OCTOBER 16 & 17 2018

S takeholder P anel on A gent D etection A ssays (SPADA)

Stakeholder Panel and Working Group Meetings 2275 Research Boulevard Conference Room #110 Rockville, Maryland, United States


OCTOBER 16: 9:00am – 4:00pm ET OCTOBER 17: 9:00am – 4:00pm ET Registration Opens 8:00AM AOAC STAKEHOLDER PANEL ON AGENT DETECTION ASSAYS (SPADA)


Welcome and Introductions (9:00am – 9:30pm)  Dave Schmidt, AOAC Executive Director & Linda C. Beck, PhD, SPADA Chair  a. Policies and Procedures  b. Introductions 


SPADA Background and History (9:30am – 10:30am) Linda C. Beck, PhD, JRAD, SPADA Chair  a. SPADA Early Work   b. SPADA DoD Initiatives   


III. Standards Development at AOAC INTERNATIONAL (10:45am – 11:30pm)  Deborah McKenzie, AOAC Sr. Director, Standards Development 

IV. Working Groups Launch:  Soil Testing Working Group (1:00pm – 4:00pm)  a. Working Group Launch Presentation  i. Goals, timelines and rosters 

b. Background Presentations 

i. Linda C. Beck, PhD, JRAD, DUSA‐TE; JPEO JPM Guardian, DBPAO  ii. Morgan Minyard, PhD, DTRA  iii. Sherry Blight, PhD, Battelle, CBRNE Defense/Aerosol 

c. Working Group Discussion 

i. Review the proposed outline of the project as presented  ii. Review existing practices for preparation and use of soil samples  iii. Identify gaps in existing practices  iv. Commence process of developing format for standard and parameters to  include

V7                                                               10/12/2018

This agenda is subject to change without notice

AOAC INTERNATIONAL   Stakeholder Panel on Agent Detection Assays (SPADA)  Working Group Sessions – Wednesday, October 17, 2018  9:00 a.m. – 4:00 p.m. Conference Room 101 


I. Working Group Kickoff Meetings:  Bacterial Strain Verification Working Group   (9:00am – 12:00pm) 

a. Working Group Launch Presentation  i. Goals, timelines and rosters 

b. Background Presentations 

i. Shanmuga Sozhamannan, PhD, Tauri Group, JPEO JPM Guardian, DBPAO  ii. David Rozak, PhD, USAMRIID 

iii. Ian Gut, PhD, NBACC  iv. Katalin Kiss, PhD, ATCC 

c. Working Group Discussion 

i. Review the proposed outline of the project as presented   ii. Identify the minimum requirements needed to verify the species/strain of a culture.    With help of the contractor and WG chair, BSVWG will prepare a draft standard for verification of  bacterial strains to be presented to SPADA for consideration of approval at the second SPADA meeting 

II. Working Group Kickoff Meetings:   In‐Silico Analysis Working Group  (1:00pm – 4:00pm) 

a. Working Group Launch Presentation  i. Goals, Timelines and Rosters 

b. Background Presentations 

i. Shanmuga Sozhamannan, PhD, Tauri Group, JPEO JPM Guardian, DBPAO  ii. Jeff Koehler, PhD, USAMRIID  iii. Trevor Brown, PhD, JPM MCS/JPEO 

c. Working Group Discussion  

i. Review the proposed outline of the project as presented   ii. Identify the different steps that can be modified/eliminated from wet lab testing by  in silico PCR  analysis. The group will review and discuss features of the different databases and determine if  one or more shall be the focus of the standard setting effort  iii. Identify approaches for in silico analyses and potential software to use  iv. Determine the optimal settings for each  in silico PCR analysis setting  v. Discuss and determine how  in silico PCR analysis can fit into existing evaluation/validation  paradigm and prescribe the procedures of the use of  in silico PCR analysis  vi. Commence preparation of a draft standard to use  in silico PCR analysis to present to SPADA for  consideration of approval at the next SPADA meeting

V7                                                               10/12/2018

This agenda is subject to change without notice

AOAC Stakeholder Panel on Agent Detection Assays (SPADA) Program Background

Linda C. Beck, PhD, SPADA Chair Joint Research and Development (JRAD) October 16-17, 2018

AOAC Staff Contacts

• David B. Schmidt, Executive Director,

• Jonathan Goodwin, Deputy Executive Director,

• Krystyna McIver, SPADA Project Executive,

• Deborah McKenzie, Sr. Dir., Standards Development,

• Scott Coates, Sr. Director, AOAC Research Institute,

• Christopher Dent, Standards Development Manager,



• SPADA Background and History

• Background on previous standards / SMPRs

• Organization

– Working Groups vs. Stakeholder Panels

• Current Initiative / Meeting Goals


SPADA Sets Standards 2007 ‐ 2013

• A voluntary consensus standards body originally established via a DHS S&T

SPADA Executive  Steering Committee


First Responder  Working Group


B. anthracis Working  Group (PCR)

B. anthracis HHA  Working Group 

• Includes representatives from DHS, CDC, DoD, DoJ, FDA, EPA, USPS, NIST, State & Local Public Health, First Responders, Industry, and Academia • Establishes method performance requirements and panels of reference materials (and validation protocols)

Y. Pestis Working Group (PCR)

Ricin HHA Working  Group 

F. tularensis Working  Group (PCR)

Burkholderia  Working Group (PCR)

Environmental  Factors Working  Group (PCR) Public Health  Actionable Assay  Working Group* 

Assay Control  Working Group (PCR )

