 3 that its water management program was adequate when in fact Legionella was found in 67% of domestic hot water and 75% of domestic cold water samples.

Table 4: Legionella test results for a hospital plumbing system. Image from LAMPS WMP Platform courtesy HC Info. Used with permission.

Table 3: Legionella test results for a hotel plumbing system. Image from LAMPS WMP Platform courtesy HC Info. Used with permission.

Location

Device

Sample

Type

Results Concentration

Mech Rm B WH

Water heater or hot water storage tank

Domestic hot water

Pre-flush

Positive 240.00

Mech Rm B WH

Water heater or hot water storage tank

Domestic hot water

Post-flush

N/A

Location

Device

Sample Type

Results Concentration

1F MRR ESK

Faucet-automatic /electronic

Domestic hot/ cold mix water

Pre-flush

Positive 980.00

Mech Rm B WH

Water heater or hot water storage tank

Domestic hot water

Pre-flush

Positive

9.00

112 EXR

Faucet-manual handle

Domestic hot water

Pre-flush

N/A

Mech Rm B WH

Water heater or hot water storage tank

Domestic hot water

Post-flush ND

N/A

202 PTRMBA

Faucet-manual handle

Domestic hot water

Pre-flush

N/A

Lobby WRR Faucet-manual handle

Domestic hot water

Pre-flush

N/A

314 PTRM

Faucet-manual handle

Domestic hot water

Pre-flush

N/A

Lobby DF Drinking Fountain

Domestic cold water

Pre-flush

Positive

8.00

416 PTRM Shower head

Domestic hot water

Pre-flush

N/A

123 Guest Faucet-manual handle Room BA

Domestic hot water

Pre-flush

Positive

8.00

402 ICU PTRM

Faucet-manual handle

Domestic hot water

Pre-flush

N/A

241 Guest Shower head Room

Domestic hot water

Pre-flush

Positive

2.00

423 BMT PTRM

Faucet-manual handle

Domestic hot water

Pre-flush

Positive 34.00

318 Guest Faucet-manual handle Room BA

Domestic hot water

Pre-flush

Positive

7.00

504 PTRM

Faucet-manual handle

Domestic hot water

Pre-flush

N/A

318 Guest Faucet-manual handle Room Bar Sink

Domestic cold water

Post-flush Positive

1.00

532 CCU PTRM

Faucet-manual handle

Domestic hot water

Pre-flush

N/A

421 Guest Faucet-manual handle Room BA

Domestic hot water

Pre-flush

Positive

1.00

6F WRR MSK

Faucet-automatic /electronic

Domestic hot/ cold mix water

Pre-flush

Positive 440.00

516 Guest Shower head Room

Domestic hot water

Pre-flush

Positive

1.00

678 PTRM

Faucet-manual handle

Domestic hot water

Pre-flush

N/A

634 Guest Faucet-manual handle Room BA

Domestic hot water

Pre-flush

Positive

4.00

721 PTRMBA

Faucet-manual handle

Domestic hot water

Pre-flush

N/A

634 Guest Faucet-manual handle Room Bar Sink

Domestic cold water

Post-flush ND

N/A

811 PTRMBA

Faucet-manual handle

Domestic hot water

Pre-flush

N/A

712 Guest Faucet-manual handle Room BA

Domestic hot water

Pre-flush

N/A

913 PTRM

Faucet-manual handle

Domestic cold water

Pre-flush

N/A

821 Guest Shower head Room

Domestic hot water

Pre-flush

N/A

2F Drinking Fountain

Drinking fountain

Domestic cold water

Pre-flush

N/A

925 Guest Faucet-manual handle Room BA

Domestic hot water

Pre-flush

Positive

3.00

314 Janitor Sink

Faucet-manual handle

Domestic cold water

Pre-flush

N/A

925 Guest Faucet-manual handle Room Bar Sink

Domestic hot water

Pre-flush

Positive

9.00

4f ICU IM Ice machine ice

Domestic cold water

Pre-flush

N/A

The team should also be aware that quantitation reported by laboratories varies significantly; another qualified laboratory may have reported concentrations >10 CFU/mL for some of the samples tested. Considering both positivity and concentration would have provided better direction for the water management program. 42

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• Do the hot water storage tanks (HWTs) operate in parallel to supply the entire building, or does one serve part of the building and the other the rest?

