Autonomous Pathogen Detection Autonomous Pathogen Detection SystemSystem
Lawrence Livermore National LaboratoryDr. John Dzenitis, 925-422-6695, [email protected]
CHI Systems Integration in Biodefense21 August 2006, Washington, DC, USA
UCRL-PRES-223111
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The APDS Team
• Hardware– Tony Makarewicz– Tom Metz– Dora Gutierrez– Chris Spadaccini– Candice Cook– Ben Hindson– Bill Benett– Dean Urone– Jeff Loge
• Assays– Julie Perkins– Corey Chinn– CDC collaborators
• Control and signals – Bruce Henderer– Dean Hadley– Todd Weisgraber
• ConOps– Wendy Wilson
• Project– John Dzenitis– Ellen Raber
This work was performed under the auspices of the U. S. Department of Energy by the University of California, Lawrence Livermore National Laboratory under Contract No. W-7405-Eng-48.
Funding from Dept. of Homeland Security. Previous funding from DOE and DoD.
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Characteristics of civilian defense against bioterrorism
• Many possible threat agents– Assays must be multiplexed
• Operation is never-ending– Operating cost must be low
• High impact of alarms– Frequency of false positives must be low
• Response time includes public health action– Speed in initial detection traded for certainty
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BASIS and BioWatch: Centralized testing of air filters for biological agents
Distributed Sampling Units
Continuous collection of aerosols
Communications Network
Radio and Internet connections
BASIS Operations Center
Links to public health and law enforcement agencies
Relocatable Field Laboratory
Aerosol testing and analyses
Receiving, Reloading, and Dispatch
Filter sample management
Lawrence Livermore and Los Alamos National Laboratories
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Autonomous Pathogen Detection System:Analysis at collector, networked reporting of results
• Aerosol collection– Particle size selection– Samples are archived, can be cultured
• Sample preparation – Sequential injection analysis platform– Flexible and expandable
• Multiplexed detection and identification– Bead-based, Luminex read-out– Any antibody or DNA sequence can be
incorporated• Data acquisition and control
– Automated, centralized monitoring– Wireless, Cellular, & Ethernet networking
• One-week autonomy at 1 sample per hour
1.2
m (4
ft)
6
APDS provides significant improvements in monitoring capabilities
• Higher temporal resolution for rapid detection and response
• Two distinct (orthogonal) testing methods for high-confidence results
• Larger number of assays to detect more threat agents
• Lower labor requirements to minimize operational costs
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APDS provides laboratory-quality answers within hours of exposure
Sample 1PCR (DNA) test
Typical Time Lines
0 hr 1 hr 2 hr 3 hr
Sample 2Immunoassay test(multiplexed)
Sample 1(If positive)
Sample 1 Sample 3Sample 2Aerosol collection
Past “PCR confirmation” process
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High flow-rate aerosol collection
• 300 – 3,000 Lpm air sample in• 4 mL liquid sample out• Multistage
– Prefractionator cap– Virtual impactor– Wetted-wall cyclone
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APDS provides laboratory-quality answers within hours of exposure
Sample 1PCR (DNA) test
Typical Time Lines
0 hr 1 hr 2 hr 3 hr
Sample 2Immunoassay test(multiplexed)
Sample 1(If positive)
Sample 1 Sample 3Sample 2Aerosol collection
Past “PCR confirmation” process
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Luminex immunoassay platform permits screening for dozens of agents
LiquidArray
The sandwich fluoroimmunoassay is one of the most credible biodetection techniques
Adding a color code (optical bar code) to the capture bead enables individual ID
Different antibodies on each bead enables deeply multiplex detection
Beads can be analyzed by flow cytometry
at rates up to 10,000/s
Many different pathogens can be detected in a single assay
Capturebead
Bioagent Labeled antibody
Color analysis
Anthrax
Plague
Smallpox
Ricin
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Testing with environmental samples shows that the assays are robust
• Wetted-wall cyclone samples obtained in the field…– Subways: DC Metro and BART– Airports: ABQ and SFO– Other: U.S. Postal Service (non-APDS), lab
• …and compared quantitatively to plain water– Immuno and PCR assays run on bench-top– Unspiked samples– Yersinia pestis and Bacillus anthracis spiked samples
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Immunoassays robust in even the worst environmental samples
• All spiked with same thing, no change in sensitivity.Immunoassay Analysis (Yp Spikes)
0
10
20
30
40
50
-28 -23 -18 -13 -8 -3 2 7 12 17 22 27 32 37 42 47
Sample #
MFI
water spikedsample spikedwater meanwater-3 sigmawater+3 sigma
USPS DC Metro BART SFO Labwater
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Example of multiplex immunoassay signals from the APDS with a BoToxoid aerosol chamber release
All agents
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
time (days)
sign
al (
MFI
)
A01
A02
MS2
Bg
BoTox
A06
Ba
A08
Yp
A10
A11
NC
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APDS provides laboratory-quality answers within hours of exposure