Variola Working  Group (PCR)

 All SPADA members volunteer their time  and expertise 

Original Objectives in 2007 - Establish standards to validate Polymerase Chain Reaction (PCR)‐based  technologies that detect aerosolized Bacillus anthracis , Yersinia Pestis , or  Francisella tularensis - Pilot the validation process with an assay that detects B. anthracis 2009 - Develop standards to validate immunoassay‐based Hand‐Held Assays (HHAs) that  detect B. anthracis or Ricin in suspicious powders - Test commercially‐available HHAs 2010 - Develop standards to validate PCR‐based technologies that detect aerosolized  Burkholderia psuedomallei and Burkholderia mallei - Develop standards to validate PCR‐based technologies that detect B. anthracis  in  suspicious powders 2011 - Develop recommendations on controls needed for field‐based assays 2013 - Develop standards to validate PCR‐based technologies that detect aerosolized  Variola - Establish First Responder Working Group - Maintain a SPADA Executive Steering Committee SPADA Sets Standards 2007 - 2013


SPADA Working Groups 2007 ‐ 2013

B. Anthracis Handheld Assay Working Group  (BaHHAWG) Marian McKee, BioReliance Corp. Ricin Handheld Assay Working Group (RicinHHAWG) Mark Poli, DoD

B. anthracis Working Group (BaWG) Paul Jackson, LLNL and Ted Hadfield,  MRI

Y. pestis Working Group (YpWG) Luther Lindler , DHS

Burkholderia Working Group (Bur WG) Paul Keim, NAU and Alex Hoffmaster, CDC

F. tularensis Working Group (FtWG) Peter Emanuel, DoD Mark Wolcott, DoD

Assay Controls Working Group (ACWG) Christina Egan, NYSDH and Larry Blyn, Ibis

Environmental Factors Working Group (EFWG) Stephen Morse, CDC

Variola Working Group (VWG) Victoria Olson, CDC and Ted Hadfield,  MRI

Public Health Actionable Assay Working Group  (PHAAWG) Peter Estacio, LLNL

SPADA Sets Standards 2014 ‐ 2016

• A voluntary consensus  standards body established  via DUSA‐TE sponsored  project  through JHU/APL • Includes representatives  from DHS, CDC, DoD, DoJ,  FDA, EPA, USPS, NIST, State  & Local Public Health, First  Responders, Industry, and  Academia • Establishes Standard  Method Performance  Requirements (SMPRs)  that  include  inclusivity/exclusivity panels


VEE Working Group 

B. anthracis Working  Group 

C. burnetti Working Group 

Brucella suis Working Group 

Burkholderia pseudomallei Working Group Botulinum Neurotoxin A  Working Group

SEB Working Group

Y. Pestis Working Group

F. tularensis Working  Group

Variola Working Group 

 All SPADA members volunteer  their time and expertise 


SPADA Sets Standards 2014 - 2016

 Under Contract with Deputy Undersecretary of the Army‐ Test and  Evaluation through The Johns Hopkins University, Applied Physics  Laboratory 2014 - Establish standards to validate technologies that detect  Venezuelan Equine Encephalitis Virus, Staphylococcus Entertoxin B, and Coxiella burnetti (Q‐fever) with emphasis on the  warfighter. 2015 – 2016 - Establish standards to validate technologies that detect Bacillus  anthracis , Yersinia Pestis ,  Francisella tularensis, Brucella suis,  Burkholderia pseudomallei, Variola, and Botulinum Neurotoxin A  with emphasis on the warfighter. 8

SPADA Working Groups 2014 ‐ 2016

Concluded at September 2015 SPADA Meeting:

Concluded at August 30, 2016 SPADA Meeting:

Venezuelan Equine Encephalitis WG James Samuel, U of Texas, A&M

Burkholderia pseudomallei WG Jay Gee, CDC

C. Burnetti WG Eileen Ostlund, USDA, ARS

Brucella suis WG Frank Roberto, Idaho Natl. Laboratory

SEB WG Sandra Tallent , FDA

Variola WG Victoria Olson, CDC

Approved at March 22 – 23, 2016 Meeting:

B. anthracis WG  Paul Jackson, LLNL and Ted Hadfield, Hadeco

Botulinum Neurotoxin A WG Sashi Sharma, FDA, HHS

Y. pestis WG Luther Lindler, DHS

F. tularensis WG Paul Keim, Northern Arizona University 

SPADA ‐ 2017

• Tasked with streamlining  the Environmental  Organisms Panel  • EOP is an evaluation of  potential cross‐reactivity to  the DNA from a wide variety  of organisms that are in the  environment


Environmental  Organisms Panel  Working Group

• SPADA streamlined this  panel for to promote  efficiency and modernity

 All SPADA members volunteer  their time and expertise 


SPADA Working Groups 2017

Concluded at April 2017 SPADA Meeting:

Environmental Organisms Panel Working Group Chair:  Linda C. Beck, JRAD (Formerly of NSWC  Dahlgren) SPADA voted to accept a revised environmental  factors panel appendix containing the final  environmental panel as an addition to all existing  DoD SMPRs for  aerosol samples.  

Environmental Organisms Panel E nvironmental Factors Annex (in all  SMPRs); three sections: • Environmental Matrix Samples – Aerosol;  Soil Testing • Environmental Organisms • Potential Interferents (operational  background)


Publication & Availability • An overview of the JHU APL/DoD/AOAC  project and all 10 SMPRs together with the  revised Environmental Organisms Panel is  being published in the Nov/Dec issue of the  Journal of AOAC INTERNATIONAL. • It will be available on the web with a DOI  (Document Object Identifier). • URL will be made available to DoD and  SPADA members.