Case Study 3: Office Building C Referring to Table 5, a consultant might advise the office building owner to take remedial action based on the Legionella concentrations and a domestic hot water positivity of 46%. At a minimum, Hot Water Storage Tank (HWT) 1 should be cleaned and disinfected.

• If HWTs serve separate parts of the building, does HWT1 supply floors 1 through 4? If not, are there differences in the system design, occupancy, operation, fixture types, temperatures, or other factors that might explain why all the Legionella-positives were on floors 1 through 4?

Before recommending long-term or costly measures, though, the consultant should investigate the following:

It is much easier to make decisions based on multiple sampling rounds rather than just one. Table 6 shows four rounds of results for the Case Study 3 building. The results shown in Table 7 were for July. After the July sampling round, the team decided to hyperchlorinate the hot and cold domestic water system and then gather more data (i.e., sample more) to determine a longer term solution. Legionella was found in just one sample collected in the August post-remediation round.

Table 5: Legionella test results for an office building plumbing system.Image from LAMPS WMP Platform courtesy HC Info. Used with permission. Location

Device

Sample

Type

Results Concentration

Mech Room A HWT 1

Water heater or hot water storage tank

Domestic hot water

Pre-flush

Positive

130.00

Mech Room A HWT 1

Water heater or hot water storage tank

Domestic hot water

Post-flush

Positive

12.00

Mech Room A HWT 2

Water heater or hot water storage tank

Domestic hot water

Pre-flush

N/A

Mech Room A HWT 2

Water heater or hot water storage tank

Domestic hot water

Post-flush

N/A

1F MRR ESK Faucet-manual handle

Domestic hot water

Pre-flush

Positive

30.00

1F Kit Water dispenser-piped

Domestic cold water

Pre-flush

Positive

23.00

2F WRR WSK Faucet-manual handle

Domestic hot water

Pre-flush

Positive

7.00

3F MRR WSK Faucet-manual handle

Domestic hot water

Pre-flush

Positive

5.00

4F WRR ESK Faucet-manual handle

Domestic hot water

Pre-flush

Positive

20.00

5F MRR WSK Faucet-manual handle

Domestic hot water

Pre-flush

N/A

6F MRR WSK Faucet-manual handle

Domestic hot water

Pre-flush

34.00

Table 6: Case Study 3—Summary of Four Rounds of Legionella Results

7F WRR ESK Faucet-manual handle

Domestic hot water

Pre-flush

N/A

8F MRR ESK Faucet-manual handle

Domestic hot water

Pre-flush

N/A

9F WRR WSK Faucet-manual handle

Domestic hot water

Pre-flush

N/A

5F DF by Drinking Fountain Elevator

Domestic cold water

Pre-flush

N/A

8F DF by Drinking Fountain Elevator

Domestic cold water

Pre-flush

N/A

6F MRR ESK Faucet-manual handle

Domestic cold water

Post-flush

N/A

Breaking down the average concentrations and positivity by device types over all four sampling rounds clearly showed that all the Legionella-positive cold water samples were from piped water dispensers (Table 5). No Legionella was found in cold water from drinking fountains or faucets. Although this may seem obvious, similar findings often go unnoticed because too little data is recorded during sampling and the test results are not entered into a database that can show average concentrations and positivity for a number of parameters. The summary of the test results over the four rounds indicates that installing a continuous disinfection system for the domestic cold water in this building is likely unnecessary if the piped water dispensers are either remediated or discontinued.

DCW Pos/Avg Conc

DHW Pos/Avg CFU/mL

Jan 25%/20

15%/8.5

May 25%/35

23%/18.3

July 25%/23

46%/34

Aug 25%/45

8%/5

All Four Rounds

23%/23.4

25%/30.8

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References

Table 7: Case Study 3—Breakdown by Outlet Type for Four Sampling Rounds

• Allen, JG, Myatt, TA, MacIntosh, DL, et al. 2012. Assessing risk of health care-acquired Legionnaires’ disease from environmental sampling: The limits of using a strict percent positivity approach. American Journal of Infection Control 40; 917-921

Positivity Highest Average Concentration Concentration Positive Sample Drinking Fountain

N/A N/A

Faucet-manual 0.20 handle

30.00

12.00

Water dispenser-piped

45.00

30.00

1.00

• Best, M, Yu, VL, Stout, J, Goetz, A, Muder, RR, Taylor, F. 1983. Legionellaceae in the Hospital Water-Supply: Epidemiological Link with Disease and Evaluation of a Method for Control of Nosocomial Legionnaires’ Disease and Pittsburgh Pneumonia. Lancet 2; 307-310.

