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  • make it tough poster resize - NEMC · NTC EDGE ToughMix 2 ... Edgewater TM vs MPN 0.7112917 0.5059 0.9663 Villa Angela EMM vs TM 0.939279 0.8822 2.919E‐14 Villa Angela EMM vs MPN
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Mark Citriglia, Manager of Analytical Services Northeast Ohio Regional Sewer District 4747 East 49th Street Cuyahoga Hts., Ohio 44125 Make It Tough: A qPCR MasterMix Comparative Study Introduction E.coli Edgewater and Villa Angela Beaches E. Coli Results Nicki Schafer, Molecular Microbiologist Northeast Ohio Regional Sewer District 4747 East 49th Street Cuyahoga Hts., Ohio 44125 The water quality of bathing beaches in Northeast Ohio is de- termined by the microbial concentration typically, the concen- tration of E. coli and/or Enterococci. The standard methods used to analyze for FIBs (fecal indicator bacteria) take 24 hours to obtain results. The use of rapid methods allows for re- al time results within 3-4 hours. Since 2007, Analytical Ser- vices has been testing various real-time quantitative polymer- ase chain-reaction (qPCR) chemistries to discover an assay which will closely correlate to the standard culture based methods, reduce environmental inhibitory effects, and ensure accuracy for predictive model impact. The use of qPCR methodology has become a critical compo- nent of the beach monitoring program at the Northeast Ohio Regional Sewer District (NEORSD). The results from the qPCR analysis have been used as a stand-alone predictor as well as a variable in predictive modeling, to determine the water quality of bathing beaches for public notification. Since timely public notification is of paramount importance, qPCR provides faster results so the public can make informed decisions about whether to refrain from recreational beach use based on data from that day and not the previous day results obtain from standard culture based methods. Method/Process Enterococci Edgewater and Villa Angela Beaches Enterococci Results E.coli calibration curves Enterococci calibration curves slope 3.3676 intercept 45.619 R2 0.9985 AF 1.969474 % Efficiency = 98.13% slope 3.291 intercept 43.475 R2 0.9996 AF 2.03859 % Efficiency 101.31% slope: 3.3388 intercept: 37.303 AF: 1.995088056 % Efficiency: 99.30% R2: R2 =0.9998 slope: 3.4708 intercept: 42.34 AF: 1.8811801 % Efficiency: 94.14% R2: 0.9988 E.coli Quality Control Data Enterococci Quality Control Data Beach Assay # sketa inhibited # IAC inhibited # (+) PBS BLK # (+) NTC EDGE ToughMix 19/121 0/121 1/7 0/7 EDGE EMM 22/121 0/121 3/6 0/6 VABE ToughMix 5/120 1/120 1/7 0/7 VABE EMM 6/120 0/120 2/6 1/6 Beach Assay # sketa inhibited # IAC inhibited # (+) PBS BLK # (+) NTC EDGE ToughMix 2/120 0/120 2/6 2/6 EDGE EMM 10/120 1/120 4/6 4/6 VABE ToughMix 0/120 0/120 1/6 0/6 VABE EMM 0/120 0/120 5/6 2/6 Accuracy 88% Accuracy 70% Sensitivity 71% Sensitivity 32% # Greater 235 42 75 3 30 12 # >3357 22 77 1 7 35 # Less than 235 78 CNE False + CE False ‐ #<3357 98 CNE False + CE False ‐ Accuracy 85% Accuracy 69% Sensitivity 70% Sensitivity 92% # Greater 190 47 69 4 33 14 # >2026 12 72 1 11 36 # Less than 190 73 CNE False + CE False ‐ #<2026 108 CNE False + CE False ‐ Accuracy 86% Accuracy 72% Sensitivity 64% Sensitivity 90% Specificity 70% #>235 42 76 2 27 15 #>3357 10 77 1 9 33 #<235 78 CNE False + CE False ‐ #<3357 110 CNE False + CE False ‐ Accuracy 85% Accuracy 75% Sensitivity 66% Sensitivity 93% # Greater 190 47 71 2 31 16 #>2026 14 77 1 13 34 # Less than 190 73 CNE False + CE False ‐ #<2026 106 CNE False + CE False ‐ Accuracy 89% Accuracy 84% Sensitivity 37% Sensitivity 50% # Greater 235 19 101 1 7 12 #>3357 2 101 1 1 18 # Less than 235 102 CNE False + CE False ‐ #<3357 119 CNE False + CE False ‐ Accuracy 89% Accuracy 85% Sensitivity 48% Sensitivity 67% # Greater 190 23 97 0 11 13 #>2026 3 101 1 2 22 # Less than 190 97 CNE False + CE False ‐ #<2026 118 CNE False + CE False ‐ Accuracy 86% Accuracy 83% Sensitivity 42% Sensitivity 33% # Greater 235 19 96 6 8 11 # >3357 3 100 2 1 18 # Less than 235 102 CNE False + CE False ‐ #<3357 118 CNE False + CE False ‐ Accuracy 91% Accuracy 80% Sensitivity 57% Sensitivity 50% # Greater 190 23 97 6 13 11 #>2026 4 95 2 2 22 # Less than 190 97 CNE False + CE False ‐ #<2026 117 CNE False + CE False ‐ Estimated Illness Rate (NGI): 32 per 1000 primary contact recreators: BAV Standard Curve Predictive Model (CCE) Standard Curve Predictive Model (CCE) Estimated Illness Rate (NGI): 32 per 1000 primary contact recreators: BAV Specificity Total Samples 121 Total Samples 121 Specificity Specificity 67% Specificity 86% 100% Total Samples 121 Specificity 85% 99% Total Samples 121 Edgewater Beach E. Coli Environmental MasterMix Assay Estimated Illness Rate (NGI): 36 per 1000 primary contact recreators: BAV Estimated Illness Rate (NGI): 32 per 1000 primary contact recreators: BAV Predictive Model (CCE) Total Samples 120 Total Samples 120 Standard Curve Predictive Model (CCE) Total Samples 120 Specificity 95% Total Samples 120 Total Samples 120 Total Samples 121 Total Samples 121 Standard Curve Predictive Model (CCE) Standard Curve Predictive Model (CCE) Total Samples 121 Total Samples 121 Specificity Specificity 100% Specificity 81% Standard Curve Predictive Model (CCE) Standard Curve Predictive Model (CCE) Estimated Illness Rate (NGI): 36 per 1000 primary contact recreators: BAV Villa Angela Beach E. Coli ToughMix Assay Total # Samples 120 97% Total # Samples 120 96% Specificity Specificity 79% Villa Angela Beach E. Coli Environmental MasterMix Assay Estimated Illness Rate (NGI): 36 per 1000 primary contact recreators: BAV Standard Curve Estimated Illness Rate (NGI): 32 per 1000 primary contact recreators: BAV Edgewater Beach E. Coli ToughMix Assay Total Samples 120 Specificity Specificity 97% 73% Specificity 94% 85% Specificity Estimated Illness Rate (NGI): 36 per 1000 primary contact recreators: BAV R² = 0.5059 0.5 1.5 2.5 3.5 0 0.5 1 1.5 2 2.5 3 3.5 4 Log TM predicted E.coli Log MPN 2014 EDGE TM predicted E.coli vs MPN R² = 0.4497 0.5 1.5 2.5 3.5 1.5 1.7 1.9 2.1 2.3 2.5 2.7 2.9 Log TM predicted E.coli Log EMM predicted E.coli 2014 EDGE TM vs EMM predicted E.coli R² = 0.4608 0.5 1.5 2.5 3.5 0 0.5 1 1.5 2 2.5 3 3.5 4 Log EMM predicted E.coli Log MPN 2014 EDGE EMM predicted E.coli vs MPN R² = 0.7081 1 2 3 4 1 1.5 2 2.5 3 3.5 4 4.5 Log TM predicted E.coli Log MPN 2014 VABE TM predicted E.coli vs MPN R² = 0.7492 1 2 3 4 1 2 3 4 Log EMM predicted E.coli Log MPN 2014 VABE EMM predicted E.coli vs MPN R² = 0.8822 1 2 3 4 1 1.5 2 2.5 3 3.5 4 Log TM predicted E.coli Log EMM predicted E.coli 2014 VABE EMM vs TM predicted E.coli Beach comparison Pearson Correlation R 2 P ‐ value Edgewater EMM vs TM 0.6705809 0.4497 0.0042 Edgewater EMM vs MPN 0.6788587 0.4608 0.0360 Edgewater TM vs MPN 0.7112917 0.5059 0.9663 Villa Angela EMM vs TM 0.939279 0.8822 2.919E‐ 14 Villa Angela EMM vs MPN 0.865581 0.7492 5.650E‐ 12 Villa Angela TM vs MPN 0.841500 0.7081 0.1193 E.coli Assay Comparison *EMM = Environmental MasterMix, TM = ToughMix, MPN = Most probable Number R² = 0.1516 0 1 2 3 0.5 1 1.5 2 2.5 3 3.5 Log TM predicted Entero Log EMM predicted Entero 2014 EDGE TM vs EMM predicted Entero R² = 0.2422 0 1 2 3 4 0.5 1 1.5 2 2.5 3 3.5 Log mEI CFU Log EMM predicted Entero 2014 EDGE EMM predicted Entero vs mEI CFU R² = 0.4474 0 1 2 3 4 0 0.5 1 1.5 2 2.5 Log mEI CFU Log TM predicted Entero 2014 EDGE TM predicted Entero vs mEI CFU Beach Comparison Pearson Correlation R 2 P ‐ value Edgewater EMM vs TM 0.3894003 0.1516 0.000001 Edgewater EMM vs CFU 0.4921501 0.2422 0.0017 Edgewater TM vs CFU 0.6688534 0.4474 0.1181 Villa Angela EMM vs TM 0.6672808 0.4453 0.000001 Villa Angela EMM vs CFU 0.7378979 0.5445 0.000062 Villa Angela TM vs CFU 0.6893558 0.4752 0.777620 Enterococci Assay Comparison *EMM = Environmental MasterMix, TM = ToughMix, MPN = Most probable Num Accuracy 81% Accuracy 71% Sensitivity 100% Sensitivity 50% #>70 27 20 11 27 0 #>1000 20 31 0 10 17 #<70 31 CNE False + CE False ‐ #<1000 ## CNE False + CE False ‐ Accuracy 76% Accuracy 78% Sensitivity 100% Sensitivity 100% #>60 27 17 14 27 0 #>640 14 31 0 14 13 #<60 31 CNE False + CE False ‐ #<640 44 CNE False + CE False ‐ Accuracy 74% Accuracy 69% Sensitivity 59% Sensitivity 62% #>70 27 27 4 16 11 #>1000 37 17 14 23 4 #<70 31 CNE False + CE False ‐ #<1000 21 CNE False + CE False ‐ Accuracy 76% Accuracy 64% Sensitivity 70% Sensitivity 65% #>60 27 25 6 19 8 #>640 37 13 18 24 3 #<60 31 CNE False + CE False ‐ #<640 21 CNE False + CE False ‐ Accuracy 78% Acc 64% Sensitivity 73% Sensitivity 38% # >70 15 35 9 11 4 # >1000 24 29 15 9 6 #<70 44 CNE False + CE False ‐ #<1000 35 CNE False + CE False ‐ Accuracy 75% Acc 63% Sensitivity 80% Sensitivity 37% # >60 15 32 12 12 3 # >640 27 27 17 10 5 #<60 44 CNE False + CE False ‐ #<640 