SPADA Meeting Book

August 13, 2019

Stakeholder Panel on Agent Detection Assays [SPADA]

Stakeholder Panel Meeting 2275 Research Boulevard First Floor Conference Room Rockville, Maryland, United States

Draft, Do Not Distribute

S TAKEHOLDER P ANEL ON A GENT D ETECTION A SSAYS Tuesday, August 13, 2019

AOAC INTERNATIONAL Headquarters First Floor Conference Room 2275 Research Blvd., Rockville, Maryland, 20850 9:00 a.m. – 4:00 p.m.

STAKEHOLDER PANEL AGENDA

I. Introductions and Call to Order (9:00am – 9:15am) Palmer Orlandi, AOAC INTERNATIONAL

II. Meeting Overview and Objectives (9:15am – 9:30am) Linda Beck, Joint Research and Development (JRAD) , SPADA Chair

III. Voluntary Consensus Standards Development at AOAC INTERNATIONAL (9:30am – 10:00am) Deborah McKenzie, AOAC INTERNATIONAL

IV. Bacterial Strain Verification Working Group (10:00am – 11:00am) Linda Beck & Shanmuga Sozhamannan, DoD JPEO JPMG DBPAO a. Working Group Overview b. For approval: Guidelines for Verifying and Documenting the Relationships Between Microbial Cultures* V. Soil Testing Working Group (11:30pm – 12:30pm) Linda Beck & Morgan Minyard, Defense Threat Reduction Agency (DTRA) a. Working Group Review b. For approval: Voluntary Consensus Standard for Collection and Use of Soils for Biothreat Agent Method Validation and Site Assessments* VI. In-silico Analysis Working Group (1:30pm – 2:30pm) Linda Beck & Shanmuga Sozhamannan a. Working Group Review b. For approval: Recommendations for Development of Molecular Assays for Microbial Pathogen Detection Using Next Generation In-Silico Analysis*

VII. Emerging Issues & Next Steps (2:30pm – 3:30pm) Linda Beck & Palmer Orlandi

VIII. Adjourn (3:30pm)

* Item requires a vote

An AOAC Provided Lunch Will be Served at 12:30pm

NO GOVERNMENT FUNDS HAVE BEEN USED IN THE PROVISION OF FOOD FOR THIS MEETING

AOAC Stakeholder Panel on Agent Detection Assays (SPADA) Meeting Overview and Objectives

Linda C. Beck, PhD, SPADA Chair Joint Research and Development (JRAD ) DUSA TE; JPEO JPM Guardian DBPAO  August 13, 2019

Meeting Overview & Objectives

• Current SPADA Initiative 

• Timeline

• Meeting Goals

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AOAC SPADA: Current Initiative

3 • Current initiative is jointly funded by: • The Department of the Undersecretary of the Army – Test  & Evaluation (DUSA T&E) • Joint Program Executive Office Joint Program Manager – Guardian Defense Biological Product Assurance Office  (JPM‐Guardian DBPAO )  (JPEO JPL ‐ CBRN Enabling  Biotechnologies) • Johns Hopkins University, Applied Physics Laboratory is prime  contractor. • AOAC INTERNATIONAL is the executing organization.   Overview of Current Initiative • In 2018, the Deputy Under Secretary of the Army,  Test & Evaluation (DUSA TE) and the  Joint Program  Executive Office for Chemical and Biological Defense,  JPM – Guardian, Defense Biological Program  Assurance Office (DBPAO) expressed interest in  developing consensus standards for the following  topics: – Preparation and use of Soil Testing Samples – Bacterial Strain Verification – In Silico PCR Analysis • The sponsors contracted with AOAC to develop these  standards under the SPADA voluntary consensus  model. 4

Bacterial 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.

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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. 

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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.