N/A

• CDC. 2003. Guidelines for Preventing Health-CareAssociated Pneumonia, 2003: Recommendations of CDC and the Healthcare Infection Control Practices Advisory Committee. Atlanta: Centers for Disease Control and Prevention. Available at http://www.cdc.gov/mmwr/ preview/mmwrhtml/rr5303a1.htm.

All hot-water Legionella positives for the four rounds were from the low system (floors 1–4). Further testing will be needed to determine whether routine cleaning and flushing of the hot water storage tanks and other physical or operational changes will result in Legionella control for floors 1–4. If not, continuous disinfection will probably be needed.

• Kool, J, Bergmire Sweat, D, Butler, J, et al. 1999. Hospital characteristics associated with colonization of water systems by Legionella and risk of nosocomial Legionnaires’ disease: A cohort study of 15 hospitals. Infect Control Hosp Epidemiol 20:798,805.

Conclusions Legionella test results can help a water management team determine the most cost effective ways to control Legionella in domestic water systems, provided that:

• Stout, JE, Muder, RR, Mietzner, S, et al. 2007. Role of Environmental Surveillance in Determining Risk for Hospital-Acquired Legionellosis: A National Surveillance Study with Clinical Correlations. Infection Control and Hospital Epidemiology; 28:7; 818-824.

• Sampling is properly planned with respect to frequency, number of samples, devices sampled, sample types and locations, hot versus cold water, and pre-flush versus postflush samples. • Samples are collected properly.

• VHA. 2014. Prevention of Healthcare-Associated Legionella Disease and Scald Injury from Potable Water Distribution Systems. VHA Directive 1061. Washington, DC: Department of Veterans Affairs.

• Sampling data is recorded to maximize useful information. • Samples are tested by a laboratory that is CDC ELITE certified and highly proficient.

• WHO. 2002. Legionella. In: Guidelines for drinking water quality. Addendum: Microbiological agents in drinking water, 2nd ed. Geneva: World Health Organization.

• Test results are entered into a database sufficient for thorough analysis over multiple sampling rounds. • Test results are interpreted based on concentration per sample as well as positivity and Legionella strains, with a breakdown of the findings by device type, hot versus cold water, pre-flush versus post-flush, and other parameters, considering equipment-specific as well as systemwide remediation.

Matt Freije is president of HC Info. He is a consultant, author, and course instructor specializing in Legionella and other waterborne pathogens. His book “Legionellae Control in Health Care Facilities: A Guide for Minimizing Risk” has sold in more than 30 countries. Mr. Freije can be reached at (760) 451-1020 or mfreije@hcinfo.com.

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Chlorine Dioxide for Control and Prevention of Biofilm and Legionella Tom McWhorter, CDG Environmental, LLC

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bacteria (including Legionella), protozoans, and other organisms that flourish in biofilm. The very nature of cooling towers creates aerosols that contain microorganisms from the biofilm. These aerosols are carried by natural air currents into neighboring spaces, where they can be inhaled by humans.

Introduction One of the greatest remaining water-related threats to human health in the United States is Legionella. Although Legionella is ubiquitous in our environment, it is especially problematic in hospitals, [1] nursing homes, and other institutions where recirculating hot water systems serve large populations of immunocompromised people.

Water parks and ornamental fountains also provide ideal growth media for biofilm and Legionella. The splashing water and spray typical of such facilities can easily aerosolize water containing Legionella.

Legionella is also problematic in cooling towers without adequate disinfection. Legionella proliferates in biofilm, from which pieces occasionally slough off and are aerosolized by the action of the cooling tower. The aerosol can then become airborne and infect people in the area surrounding the cooling tower.