32 CNE False + CE False ‐ Accuracy 80% Accuracy 81% Sensitivity 60% Sensitivity 60% #>70 15 38 6 9 6 # >1000 20 36 8 12 3 #<70 44 CNE False + CE False ‐ #<1000 38 CNE False + CE False ‐ Accuracy 81% Accuracy 80% Sensitivity 67% Sensitivity 65% #>60 15 38 6 10 5 # >640 20 34 10 13 2 #<60 44 CNE False + CE False ‐ #<640 38 CNE False + CE False ‐ 59 Specificity 84% Total # Samples 59 Specificity 73% Total # Samples 58 Specificity 89% Edgewater Beach Enterococci Environmental MasterMix Assay Standard Curve Predictive Model (CCE) Total # Samples 59 Specificity 80% Total # Samples 59 Specificity 83% Standard Curve Predictive Model (CCE) Total # Samples 59 Specificity 86% Total # Samples Edgewater Beach EnterococciToughMix Assay Estimated Illness Rate (NGI): 36 per 1000 primary contact recreators: BAV Standard Curve Predictive Model (CCE) Total # Samples 59 Specificity 86% Total # Samples 58 Specificity 95% 58 Specificity 62% Villa Angela Beach Enterococci Environmental MasterMix Assay Estimated Illness Rate (NGI): 36 per 1000 primary contact recreators: BAV Standard Curve Predictive Model (CCE) Total # Samples 58 Specificity Total # Samples 58 Specificity 31% Estimated Illness Rate (NGI): 36 per 1000 primary contact recreators: BAV Total # Samples 58 Specificity 81% Total # Samples Villa Angela Beach Enterococci ToughMix Assay Estimated Illness Rate (NGI): 36 per 1000 primary contact recreators: BAV Standard Curve Predictive Model (CCE) Total # Samples 58 Specificity 87% Total # Samples 58 Specificity 81% Estimated Illness Rate (NGI): 32 per 1000 primary contact recreators: BAV Estimated Illness Rate (NGI): 32 per 1000 primary contact recreators: BAV Standard Curve Predictive Model (CCE) Estimated Illness Rate (NGI): 36 per 1000 primary contact recreators: BAV Estimated Illness Rate (NGI): 32 per 1000 primary contact recreators: BAV Total # Samples 58 Specificity 55% Total # Samples 58 Specificity 70% This comparison was done to identify a qPCR assay that correlated with the culture based reference methods, reduced inhibi- tion in the sample matrices, had minimal residual E. coli contaminates in the mix, and could be used for multiple fecal indicat- ing bacteria. The assays tested were the Environmental MasterMix and ToughMix, ROX. The results of the comparison study indicated that ToughMix had a reduction the number of controls with elevated Ct value, as well as a reduction in the number of samples showing inhibition. Both assays had comparable results when statistically analyzed against samples from two different beaches, and two separate organisms. The results for the ToughMix were highly correlated with and not signiϐicantly different from the culture based methods. Based on the results of this study NEORSD has decided to utilize the ToughMix ROX assay for qPCR analysis of both Enterococ‐ ci and E.coli for samples collected at recreational beaches monitored by the NEORSD. Conclusion and Summary