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Current SPADA Timeline

2019

2018

Aug

Oct

Dec

Feb

Apr

Jun

Aug

Oct

Dec

Today

Preparatory Work

SPADA Meeting: Working Groups Launch

10/15/2018

In‐PersonWorking GroupMeetings

10/16/2018

Working Group Teleconferences

11/2/2018

5/17/2019

Public Comment Period 7/12/2019

6/7/2019

Public Comment Discussion

7/8/2019

Preparation of Final Drafts by WG Chairs 8/9/2019

7/15/2019

SPADA Meeting 2

8/13/2019

Publication Period 

8/14/2019

12/31/2019

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August 13 SPADA Meeting Goals

• Review work to date • Discussion and vote on the three  documents:

– Recommendations for Development of Molecular Assays for  Microbial Pathogen Detection Using Modern  In‐Silico Approaches – Guidelines for Verifying and Documenting the Relationships  Between Microbial Cultures – Guidance for Soil Collection, Characterization and Application for  Biothreat Agent Detection Method and Site Evaluations • Discuss potential future topics for SPADA

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AOAC Staff Contacts

• David B. Schmidt, Executive Director, dschmidt@aoac.org

• Palmer Orlandi, Deputy Executive Director / Chief Science Officer,  porlandi@aoac.org

• Deborah McKenzie, Sr. Dir., Standards Development,  dmckenzie@aoac.org

• Sharon Brunelle, AOAC Technical Consultant,  sbrunelle@aoac.org

• Christopher Dent, Standards Development Manager,  cdent@aoac.org

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Questions?

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AOAC STAKEHOLDER PANEL ON  AGENT DETECTION ASSAYS (SPADA) Bacterial Strain Verification Working Group Co‐Chairs:  Linda Beck, JRAD and Shanmuga Sozhamannan, DoD, JPEO, JPMG, DBPAO Presentation: Guidelines for Verifying and Documenting the  Relationships Between Microbial Cultures  August 13, 2019

AOAC Headquarters 2275 Research Blvd. Rockville, Maryland, USA

Bacterial Strain Verification Working Group Scope of Work

Develop a consensus standard to provide confidence and assurance in the identification of the strains being utilized in testing. A consensus standard would improve the credibility of validation test results since evaluators will have a standard procedure recognized by the community that can be used to authenticate the strain of the cultures used in assay validation.

Bacterial Strain Verification

Working Group Members

Linda Beck, JRAD (Co‐Chair) Shanmuga Sozhamannan, DoD, JPEO, JPMG, DBPAO (Co‐Chair)

Brian Bennett, Dugway Proving Ground Cory Bernhards, ECBC Rick Blank, NBACC Trevor Brown, JPEO Ryan Cahall, Censeo Insight /DUSA T&E Randy Hoffman, ECBC Paul Jackson, LLNL (Ret.)

Scott Jackson, NIST Katalin Kiss, ATCC Nancy Lin, NIST Timothy Minogue, USAMRIID Jason Opdyke, JPEO David Rozak, USAMRIID Mark Scheckelhoff, Armed Forced Surveillance Branch Sanjiv Shah, EPA Chuck Young, JHU/APL

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Strain Verification Working Group Work to Date

• Working Group Launch (October 16‐17, 2018) • Six teleconferences (October 2018 – April 2019) • Drafted   “ Guidelines for Verifying and Documenting the Relationships Between Microbial Cultures ” • Public comment period (June 4, 2019 – July 12, 2019) • Comments reviewed and document prepared for  SPADA review and approval

Background

• Microbial cultures are dynamic systems, which can undergo  significant changes as they are handled and propagated in  laboratory settings. • While storage, propagation (passing), and distribution is  necessary to ensure the availability of the organism for  basic and applied research, these processes can make it  difficult and misleading to compare experimental results  obtained from different locations and times. • The careful handling and verification of microbial cultures is  essential for comparing experimental results.

Document Key Points

• Definitions

• Roles and 

Responsibilities – Study sponsor – Culture producer – Performer • Culture Verification  Statement

– Extensible study – Test and index  cultures

– Culture verification – Propagation history – Orthogonal tests – Application oriented  tests

– Propagation history – Orthogonal testing – Application oriented  testing

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Discussion?