AOAC Standard Methods Performance Requirements • A standard for analytical methodology. – the traditional standard was a description of a method. – an SMPR specifies the minimum performance requirements for a methodology.

• Documents a community’s analytical needs

• Description of the analytical requirements

• Includes method acceptance requirements


AOAC Standard Methods Performance Requirements

• Previous SPADA  initiatives have  developed 19 sets of  AOAC  Standard  Method Performance  Requirements® (SMPR®)

Standard Methods Performance Requirements

General Format

– Intended Use – Applicability – Analytical technique – System suitability

– Reference materials – Validation guidance – Maximum time-to-determination – Method performance requirements table – Inclusivity/exclusivity/environmental contaminants

Standard Methods Performance Requirements

Use of SMPRs • Guidance to developers for the development of new assays. • Advance the state-of-the-art in a particular direction. • Address specific analytical needs. • Specifications for acquisition. • Vendor self-qualification. • Basis for method acceptance and AOAC approval.

Organization: AOAC Stakeholder Panels

• Standard adopting bodies for AOAC • Meetings are open to all interested parties. • Oversee working groups • Typically 50+ members. • Voting members are vetted based on: • Expertise • Perspective/sector (government, academia, industry, etc.) • Availability


October 2018 Kick- Off for New AOAC SPADA Initiative This work is collaboratively funded by:  The Department of the Undersecretary of the      Army ‐ Test and Evaluation   (DUSA‐TE)

Joint Program Executive Office Joint Program Manager – Guardian         Defense Biological Product Assurance  Office (JPM‐ Guardian DBPAO) 20

October 2018 Meeting Goals

SPADA will launch new working groups on:

 Bacterial Strain Verification  Co-Chairs Linda C. Beck, JRAD and Shanmuga Sozhamannan, ECBC  In-Silico Analysis  Co-Chairs Linda C. Beck, JRAD and Shanmuga Sozhamannan, ECBC  Soil Analysis  Co-Chairs Linda C. Beck, JRAD and Morgan Minyard, DTRA

This work is funded by  DUSA‐TE and JPEO JPM Guardian, DBPAO


Bacterial Strain Verification

Bacteria Strain Verification Working Group It is not an uncommon occurrence for an assay  developer, researcher, or evaluator to discover that a  bacterial culture is not what it is purported to be in  terms of species or strain. There is no consensus on  the process to authenticate bacterial strain. This group  will work to develop guidelines for the characterization  and authentication of bacterial strains to provide  confidence in the identification of material being used.


In-Silico Analysis

In Silico Analysis Working Group

With the advantages of  in silico PCR analysis, the building  of confidence in the results can been enhanced by  establishing standards and recommendations for use as a  complimentary tool to wet testing. The goal of the AOAC  SPADA Working Group is to draft standard procedures for  the use of in silico PCR analysis, so different analysts  working across separate laboratories will achieve  equivalent results, and thus build confidence in the data  from  in silico PCR analysis. 


Soil Analysis

Soil Testing Working Group

Increased confidence and reliability in performance of assays  that meet the operational needs when deployed in the field  requires evaluating inhibition, interference, and cross‐ reactivity of an assay. Currently there is a lack of consensus  standards for the preparation and use of soil testing samples.  The goal of the AOAC SPADA Working Group is to draft  standard procedures for preparation and characterization of  soils to be used in the evaluation of candidate biothreat  detection assays.





AOAC Standards Development: An Overview

AOAC SPADA Meeting Rockville, MD USA Tuesday, October 16, 2018

Deborah McKenzie Sr. Director, Standards and Official Methods SM AOAC INTERNATIONAL

AOAC began in Washington, DC as the Association  of Official Agricultural Chemists (1884)

• Federal and state departments of  agriculture through the USDA  Bureau of Chemistry.   Initially to standardize  methodology to be used for  composition of fertilizers by state  laboratories  Directed by Harvey Washington  Wiley who wrote the 1906 law  that began the US Food and Drug  Administration (FDA) 

• By the 1980s AOAC’s membership  included microbiologist, food  science professionals • In 1991, Association of Official  Agricultural Chemists  legally  changed its name to AOAC  INTERNATIONAL  • Often referred to as Association of  Analytical Communities ‐ used to  encompass all of the scientific  disciplines involved in AOAC’s work.



AOAC Leverages the Power of Many

AOAC Leverages Networks to Assemble Stakeholders & Experts


• Develop international voluntary consensus standards method performance requirements

• Discuss & adopt methods that are published in the Official Methods of Analysis of AOAC INTERNATIONAL using judgment of the world’s leading experts. Providing fit for purpose methods through standards development

GeneralLocationsof AOAC stakeholderpanelparticipants GeneralLocationsof the16 AOAC INTERNATIONALcurrentSections

AOAC ® INTERNATIONAL (AOAC) is an independent third‐party international standards  developing organization and AOAC has no vested interest in the development of standards or in  the evaluation of methods of analysis.

AOAC Stakeholder Panels

Strategic Food  Analytical  Methods SPSFAM

Agent  Detection 

Alternative  Methods ISPAM

AOAC   Standards  Developing  Communities

Assays SPADA

Infant Formula  and Adult  Nutritionals SPIFAN

Dietary  Supplements SPDS



What Are Standards?

Common and repeated use of rules, conditions, guidelines  or characteristics for products or related processes . 

