Autonomous Pathogen Detection Autonomous Pathogen Detection SystemSystem

Lawrence Livermore National LaboratoryDr. John Dzenitis, 925-422-6695, john.m.dzenitis@llnl.gov

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)

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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