biodetection challenges: broad perspectives on sampling and … · 2016-05-17 · sanjiv shah,...

Office of Research and DevelopmentNational Homeland Security Research Center

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Biodetection Challenges:Broad perspectives on Sampling and Detection

Sanjiv Shah, Ph.D. National Homeland Security Research Center

2012 APHL Annual Meeting

May 20, 2012

Seattle, Washington


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Pre-9/11/2001 Biodetection Efforts

Defense against Biological Weapons

Protect our Warfighters on OCONUS lands

Mostly, DOD and DOE (National) laboratories efforts

Limited private industry interest and participation

Mostly, aerosol sampling and detection of Biological Warfare Agents (BWAs)

Establishment of CDC-Laboratory Response Network (LRN) – Public Health Focus


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Pre-9/11/2001……Continued

Aerosol/Environmental sampling, and identification of BWAs

Aerosol sampling, sample in liquid buffer, and identification

Traditional microbiological methods

Traditional mass spectrometric methods

Traditional immunoassays (ELISA)

Traditional PCR methods

BIDS, JBPDS

Advanced methods

• Improved Sample Collection Devices

• Better Selective Culture Media and Methods for some BWAs

• Better Immunoassays (ECL)

• Real-Time PCR Methods (TaqMan-PCR)

• Multiplex TaqMan PCR assays

Initiation of development of automated and integrated sample

preparation and detection biosensor systems (MIDAS)


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October 2001 Anthrax Events

Environmental and clinical samples within CONUS – Public Health at Risk

Heavy sample analysis burden on CDC, limited DOD and DOE Labs, and limited state public health and other labs for Many Months

Samples in a variety of environmental matrices

No systematically validated methods

“Use the Best Methods You Have” for Real-World Samples

Sampling methods and sampling materials

Real-time PCR

ELISA-HHAs

ECL-Immunoassays

Microbiological methods

We managed!


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Post-9/11 and October 2001 Anthrax Events

Analysis? What could have been done better? How?

Lessons learnt and improvements began and continue

Homeland Security Presidential Directives (HSPDs)

Establishment of the EPA’s National Homeland Security Research

Center (NHSRC) - 2002

Establishment of the Department of Homeland Security (DHS) - 2003

Systematic efforts on preparedness for bioterrorism events began

Ten Years Later, We are Better Prepared than Before!


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Post-9/11 and October 2001 Anthrax Events..

….Continued

Ten Years Later, We are Better Prepared Than Before – Why?

Many Government agencies work together better than before

Effective programs for protection, response, and remediation of infrastructure including Water

Explosion of Government funding and private industry participation

Development of many Biosensor Technologies and Methods

Many Commercially Available Biothreat Agent Assays/Reagents Kits

Many academicians contribute

Increased public awareness


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Integrated Consortium of Laboratory

Networks (ICLN)

Laboratory Response Network (LRN) – CDC

Environmental Response Laboratory Network (ERLN) – EPA

National Animal Health Laboratory Network (NAHLN) – USDA-APHIS

Food Emergency Response Network (FERN) – FDA

National Plant Diagnostic Network (NPDN) USDA-APHIS

Department of Defense Laboratory Network (DLN) – DOD

Monthly NCG Meetings


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Sampling and Analyses


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Re-drawn from 2007 Biowatch document

Sample Load

Need for adequate laboratory capacity to analyze a large

number of samples during response and recovery


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Pre-Decontamination Phase

Sampling – Hundreds-to-Thousands of Samples!

Sample processing

Real-Time PCR and/or immunological methods to determine extent of contamination

Limited culture and standard microbiology and serology methods for ID


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Re-drawn from 2007 Biowatch document

Sample Load

Need for adequate laboratory capacity to analyze a large

number of samples during response and recovery


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Post-Decontamination/Remediation Phase

Sampling – Thousands of Samples!

Sample processing

Microbiological methods to determine viability

OR

Rapid Viability-PCR method (RV-PCR)


Sampling (Sample Collection)

Sampling considerations

Sample can be air, water, any surface, carpet, clothes, white powder,

vegetation, soil, etc.