Biofilm and the associated Legionella can readily grow in the dead legs inherent in many water systems. For example, water lines to eyewash or safety shower stations may be unused or used very infrequently for months or years and, when activated, can aerosolize biofilm that is growing in the line. Water lines to unused rooms and wings of hospitals, hotels, and institutions are often breeding grounds for biofilm and Legionella. Even in active rooms, water lines to infrequently used facilities or internal piping in facilities such as whirlpool baths can be growth sites for biofilm.

Cooling towers often are associated with closed-loop chilled water cooling systems. These closed-loop systems are usually not a concern with regard to Legionella because the water contained in the systems cannot escape into the environment. However, biofilm can grow in closed-loop systems where it impedes water flow and insulates heat transfer surfaces, creating a strong negative impact on efficiency of the cooling system.

Some companies and institutions recognized this problem and pioneered the use of chlorine dioxide for biofilm/Legionella control in the early/mid-2000s [3] with Beta-systems in major hospitals. These systems reinforced the knowledge in water treatment literature that chlorine dioxide is a safe and reliable way to control biofilm and mitigate the threat of Legionella.

Decorative fountains are another potential breeding ground for Legionella, and many potential Legionella sources exist in water systems that are not normally considered. Examples include eyewash and safety shower water lines, spas and hot tubs, water parks, and water lines that are infrequently used. Recent publications suggest that water systems in automobiles (such as windshield washers) are sources of Legionella. Scarcely a day goes by without a news announcement concerning another Legionella outbreak.

Chlorine Dioxide Chlorine dioxide is a broad-spectrum biocide that has major advantages over other biocides in a variety of applications.

In hospitals, hotels, and institutions such as nursing homes, the hot water serving the patients’ rooms, laundry, food facilities, etc. is usually served by a recirculating system that heats water in a central boiler and recirculates the hot water so that it is immediately available at the point of use. The nature of the recirculating system is often such that conditions favor heavy growth of biofilm in the pipelines and water heaters. Precautions against scalding often limit hot water temperatures to below the limits required to control biofilm and Legionella. Legionella and other pathogens embed themselves in the biofilm, which sloughs off from time to time, creating a Legionella-laced aerosol in sinks, showers, and other use points. Legionella bacteria infect single-celled amoebae, [2] where they are protected from many disinfectants. The infected protozoans escape the biofilm and die, releasing Legionella into the flowing water.

• Chlorine dioxide is well established as a disinfectant for drinking water, where it has been used for more than 70 years in the United States. • At very low doses and contact times, it rapidly kills bacteria and viruses. • It also kills waterborne pathogenic parasites like Cryptosporidium and Giardia. • It functions as a biocide by oxidizing with a unique singleelectron-transfer process that does not produce chlorinated organic byproducts such as THMs or HAAs. It kills microorganisms by destroying cell walls and interfering with the metabolic processes; it does not just force them into a dormant stage. • It is a selective oxidant and does not react with chemicals such as ammonia that react with other disinfectants.

Recently, public awareness of the dangers of Legionnaires’ disease has been enhanced by news articles about people infected by Legionella from poorly maintained cooling towers in New York City and surrounding areas. The warm water in cooling towers provides an ideal medium for growth of biofilm and the

• It can be used intermittently in conjunction with other biocides. • It does not need dispersants to remove biofilm.

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capital equipment. However, batch mixing requires expertise in handling hazardous materials, and sometimes can expose operators and other people to fumes or hazardous chemicals. Errors in batch mixing can result in explosive concentrations of chlorine dioxide in gaseous head spaces. Batch processes utilize the chemistries described below and have the same chemistry-related advantages and drawbacks as the continuous reactors that use the same chemistry.