The definition of terms; classification of components;  delineation of procedures; processes, products, systems,  services, or practices;  test methods and sampling  procedures. A Performance standard is a standard that states  requirements in terms of required results with criteria for  verifying compliance but without stating the methods for  achieving required results. Voluntary Consensus standards are standards developed or  adopted by voluntary consensus standards bodies , both  domestic and international.

US OMB Circular A‐119

AOAC Consensus Standards

Examples: AOAC  Standards

Basic Principles

• Transparency • Openness • Balance of Interests • Due Process • Consensus • Appeals

• Performance  Requirements • Guidelines

• Sampling Standards •Methods of Analysis

• Best Practices • Operational  Documents



Advisory Panel, Stakeholder Panel & Working Groups

Advisory  Panel


• Community of experts and key stakeholders • Balanced representative subset of vetted voting stakeholders • Anyone with a material interest can participate

Stakeholder Panel

• Consists of technical experts from Industry, Government, CRO’s, and Academia to develop method performance criteria required for needed methods • Present background and history on analytical method needs for stakeholder panel • Members approved by SPADA Chair • Begin drafting standards

Working  Groups

Stakeholder Panel Role and Output

• Defines specific analytical issue(s)

1 st

• Forms working groups to draft standard(s)  that address the issue(s)

2 nd

• Comments on draft standard(s)

3 rd

• Adopts voluntary consensus standard(s)

4 th

AOAC Voluntary Consensus Standards  – Published in Official Methods of Analysis of AOAC INTERNATIONAL – Manuscript published in  Journal of AOAC INTERNATIONAL



Working Groups

• SPADA has had 18 working groups – 19 standards developed across 11 biological threat agents

– 3 new working groups formed

• Soils Testing ‐ (Dr. Beck and Dr. Minyard) • Bacterial Strain Verification – (Dr. Beck & Dr. Sozhamannan) • In Silico Analysis – (Dr. Beck & Dr. Sozhamannan)

– Working groups to begin work during this SPADA meeting on  draft standards • Continue over next 8 months

Standards Drafting Overview

Working  Group  Meets 

• Draft 


• Public 

Post draft  standard

Comment  Period        (≥ 30 days)

Reconcile  Comments 

• Recommend  final draft to  SPADA



Establish AOAC Standards

Establish  Standards

• SPADA demonstrates consensus on any standard • Documents a community’s needs • Very detailed description of the analytical requirements • Published by AOAC as a standard

Second SPADA meeting

• Working Groups present draft standard

• Vetted Representative Voting Members  selected based on registration and  organizational or subject matter perspective – represents balance of perspectives – Demonstrates consensus on behalf of  stakeholders – 2/3 vote in favor of a motion establishes an  AOAC standard



Documentation and Communication

• AOAC carefully documents the actions of Stakeholder Panel,   Working Groups, and ERPs

• AOAC will prepare summaries of the meetings  – Communicate summaries to the stakeholders – Publish summaries in the Referee section of AOAC’s  Inside  Laboratory Management • AOAC publishes its voluntary consensus standards and Official  Methods – Official Methods of Analysis of AOAC INTERNATIONAL – Journal of AOAC INTERNATIONAL • AOAC publishes the status of standards and methods in the Referee  section of AOAC’s  Inside Laboratory Management



AOAC Standards Published Since 2011

OTHER 3% AOAC Standards Developed




SPDS 30%





Soil Standards for Assay  Development and T&E

Linda C. Beck, Ph.D. Principal Biological Scientist Joint Research and Development, Inc. Supporting DUSA –TE and JPEO JPM Guardian

This work was funded by  DUSA-TE and JPEO JPM Guardian, DBPAO


• AOAC Stakeholder Panel on Agent Detection Assays (SPADA) Standards Project: • Standard Methods Performance Requirements: SMPR (10) • Documents community’s analytical needs • Descripts analytical requirements • Includes method acceptance requirements • Panel – consensus standards body:  • DoD, DHS, CDC, FDA, EPA, USPS, NIST, ATCC, State and Local PH, Industry, Academia,  First Responders

• Most recent panel WG effort:  • Environmental Factors Annex (in all SMPRs); three sections: • Environmental Matrix Samples – Aerosol; Soil Testing • Environmental Organisms • Potential Interferents (operational background) Soil testing has always generated the most  questions


• Recommendations to evaluate candidate assays using soil  samples was added to Part 2 of the Environmental Factors  appendix in 2017: • 2.2:  Soil Testing • “Airborne soil particles may constitute a significant challenge  to the analysis of collected aerosol samples by polymerase  chain reaction (PCR) assays.  Soils contain genomic materials  or nucleic acid fragments of countless archaebacterial,  bacterial, and eukaryotic organisms.  Some of the more  common soil organisms can be anticipated.  Soils may also  contain unanticipated components that interfere with  extraction, denaturation, polymerization, or annealing  reactions. Therefore, determining the effect of a variety of  representative soils on the robustness of a PCR assay is an  important first step.”  • However, instructions in the Soil Testing section are  extremely limited because  there was not a consensus on  how to conduct soil testing , nor what kinds of soil samples  to use (T&E; assay development; industry/gov’t )


• Lack of uniformity for assay development and comparison of performance  across vendors for the USG

• Limited soil representations 

• Unknown soil composition as relates to bio assay performance testing 

• The community does not evaluate an assays performance using consistent  standard soil samples and standard methodology for testing

• Testing for cross reactivity and inhibition limited to the near neighbors 

• Testing interferents conducted with identified operational interferents (i.e.  diesel exhaust, sea salt, etc.) during DT, DT/OT and O


Develop specific soil standards to support assay development  and T&E testing (Bio detection/identification)

Standards for: • Characterization of soil – parameters • Representative soils for testing

PATH FORWARD  • Develop the process for a standard method of soil  collection  • Identify/characterize types of soil needed • Create soil standards for material development  • Standardize the characterization of soil • Support the development and archival of test  materials


JRAD Proprietary


Soil 101

Morgan Minyard

Soil 101 Soils are very complex and unique They are an important natural resource that is decreasing over time In US alone, over 19,000 soils have been identified The study of soil science includes taxonomy, chemistry, physics and  biology in addition to agricultural and conservation practices.