Different sampling devices, sampling materials and physical structure and

chemical composition of materials

Different ways/techniques of sampling, and area and/or amount to be

sampled

Sample transport and maintenance of integrity of sample/bioagent

Sample storage before analysis

Sample archival

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Sampling (Sample Collection) Challenges

Challenges

No Single technique would work for all different types of samples: air,

water, any surface, carpet, clothes, white powder, vegetation, soil, etc.

No Single technique would work for all scenarios: Different

ways/techniques of sampling, and area and/or amount to be sampled

Optimum procedures and conditions not established for transport and

maintenance of integrity of samples in different environmental matrices

and for different bioagent types

Validation of sampling methods for different types of biothreat agents

in different sample types/environmental matrices, potential incidence

areas and environmental conditions, and quality considerations

Gov agency/Lab-Network-specific requirements!

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Sample Processing

Sample Processing Considerations

Different Forms of Biothreat Agents: Spore, Vegetative Cell, Virus

(DNA/RNA Virus), Toxin

Analytical Method-Conducive Sample Prep to purify target molecules:

DNA, RNA, Protein/antigen

Sample Prep Method and Limit of Detection (LOD) of Analytical Method

Storage and archival of extracted bioagent target material

Significant innovations and improvements have occurred: Many ready-

to-go kits are available for some sample types, better DNA/RNA purification

devices, methods and kits have been developed

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Sample Processing Challenges

……Still a Bottleneck!!

No standardized methods for pre-processing of different types of sample articles and samples in different environmental matrices

No standardized single method and kit for extraction and purification of DNA, RNA, and protein from the single/same aliquot of sample

Spore lysis and extraction of DNA is still a challenge: Improvements need to be made for either quick physical disruption of spores or quick spore germination

High throughput?

Concentration of sample and post-sample prep concentration of target material are still challenges for a better LOD.

Validation of sample prep methods for different types of biothreat agents in different sample types/environmental matrices and quality

Automation and integration with sensor device need more efforts

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Analytical Methods for

Biothreat Agent Detection

Real-time PCR assays and biosensors/thermal cyclers to detect and

identify biothreat agent based on specific gene markers on their DNA

or RNA

Highly sensitive and specific Real-time PCR assays (DNA/RNA) for

multiple biosensors are available for many biothreat agents : Also, for

high throughput biosensors

Limited multiplexing capability

HHAs and ECL-based immunoassays to detect and identify biothreat

agent based on specific protein markers

Better HHAs, and high throughput and improved ECL-immunoassays are

available

GC-MS and LC-MS methods for biological toxins

Better instruments and sensitive methods have been developed

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Analytical Methods Challenges

No standardized Real-time PCR, immunoassay, GC-MS or LC-MS

methods for biological select agents that is available to all Gov

Agencies

Standardized reference material (DNA, RNA, inactivated or live

agents) for assay development and validation is not easily available:

Additionally, safety, regulatory, and other challenges

Validation of different analytical methods for different types of

biothreat agents in different sample types/environmental matrices and

quality considerations

Gov agency/Lab-Network-specific requirements!

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Current Government Efforts Against

Challenges

Sampling

Validated Sampling Plan (VSP) Inter Agency effort led by DHS

Testing exercises at Idaho National Lab (INL): INL-1 (2007), INL-2 (2008), and BOTE (2011)

Sampling method validation efforts at CDC

Development of improved sampling methods – Various Gov agencies

Sample preparation

Search for new/better/improved technologies, methods, and reagents continues

Analytical methods

Search for new/better/improved technologies, methods, and reagents continues

Standards for validation of Real-time PCR assays for few select agents established (DHS-led effort), further refinement continues (Interagency effort)

Biodetection Standards – White House OSTP effort

Development of Rapid Viability PCR (RV-PCR) method

Multiple Government agencies are addressing these challenges

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Thank You…

Questions???

Sanjiv R. Shah, PhDNational Homeland Security Research Center

Office of Research and Development

U. S. Environmental Protection Agency

Washington, DC

(202) 564-9522

[email protected]

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biodetection challenges: broad perspectives on sampling and … · 2016-05-17 · sanjiv shah,...

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