Guidelines for Verifying and Documenting the Relationships betweenMicrobial Cultures 1

1.0 Background

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Microbial cultures are dynamic systems that can accumulate inheritable changes when 3 propagated or stored in laboratory environments. These changes often affect key virulence traits 4 that are targeted during the development and testing of medical countermeasures and pathogen 5 detection assays. For example, laboratory-propagated Francisella tularensis and Coxiella 6 burnetii tend to lose distinctive surface antigens that protect them from the host immune 7 response (1, 2, 3). When cultured at 37°C, Yesinia pestis frequently jettisons the pCD plasmid, 8 which encodes a number of key virulence genes associated with the bacteria’s type II secretion 9 system (4, 5). In yet another example, laboratory-acclimated Bacillus anthracis is less likely to 10 sporulate (6). 11 These laboratory-acquired mutations have the demonstrated potential to generate conflicting 12 results in laboratories that are working nominally with the same strain. For example, 13 investigators at the United States Army Medical Research Institute of Infectious Diseases 14 (USAMRIID) found as much as a 16-fold difference in virulence of internally and externally 15 sourced F. tularensis Schu S4 cultures (David Waag, personal communications) that appears to 16 be attributed to a laboratory-acquired frame shift deletion in the known virulence determinant, 17 FTT_0615C (7). Similarly, Molins et al. (8) noted that their version of F. tularensis Schu S4 18 exhibited decreased virulence compared to other Type A isolates. 19 Consequently, the research community would benefit from a consensus standard for tracking 20 provenance of microbial stocks used in different applications. This need is especially critical for 21 microbiologists involved in developing various health care applications such as diagnostics, 22 vaccines and therapeutics. Microbial reference materials used in these applications are obtained 23

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from different sources and are often not qualified/certified to the same set of standards, making it 24 difficult for results to be confidently compared. 25 One way to limit and monitor the genetic drift in laboratory handled strains is by encouraging 26 researchers and culture producers to carefully document the histories of bacterial cultures and 27 routinely screen them for divergent genotypic or phenotypic signatures. This guideline 28 establishes a framework for investigators to use in documenting the relationships among 29 microbial cultures used in well-documented studies. 30

2.0 Objectives

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These guidelines establish the roles and responsibilities for sponsors, performers, and culture 32 producers with respect to the verification of relatedness among test and index cultures used in an 33 extensible study. While not broadly enforceable, the guidelines are intended to create a 34 framework and set of expectations for properly qualifying and documenting the provenance of 35 microbial cultures used in scientific studies. 36

3.0 Concepts and Definitions

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( a ) An extensible study is a research program whose results and conclusions are expected to

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apply equally to test and index cultures.

( b ) In an extensible study, the test culture is the microbial culture that is being evaluated. 40 The index culture is the culture to which the assay results are to be applied. 41 42 well-documented number (e.g., lot/batch/subculture, etc., as appropriate) and propagation 43 history. 44 ( d ) Extensible studies are generally supported by: 45 ( c ) Both the test and index cultures must be traceable cultures , meaning that each has a

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( 1 ) sponsors , who establish the experimental objectives;

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( 2 ) culture producers, who manufacture and characterize the study’s traceable cultures; and

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( 3 ) performers, who conduct the study.

48

49 specific responsibilities with respect to the culture verification process. 50

These roles can be filled by the same or different organizations. However, each role has

( e ) Culture verification is the process by which the species in a test culture is shown to be 51 sufficiently related to that in an index culture to allow the meaningful extension of experimental 52 results from one culture to the other. The relationship between the test and index cultures should 53 be established via propagation history and orthogonal testing. It may also be desirable to use 54 application-oriented testing to ensure study-specific similarities between the cultures. 55 ( f ) Propagation history describes a test culture’s step-by-step derivation from the index 56 culture via a series of propagation events. These data are an essential part of the culture 57 verification process because a culture’s propagation history is impossible to recover through 58 empirical means. Furthermore, production and handling details provide important clues to the 59 health and disposition of the culture that may not be evident through empirical observations, 60 including potential changes in the genetic makeup. 61 ( g ) Orthogonal testing is the use of functionally independent assays to verify the genotypic 62 and phenotypic relatedness of test and index cultures. Orthogonal testing is important for 63 identifying genetic or physical changes that might have resulted from laboratory handling and 64 could impact the validity of an extensible study. 65 ( h ) Application-oriented testing is designed to assess the relationship between the test and 66 index cultures with respect to the specific genotypic or phenotypic phenomena being evaluated 67

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in the extensible study. For example, if the study relates to microbial virulence, some effort 68 should be made to show that the virulence of the test strain resembles that of the index strain. 69 ( i ) Culture verification statements provide a convenient mechanism for documenting the 70 relatedness of microbial cultures used in extensible studies. 71