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Page 1: make it tough poster resize - NEMC · NTC EDGE ToughMix 2 ... Edgewater TM vs MPN 0.7112917 0.5059 0.9663 Villa Angela EMM vs TM 0.939279 0.8822 2.919E‐14 Villa Angela EMM vs MPN

Mark Citriglia, Manager of Analytical Services

Northeast Ohio Regional Sewer District

4747 East 49th Street

Cuyahoga Hts., Ohio 44125

MakeItTough:AqPCRMasterMixComparativeStudy

Introduction

E.coliEdgewaterandVillaAngelaBeaches

E. Coli Results

Nicki Schafer, Molecular Microbiologist

Northeast Ohio Regional Sewer District

4747 East 49th Street

Cuyahoga Hts., Ohio 44125

ThewaterqualityofbathingbeachesinNortheastOhioisde-terminedbythemicrobialconcentrationtypically,theconcen-trationofE.coliand/orEnterococci.ThestandardmethodsusedtoanalyzeforFIBs(fecalindicatorbacteria)take24hourstoobtainresults.Theuseofrapidmethodsallowsforre-altimeresultswithin3-4hours.Since2007,AnalyticalSer-viceshasbeentestingvariousreal-timequantitativepolymer-asechain-reaction(qPCR)chemistriestodiscoveranassaywhichwillcloselycorrelatetothestandardculturebasedmethods,reduceenvironmentalinhibitoryeffects,andensureaccuracyforpredictivemodelimpact.