• As a dissolved gas, it rapidly and fully penetrates and destroys biofilm that is resistant to chlorine and many other disinfectants. • Chlorine dioxide is much less corrosive than chlorine at the doses that are typically used for water treatment. [4] • It functions synergistically with chlorine that is already present in municipal water that feeds most water supply systems. When chlorine dioxide reacts with contaminants in water, the primary initial breakdown product is chlorite ion (ClO2-). That ion reacts with any chlorine that is present to regenerate chlorine dioxide, creating a double “bang for the buck.” Chlorite can also react with acid produced by bacteria to generate chlorine dioxide. Hence, chlorite ion is a mild disinfectant in its own right. The chlorite ion formed from the breakdown of chlorine dioxide can significantly retard the rate of bacterial regrowth in water systems. [5]

Onsite generators react various combinations of chemicals to produce chlorine dioxide for drinking water treatment and other small applications. This requires generators that are sometimes unreliable and require frequent adjustment and service. Generators are especially difficult to control in applications such as disinfection of institutional water supply systems where the generator must often function unattended, and the demand for chlorine dioxide varies by several orders of magnitude, as water consumption varies during the day. Many generators are also unreliable in applications that require start and stop operation since continuous generators typically require adjustment at startup.

• The chlorine dioxide in water that does not form chlorite forms environmentally harmless chloride ion (Cl-). Eventually, all of the breakdown products of chlorine dioxide form chloride.

Two-reagent generators often employ acid/chlorite chemistry, as shown in equation 1:

• Unlike chlorine, chlorine dioxide is effective as a biocide over a wide range of pH (pH = <5 to >9)

5NaClO2 + 4HCl

4ClO2 + 5NaCl + 2H 2O

(1)

Recent literature as well as our own testing indicates that much of the product from such reactions is not chlorine dioxide, but chlorous acid (HClO2). Chlorous acid is an oxidant that behaves like chlorine dioxide in many analytical techniques. Chlorous acid is also a disinfectant and dissociates over time to produce chlorine dioxide. But in some applications, chlorous acid behaves very differently than chlorine dioxide. Therefore, it is difficult to establish how much of the product of such a reaction is chlorine dioxide, and it is not established how effective this type of product is compared to products produced in other generators. For example, chlorous acid is dissolved in water as ions, whereas chlorine dioxide dissolves in water as a dissolved gas. Therefore, the ability of chlorine dioxide to penetrate biofilm and water purification membranes may be much greater than that of chlorous acid.

Sources of Chlorine Dioxide Historically, the primary drawback to chlorine dioxide as a disinfectant was that chlorine dioxide could not be shipped or stored for long periods due to its instability. In the past, chlorine dioxide had to be produced near the point of use and used almost immediately. Onsite generation of chlorine dioxide was performed in various ways, each with its own advantages and drawbacks. Recently, ready-to-use, storage stable, shippable aqueous solutions of pure chlorine dioxide have become available in the marketplace. The following paragraphs summarize some of the advantages and concerns surrounding the most common techniques for generating chlorine dioxide. These are not comprehensive discussions, but an attempt to highlight key things to consider. Paper pulp bleaching is the largest use for chlorine dioxide. In that industry, chlorine dioxide is produced from sodium chlorate (NaClO3) in large sophisticated reactors. This process is not amenable to production of chlorine dioxide at a scale useful in municipal water treatment or disinfection applications.

Acid/chlorite generators require a holding tank to provide time for the acid/chlorite reaction to be completed. Three-reagent generators usually employ acid/chlorite/hypochlorite chemistry such as shown in equation 2: 2NaClO2 + NaOCl + HCl

For smaller uses (up to about one ton per day), chlorine dioxide may be produced by reacting chemicals near the point of use in either a batch or continuous mode. There are a few chemical reactions that are commonly used for production of chlorine dioxide in either the batch or continuous mode.

2ClO2 + 2NaCl + NaOH

(2)

This reaction is a much faster reaction than the reaction described in equation 1 and typically does not produce chlorous acid. However, three-reagent generators often produce free chlorine in the process, which is detrimental for some applications. The process also requires handling and metering three hazardous liquid chemicals.

Batch generation is typically very simple and requires little

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(Smaller containers are reportedly being introduced.) It requires no mixing or adjustment except for dosing by a conventional chemical feed pump. Since the product consists of pure chlorine dioxide dissolved in pure water, it contains very little byproduct or contamination. Dose can be controlled by any control scheme that can control a chemical feed pump. Containers can be supplied with a special dip tube with foot valve and a noleak connection so that workers are never exposed to liquid or gaseous chemicals (there are no solid chemicals). The solution is shippable and storage stable for nine months in the larger container sizes.

