xTAG® Gastrointestinal Pathogen Panel (GPP) · xTAG GPP has been in development for the last two years Undergone extensive analytical and clinical evaluation CE stmarked April 21

xTAG® Gastrointestinal Pathogen Panel (GPP)

Prospective performance at Guys/St Thomas’s Hospitals Spring 2011Dr. Richard JaneczkoVice President LMD

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Gastrointestinal infection & the NHS

Gastroenteritis outbreaks cost the NHS £115 million (2002 to 2003)

Among the most costly healthcare‐acquired infections to NHS ≈1% of the total inpatient services budget.

The cost of bed‐days lost plus staff absence calculated to be £635,000 per 1,000 beds. 

Outbreaks were contained faster (7.9 vs. 15.4 days p = 0.0023) when units were rapidly closed to new admissions(<4 days). 

Current testing algorithms are slow, laborious and exhibit low diagnostic yield

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Noro G1/2

Non-SpecificGastroenteritis

“Patient Presentation”

Clinical investigation of Gastroenteritis

ManyPotential Pathogens

E.Coli

Shigella

Campy

Salm.

Giardia

Crypto.

Enta. His.

Noro G1/2

Rota

Adeno

Selectionof many

sequentialtests for

one potential pathogen

InfectiveGastroenteritis

???

C. Diffe.

Crytpo.

Decision on:Infection Control

Bed/Ward ManagementPatient Treatment

Local disease epidemiology

Average time to negative result is 3days

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GSTS Gastroenteritis Workflowfor cohort of 450 stools February  2011

50-100 Stool Samples

Received in GSTSper day for

Gastroenteritis investigation

MicrobiologyReceives and analyses

All stool samples

Virology 39% of daily

Stool samples

Norovirus EIA on all samples

Rota/Adenovirus EIA 2%

89%Cultured forSalmonella

ShigellaCampylobacter

E.Coli 0157

0%Cultured for

Vibrio choleraeYersinia enterocolitica

37%Clostridium DifficileEIA 10-20 per dayCephid GeneXpert

2-5 per day

34%Tested for

CrytosporidiumGiardia

Entamobea His.

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Current Gastroenteritis Labour Requirements at GSTS

Total Staffing GastroenteritisInfection Sciences

2.5 BMS WTE 1.0 MLA WTE Monday-Friday

( Peak Seasonal)

Microbiology2 BMS1MLA

Setup,Read,InputResults

Virology 0.5 BMS

Setup,Read,InputResults

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The xTAG Gastrointestinal Pathogen Panel

Unmet need for a rapid, comprehensive test with high diagnostic yield 

xTAG GPP has been in development for the last two years

Undergone extensive analytical and clinical evaluation

CE marked April 21st 2011

Commercial launch May 7th 2011

FDA trials completed, submission scheduled Q4, 2011

6For In Vitro Diagnostic Use. Products are region specific and may not be approved in some countries/regions. Please contact Luminex to obtain the appropriate information for your country of residence.

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xTAG® GPP Description

Assay Characteristics

detects 15 bacterial, viral and parasitic targets in a single tube

representing > 95% coverage of clinical GI infections

xTAG FAST chemistry with ~ 5 hour TAT (including extraction)

Platforms

LX200 and new MAGPIX platform

xPONENT 3.1 & 4.1 software

All required components provided

Primer mix

Magplex‐TAG bead Mix

RT‐PCR enzyme/ buffer

BSA

SAPE & Buffer

Extraction/PCR inhibition control (MS2)

Reportable Targets                    Number of Analytes

Adenovirus (40 and 41) 1

Rotavirus (A) 1

Norovirus GI/GII 2

Clostridium difficile: Toxin A and B 2

Salmonella spp. 2

Shigella spp. 1

Campylobacter spp.(jejeuni, lari, coli) 1

E. coli O157 1

E. Coli (ETEC) (ST/LT) 2

Yersinia enterocolitica 1

Vibrio cholerae 1

Shiga‐like toxin  E. coli (STEC) (stx 1/stx2) 2

Giardia lamblia 1

Cryptosporidium sp. 1

Entamoeba histolytica 1

Internal control (MS2) 1

Total 21

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Universal pre‐treatment and extraction

Maximizes efficiency of extraction for parasites within the same sampleViruses, Bacteria and parasites able to be handled in the same step