4.0 Roles and Responsibilities

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An extensible study’s sponsor, culture producer, and performer have distinct roles and 73 responsibilities with respect to the culture verification process. Each of these roles and their 74 associated responsibilities are described below and can be filled by the same or different 75 organizations. 76

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4.1 Study Sponsor

The sponsor is the entity that defines the study’s objectives. The sponsor’s principal 78 responsibilities with respect to culture verification are to approve the index cultures to which 79 each of the test cultures must relate and to determine the acceptable level of relatedness. These 80 responsibilities are generally accomplished by specifying the culture producer and lot number (or 81 equivalent designator) of each of the study’s index cultures and ruling on the adequacy of 82 performer-supplied culture verification statements. 83 When selecting index cultures and approving culture verification statements, the sponsor 84 should be aware of how their decision will impact project costs. Limited availability of singled- 85 sourced cultures or capable culture providers can drive verification costs upwards. Furthermore, 86 the paucity of producer-supplied provenance records and test data could make it costly, if not 87 impossible, for the performer to adequately demonstrate the relationship between the test and 88 index cultures. 89

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90

4.2 Culture Producer

Under these guidelines, it is the responsibility of the culture producers, who manufacture the 91 test and index cultures, to provide the performer with any nonproprietary information that can 92 help the performer demonstrate the relatedness of the test and index cultures. It is understood that 93 the culture producer may not have or be able to release all of the information that the performer 94 needs to complete a culture verification statement. However, the lack of supporting data may 95 drive the sponsor or performer to select different sources for their index and test cultures. 96 Internal proprietary information and HIPAA-related materials are examples of content that may 97 not be sharable with the performer. 98 100 It is the performer’s responsibility to demonstrate to the sponsor’s satisfaction that they are using 101 test cultures that are sufficiently related (as determined by the sponsor) to the index cultures to 102 support meaningful comparisons and conclusions within the scope of the extensible study. The 103 performer meets this obligation by providing the sponsor with a culture verification statement for 104 each of their test cultures. The verification statement summarizes and references enough data to 105 convincingly demonstrate the provenance and empirical equivalence of the test and index 106 cultures. 107 Most, if not all, of the information contained in the culture verification statement should be 108 available from the culture producer, who likely generates provenance and test data as part of 109 their production and quality control processes. However, in some cases the performer may be 110 required to complete additional orthogonal or application-based testing on the test culture, as 111 dictated by the specific study. 112 4.3 Study Performer 99 Organization(s) that are tasked with conducting the scientific study fill the role of performer.

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5.0 Implementation

113

The performer is expected to demonstrate, to the sponsor’s satisfaction, that their test cultures 114 are sufficiently related to the study’s index cultures by documenting passage/subculture history, 115 orthogonal test results, and application-specific assay outcomes in a culture verification 116 statement. Typically, the performer will not have independent access to the records or resources 117 necessary to fully demonstrate culture relatedness at the level recommended by this guide. 118 Rather, they will often rely on information available from the culture producer to verify the 119 relatedness of the test and index cultures. The culture producer will generally transmit this 120 information to the performer via certificates of analysis, product information sheets, and direct 121 communications. 122 5.1 Culture Verification Statement 123 As illustrated in Appendix A, a well-constructed culture verification statement should relate 124 the test culture to the index culture via the culture’s passage/subculture history, orthogonal test 125 results, and optionally application-oriented test results. 126 128 production and handling of the test and index cultures as well as any cultures that constitute the 129 intervening lineage. Each of these cultures should be linked to its predecessor via a documented 130 production method. The performer should specifically identify the manufacturer, lot number, 131 production date, and production method of each culture in the passage history. With respect to 132 the production method, the performer should describe the materials and methods used to derive 133 the referenced culture from its predecessor in the lineage declaration. If the current culture is the 134 first in the chain, the production method should clearly describe the clinical, environmental, or 135 5.1.1 Propagation History 127 The culture verification statement should contain a propagation history, which describes the

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laboratory origin of the culture and the method used to propagate the traceable culture from that 136 origin. 137

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5.1.2 Orthogonal Test Results

It is also important that the culture verification statement describe how available orthogonal 139 test results address the relatedness of the test and index cultures. Orthogonal testing relies on 140 multiple analytic techniques to compare one culture to another. Cultures can be compared with 141 respect to morphology; genotypic and phenotypic properties; metabolic, immunological, and 142 molecular features; molecular functions; and virulence. However, the quality of microbial 143 verification and confidence associated with it ultimately depends on the number, type, and 144 diversity of applied assays. Culture producers should strive for more comprehensive approaches 145 to orthogonal testing. Minimally, orthogonal testing should include a mix of genotypic and 146 phenotypic assays. Table 1 provides examples of various tests and the largely orthogonal 147 categories to which they apply.

