Tetracore Profile T-COR 8TM Molecular Diagnostics Platform ...• Send text/e-mail notifications •...
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Tetracore ProfileT-COR 8TM
Molecular Diagnostics Platform
William M Nelson MD
May 3, 2017
TetracoreOverview
• Founded in 1998
• Four principal founders formerly with Biological Defense Research Program (Naval Medical Research Institute)
• Small business: 70+ employees
• Facility is FDA cleared, USDA certified– cGMP manufacturing
• All Assays produced under QMS
• We have our own BSL-3 suite – CDC certification, Select Agent Permits – Extensive collect of Viruses and Bacteria
TetracoreField PCR Experience
• Areas of expertise• Molecular biology: real-time PCR and isothermal assays
• Bio-detection platforms: T-CORTM, Biothreat Alert®
• Microbiology: BSL-3 CDC licensed; bacteriology and virology
• Immunology: monoclonal/polyclonal antibody production
• Mobile Laboratory Development
• Privately Held no outside funding
TetracoreField PCR Experience
• First use of PCR (gel-based) in a forward deployed lab• Navy Forward Lab, Operation Desert Shield/Storm, 1991
TetracoreCanal Hotel (UNSCOM Headquarters)Baghdad, Iraq
• First use of PCR (IGEN detection) for the forensic analysis of bio facilities in Iraq – 1993
TetracorePCR for smallpox simulate in a C-141ANAA (LLNL) – IGEN 1998
Develop Small Battery Operated Real-Time PCR Instrument for POC/Penside Testing
• First instrument was the TCOR-4™– 4 Independently Programmable Amplification Wells
– Two Color detection FAM/CY5
– 8 hour battery life
– 7 pounds
– Required extracted samples
T-COR 4Detailed Description
• Stand-alone mode• Self-contained; operated via
user interface on T-COR 4
• Simple software runs basic functions
• Data export using USB port
• PC (laptop) mode• T-COR 4 connected to
computer via a USB cable
• Fully featured T-COR software
• Can run multiple systems to increase capability
Field trial for portable PCR laboratory testing for African swine fever virus in Gulu District, Uganda
Off the grid, one hour drive to nearest place with standard electricity
Solar panels only
proximate source, used
for cell phones, lamps,
etc...
No practical
power source
to run a
laboratory
Lab Procedures
• Three extraction methods were tested:– Magnetic Beads
– FTA Elute cards
– Tego cards with lysis and neutralization buffer
• All three methods worked; which method is best depends on purpose of laboratory
T-COR 4 used as the detection device
• Assays were run simultaneously on two instruments
• When needed, instruments were recharged using the 12 volt car adapter
Requirements for Remote rtRTPCRTesting
• Room temperature stable reagents
• Sample - Blood, Sera, Oral Fluids, or NS
• Portable PCR cycler (battery or mains power free)
• Remote connectivity (Expert review of results or results communication)
• Field capable extraction
Needed for a better portable MolDx platform
• Limitations of Existing PCR Systems– Complex large multi-laboratory based instruments limits global use
– High capital cost of systems & licensing fees
– Highly trained operators & requirement for separate laboratories