May be omitted if only looking at bacterial or viral pathogens

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xTAG® FAST Chemistry

LX200 Read

BB

II. xTAG bead hybridization

MAGPIX

~5 hour TAT

Extracted pathogen NA

B

Tag 1

B

BB

Target 1B

Target 1

Tag 1

I. Multiplex Tagged Target Specific RT-PCR

PEB PEBIII. Sorting and Detection

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Comparison of gastroenteritis pathogens detection rate:evaluation cohort of 458 stools February  2011

Target GSTS Micro/Viro Percentage Positive xTAG‐GPP Percentage Positive 

Norovirus G1/G2 10 2.2% 40 8.9%

Rotavirus A 4 0.9% 14 3.1%

Adenovirus 40/41 0 0% 5 1.1%

Salmonella 5 1.1% 11 2.4%

Shigella 4 0.9% 14 3.1%

Campylobacter 5 1.1% 11 2.4%

C.difficile(ToxA/B) 7 1.5% 7 1.5%

E. coli O157 1 0.2% 3 0.7%

Yersinia  0 N/A 0 N/A

Vibrio cholerae 0 N/A 0 N/A

Giardia 1 0.2% 5 1.1%

Entaemoba hist. 0 0% 3 0.7%

Cryptosporidium 1 0.2% 5 1.1%

Overall 38 8.3% 118 26.2%

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Comparison of gastroenteritis pathogens detection rate:evaluation cohort of 458 stools February  2011

Noro

viru

s G

1/G

2Ro

tavir

us A

Aden

oviru

s 40

/41

Salm

onel

laSh

igel

laCa

mpy

loba

cter

C.di

ffici

le(T

oxA/

B)E.

col

i O15

7Ye

rsin

ia

Vibr

io c

hole

rae

Gia

rdia

Enta

emob

a hi

st.

Cryp

tosp

orid

ium

0

5

10

15

20

25

30

35

40

GSTS Micro/ViroGPP

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GSTS

Norovirus G1/2

Rotavirus

GPP

46

0412

005

34

Adenovirus

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Sample GPPNoro

Noro EIA  Rt‐PCR

Stool 1 388 not done 32.5

Stool 2 265 not done 30.1

Stool 3 1890.5 not done 28.8

Stool 4 1357 not done 27.9

Stool 5 1088 pos 27.3

Stool 6 386 not done 26.4

Stool 7 2953 not done 25.9

Stool 8 4336 not done 21.4

Stool 9 3750 not done 20.2

Stool 10 6447.5 not done 19.7

Stool 11 6106 not done 19.4

Stool 12 4493.5 not done 18.4

Stool 13 4101.5 not done 18.3

Stool 14 2954 pos 16

Stool 15 4385 not done 15.3

Stool 16 6708 pos 14.8

Stool 17 5018 not done 14.4

Stool 18 5043.5 pos 13.9

Stool 19 3371 pos 13.1

Stool 20 4511.5 not done 10.7

Stool 23 178 not done 0

Stool 24 151 not done 0

Stool 25 39 pos 0

Stool 26 41 pos 0

Sample GPP RotaA EIA Rota  Rt‐PCR Rota 

Stool 27 167 not done 31.2

Stool 28 4374.5 not done 25.2

Stool 29 3705.5 not done 23.6

Stool 30 2824 not done 20.1

Stool 31 2540 not done 19.8

Stool 32 2541 not done 19.3

Stool 33 3767 pos 19.2

Stool 34 3653 pos 18.8

Stool 35 2431.5 not done 18.5

Stool 36 5112 not done 17.8

Stool 37 4341.5 not done 17.7

Stool 38 3630 not done 17.6

Noro GII Rotavirus

Sample GPP Adeno EIA Adeno Rt‐PCR Adeno

Stool 39 2072.5 not done 23.8

Stool 40 986 not done 21.4

Stool 41  1295 not done 20.5

Stool 42 2060 not done 15.8

Stool 43 1951.5 not done 14.4

Adenovirus 40/41

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invasive liver abscess

Entamoeba His. 003

Cryptosporidium

Giardia

114

GPP GSTS

11

MFI 121

4

MFI147

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GPP GSTS

Salmonella

Shigella

Campylobacter

147

0410

147

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C.Difficile 434

GPP GSTS

E.Coli 0157 012

Stool Sample

GDH/GXprtC.difficile toxin 

testGPP ToxA (C. difficile)