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Table 1. Examples of orthogonal assays

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Category

Example assays

Morphology

Colony plating, Gram stain

Genotypic properties Phenotypic properties

Next generation sequencing, RFLP a , MLVA b , MLST c Fatty acid-based microbial identification, Mass spectrometry

Metabolism

Biochemical arrays

Immunological assay response Molecular assay response

ELISA d , Bead-based multiplex assays, DFA e , IFA f

Real-time PCR g

Molecular function

Phage sensitivity, motility, hemolysis In vivo studies using animal models

Virulence

151 152 153 154 155 156 157

a RFLP = Restriction fragment length polymorphism b MLVA = Multilocus variable number tandem repeat analysis

c MLST = Multilocus sequence typing

d ELISA = Enzyme-linked immunosorbent assay e DFA = Direct fluorescent antibody assay f IFA = Indirect immunofluorescence assay

g PCR = Polymerase chain reaction

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5.1.3 Application-Oriented Test Results

While orthogonal testing is intended to uncover unexpected changes that might occur in 160 cultured bacteria during laboratory passage and handling, application-oriented tests are used to 161 confirm that laboratory propagation did not adversely affect properties that relate directly to the 162 planned extensible study. Although application-oriented test results do not need to be included in 163 the culture verification statement, it may be helpful to include such data to show that the test and 164 index samples perform comparably on assays that relate to the specific extensible study. Table 2 165 provides examples of application-oriented tests that can be applied under different study 166 objectives.

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Table 2. Examples of application-oriented assays

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Application

Example assays

Molecular assays Immunoassays

Target-specific sequencing, real-time PCR a

Target-specific ELISA b , Bead-based multiplex assays, DFA c , IFA d

Therapeutics

Virulence, antimicrobial resistance or sensitivity Virulence, gene expression, host immune response

Vaccines

170 171 172 173

a PCR = Polymerase chain reaction

b ELISA = Enzyme-linked immunosorbent assay c DFA = Direct fluorescent antibody assay d IFA = Indirect immunofluorescence assay

6.0 References 174 (1) Hartley, G., Taylor, R., Prior, J., Newstead, S., Hitchen, P.G., Morris, H.R., Dell, A., & 175 Titball, R.W. (2006) Vaccine 24 , 989-96. doi: 10.1016/j.vaccine.2005.08.075 176 (2) Soni, S., Ernst, R.K., Muszyński, A., Mohapatra, N.P., Perry, M.B., Vinogradov, E., Carlson, 177 R.W., & Gunn, J.S. (2010) Front. Microbiol . 1 , article 129. doi: 10.3389/fmicb.2010.00129 178

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(3) Beare, P.A., Jeffrey, B.M., Long, C.M., Martens, C.M., & Heinzen, R.A. (2018) PLoS 179 Pathog. 14 , e1006922. doi: 10.1371/journal.ppat.1006922 180 (4) Cornelis, G.R., Boland, A., Boyd, A.P., Geuijen, C., Iriarte, M., Neyt, C., Sory, M.P., & 181 Stainier, I. (1998) Microbiol. Mol. Biol. Rev. 62 , 1315-1352. 182 (5) Higuchi, K., Smith, J.L. (1961) J. Bacteriol. 81 , 605-608. 183 (6) Leiser, O.P., Blackburn, J.K., Hadfield, T.L., Kreuzer, H.W., Wunschel, D.S., & Bruckner- 184 Lea, C.J. (2018) PLoS One 13 , e0209120. doi: 10.1371/journal.pone.0209120 185 (7) Russo, B.C., Horzempa, J., O'Dee, D.M., Schmitt, D.M., Brown, M.J., Carlson, Jr., P.E., 186 Xavier, R.J., & Nau, G.J. (2011) Infect Immun. 79 , 3665-3676. doi: 10.1128/IAI.00135-11 187 (8) Molins, C.R., Delorey, M.J., Yockey, B.M., Young, J.W., Belisle, J.T., Schriefer, M.E., & 188 Petersen, J.M. (2014) BMC Infect Dis. 14 , article 67. doi: 10.1186/1471-2334-14-67