Soil are:  •Plant growth medium •Regulator for water supply •Recycler of raw materials •Habitat for Soil Organisms •Engineering Medium •Environmental interface

Soil horizons:  •Consist of the O, A, E, B and C horizon •Not all horizons are present in a soil •Each horizon has different properties •The soil horizons and their properties identify  the soil •Soil on top of another soil is possible, usually  due to flooding

The 12 Soil Orders

• Entisol – Youthful soil with little profile development • Andisol – Not a well developed clay layer but prominent organic‐rich A  • Gelisol – Permafrost within 1 m of soil surface • Histosol – Organic accumulation of great depth due to anaerobic conditions • Inceptisol – More well developed clay layer or B horizon • Aridisol – Arid to semi‐arid ecosystems of shrub and short grasses • Vertisol – Highly‐active and swelling clay  • Mollisol – Semi‐arid or moist grassland ecosystem and deep organic‐rich A • Alfisol – Clayey B horizon with low acidity • Spodosol – Humic B horizon with high acidity and coarse texture (sandy)

• Ultisol – Clayey B horizon with high acidity • Oxisol – Oxic B hoizon with low‐activity clay

5 Soil forming factors

• Climate – Weather regime • Parent Material – includes bedrock and deposited sediments • Organisms – biota includes bacteria to trees to animals • Relief – topography makes a deference • Time – weathering time not actual

Soil profile includes the Picacho inceptisol that is underlaid by 4 ‐ 8 m of saprolite

One of the fastest weathering granodiorite in  the world.  Forms a unique soil profile  Quick percolation so a well drained soil

Granodiorite exposed road cut demonstrates that bedrock undergoes spheroidal  weathering to saprock

Bedrock Minerals: Quartz (20‐24%) Plagioclase (50%) Biotite (9%)  Hornblende (6%) K‐feldspar (2%) Fe, Ti oxides (2%) Apatite (0.6%)

Saprolite Minerals: Mica Iron oxides – Goethite and Hematite Halloysite Kaolinite Quartz

Saprock (Cr)

White et al. 1998; Buss et al., 2008

Soil Texture

Soil Texture

Soil Texture: • Critical to understanding soil behavior • Varies little with time • Impacts water retention  • Impacts gas exchange • Impacts nutrient retention • active fraction of soil (clay) • Silt includes bedrock material and minerals • Sand lest active and less surface area

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Habitat for Soil Organisms

• Primary Producers • Plants • Moss • Algae • Lichens • Primary Consumers • Herbivores

• Photosynthetic Micobes

• Detritivores (dead leaves) • Saprophytic (dead tissue)

• Secondary Consumers

• Consume Primary Consumers

• Tertiary Consumers • Predators • Microbial feeders • Ecosystem Engineers • Earthworms • Burrowing animals 

• Certain beetles – dung beetle

Habitat for Soil Organisms

Organisms Microflora Bacteria

Number per m 2

Number per gram soil

10 13 ‐10 14 10 12 ‐10 13 10 10 ‐10 11 10 9 ‐10 10 10 9 ‐10 10 10 6 ‐10 7 10 3 ‐10 6 10 3 ‐10 6 10‐10 3 10 2 ‐10 4

10 8 ‐10 9 10 7 ‐10 8 10 5 ‐10 6 10 4 ‐10 5

Actinomycetes Fungi Algae


Protozoa Nematodes Mites Collembola Earthworms Other fauna

10 4 ‐10 5 10‐10 2

1‐10 1‐10

KSU image

Soil porosity and soil drainage

• Soil porosity impacts:

• Water flow and therefore  drainage • Gas exchange • Mineral weathering • Root growth

Anaerobic microbial  processes, reduced iron

Oxic conditions,  oxidation of iron to iron  oxides

Soil pH • Impacts nutrient availability • Acidic soils Al3+ exchangeable and heavy metals • Neutral and basic soils Cations like Calcium and potassium exchangeable • Biota diversity • Soil horizons and structure

pH range Ultra acidic < 3.5   Extremely acidic 3.5–4.4   Very strongly acidic 4.5–5.0   Strongly acidic 5.1–5.5   Moderately acidic 5.6–6.0   Slightly acidic 6.1–6.5   Neutral 6.6–7.3   Slightly alkaline 7.4–7.8   Moderately alkaline 7.9–8.4   Strongly alkaline 8.5–9.0   Very strongly alkaline > 9.0  

Red more acidic Blue more basic Yellow neutral

Soil Internet Resources

• Web Soil Survey •

• Critical Zone Exploration Network and Critical Zone Observatories •‐zone‐exploration‐ network/

• Soil Testing Labs •‐testing

Soil experiment guidelines • OECD 106 Guideline • Provides Guidance on: • Chemical properties that should  be known • Soil characteristics • Soil sampling • Soil storage Soil type

pH range (in 0.01 M  CaCl2)

