Tetracore Profile T-COR 8TM Molecular Diagnostics Platform ... · Molecular Diagnostics Platform William M Nelson MD May 3, 2017. Tetracore Overview ... • Send text/e-mail notifications

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



5 OF #4- directInf Direct 50cy 27




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



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


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




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

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Tetracore Profile T-COR 8TM Molecular Diagnostics Platform ... · Molecular Diagnostics Platform William M Nelson MD May 3, 2017. Tetracore Overview ... • Send text/e-mail notifications