Biodetection Challenges: Broad perspectives on Sampling and Detection

Transcription

1 Biodetection Challenges: Broad perspectives on Sampling and Detection Sanjiv Shah, Ph.D. Photo image area measures 2 H x 6.93 W and can be masked by a collage strip of one, two or three images. The photo image area is located 3.19 from left and 3.81 from top of page. Each image used in collage should be reduced or cropped to a maximum of 2 high, stroked with a 1.5 pt white frame and positioned edge-to-edge with accompanying images APHL Annual Meeting May 20, 2012 Seattle, Washington

2 1

3 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 2

4 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) 3

5 4

6 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! 5

7 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) Establishment of the Department of Homeland Security (DHS) Systematic efforts on preparedness for bioterrorism events began Ten Years Later, We are Better Prepared than Before! 6

8 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 7

9 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 8

10 Sampling and Analyses 9

11 Sample Load Re-drawn from 2007 Biowatch document Need for adequate laboratory capacity to analyze a large number of samples during response and recovery 10

12 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 11

13 Sample Load Re-drawn from 2007 Biowatch document Need for adequate laboratory capacity to analyze a large number of samples during response and recovery 12

14 Post-Decontamination/Remediation Phase Sampling Thousands of Samples! Sample processing Microbiological methods to determine viability OR Rapid Viability-PCR method (RV-PCR) 13

15 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 14

16 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! 15

17 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 readyto-go kits are available for some sample types, better DNA/RNA purification devices, methods and kits have been developed 16

18 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 17

19 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 18

20 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! 19

21 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 20

22 Thank You Questions??? Sanjiv R. Shah, PhD U. S. Environmental Protection Agency Washington, DC (202)