Often, the sodium chlorite for two-reagent and three-reagent generators is supplied as an aqueous solution labeled “stabilized chlorine dioxide,” and the acid is labeled “activator.” In other cases, the acid and sodium chlorite are given proprietary brand names. Batch-mixed acid/chlorite reactors using liquid feedstock require operators to mix concentrated sodium chlorite solution with acid (typically hydrochloric acid), and sometimes hypochlorite solution to produce chlorine dioxide. Solid reagents for batch two-part reactors are sold as tablets or granules/powder. Solid-based batch reactions are often useful for making small batches of chlorine dioxide solution for certain applications. Solid reagents typically contain sodium chlorite and acidic salts that produce chlorine dioxide when added to water. In some products, the acid salt and the sodium chlorite are combined in a single package or tablet that is desiccated and in a watertight package. Most, if not all, of these products employ the acid/chlorite reaction, so users should note whether the reaction produces chlorine dioxide or chlorous acid.

Compared to onsite generation in either continuous or batch mode, this product tends to be more expensive in terms of cost per pound of chlorine dioxide. It is primarily used in applications where the benefits of ready-to-use chlorine dioxide outweigh the increased cost per pound. Factors to consider in evaluating ready-to-use chlorine dioxide include low capital cost, minimal labor requirements for operation and maintenance, startup and shutdown issues for generators, and purity of chlorine dioxide. Continuous application of chlorine dioxide typically involves continuous dosing at a rate of 0.3–0.75 mg/L to eliminate existing biofilm and prevent regrowth. However successful prevention of Legionella infestations has been reported with doses as low as 0.1 mg/L chlorine dioxide.

It is important to mix the correct solid/water ratio since too much solid can produce explosive concentrations of chlorine dioxide in the headspace above the liquid product. Mixing the solid with water can expose workers to chlorine dioxide gas that escapes through any openings in the reaction container during mixing. Devices exist for continuously dissolving solid reagents, but control of such devices is often problematic.

Either chlorine dioxide generated on site or ready-to-use solutions of chlorine dioxide can be used effectively in continuous application. The relative economics of different types of supply depends on a number of factors, such as capital versus operating cost, reliability and ease of control, and the ability to provide the appropriate dose if water flow rates change during operation of the water system.

Continuous reactors usually employ a two-part or three-part chemical mixture that requires frequent balancing and adjusting. Small generators using liquid feedstocks are especially difficult to control because small flows of liquids require small valves and flow control devices that are easily clogged by precipitated salt granules or gas bubbles. Generators also often produce undesirable byproducts, especially when not perfectly adjusted, and two-component systems require a time delay for the reaction to occur, causing additional operational complications.

Intermittent application of chlorine dioxide can be an effective and economic way to prevent the formation of biofilm on an ongoing basis after an initial flush. For example, chlorine dioxide may be dosed at 0.5–0.75 mg/L for a few hours per day, ideally during low water flow times. For example, cooling tower operators could consider dosing in the early morning hours when the system may be shut down. At such times, exposure of cooling water to sunlight (if any) is likely to be minimal. At such times, the water with chlorine dioxide should be circulated through the system, but if operational considerations permit, the air fans should be shut down to minimize the loss of chlorine dioxide. Some water systems successfully prevent the formation of biofilm with application of 0.3–0.5 mg/L of chlorine dioxide for a few hours two or three times per week.

Depending on the generator used, byproducts are unavoidable, including chlorite (ClO2-), chlorate (ClO3-), chlorous acid (HClO2), chlorine in various forms (HCl, OCl-, HOCl and Cl 2), and various types of acid. These byproducts typically flow directly into the treated water stream where they can create various problems, including increased corrosion. Efficiency of the generator and production of byproducts depend strongly on the type of generator used and the design and adjustment of the generator. Ready-to-use chlorine dioxide is a pure solution of 3,000 mg/L chlorine dioxide in water. The product is sold under various label names by chemical distributors. It is sold in DOT-approved plastic containers ranging in size from 5 gallons to 330 gallons.

If continuous operation or intermittent application is employed without an initial flush, care must be taken because biofilm will slough off as it dies, and pieces will be carried into the downstream processes and use points of the water.

the Analyst Technology Supplement 2017

Control and Prevention continued

Special Considerations

References

Chlorine dioxide exists as a dissolved gas in water. As such, it can be stripped out of solution by contact with air. The rate at which chlorine dioxide is removed from water is a function of many variables, including temperature, chlorine dioxide concentration in the water, and area of the air/liquid interface.