GPP: Tox B (C. difficile)

66 Positive 1320 527

55 positive 687 298

84 positive 161 89

89 Positive 121 81.5

28 Positive 120 91

69 positive 39 45

68 Positive 37 41

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C.difficile evaluation 

Retrospective cohort of Clostridium Difficile validated by GDH‐EIA,Genexpert C.Diffe and Culture

Extracted DNA from 150 specimens 

Data on 96 presented here (74 positives 21 negatives)

MolecularAssay

C.DiffePositive

ConfirmedBy Culture % TP %FP

Cepheid 74 59 79% 20%

GPP 51 47 92% 7.8%

StoolGPP Toxin A

GPP Toxin B 

OriginalCepheid Toxin B

Rpt Cepheid  Toxin B 

1 1564 1410 21.8 29

2 51 48.5 24.9 33.5

6 45 46 24.2 34.2

7 120 54 27.2 30.4

3 46 44.5 35.6 0

4 45.5 46 22.6 0

8 47 53 33.2 33

5 58 45 34 34.6

C.Diffe Assay Average Ct Stnd. Dev. Ct

GPP Positive 25.7 3.7

GXprt Positive 31.4 6.0

GeneExpert C.Diffe Assay

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Culture dilutions GPP Tox A GPP Tox B G-xpert [ct] cell countNCTC027 S1 2623 2390 113414/rtnNCTC027 S2 2429 2353 11341NCTC027 S3 1382 1264 1134NCTC027 S4 236 262.5 113NCTC027 S5 103 50 tox b [34.2] 11.3NCTC027 S6 46 46NCTC027 S7 47 50UCH027 S1 2920 2626 155170/rtnUCH027 S2 2680 2447 15517UCH027 S3 1786.5 1753 1551UCH027 S4 408.5 301 155UCH027 S5 42 55 tox b [34.5] 15.5UCH027 S6 39 46UCH027 S7 43 39UCH 106 S1 2844.5 2591 48255/rtnUCH 106 S2 2201 2198 4825UCH 106 S3 729 781 482UCH 106 S4 52.5 59 tox b [32.4] 48UCH 106 S5 51 61 4.8UCH 106 S6 40 47UCH 106 S7 38 42

C.difficile prospective clinical samples & reference panels

‘Inhibited’ FN re-extracted after diln

GSTS ‘Homebrew’C.Diffe proficiency Panel

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GI Targets implicated in co‐infections

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Target N Percentage

Adenovirus 40 and 41 4 4.6%

Rotavirus A 4 4.6%

Norovirus 23 26.7%

Salmonella 18 20.9%

Shigella 21 24.4%

Campylobacter 22 25.6%

Toxin A/B (C. difficile) 26 30.2%

ETEC 13 15.1%

E. coli O157/ STEC 11 12.8%

Yersinia enterocolitica 0 N/A

Vibrio cholerae 0 N/A

Giardia 18 20.9%

Entaemoba histolytica 7 8.1%

Cryptosporidium 7 8.1%

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GPP NPV

Target NPV

Salmonella 97.22% 421/433

Shigella 99.78% 451/450

Campylobacter 99.32% 438/441

C. diff Tox A/B 99.76% 424/425

ETEC 100% 287/287

E. coli 0157 99.75 406/407

STEC 100% 291/291

Yersinia 100% 400/400

Vibrio Cholera 99.75% 414/415

Giardia 100% 835/835

E. histolytica 100% 643/643

Cryptosporidium 99.76% 853/855

Rotavirus 99.88% 852/853

Adenovirus 100% 235/235

Norovirus (GI/GII) 99.35% 769/771

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Summary and Conclusions

The xTAG GPP assay provides rapid and comprehensive detection and identification of pathogens  in stool specimens

Exhibits high NPV for all targets

Overall and individual positivity rates are 2‐3 fold higher than current testing algorithms …………………. about 70% are missed!!

Detects about 3‐4% co‐infections

Provides the fastest and most accurate data for patient management and infection control

Implementation should result in improved outcomes, better use of resources and overall cost savings

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Thank Youwww.luminexcorp.com

Eugene Halligan*

John Bible*

Simon Goldenberg*

Sevana Yaghoubian**

*GSTS, **[email protected]