TheuseofqPCRmethodologyhasbecomeacriticalcompo-nentofthebeachmonitoringprogramattheNortheastOhioRegionalSewerDistrict(NEORSD).TheresultsfromtheqPCRanalysishavebeenusedasastand-alonepredictoraswellasavariableinpredictivemodeling,todeterminethewaterqualityofbathingbeachesforpublicnotification.Sincetimelypublicnotificationisofparamountimportance,qPCRprovidesfasterresultssothepubliccanmakeinformeddecisionsaboutwhethertorefrainfromrecreationalbeachusebasedondatafromthatdayandnotthepreviousdayresultsobtainfromstandardculturebasedmethods.

Method/Process

Enterococci

EdgewaterandVillaAngelaBeaches

Enterococci Results

E.colicalibrationcurvesEnterococcicalibrationcurves

slope 3.3676 

intercept 45.619 

R2  0.9985 

AF 1.969474 

% Efficiency = 98.13%  

slope 3.291 

intercept 43.475 

R2 0.9996 

AF 2.03859 

% Efficiency 101.31% 

slope: 3.3388 

intercept: 37.303 

AF: 1.995088056 

% Efficiency: 99.30%  

R2:  R2 =0.9998

slope: 3.4708 

intercept: 42.34 

AF: 1.8811801 

% Efficiency: 94.14%  

R2:  0.9988

E.coliQualityControlDataEnterococciQualityControlData

Beach  Assay # sketa inhibited 

# IAC inhibited 

# (+) PBS BLK 

# (+) NTC 

EDGE  ToughMix  19/121  0/121  1/7  0/7 

EDGE  EMM  22/121  0/121  3/6  0/6 

VABE  ToughMix  5/120  1/120  1/7  0/7 

VABE  EMM  6/120  0/120  2/6  1/6 

Beach  Assay # sketa inhibited 

# IAC inhibited 

# (+) PBS BLK 

# (+) NTC 

EDGE  ToughMix  2/120  0/120  2/6  2/6 

EDGE  EMM  10/120  1/120  4/6  4/6 

VABE  ToughMix  0/120  0/120  1/6  0/6 

VABE  EMM  0/120  0/120  5/6  2/6 

Accuracy 88% Accuracy 70%

Sensitivity 71% Sensitivity 32%

# Greater 235 42 75 3 30 12 # >3357 22 77 1 7 35

# Less than 235 78 CNE False + CE False ‐ #<3357 98 CNE False + CE False ‐

Accuracy 85% Accuracy 69%

Sensitivity 70% Sensitivity 92%

# Greater 190 47 69 4 33 14 # >2026 12 72 1 11 36

# Less than 190 73 CNE False + CE False ‐ #<2026 108 CNE False + CE False ‐

Accuracy 86% Accuracy 72%

Sensitivity 64% Sensitivity 90% Specificity 70%

#>235 42 76 2 27 15 #>3357 10 77 1 9 33

#<235 78 CNE False + CE False ‐ #<3357 110 CNE False + CE False ‐

Accuracy 85% Accuracy 75%

Sensitivity 66% Sensitivity 93%

# Greater 190 47 71 2 31 16 #>2026 14 77 1 13 34

# Less than 190 73 CNE False + CE False ‐ #<2026 106 CNE False + CE False ‐

Accuracy 89% Accuracy 84%

Sensitivity 37% Sensitivity 50%

# Greater 235 19 101 1 7 12 #>3357 2 101 1 1 18

# Less than 235 102 CNE False + CE False ‐ #<3357 119 CNE False + CE False ‐

Accuracy 89% Accuracy 85%

Sensitivity 48% Sensitivity 67%

# Greater 190 23 97 0 11 13 #>2026 3 101 1 2 22

# Less than 190 97 CNE False + CE False ‐ #<2026 118 CNE False + CE False ‐

Accuracy 86% Accuracy 83%

Sensitivity 42% Sensitivity 33%

# Greater 235 19 96 6 8 11 # >3357 3 100 2 1 18

# Less than 235 102 CNE False + CE False ‐ #<3357 118 CNE False + CE False ‐

Accuracy 91% Accuracy 80%

Sensitivity 57% Sensitivity 50%

# Greater 190 23 97 6 13 11 #>2026 4 95 2 2 22

# Less than 190 97 CNE False + CE False ‐ #<2026 117 CNE False + CE False ‐

Estimated Illness Rate (NGI): 32 per 1000 primary contact recreators: BAV

Standard Curve Predictive Model (CCE)

Standard Curve Predictive Model (CCE)

Estimated Illness Rate (NGI): 32 per 1000 primary contact recreators: BAV

SpecificityTotal Samples 121

Total Samples 121 Specificity

Specificity 67%

Specificity 86%100%

Total Samples 121 Specificity 85%99%

Total Samples 121

Edgewater Beach E. Coli   Environmental MasterMix AssayEstimated Illness Rate (NGI): 36 per 1000 primary contact recreators: BAV

Estimated Illness Rate (NGI): 32 per 1000 primary contact recreators: BAV

Predictive Model (CCE)

Total Samples 120 Total Samples 120

Standard Curve Predictive Model (CCE)

Total Samples 120 Specificity 95% Total Samples 120

Total Samples 120

Total Samples 121 Total Samples 121

Standard Curve Predictive Model (CCE)

Standard Curve Predictive Model (CCE)

Total Samples 121 Total Samples 121 Specificity

Specificity 100% Specificity 81%

Standard Curve Predictive Model (CCE)

Standard Curve Predictive Model (CCE)

Estimated Illness Rate (NGI): 36 per 1000 primary contact recreators: BAV

Villa Angela Beach E. Coli  ToughMix Assay

Total # Samples 12097%Total # Samples 120

96%Specificity Specificity 79%

Villa Angela Beach E. Coli   Environmental MasterMix AssayEstimated Illness Rate (NGI): 36 per 1000 primary contact recreators: BAV