– Separate sample preparation
– Frozen reagents require cold storage chain
– High training & logistical support burden
• Point-of-Care or “Test & Treat” capability– POC market requires an ‘immediate results’ system (ie diagnostic
results ~60 minutes or less)
– Molecular diagnostic test accuracy represents substantial improvement from current lateral flow solutions
T-COR 8TM Real-Time PCR System
• Innovative diagnostic tools– Hardware
– Software
– Assays
• Collect to test (“C2T”) system comprised of:– Inexpensive plastics
– Dry reagents in the tube
– Assay solution
• Uniform system approach:– CLIA Waive-able
– Common collection/processing device
– Bi-directional communication and push capabilities• LIS interface-able/EMR interface-able
• Send text/e-mail notifications
• Can operate in real-time server upload mode– Provides real-time monitoring of disease incidence
– Health systems can link entire network of instruments to provide greater data access
Collection Device /C2T Packaging
T-COR 8
• Innovative “Collect-to-Test” device
– Eliminates pipetting
– Eliminates extraction
– No transfer of materials
– Test immediately or ship to alternative location to test
T-COR 8TM Assays
C2T Cartridge
T-COR 8TM Candidate Workflow: Sample to Result - under an hour
1. Swab Patient 2. Snap / Lyse / Dispense / Close 3. Squeeze bottom to pull under filter
4. Squeeze 3 drops in C2T 5. Cap C2T 6. Follow Software Prompts
Oral fluids extracted vs. directly tested on the TCOR-8
Clarified by centrifugation – low speed/portable - 5µL added
Well Sample Assay Protocol FAM
1 OF #3- extractedInf Direct 50cy 26.1
2 OF #3- directInf Direct 50cy 27.7
3 OF #3- directInf Direct 50cy 26.7
4 OF #4- extractedInf Direct 50cy 26.8
5 OF #4- directInf Direct 50cy 27
6 OF #4- directInf Direct 50cy 26.9
7 OF #5- extractedInf Direct 50cy 33.3
8 OF #5- directInf Direct 50cy 32.3
T-COR 8TM Hardware, Design and Architecture
• Portable real-time PCR thermocycler– 11.81” x 10.71” x 3.19”
– Under 10 lbs
– Fits in a backpack
– 4.5 hour battery life
– 5 full PCR runs
– Full day with extra battery
– Integrated Bar Code Reader
T-COR 8TM Highlights
• Fully automated raw sample to result systems• 8 independent wells• Open & Closed system settings
– Run most real time assays
• Runs Multiple Chemistry Types– Real-Time PCR– Isothermal
• Expandable up to 6 dye channels– 4 channel instruments used in development work
• Random Access– Multiple protocols simultaneously
• C2T consumable producible at very low cost• Touch Screen user interface & connectivity • Cloud driven software interface • Small footprint, lightweight enhance portability
– Battery powered, with power backup – no system downtown
• CLIA “waive-able” design with intention of seeking CLIA waiver
VETERANARY ASSAYS
• VETERANARY ASSAYS (ALL INCLUDE AN IC/Extraction control)– PED, TGE, Delta corona Virus– PRRSV - UNIVERSAL (ALL SEROTYPES)– PRRSV, INF-A– AFRICAN SWINE FEVER– FOOT-AND-MOUTH – UNIVERSAL (FMD) (ALL SEROTYPES)– SENECA VALLEY VIRUS (SVA)– SWINE VESICULAR DISEASE (SVD)– FMD, SVD, SVA– INFLUENZA C and D– CLASSICAL SWINE FEVER (CSF)– CAPRIPOX– PESTE DES PETITS RUMINANTS– RINDERPEST
