xtag® gastrointestinal pathogen panel (gpp) · xtag gpp has been in development for the last two...
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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
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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]