Standard Curve

Estimated Illness Rate (NGI): 32 per 1000 primary contact recreators: BAV

Edgewater Beach E. Coli   ToughMix Assay

Total Samples 120 Specificity Specificity97% 73%

Specificity 94% 85%

Specificity

Estimated Illness Rate (NGI): 36 per 1000 primary contact recreators: BAV

R² = 0.5059

0.5

1.5

2.5

3.5

0 0.5 1 1.5 2 2.5 3 3.5 4

Log TM

 predicted

 E.coli

Log MPN

2014 EDGE TM predicted E.coli vs MPN

R² = 0.4497

0.5

1.5

2.5

3.5

1.5 1.7 1.9 2.1 2.3 2.5 2.7 2.9

Log TM

 predicted

 E.coli

Log EMM predicted E.coli

2014 EDGE TM vs EMM predicted E.coli

R² = 0.4608

0.5

1.5

2.5

3.5

0 0.5 1 1.5 2 2.5 3 3.5 4

Log EM

M predicted

 E.coli

Log MPN

2014 EDGE EMM predicted E.coli vs MPN

R² = 0.7081

1

2

3

4

1 1.5 2 2.5 3 3.5 4 4.5

Log TM

 predicted

 E.coli

Log MPN

2014 VABE TM predicted E.coli vs MPN

R² = 0.7492

1

2

3

4

1 2 3 4

Log EM

M predicted

 E.coli

Log MPN

2014 VABE EMM predicted E.coli vs MPN

R² = 0.8822

1

2

3

4

1 1.5 2 2.5 3 3.5 4

Log TM

 predicted

 E.coli

Log EMM predicted E.coli

2014 VABE EMM vs TM predicted E.coli

Beach comparisonPearson

CorrelationR2 P‐value

Edgewater EMM vs TM 0.6705809 0.4497 0.0042

Edgewater EMM vs MPN 0.6788587 0.4608 0.0360

Edgewater TM vs MPN 0.7112917 0.5059 0.9663

Villa Angela EMM vs TM 0.939279 0.8822 2.919E‐14

Villa Angela EMM vs MPN 0.865581 0.7492 5.650E‐12

Villa Angela TM vs MPN 0.841500 0.7081 0.1193

E.coli Assay Comparison

*EMM = Environmental  MasterMix, TM = ToughMix, MPN = Most probable Number

R² = 0.1516

0

1

2

3

0.5 1 1.5 2 2.5 3 3.5

Log TM

 predicted Entero

Log EMM predicted Entero

2014 EDGE TM vs EMM predicted Entero

R² = 0.2422

0

1

2

3

4

0.5 1 1.5 2 2.5 3 3.5

Log mEI CFU

Log EMM predicted Entero

2014 EDGE EMM predicted Entero vs mEI CFU

R² = 0.4474

0

1

2

3

4

0 0.5 1 1.5 2 2.5

Log mEI CFU

Log TM predicted Entero

2014 EDGE TM predicted Entero vs mEI CFU

Beach ComparisonPearson

CorrelationR2 P‐value

Edgewater EMM vs TM 0.3894003 0.1516 0.000001

Edgewater EMM vs CFU 0.4921501 0.2422 0.0017

Edgewater TM vs CFU 0.6688534 0.4474 0.1181

Villa Angela EMM vs TM 0.6672808 0.4453 0.000001

Villa Angela EMM vs CFU 0.7378979 0.5445 0.000062

Villa Angela TM vs CFU 0.6893558 0.4752 0.777620

Enterococci Assay Comparison

*EMM = Environmental  MasterMix, TM = ToughMix, MPN = Most probable Num

Accuracy 81% Accuracy 71%

Sensitiv ity 100% Sensitiv ity 50%

#>70 27 20 11 27 0 #>1000 20 31 0 10 17

#<70 31 CNE Fa lse  + CE Fa lse  ‐ #<1000 ## CNE Fa lse  + CE Fa lse  ‐

Accuracy 76% Accuracy 78%

Sensitiv ity 100% Sensitiv ity 100%

#>60 27 17 14 27 0 #>640 14 31 0 14 13

#<60 31 CNE Fa lse  + CE Fa lse  ‐ #<640 44 CNE Fa lse  + CE Fa lse  ‐

Accuracy 74% Accuracy 69%

Sensitiv ity 59% Sensitiv ity 62%

#>70 27 27 4 16 11 #>1000 37 17 14 23 4

#<70 31 CNE Fa lse  + CE Fa lse  ‐ #<1000 21 CNE Fa lse  + CE Fa lse  ‐

Accuracy 76% Accuracy 64%

Sensitiv ity 70% Sensitiv ity 65%

#>60 27 25 6 19 8 #>640 37 13 18 24 3

#<60 31 CNE Fa lse  + CE Fa lse  ‐ #<640 21 CNE Fa lse  + CE Fa lse  ‐

Accuracy 78% Acc 64%

Sensitiv ity 73% Sensitiv ity 38%

#  >70 15 35 9 11 4 #  >1000 24 29 15 9 6

#<70 44 CNE Fa lse  + CE Fa lse  ‐ #<1000 35 CNE Fa lse  + CE Fa lse  ‐

Accuracy 75% Acc 63%