Organic carbon  content (%)

Clay content (%)

Soil texture *

1 2 3 4 5 6 7


1.0‐2.0 3.5‐5.0 1.5‐3.0 3.0‐4.0

65‐80 20‐40 15‐25 15‐30


> 7.5 

clay loam silt loam

5.5‐7.0 4.0‐5.5


< 4.0‐6.0

< 0.5‐1.5 < 0.5‐1.0

< 10‐15

loamy sand

> 7.0  < 4.5 

40‐65 < 10 

clay loam/clay

> 10 

sand/loamy sand

Protein Extraction from Soils

Sherry Blight, Ph.D. Principal Research Scientist Battelle Memorial Institute October 2018

Distribution Statement D: Distribution authorized to Department of Defense and their contractors in order to protect technical data or information from automatic dissemination. Other requests for this document shall be referred to the Joint Project Manager Guardian, 2800 Bush River Road, Aberdeen Proving Ground, MD 21010-5424.


Common Analytical Laboratory System (CALS) • Since 2015 • ALS since 2012 • MSD PR2-1800 is the toxin detector used


National Guard Response Sectors


Soil Testing on the PR2 • 1X Phosphate buffered saline + 0.1% Triton X-100 (PBST) • Purified botulinum neurotoxin type A (BoNT/A), ricin, and Staphylococcus aureus enterotoxin B (SEB) • NIST Soils: Montana I, New York/New Jersey Waterway Sediment, Flint Clay • 4 extraction protocols/buffers were tested  Current CST  10X PBST  MSD proprietary buffer (PBST with BSA and azide)  MO BIO NoviPure TM Soil Protein Extraction Kit (garnet beads + vortex)


Results – BoNT/A (ng/mL)




NY/NJ Flint Clay Montana 1


Average ECL



10X PBST MSD Buffer MO BIO Buffer CST (PBST)

Extraction Method


Results – SEB (ng/mL)





NY/NJ Flint Clay Montana 1


200 Average ECL



10X PBST MSD Buffer MoBio Buffer CST (PBST)

Extraction Method


Results – Ricin (ng/mL)




NY/NJ Flint Clay Montana 1



Average ECL


10X PBST MSD Buffer

MoBio Buffer


Extraction Method


Other Soils Tested on the PR2

• Sigma - clean loam (RTC) • Sigma also has a few other “clean” soils (RTC)  Clay loam, clay, sandy loam, sand


Naval Medical Research Center (NMRC) PR2 Confidence Testing • 98 different soil samples were purchased from the North American Proficiency Testing (NAPT) Program of the Soil Science Society of America • One potting soil purchased locally • Same CST PBST extraction utilized • Toxin LOD determination possible, but at some cost to sensitivity (but specificity gained)


Final Thoughts • Clean soil background (or “why we are here”)  CSTs currently using PBST to determine thresholds

 Storage conditions?  How do we obtain? • Potentially a standardized protein extraction protocol  PBST is not the ideal extraction buffer  Is this possible? Instrumentation, procedural issues  Varies from protein to protein of interest • National Ecological Observatory Network (NEON) (National Science Foundation [NSF]) – collect field data from 81 sites (47 terrestrial)




800.201.2011 | |

Defense Biological Product Assurance Office Presented to SPADA on October 17, 2018 SPADA- Strain Verification Standards for Assay Development

Shanmuga Sozhamannan, Technical Coordinator, DBPAO Joint Program Executive Officer for Chemical, Biological, Radiological and Nuclear Defense This work was funded by DUSA-TE and JPEO JPM Guardian, DBPAO

UNCLASSIFIED Distribution Statement A:Approved for public release; distribution isunlimited.

Why do we need a Standard for strain verification and authentication; i.e., characterization?-

There is no such consensus standard agreed upon by the community



Defense Biological Product Assurance Office (The of DoD) Dugway Proving Ground anthracis debacle CRP/DBPAO Major Milestones




DBPAO Product portfolio

Ordering System For Critical Assays and Reagents (OSCAR)- “” for Biodefense products




All hell broke loose in the summer of 2015 (May 22 nd to be precise)

News about anthrax mailings




The Solution to the problem- Risk Mitigated Reference Materials (RM2)

SPARK: Guaranteed Risk free because the organism does not have the ability to cause disease

This effort is unique and to the best of our knowledge does not exist in JPEO or other agencies




Lesson from Genome Sequencing Standards • Genomics Standards Consortium • MIGS • Minimum Information about a Genome Sequence- captures information about a genome sequence- e.g., depth and breadth of coverage, sequencer type, organism information etc (Chain et al: Science 2009 326 (5950:236-237); Ladner et al. Mbio 2014 Jun 17;5(3):e01360-14) • MIMS • Minimum Information about a Metagenomic Sequence/Sample; metadata on samples and sequences (Kottmann et 1l 2008. OMICS 12 (2):115-121). • Why not a standard for microbial strains? • MIBS • Minimum Information about a Bacterial Strain. What are the information to be captured? • MIVS (not the focus now) • Minimum Information about a Viral Strain. What are the information to be captured?




