Water Institute - Legiolert, for the Determination of Legionella ......2017/05/15 · Legiolert vs....

A New Enzymatic Method, Legiolert, for the

Determination of Legionella pneumophila in Water

2 Copyright 2017 IDEXX Laboratories

Unique 100 mL “Quanti-Tray”

device

6 large wells (overflow)

90 small wells (resolution)

Counts L. pneumophila; from

1-2272 MPN/ Quanti-Tray

Blister pack reagent

Reaction with L. pneumophila

Legiolert platform

Negative Sample


Count @ 7 days

=

Confirmed result

Legiolert protocols: Potable and nonpotable water

Pour into Quanti-

Tray, seal, and

incubate

Application Incubation

Nonpotable 37°C

Potable 39°C

90 mL

sterile

diluent

Add Legiolert reagent to

90 mL sterile diluent

Add 10 mL water sample

100mL

Legiolert

+

sample

100mL

sterile

diluent

Add Legiolert reagent to

100 mL sterile diluent

Mix 0.2 mL sample with 0.2 mL

pretreatment, wait 60 seconds

Legiolert

pretreat-

ment

Water

sample

Add 0.2 mL treated water

sample

100mL

Legiolert

+

sample

Pota

ble

Nonpota

ble


Legiolert performance ─ ISO 13843 Method Validation

Validation of Legiolert

ISO/TR 13843:2000(E) Water Quality – Guidance on validation of

microbiological methods

Metric Value

Sensitivity 98%

Specificity > 99%

Repeatability < 0.01

Reproducibility < 0.01

Sensitivity =

Specificity =

True Positives (TP)

TP + False Negatives (FN)

True Negatives (TN)

False Positives + TN

https://www.idexx.com/resource-library/water/legiolert-validation-report-iso-13843.pdf

https://www.idexx.com/resource-library/water/legiolert-validation-report-iso-13843.pdf


Potable and Nonpotable water

Independent laboratories

Regions

Germany

North America (U.S. and Quebec)

All methods confirmed by secondary culture and serotyping

when necessary

Split sample analyses

Samples were obtained from sites/customers during routine

business/laboratory operations in order to mimic performance in

a real world scenario

Data segregated for L. pneumophila by isolate speciation

Legiolert Field Trials


Expanded uncertainty (95%)

Variability chart

Mean Relative Difference

vs. 100mL - membrane filtration

Legiolert is more sensitive than the

ISO membrane filtration method for

Potable water

Total samples analyzed = 1604

290 nonzero, non-TNTC data pairs

Mean relative difference (MRD) analysis:

ln (ISO std method) – ln (exp method)

Statistic Statistic vs. ISO-11731-2

Mean Relative

difference

MRD + 35.3%

High 95% Conf. Interval + 54.8%

Low 95% Conf. Interval + 15.7%

Specificity = 96.5%

Beta Trial Evaluation – Germany, Potable Water

Legiolert vs. ISO-11731-2

Sartory DP, Spies K, Lange B, Schneider S, Langer B. Evaluation of a most probable number

method for the enumeration of Legionella pneumophila from potable and related water samples.

Lett Appl Microbiol. 2017 Apr;64(4):


100 mL 1 mL

Statistic Mean Lower 95% Upper 95% n

Rel. Diff. 100mL 89.5% 72.7% 106.4% 444

Rel. Diff. 1mL -2.0% -14.5% 10.6% 277

Specificity = 97.8%

Expanded uncertainty (95%)

Legiolert is more sensitive than the

ISO membrane filtration method for

Potable water• Total samples analyzed = 844

• 6 participating laboratories

• 100mL vs. 100mL

• 1mL vs. 1mL

Regulatory Trial Evaluation – Germany, Potable Water

Legiolert vs. ISO-11731-2



Presence/absence evaluation – McNemar’s analysis

Combined data set – 100mL volume

100 mL Legiolert

Total 836 positive negative TNTC

ISO

positive 349 23 3

negative 73 283 1

TNTC 79 1 24

McNemar

X²-values: exact 26.0 (diff, p= 6.5 *10-7)

Yates 25.5 (diff, p= 8.5 *10-7)

Edwards 25.0 (diff, p= 1.1 *10-6)

Combined data set – 1mL

1 mL Legiolert


ISO

positive 178 55 0

negative 44 484 0

TNTC 2 1 0

McNemar

X²-values exact 1.2 (not diff., p=0.27)

Yates 1.1 (not diff., p=0.29)

Edwards 1.0 (not diff., p=0.32)