EPIZOOTIC
• INFLUENZA A – UNIVERSAL (ALL SEROTYPES)• JAPANESE ENCEPHALITIS• WEST NILE VIRUS• VENEZUELAN EQUINE ENCEPHALITIS – (VEE)• JUNIN VIRUS• MACHUPO VIRUS• RIFF VALLEY FEVER VIRUS• NIPAH VIRUS• CAMEL POX VIRUS• MONKEY POX VIRUS• MERS VIRUS• NORO VIRUS• ORTHOPOX• BACILLUS anthracis• BRUCELLA • BURKHOLDERIA
HUMAN ASSAYS
• CONGO CRIMEAN HEMORAGIC FEVER VIRUS (CCHF)
• DENGUE VIRUS - GROUP
• DENGUE VIRUS SEROTYPE SPECIFIC
• DENGUE, CHIKUGUNYA, and ZIKA VIRUS
• YELLOW FEVER VIRUS
• INFUENZA A/B, RSV
• SIN NOMBRE VIRUS (HANTA VIRUS)
• YERSINIA pestes
• MALARIA
Direct Blood RT-PCR
Pan Dengue
Whole Blood / Dengue Assay
• Whole Blood– Venipuncture collection
• Vacutainer test tube (purple)
• EDTA
– Finger Stick
• Lancet
• Dengue, Chikungunya, Zika Assay (DCZ)– Pan Dengue
• Detects all serotypes in FAM
– Internal Control
• Detected in Cy5/Q670
Direct Blood Process Protocol
• Whole Blood– Add 25µL of blood to 600µL of RPDB (Rapid Plasma Dilution Buffer)
– Filter sample through glass fiber filter
– Use 25µL directly in dried RT-PCR reaction
– Add 1μl directly to reaction volume 24μl
• Serum – Add 10μl to 90μl Buffer – add to C2T close
Extraction vs RPDB
• Dengue 2 Virus was diluted into blood
• Samples were processed:– 25µL into 600µL RPDB, glass fiber filtered, PCR 25µL
– 1µL blood (no processing) tested directly in PCR
– Extracted blood (Qiagen Mini Prep Kit)
• 200µL blood, eluted with 200µL
• PCR with 1µL
Run: 29/254
Confirmation of Ratio of Blood to Buffer
• Volume of RPDB 600µL
• Varied the volume of Zepto Spiked Blood– 25µL (standard volume)
– 0.25, 0.5, 2x, 4x, and 8x the volume (6.25, 12.5, 50, 100 and 200µL)
AmtBlood
Vol.Blood
Dengue,Rep 1
Dengue,Rep 2
IC, Rep 1
IC, Rep 2
1x 25µL 28.6 28.3 27.8 Pos
0.5x 12.5µL 28.6 29.2 27.6 28.3
0.25x 6.25µL 30.1 29.5 28.8 28.5
2x 50µL 27.8 27.0 0 0
4x 100µL 26.9 26.3 0 0
8x 200µL 0 0 0 0
Contrived Dengue Samples
• Ten microliters of the contrived sample was added to 90 µl of C2T buffer
• The whole 100µl sample was loaded into a C2T cartridge that contained the dry DCZ triplex assay.
• The DCZ assay on the T COR8™ is thermally cycled with a proprietary profile that is designed for maximizing sensitivity and has a tendency skew the low end linearity.
• This is evident in the last two dilutions below – dengue 2 and 3 have lower Cts, dengue 1 is flat, and dengue 4 continues to rise. If these samples had been cycled with a traditional profile they would be approximately 10 Cts later.
Contrived specimen evaluation for Dengue Virus
15
20
25
30
35
2 3 4 5 6 7 8 9
Ct
LOG DILUTION FROM TISSUE CULTURE SUPERNANT AT 109 VIRAL PARTICULES PER ML
Dengue
Dengue 1
Dengue 2
Dengue 3
Dengue 4
Zika Samples from Grenada
• Ten microliters of the contrived sample was added to 90 µl of C2T buffer
• The whole 100µl sample was loaded into a C2T cartridge that contained the dry DCZ triplex assay.
• The DCZ assay on the T COR8™ is thermally cycled with a proprietary profile that is designed for maximizing sensitivity and has a tendency skew the low end linearity.
• If these samples had been cycled with a traditional profile they would be approximately 10 Cts later.