The impetus for this work (DoD specific) • May 2015 DPG debacle; In its aftermath, many committees were established and investigations were carried out to understand and fix DPG like problems. • Moratorium on Select agent work within DoD; Sec Army Directive to fortify safety of BAST activities within DoD and implementation of recommendations. Establishment of BBPO (BSAT Bio Safety Program Office). • Prevent indiscriminate transfer of iBSATs or dBSATS (derivative) that are not 100% guaranteed for inactivation. • Uncertainty of 100% guarantee of iBSATs safety and security • Need for materials BSAT derivatives across the biodefense enterprise; however, DBPAO divested BSAT business. • But leadership wanted DBPAO involvement in assurance of and confidence in materials from other sources by other means across the enterprise. So, setting the standard for strains used in assay development, test and evaluation




The impetus for this work (Interagency specific)

• Bio Alliance • Interagency collaboration for a network of repositories for various bacterial and viral reference materials • Networking different repositories • Common standards a must for reliability and confidence in materials • The materials are used for various applications: assay development, Vaccines and therapeutics development




• Blindly trusting the information provided by the donor of a strain • Discrepancies in genotypes; end up storing in the wrong containment spaces (PAK1 BACI 224 in BSL3 and YERS 003 in BSL2); CDC is not happy about this. • Lab specific growth conditions may affect the strain’s properties and genetic make up (surface antigen expression in Yp is heavily influenced by growth temperature); major issues: wrong classification of BSATs • The sources for strains having the same designations are many. But they may not have the same genotypic/phenotypic characteristics due to different pedigrees, laboratory propagation and growth conditions (Sterne, BG examples) • Set the standards so that those that are authorized to use BSATs can follow and make sure the strain meets the standards and adds confidence to the identity and authenticity of strains. DBPAOs assurance role !! • Once you characterize the strain the stock is aliquoted, frozen and further distributed from the repository or any other source. You can always trace it back to the stock. Further growth is according to the same protocol. [QR coding vials and electronic tracking] The impetus for this work (Global Issues)




Blindly trusting the information provided by the donor of a strain




Error in published sequence

• Error discovered in published sequence

Multilocus sequence typing ‐ seven housekeeping genes ‐ evolutionary history generated ‐ Neighbor‐joining method, MEGA7

Johnson 2015 Genome Announc 3, e00151 Johnson 2018 Genome Announc 6, e01144

Previous researchers given wrong sample Current sample is correctly identified as Al Hakam





FOUO – not approved for public distribution

Blindly trusting the information provided by the donor of a strain • BACI224 • PAK-1: supposed to be wild type Ba with both plasmids. Customer (assay developer) spent a bit of time trying to figure out the reason for PCR failure against one of the plasmids. Turned out, the stock had either lost one of the plasmids or a mixed population. Had been stored in BSL3 • YERS003 • CO92: Based on a PCR result, deemed to have a missing plasmid and was stored in BSL2. Other PCRs showed weak positive for the target plasmid. Sequencing could unequivocally resolve it. Again a problem of mixed population of both genotypes? Growth conditions (media and temperature can make a big difference)




Strains with the same designations from different labs are not the same (phenotypically and genotypically) • Bacillus anthracis Sterne (different SNPs in WG sequences) • Phage gamma sensitivity is different for different Sterne strains and growth conditions • Bacillus globigii




Genomic changes in same strains from different sources




Metabolic changes in same strains from different sources




• There is no standard on the level/granularity of strain characterization needed to ensure the confidence on the identity of a bacterial or viral strain used in assay development. Each repository/laboratory has their own set of procedures- or they don’t or they do “ad hoc” what is needed. • The cost of extensive characterization may vary widely depending on what assays are done and exorbitant cost can be prohibitive; so need minimum standard, to start with. Do we create tiered standards analogous to sequencing? • Is WGS sequencing good enough? • No- Knowing the genetic make up (genotype) without the phenotype is no use. • The jury is still out there on level of genome sequencing – to finish or not to finish the genome? The impetus for this work (General issues)




Microbiological analyses essential for strain verification and validation- the B. anthracis like bacteria- B. cereus biovar anthracis B. Ce var anthracis Classic anthracis Klee et al 2006. J Bac 188: 5333-5344




Even Whole Genome Sequencing Cannot Resolve Some Genomic Rearrangements

Tandem Repeats in Vibrio cholerae Strains

Optical Mapping showed tandem repeats of (~160 kb), read mapping showed mixed results, PCR could not identify the junctions unequivocally




Cost of Characterization

Strains in CRPµTIC full characterization: ~$100 k/strain




Why SPADA for this?

• SPADA is an ideal forum to develop a standard because of its history in developing standards and developing a consensus thorough brining in SMEs from across the domains (Sponsor, government, industry, academia- national and international). • SMPR: • 6 Validation Guidance • AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Biological Threat Agent Methods and/or Procedures (Official Methods of Analysis of AOAC INTERNATIONAL, 2016, 20th Ed., Appendix I). • Inclusivity and exclusivity panel organisms used for evaluation must be characterized and documented to truly be the species and strains they are purported to be.




What is the objective for the WG • Evolve a standard for bacterial strain characterization • Set minimal information needed for such a strain verification and validation • Write the document and publish • What are the broad categories of information to be captured? Process and not the specific method/instrument • What is the outline of the document- this is not the usual run of the mill SMPR. • What is the forum/journal to publish? • Developed many SMPRs for agent specific assays and user scenarios that help the assay developers and stakeholders who use the assays




What would be an ideal strain characterization? e.g., rBaSwAT

VEGETATIVE CELLS • Phenotypic assays  Microbiological tests

 Phage sensitivity (  and AP50c)  Antimicrobial resistance  Spore formation  Immunological tests (LFI, PR2, Magpix and Toxin Assay) • Genotypic assays  Toxin deletion verification by PCR  Optical mapping and Whole genome sequencing  Molecular tests for signatures (PCR) SPORES • Production, purification and irradiation inactivation (using new protocol established by the working group)  Physical properties  Molecular and Immuno assays and bridging studies  Animal studies Tab: ~$800K/~8 strains




Critical Reagents Program Microbial Threat Information Center (CRPµTIC)




Grades of Characterization Gold- all types of applications including vaccines and therapeutics reference materials Silver- detection assay development Bronze- minimal information for making sure it is the right strain




What is the format of the standards document Standards Guidelines Opinion document Manuscript What is the forum for publication?