1) Z hang, Zhe, et al., “Legionella Control by Chlorine Dioxide in Hospital Water Systems,” Journal of AWWA, 2009. 2) D eclercka, Priscilla et al., “Replication of Legionella Pneumophila in Biofilms of Water Distribution Pipes” Microbiological Research v.164 p. 593-603.

At concentrations of 10 to 50 mg/L sometimes used for batch treatment, chlorine dioxide can be rapidly stripped from solution by contact with air. At lower concentrations (typically <1 mg/L) used for continuous treatment or intermittent treatment, stripping occurs much more slowly.

3) Bova, Gregory, Paul Sharpe, Tim Keane, “Evaluation of Chlorine Dioxide in Potable Water Systems for Legionella Control in an Acute Care Hospital Environment,” presented as part of the 65th Annual International Water Conference, 2004.

Ultraviolet light from the sun or, to a lesser extent, fluorescent bulbs will cause decomposition of chlorine dioxide. The rate of decomposition from UV light depends on the intensity and wavelength of the light as well as pigments and UV blockers in the walls of the containers. Decomposition from direct sunlight is many times greater than from fluorescent or other common forms of artificial lighting.

4) Gupta, Amit and Stefan Muench, “Using Chlorine Dioxide for Effective Water Treatment,” Process Cooling, March 2016. 5) S impson, Greg D. Ph.D., “Practical Chlorine Dioxide,” Volume 1. 6) Ferretti , Emanuele, “Use of Chlorine Dioxide in Legionella Disinfection,” Seminar on “Legionella: sampling, monitoring and treatments, Warsaw, 11-12.07.2007.

To optimize the use of chlorine dioxide, consideration should be given to these factors. For example, batch or intermittent treatment may be more efficient if carried out at night when ambient temperatures are lower, heat load (for example, on cooling water systems) may be lower, and sunlight is not a factor.

Tom McWhorter is employed at CDG Environmental, LLC. He can be reached at (888) 610-2562 or tmcwhorter@cdgenvironmental.com.

Be sure to follow safety instructions provided by the chlorine dioxide supplier and provide adequate ventilation and/or personal protective equipment. Chlorine dioxide stripped from solution may enter the work space air, where it can be harmful to workers.

Advertising Index Albemarle Corporation....................... 5 AMSA, Inc........................................... 25

No two water systems are the same, and no single treatment strategy applies to all water systems. Legionella can enter a system at any time from the air or even from chlorinated municipal drinking water. Ongoing continuous or intermittent dosing is essential to ensure no recurrence of Legionella. Intermittent dosing with chlorine dioxide can ensure that biofilm does not grow and Legionella does not proliferate in the system. It does not inactivate Legionella or other microorganisms that enter the system between dosing times.

Browne Laboratories, Inc................. 12 Bulk Systems, Inc.............................. 19 Chem-Met Company......................... 24 IDEXX.................................................. 54 Myron L Company........................26-27 Pulsafeeder, Inc................................... 2

Water systems should be tested for Legionella before, during, and after treatment with chlorine dioxide, and dosing should be adjusted to make sure that the treatment strategy is effective. In some reported cases Legionella was undetectable after a single treatment. In other cases, Legionella was reduced dramatically— to a level well below that specified by regulatory standards—after one treatment, but chlorine dioxide had to be used continuously or intermittently over periods of months [6] before no remaining Legionella could be detected in the system.

QualiChem, Inc.................................... 6 Sanipur US LLC................................. 16 Special Pathogens Laboratory......... 56 Walchem, IWAKI America Inc........... 31 Water Science Technologies............ 55 WaterColor Management.................. 43

the Analyst Technology Supplement 2017

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Reference 1. Data on file at IDEXX Laboratories, Inc. Westbrook, Maine USA. © 2017 IDEXX Laboratories, Inc. All rights reserved. • 110397-03 All ®/TM marks are owned by IDEXX Laboratories, Inc. or its affiliates in the United States and/or other countries. The IDEXX Privacy Policy is available at idexx.com.

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