Zika Testing Direct vs ExtractedGrenada Samples
15
20
25
30
35
40
0 5 10 15 20 25 30 35 40 45 50
Re-direct
Extracted
Direct-HiIC
Samples collected in Grenada tested by PCR and multiplex serology assay
Total number of samples and IgM serology results
Serological Data Categories# of
samples
CSF- not tested 2
urine-not tested 207
Not Tested/hemolyzed 12
Presumptive recent Zika virus infection 192
Presumptive recent flavivirus infection 174
Presumptive recent arbovirus infection 11
Possible recent Zika virus infection 30
Possible recent flavivirus infection 147
Possible recent arbovirus infection 18
Possible recent CHIKV infection 10
No evidence of recent flavivirus or arbovirus infection 256
Possible Cross-reactive arboviral IgM 30
Total number of samples 1089
Correlation of IgM serology with PCR results
Serological Data Categories# Zika PCR
Positive CHIKV DENVCSF- not tested 0 0 0
urine-not tested 13 0 0
Not Tested/hemolyzed 8 0 0
Presumptive recent Zika virus infection 21 0 0
Presumptive recent flavivirus infection 26 0 0
Presumptive recent arbovirus infection 4 0 0
Possible recent Zika virus infection 1 0 0
Possible recent flavivirus infection 19 0 0
Possible recent arbovirus infection 2 0 0
Possible recent CHIKV infection 4 0 0
No evidence of recent flavivirus or arbovirus infection 32 0 0
Possible Cross-reactive arboviral IgM 2 0 0
Total number of PCR positive with ct 132
Sample ZIKV1 ZIKV2 ZIKV3 DENV1 DENV2 DENV3 DENV4 WNV JEV YFV TBEV CHIKV1 CHIKV2 IC NC SCM SCG FC IgM Calls
0110 672 2290 164 120 527 42 172 26 1754 43 21 95 73 2944 25 6660 57 1348 Presumptive recent Zika virus infection
0112 124 2796 846 125 185 77 82 42 20 37 12 28 45 2803 19 5373 27 1252 Presumptive recent Zika virus infection
0113 120 78 36 44 81 16 41 17 23 27 11 19 32 2809 17 5779 24 1210 Presumptive recent Zika virus infection
0114 61 119 273 64 340 29 147 17 19 94 11 26 36 2799 18 7082 25 1177 Possible recent flavivirus infection
0130 54 55 1421 119 690 91 170 21 20 182 12 21 50 2910 17 6119 63 1184 Presumptive recent flavivirus infection
0131 36 125 160 119 176 128 91 19 20 19 13 25 34 2804 18 5348 88 1264 Possible Cross-reactive arboviral IgM
0132 49 193 146 63 144 33 74 24 28 36 21 44 82 2843 18 5876 95 1284 Possible Cross-reactive arboviral IgM
0119 129 221 99 30 80 20 38 17 17 20 13 23 40 2789 18 5867 26 1249 Presumptive recent Zika virus infection
0122 102 36 53 48 68 44 58 21 54 27 108 28 177 2930 16 5521 24 1228No evidence of recent flavivirus or
arbovirus infection
0123 32 42 66 108 202 22 130 18 25 126 11 21 53 2913 16 5093 24 1249 Presumptive recent flavivirus infection
0124 574 93 142 132 188 68 124 22 26 26 19 51 95 2884 20 6119 36 1353 Presumptive recent Zika virus infection
0125 5114 4956 177 111 614 27 152 21 48 71 17 149 104 2884 18 6915 27 1272 Presumptive recent Zika virus infection
0126 571 483 681 380 621 264 220 41 33 25 11 23 32 2838 19 5845 20 1162 Presumptive recent Zika virus infection
0127 17 155 622 43 57 28 35 17 15 15 10 13 38 2877 15 4897 19 1193 Possible recent flavivirus infection
0128 17 23 79 62 113 37 46 20 17 15 10 20 34 2945 17 5130 21 1235No evidence of recent flavivirus or
arbovirus infection
EXAMPLE DATA FROM CLINICAL SAMPLES TESTED FROM GRENADA
Assay Development
The recombinant antigens for following agents added to the assay• Zika• Dengue 1-4• Chikungunya• West Nile• Japanese encephalitis• Tick-borne encephalitis• Yellow feverInternal controls included to monitor the assay performance
Initial testing of assay performance was performed with various commercially obtained serum and plasma samples.
Non human primate samples obtained from University of Wisconsin were also used defining the assay characteristics for both IgMand IgG detection in experimentally infected animals.