Sensitiv ity 80% Sensitiv ity 37%

#  >60 15 32 12 12 3 #  >640 27 27 17 10 5

#<60 44 CNE Fa lse  + CE Fa lse  ‐ #<640 32 CNE Fa lse  + CE Fa lse  ‐

Accuracy 80% Accuracy 81%

Sensitiv ity 60% Sensitiv ity 60%

#>70 15 38 6 9 6 #  >1000 20 36 8 12 3

#<70 44 CNE Fa lse  + CE Fa lse  ‐ #<1000 38 CNE Fa lse  + CE Fa lse  ‐

Accuracy 81% Accuracy 80%

Sensitiv ity 67% Sensitiv ity 65%

#>60 15 38 6 10 5 #  >640 20 34 10 13 2

#<60 44 CNE Fa lse  + CE Fa lse  ‐ #<640 38 CNE Fa lse  + CE Fa lse  ‐

59 Specific ity 84%Tota l #  Sam ples 59 Specific ity 73% To ta l #  Sam ples

58 Specific ity 89%

Edgew ater  Beach  Enterococci Environm enta l MasterM ix  Assay

Standard  Curve Pred ictive  Model (CCE)

To ta l #  Sam ples 59 Specific ity 80% To ta l #  Sam ples 59 Specific ity 83%

Standard  Curve Pred ictive  Model (CCE)

To ta l #  Sam ples 59 Specific ity 86% To ta l #  Sam ples

Edgew ater  Beach  EnterococciToughM ix  AssayEstim ated  Illness  Rate  (NG I):  36  per  1000  prim ary  contact  recreato rs: BAV

Standard  Curve Pred ictive  Model (CCE)

To ta l #  Sam ples 59 Specific ity 86% To ta l #  Sam ples 58 Specific ity 95%

58 Specific ity 62%

V illa  Ange la  Beach  Enterococci Environm enta l MasterM ix  AssayEstim ated  Illness  Rate  (NG I):  36  per  1000  prim ary  contact  recreato rs: BAV

Standard  Curve Pred ictive  Model (CCE)

To ta l #  Sam ples 58 Specific ity To ta l #  Sam ples 58 Specific ity 31%

Estim ated  Illness  Rate  (NG I):  36  per  1000  prim ary  contact  recreato rs: BAV

Tota l #  Sam ples 58 Specific ity 81% To ta l #  Sam ples

V illa  Ange la  Beach  Enterococci ToughM ix  AssayEstim ated  Illness  Rate  (NG I):  36  per  1000  prim ary  contact  recreato rs: BAV

Standard  Curve Pred ictive  Model (CCE)

To ta l #  Sam ples 58 Specific ity 87% To ta l #  Sam ples 58 Specific ity 81%

Estim ated  Illness  Rate  (NG I):  32  per  1000  prim ary  contact  recreato rs: BAV

Estim ated  Illness  Rate  (NG I):  32  per  1000  prim ary  contact  recreato rs: BAV

Standard  Curve Pred ictive  Model (CCE)

Estim ated  Illness  Rate  (NG I):  36  per  1000  prim ary  contact  recreato rs: BAV

Estim ated  Illness  Rate  (NG I):  32  per  1000  prim ary  contact  recreato rs: BAV

Tota l #  Sam ples 58 Specific ity 55% To ta l #  Sam ples 58 Specific ity 70%

ThiscomparisonwasdonetoidentifyaqPCRassaythatcorrelatedwiththeculturebasedreferencemethods,reducedinhibi-tioninthesamplematrices,hadminimalresidualE.colicontaminatesinthemix,andcouldbeusedformultiplefecalindicat-ingbacteria.TheassaystestedweretheEnvironmentalMasterMixandToughMix,ROX.

TheresultsofthecomparisonstudyindicatedthatToughMixhadareductionthenumberofcontrolswithelevatedCtvalue,aswellasareductioninthenumberofsamplesshowinginhibition.

Bothassayshadcomparableresultswhenstatisticallyanalyzedagainstsamplesfromtwodifferentbeaches,andtwoseparateorganisms.TheresultsfortheToughMixwerehighlycorrelatedwithandnotsigni icantlydifferentfromtheculturebasedmethods.

BasedontheresultsofthisstudyNEORSDhasdecidedtoutilizetheToughMixROXassayforqPCRanalysisofbothEnterococ‐ciandE.coliforsamplescollectedatrecreationalbeachesmonitoredbytheNEORSD.

Conclusion and Summary

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