Methods different Methods not different





100 mL Legiolert


ISO

positive 349 23 3

negative 73 283 1

TNTC 79 1 24

McNemar


Yates 25.5 (diff, p= 8.5 *10-7)

Edwards 25.0 (diff, p= 1.1 *10-6)


1 mL Legiolert


ISO

positive 178 55 0

negative 44 484 0

TNTC 2 1 0

McNemar






Beta Trial Evaluation – N. America, Potable Water

Legiolert vs. Standard Methods SM9260J: Detection of

Pathogenic Bacteria: Legionella

Legiolert is equal to or more

sensitive than standard methods

for Potable water

Statistical method Statistic

vs. SM9260J:

BestT-Test N 74

Prob > |t| 0.120

Prob > t 0.060

Prob < t 0.940Wilcoxon Signed Rank Test* Prob > |S| <0.0001

Prob > S <0.0001

Prob < S 1

p < 0.05 indicates a significant difference

% False positive = 0%

*Statistical method for non-parametric data

All Data

Zoom


Beta Trial Evaluation – N. America, Nonpotable Water

Sensitivity statistics for all trial sites

Specificity: True/false positivity

Statistical method Statistic

vs.

CDC: Best

vs.

SM9260J: Best

vs.

CDC/AFNOR:

Best

vs.

Laboratory

custom method

Best

T-test N 92 49 74 76

Prob > |t| 0.076 0.201 0.326 0.130

Prob > t 0.038 0.100 0.163 0.935

Prob < t 0.962 0.899 0.837 0.065

Wilcoxon Signed Rank

Test*

Prob > |S| 0.258 0.728 0.924 <0.0001

Prob > S 0.129 0.636 0.462 1.0000

Prob < S 0.871 0.363 0.363 <0.0001

p < 0.05 indicates a significant difference

*Statistical method for non-parametric data

Legiolert wells sampled 671

Legiolert wells confirmed as L. pneumophila 652

Cumulative false positivity rate 3.6%


Lab A CDC

GVPC

Lab B SM9260J

CCVC

Lab C Custom

PCV

Lab D Custom

DGVP

------------------------------------------------------------- All Data ------------------------------------------------------------

------------------------------------------------------------- Zoom -------------------------------------------------------------

Beta Trial Evaluation – N. America, Nonpotable Water


Specifically and robustly detects the pathogen, Legionella

pneumophila

No other species interfere

No other species are detected

No confirmation required: positive = confirmed

Few to no FP or FN rxns (overgrowth)

Low subjectivity and counting uncertainty

Larger counting range, very few TNTC – no need to

resample and retest

Easy test means

More labs will offer this

Premise owners can react with the best information to protect the

public as soon as Legionella pneumophila is found

Legiolert summary


Backup

Slides


Read after 7 days

=

Confirmed result

Pour into Quanti-

Tray, seal, and

incubate at 39°C

with humidity

Determine

water

hardness

Add

Legiolert

reagent

100 mL

water

sample

100 mL

Legiolert

+

sample

100 mL

water

sample

Add 0.33 or 1.0

mL hardness

supplement

Hardness

Supple-

ment

Legiolert – Potable Water Protocol Germany


Quanti-Tray/Legiolert: Serotyping


Serotyping from Quanti-Tray LegiolertExtract

material from

positive wells Microfuge

Resuspend to

concentrate

Serotype

Or DFA

microscopy

Extract sample

from positive

wells

Resuspend

DFA Microscopy Serotype

OR

Microfuge to

Concentrate

https://www.google.com/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&ved=0ahUKEwi0_vXtvIfMAhWJRSYKHZQnB0gQjRwIBw&url=https://www.thinglink.com/scene/632248132972838912&bvm=bv.119028448,d.eWE&psig=AFQjCNFQG9T0_fMP8_tgHSQozXiMmc3OCw&ust=1460494692422080

http://www.google.com/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwiOqM3l1onMAhXMbD4KHTtuAKoQjRwIBw&url=http://www.pocdscientific.com.au/eppendorf_centrifuge_5427R.php&psig=AFQjCNG1XjQ3gKKeW7mXX5O4r0cG2xeveQ&ust=1460570304318206


MPN vs. CFU = no technical difference


Legiolert – Proficiency Testing

NSI Lab solutions

• Accredited PT provider

• ISO GUIDE 34:2009, ISO/IEC 17025:2005, ISO/IEC 17043:2010, ISO

9001:2008

Water Institute - Legiolert, for the Determination of Legionella ......2017/05/15 · Legiolert vs....

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