Assay Development – results from a set of 481 results obtained from various human samples
D09*
D21
D28
D35
D42
1
1 0
1 0 0
Fo
ld
Ch
an
ge
(M
FI)
PR
NT
90
Tit
er
Ig G P R N TIg M
2 0
4 0
8 0
1 6 0
3 2 0
6 4 0
1 0
3 0
3 0 0
M o n k e y 6
Comparison of PRNT to IgM and IgG data in primates shows that PRNT co-relates directly with IgG even though IgM comes down on day 42, both IgG and PRNT are rising.
Multiplex Arbo Viral Serology Assay
• IgM response to 10 different viruses can be evaluated using 13 different antigens in a single well.
• Very small sample volume required
• Simple sample preparation
• Simple easy to use protocol
• Data analysis algorithm provided for easy interpretation of multiplex data
• Each run can accommodate up to 96 sample including controls
• Each well includes 5 different internal controls that are used ensuring the sample and reagent and process quality
Multiplex Arbo Viral Serology Assay
6.5 µ Magnetic, discrete magnetic fluorescent microsphere, Array
Instrument : Luminex 100/200™ and MAGPIX®
Recombinant Antigens on Microspheres
Add Sample Add Reporter Reagent
ZeptoMetrix Dengue • Dengue Virus Type 2 Culture Fluid
• TCID50 Endpoint dilution 1 x 105.15 U/mL
• Serial Dilutions of ZeptoMetrix into whole blood– 1µL of blood (no process), PCR directly
– 25µL blood with 600µL buffer, filter thru glass fiber and PCR (25µL)
Dilution of Zepto in
Blood1µL, no process
25µL processed
10-2 23.1 24.4
10-3 25.5 27.4
10-4 29.2 30.0
10-5 31.6 31.7
10-6 0 0
ZeptoMetrix Graphs
Titer Dengue IC
10-2 25.2 0
10-3 27.3 28.4
10-3 27.5 28.6
10-4 28.5 29.2
10-4 30.9 0*
10-5 33.1 30.1
10-5 33.4 28.7
NTC 0 31.2
Run: 29/400
*Sample hemolyzed
Zepto Titer, 2 techs, 3 reps, 3 instruments
• Zeptometrix diluted into blood– 25µL blood into 600µL RPDB, filter glass fiber, PCR 25µL
– 2 technicians
– Each sample in triplicate
– 5 logs of dilutions
– PCR on 3 TCOR-8
TCOR-8Tech 1 Tech 2
Rep 1 Rep 2 Rep 3 Rep 1 Rep 2 Rep 3
10-1 28 20.2 20.4 20.4 20.6 21.7 22.4
10-2 28 24.3 24.5 24.0 24.9 25.5 26.2
10-3 29 27.5 28.0 28.4 27.2 27.2 28.1
10-4 29 31.3 31.0 31.2 29.4 30.3 30.2
10-5 44 ** 32.0 0 31.3 ** **
10-6 44 0 0 0 32.2 31.7 0
NTC 28/29/44 0 0 0 0 0 0
**Positive but not called by SmartCt
IC not displayed; negative for 10-1, 10-2 and 10-3. All others positive at Ct ~30
Runs Tech 1: 28/807, 29/299, 44/590Runs Tech 2: 29/300, 28/808, 44/591
Additional Multiple Reps, 10-5
• 18 reps, tested over 4 days, 2 techs
• 17 pos / 18 tested
• Runs: 44/615, 44/621, 28/x
NTC
Run: 44/615
Reps
Reps, 10-6 (below LOD)
• Tested 6 reps to ensure next log dilution is below LOD
• 2/6 amplified (confirmed below LOD)
Rep Dengue IC Comments
1 ** 30.1 Positive / Very Low Fluorescence
2 0 30.4
3 ** 30.9 Positive / Very Low Fluorescence
4 0 30.0
5 0 29.7
6 0 30.3
NTC 0 31.4
Run: 44/616
Wick for Sample Collection
• Sample collection via wick
• Zepto spiked into blood
• Use of blood either standard format (pipet 25µL) or collection via wick (16µL)
Run: 32/82