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Legiolert for Quantification of

Legionella pneumophila
IDEXX Water Testing Solutions
• Worldwide market leader and
specialist in water microbiology

• Dedicated, established channel

in worldwide water testing
market – more than 20 million
tests serving customers in 115
countries on all 7 continents

• Accepted or approved in 40+

countries with more than 100
different approval/acceptances
by more than 20 entities around
the world

• Help protect water quality for an

estimated 2 billion people
around the globe every day

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Innovative Water Microbiology Tests
Coliform and
Enterococci P. aeruginosa L. pneumophila Quality Control
E. coli
Colilert® Enterolert® Pseudalert® Legiolert® Quanti-Cult®
Colilert®-18 Enterolert®-E Pseudalert® 250 IDEXX-QC
Colisure® Enterolert®-DW
Colilert ® 250 Enterolert® 250

Cryptosporidium/ Heterotrophic Plate

Quantification Accessories
Giardia Count
Filta-Max® SimPlate® for HPC Quanti-Tray® EZ-DPD™
Filta-Max xpress® HPC for Quanti-Tray® Quanti-Tray®/2000 Collection vessels
Invitrogen Quanti- Incubators
Dynabeads® for IMS Tray®/Legiolert Sealers

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 Simple quantitative method suitable for both potable and non-

potable matrices.
 Legiolert reagent consists of a rich supply nutrients to allow
Legionella to grow as rapidly as possible - High Sensitivity
 Legiolert contains a substrate for an enzyme diagnostic for
Legionella pneumophila which produces a brown colour - High
 Confirmed results in 7 days

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 Unique 100 mL Quanti-Tray device
 two well types
 6 large wells (overflow)
 90 small wells (resolution)
 counts L. pneumophila; from 1-
2276 MPN/ Quanti-Tray

 Blister pack reagent

 additional supplement required
for 100ml Potable analysis
 additional pre-treatment reagent
required for Non-potable
Reaction with L. Negative Sample
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Water Types
Potable Water Non-Potable Water
Definition Water which is fit for consumption by Water that is not of drinking water
humans, also called drinking water in quality, but may be used for other
reference to it’s intended use. purposes depending upon the quality.
Common Drinking Water Cooling Towers
Examples Drinking Fountains Evaporative Condensers
Kitchen taps/sinks Fluid Coolers
Bathroom taps/sinks Chilled-water Return tanks
Showers Header tanks
Eyewash Stations Air Conditioners
Bath Taps Machine/utility waters
Ice Machines Whirlpools
Decorative fountains
Untreated waters (springs, ponds,
surface waters)
Car Washes
Rainwater harvest storage tanks

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Potable Water - 100mL Test Procedure

100mL Determine 0.33 – 1mL Add Legiolert /

sample Hardness supplement Legiolert Quanti-Tray

Generate MPN Count at 7 days (39°C) Quanti-Tray Sealer Plus

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Potable Water - 10mL Test Procedure

90mL DI Add 10mL Add Legiolert /

Water Sample Legiolert Quanti-Tray

Generate MPN Count at 7 days (39°C) Quanti-Tray Sealer Plus

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Non-potable Water - 1mL Test Procedure

Water 2ml

2ml Legiolert
+ Sample

treatment 2ml Add 100mL
60±5s Legiolert Diluent

Generate MPN Count at 7 days (37°C) Quanti-Tray Legiolert /

Sealer Plus Quanti-Tray
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Legiolert: The Results

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Quanti-Tray/Legiolert – MPN Table

• Positive Wells: 6 Large and 24 Small
• MPN: 156.6

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Limits of Detection

Limit of Detection
Protocol /
Volume examined (mL)
100 mL potable 0.01 10
10mL potable 0.1 100
1.0 mL non-potable 1 1,000

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IDEXX Confidential
Optional Serotyping Procedure

Extract material Add material Centrifuge to Resuspend the

from positive wells to tube concentrate cells and serotype
5,000xg / 1 min

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ISO 11731 is more labor intensive and time
consuming than Legiolert

Hands-on time in the lab (in min) ISO 11731 Legiolert

Setup 15 3
Reading/scoring 1-5 1
Confirmation 1-5 0
TOTAL time / Sample 22-30 4

Time to results (in days) ISO 11731 Legiolert

For negatives 10 7
For positives 14 7

Material cost estimate (no labor) $25 $20

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Technical Performance – ISO 13843 Method


Metric Value
Tactics Sensitivity 98%
Specificity > 99%
False positive rate < 0.01%
False negative rate 4.20%
Efficiency > 99%
Repeatability < 0.01 (99%)
Reproducibility < 0.01 (99%)