AOAC Journal ASM journals Science Other Microbiology Journals





Bruce Goodwin Joint Product Leader Bryan Necciai

Assistant Product Manager Shanmuga Sozhamannan, Ph. D Technical Coordinator email:

This work was funded by DUSA-TE and JPEO JPM Guardian, DBPAO




Bacterial Strain Verification in  the Unified Culture Collection David Rozak USAMRIID 17 October 2018

The Unified Culture Collection

Bacteriology Section

Escherichia, 71 Clostridium, 57

Staphylococcus, 55 Pasteurella, 45

Brucella, 104

Streptococcus, 26 Coxiella, 19

Mycoplasma, 8

Yersinia, 107

Listeria, 6

Enterococcus, 11 Lactobacillus, 11 Neisseria, 11 Enterobacter, 9 Haemophilus, 9

Ralstonia, 6

Burkholderia, 194

Salmonella, 6 Arcanobacterium, 5 Klebsiella, 5 Mannheimia, 5 Mycobacterium, 5 Pseudomonas, 5

Other, 51

Bacteroides, 8 Corynebacterium, 8

Francisella, 241

Bacillus, 382

Numbers of accessioned strains

ISO‐accredited BSAT, exempt BSAT, and near‐neighbor bacterial reference  materials for the biodefense community: • Fully characterized bacterial inoculum • Quantified animal challenge doses • Antigen and nucleic acid reference standards

Key Assertions

Production and Characterization


• Untouched original

• Single colony pick

• Colony morphology • Gram Stain • Vitek • Biolog • PCR • Riboprinter • MLST • MiSeq • Colony morphology • Gram Stain • Phenotypic and  genotypic assay




Assay Accuracies

Genus Bacillus Brucella


Wrong Genus

Wrong Species



5% 3% 2% 9% 7% 8% 1% 0% 0%


37% 21% 46% 12% 69% 41%

54% 256 62% 34 51% 191 78% 97 21% 29 14% 138 6% 104 10% 52 44% 32 36% 105 94% 17 17% 103 43% 132 55% 40 52% 127 45% 73 57% 14 67% 36 95% 20 0 0



2% 0% 3%


Yersinia Bacillus Brucella MLST MIDI Ribotype Biolog Vitek Next Gen Sequencing Procedure • Extract DNA using EZ1 • Measure concentration with Qubit • Nextera DNA Flex Library prep kit • Run library prep on MiSeq using the v2  500 cycle kit • Run FASTQ files through EDGE  bioinformatics pipeline 37% ‐‐ ‐‐ ‐‐ ‐‐ Burkholderia 88% 87% 6% 4% Francisella Yersinia Bacillus Brucella 9% 47% 27% 23% 14% 6% 0% 2% 0% 8% 2% 0% Burkholderia 40% 51% 33% 18% 42% Francisella 6% 5% Yersinia Bacillus Brucella 28% ‐‐ ‐‐ ‐‐ ‐‐ Burkholderia 0% 4% 51% Francisella 21% 21% 25% 0% 6% 5% Yersinia Bacillus Brucella 3% 0% 0% ‐‐ ‐‐ ‐‐ ‐‐ Burkholderia 12% 0% 2% Francisella ‐‐ ‐‐ ‐‐ ‐‐ ‐‐ ‐‐ ‐‐ ‐‐ Yersinia


86% 103

0 0

EDGE Bioinformatics Pipeline • Developed by LANL and Navy • Quality trim and filter • Host removal • Map reads to reference • Assembly • Contig‐based taxonomy  classification • MetaPhlAn • Kraken mini • Phylogenetic analysis

• Reads‐based taxonomy classification • GOTTCHA bacterial and viral • BWA

• Result reported from consensus of  BWA, Kraken mini, and contig‐based

Validation Results

• 15/16 organisms correctly identified • Inter‐assay repeatability was 100% • Intra‐assay reproducibility was 100%

Accuracy of  Platform (%)

UCC Number

Reference Sequence ID

EDGE Result


Bacillus anthracis

Bacillus anthracis


Bacillus thuringiensis

Bacillus thuringiensis


Bacillus cereus Brucella abortus Brucella canis

Bacillus cereus Brucella abortus Brucella canis

100 100 100 100 100 100 100 100 100 100 100 100 100

Burkholderia thailandensis Francisella philomiragia

Burkholderia thailandensis Francisella philomiragia

Francisella tularensis

Francisella tularensis

Yersinia pseudotuberculosis Yersinia pseudotuberculosis

Yersinia pestis

Yersinia pestis

Acinetobacter baumannii

Acinetobacter baumannii

Bacillus anthracis

Bacillus anthracis

Staphylococcus aureus

Staphylococcus aureus

Pseudomonas aeruginosa Pseudomonas aeruginosa

Salmonella enteritidis Burkholderia cepacia

Salmonella enteritidis

Burkholderia lata


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