Sample 1PCR (DNA) test
Typical Time Lines
0 hr 1 hr 2 hr 3 hr
Sample 2Immunoassay test(multiplexed)
Sample 1(If positive)
Sample 1 Sample 3Sample 2Aerosol collection
Past “PCR confirmation” process
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TaqMan PCR is used for confirmation of identified pathogens
• Excellent selectivity and sensitivity• BASIS and CDC/ LRN signatures currently used
– Cross-reactivity has been screened out
• Internal controls are used on every sample for high-confidence results
• System error rate is minimized by having orthogonal tests
FalsePCRctiveFalseIAReaiveFalsePosit ppp =
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Orthogonal identification using TaqMan PCR
• Uses DNA instead of protein recognition– Looking for different signature, so “orthogonal”– Tremendous amplification gives great sensitivity
• TaqMan used for confirmatory PCR
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Flow-through PCR module uses a custom but simple surface-mount approach
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An internal positive control gives confidence in PCR negatives
1.0
1.5
2.0
2.5
3.0
3.5
4.0
0 5 10 15 20 25 30 35 40 45
cycle number
PCR
sig
nal (
V)
Agent signal no Ba
Agent signal w/ Ba
Control signal no Ba
Control signal w/ Ba
control
B. anthracis +
B. anthracis -
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APDS network allows remote access to a wide range of system functions and reports
Data-rich command/control helps evaluate alerts
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APDS and its components have undergone extensive performance testing on the bench and in the field
• Laboratory– Aerosol collection– Immunoassay, including environmental samples– PCR, including environmental samples
• Dugway Proving Ground– Live-agent challenges of immuno. system– Killed-agent challenges of immuno.+PCR system
• In-field– Airports– Subways– Facilities
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Dugway testing in 2002 and 2003 demonstrated multiplexed immunoassay and PCR confirmation capabilities
• Objectives– End-to-end system tests
with aerosolized agents– Identify multiple agents
with a single panel– Automatically confirm
DNA with PCR– Demonstrate DNA
extraction module in system
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Identification and PCR confirmation of a B. anthracisrelease
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8time (days)
sign
al
A01A02
MS2Bg
BoTox
A06
Ba
A08
Yp
A10
A11
NC
Anthracis identified
0.8
1
1.2
1.4
1.6
1.8
2
0 5 10 15 20 25 30 35 40cycle
PCR
sig
nal
Ba releaseBlank
Anthracis confirmed
Immunoassay
PCR
Included DNA extraction module
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Proven in fully autonomous field testing
• 3 subways• 2 airports• Other facilities• Continuing tests across
the country– Over 19,000 field
samples– Over 95,000 assays run
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1.E-09
1.E-07
1.E-05
1.E-03
1.E-01
1.E+01
0 1 2 3 4 5 6
scaled threshold or signal
prob
. fal
se im
mun
o. r
eact
ive
0.E+00
1.E+04
2.E+04
3.E+04
4.E+04
5.E+04
6.E+04
Lim
it O
f Det
ecti
on
Sensitivity is inextricably linked to error rate through Receiver Operating Characteristic (ROC) curves
Note: Immuno. reactive would normally be checked with PCR
FalsePCRo.FalseImmunmFalseSyste ppp =
Theoretical example
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Comparing instrument data by analyzing immunoassay positives vs. threshold
1.00E-06
1.00E-05
1.00E-04
1.00E-03
1.00E-02
1.00E-01
1.00E+00
0 1 2 3 4 5 6
N SIgma
Pfa
151 Instrument
152 Instrument
153 Instrument
154 Instrument
155 Instrument
158 Instrument
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High-quality assays are the foundation of the APDS
• Luminex multiplexed immunoassay screening test– CDC Laboratory Response Network assays – Includes internal controls for high-confidence results
• TaqMan PCR secondary test– CDC Laboratory Response Network signatures – Includes internal positive control for high-confidence
results
• New assay developments are being added...
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We are integrating a new Multiplex PCR (MuxPCR) capability
• Tests for many DNA signatures at once – Signature sets can be based on TaqMan probes & primers
• Readout by hybridization to Luminex beads instead of electrophoresis or microarrays
• Much of the process and hardware are similar to the immunoassay
Encodedbead
Labeled DNAfrom PCR
Color analysis
Multiplex PCR process
Encodedbead
Labeledantibody
Color analysisBioagent
Current Multiplex Immunoassay process
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MuxPCR will be a step-change in monitoring capability for the APDS
• Allows running full CDC/ LRN PCR panel on every sample• Assay development and validation already underway for BioWatch• Better sensitivity • Easier reagent sourcing
(chemical synthesis, not animals) • APDS already has all required types of hardware and software
Collection
MultiplexImmunoassay
ConfirmatoryPCR assay
Current process
Collection
MultiplexPCR Assay
MultiplexImmunoassay
Improved process
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The APDS Team
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