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Excellent Performance in Studies
Equal or greater sensitivity than standard methods
3,570+ matched samples to-date in beta, regulatory and validation studies
• Two peer reviewed studies
1. Evaluation of a most probable number method for the enumeration of Legionella
pneumophila from North American potable and nonpotable water samples. Petrisek
R, Hall J. J Water Health. 2018 Feb;16(1):25-33.
2. Evaluation of a most probable number method for the enumeration of Legionella
pneumophila from potable and related water samples. Sartory DP, Spies K, Lange B,
Schneider S, Langer B. Lett Appl Microbiol. 2017 Apr;64(4):271-275. Epub 2017 Feb
• Three additional studies pending publication
• 17 Independent studies & validations
 Completed or in progress: US, Canada, Germany, UK, Australia, Austria, France,
Portugal, Italy, Singapore, Netherlands
 Split samples analyses from customers during routine business/laboratory
 All methods confirmed by secondary culture and serotyping when needed
 Data segregated for L. pneumophila by isolate speciation
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German Regulatory Trial Evaluation – Potable Water
Legiolert vs ISO-11731-2
• 6 labs
• 846 samples
• 100mL and 1mL analyzed using
both methods
• 1991/2034 wells confirmed
• 97.9% true positive
Relative Lower 95% Upper 95%
Statistic N
difference CI CI
Legiolert 100mL
89.5% 73.0% 106.1% 444
vs. ISO 100mL Lp
Legiolert 1mL vs.
-2.0% -14.4% 10.4% 277
ISO 1mL Lp
Legiolert 100mL
6.6% -8.0% 21.1% 527
vs. Best Lp
Legiolert 100mL
-8.1% -24.1% 7.9% 537
vs. Best all Lspp
Legiolert detection of L. pneumophila only is still not statistically different
compared to detection of all L. spp by ISO-11731-1/2 in this study
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German Regulatory Trial Evaluation – Potable Water
Legiolert vs ISO-11731-2
• McNemar’s Analysis (presence/absence)
• Higher incidence of false negatives found with ISO method
Legiolert 100mL vs. MF 100mL - L. pneumophila
Legiolert chi2 p-value Reject/Accept Null
+ - 27.38 0.0000 Reject
+ 349 22
- 73 284

Legiolert 1mL vs. MF 1mL - L. pneumophila

Legiolert chi2 p-value Reject/Accept Null
+ - 1.222 0.2689 Accept
+ 178 55
- 44 484

Legiolert 100mL vs. ISO best - L. pneumophila

Legiolert chi2 p-value Reject/Accept Null
+ - 10.98 0.0009 Reject
+ 445 26
- 56 281

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Challenges with ISO 11731:2017

• ISO 11731 was updated to

improve performance, but it
created even more protocol
decisions for the laboratory

• Decision matrix is impractical

for routine monitoring

• A new method Appendix was

added that focuses on
measurement uncertainty and
reliable vs. unreliable data (not

O = Optional step
R = Required step

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Challenges with ISO 11731:2017
Cumulative measurement uncertainty is significant





Concentration Total
Method Measurement
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Challenges with ISO 11731:2017
Subjectivity contributes to measurement uncertainty
• Subjectivity of colony differentiation
• Interference of non-target growth
• Analyst experience level



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Challenges with ISO 11731:2017
Readability can vary greatly
One sample was plated 4 times at 3 dilutions on the same media
 The impact of the non-target interference can vary dramatically

1. Sample A, 1:2 dilution 2. Sample A, 1:4 dilution 3. Sample A, 1:6 dilution

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Challenges with ISO 11731:2017
Measurement Uncertainty Reduces Usefulness of Routine
Testing Data for Public Health Decision-Making
High potential for High potential for
inconsistency in analyst to analyst and
results over time lab to lab variability

• Reduces data users’ trust in the results

• Reduces confidence in what actions should

be taken on the basis of trending

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For routine testing, the best decisions come from
data that is:

• Targeting the disease-causing agent

• Accurate

• Highly reproducible, with minimal measurement uncertainty, so

that trending is accurate, no matter who is processing the sample

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Reasons IDEXX Focused on L. pneumophila

• Higher prevalence of L. pneumophila in potable and nonpotable

water systems

• Goal of specifically identifying and quantifying the pathogen

• No masking of the pathogen by other species

• Cost/benefit of having a much easier, reliable, and accurate test

vs. missing the low frequency/ low clinical impact isolates

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L. pneumophila is by far the most pathogenic
species of Legionella
All legionellosis, including Pontiac Fever

CDC Waterborne Disease & Outbreak Surveillance Report data 2009-2014

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L. pneumophila is recognized by WHO as the
pathogen of concern

”The most common cause of illness is

the freshwater species L. pneumophila”

“Focus verification monitoring on L.

pneumophila instead of Legionella
spp. …L. pneumophila is the most
significant causative agent of
legionellosis in Europe.”
Bonn, 17 September 2017
“Legionella pneumophila is the WHO Recommendation
aetiological agent responsible for most Paper
reported cases of Legionnaires'

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Summary – Why Legiolert?
 Faster results means better protection
• Confirmed result 3-7 days faster than plate methods

 Accurate data for trending and process control

• 99% reproducible & 99% repeatable
• No method uncertainty

 Finds the dangerous pathogen when standard methods do not

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Related Interests