189 190

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Appendix A: Example of a Culture Verification Statement 191 192 Culture Verification Statement for Francisella tularensis LVS test culture lot 2425-3243 193

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Summary

This culture verification statement documents the relatedness of Francisella tularensis LVS 195 test culture lot 2425-3243 (Manufacturer A; Boston, MA) to the sponsor-specified index culture 196 lot 9210-2349 (Manufacturer A). 197

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Passage History

F. tularensis LVS test culture lot 2425-3243 was derived from lot 9210-2349 via an 199 intermediate culture (lot 8434-6286). The production and handling of the test and intermediate 200 cultures are summarized as follows. 201 Lot 2425-3243 was derived from lot 8434-6286 by Manufacturer A on 20 August 2019 202 according to the following procedure. A 10 µL aliquot of thawed lot 8434-6286 was spread onto 203 Sheep’s Blood Agar (Remel) and incubated at 35°C with 5% CO 2 for 48 h. The bacterial growth 204 was suspended to 1.0 McFarland unit in Tryptic Soy Broth (Remel) supplemented with 12.5% 205 glycerol. The resulting suspension was aliquoted into 1 mL cryotubes and stored at -80°C. 206 Lot 8434-6286 was derived from the index culture (lot 9210-2349) by Manufacturer A on 14 207 July 2016 using a method identical to the one described above. 208

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Orthogonal Testing

Manufacturer A used whole genome sequencing along with Biolog and Vitek 2 GN 210 phenotypic assays to compare F. tularensis LVS test culture lot 2425-3243 with index culture lot 211 9210-2349. These results, which are reported in the attached Certificate of Analysis (1) and 212

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detailed on the manufacturer’s website (2), identified 5 single nucleotide polymorphisms in 213 noncoding regions of the bacterial genomes and generated identical genus and species calls on 214 the Biolog and Vitek 2 GN assays. Based on these results, we conclude that the test and index 215 cultures are sufficiently related for the test culture to be used as a surrogate for the index culture 216 in the sponsored study. 217 The manufacturer’s Certificate of Analysis (1) reports that the test and index lots exhibit 219 identical responses to a proprietary fluorescent tagged monoclonal antibody that similarly targets 220 the O-antigen used in the current study’s lateral flow assay. 221 222 (1) Certificate of Analysis for F. tularensis Lot 2425-3243, 9 SEP 2018, Manufacturer A, 223 Boston, MA. 224 (2) Supporting Data, Manufacturer A, http://ManA.com/24253243/SNP.html References Application-Oriented Testing 218

225 226

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AOAC STAKEHOLDER PANEL ON  AGENT DETECTION ASSAYS (SPADA) Soil Testing Working Group Co‐Chairs:  Linda Beck, PhD and Morgan Minyard, PhD Presentation: Guidance for Soil Collection, Characterization and  Application for Biothreat Agent Detection Method and  Site Evaluations

August 13, 2019 AOAC Headquarters 2275 Research Blvd. Rockville, Maryland, USA

Soil Testing Working Group Members

Linda Beck, JRAD – DUSA TE; JPEO JPM Guardian DBPAO  (Co‐Chair) Morgan Minyard, DTRA (Co‐Chair) Brian Bennett, Dugway Proving Ground Sherry Blight, Battelle Chris Bradburne, JHU/APL Ryan Cahall, Censeo Insight/DUSA T&E

Don Cronce, DTRA Jason Gans, LANL Paul Jackson, LLNL (Ret.)

Scott Jackson, NIST Kevin Kearns, JPEO Jeff Koehler, USAMRIID Nancy Lin, NIST Richard Ozanich, PNNL Frank Schaefer, EPA (Ret.) Sanjiv Shah, EPA Shanmuga Sozhamannan, JPEO, JPMG, DBPAO Randy Vines, ATCC

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Soil Testing Working Group

Scope of Work • Develop standards to aid both assay developers  and evaluators • Provide an increased confidence level for the  robustness of assays with regards to soil  contamination  • Develop the standard methodology for soil  collection and characterization  • Develop or identify protocols/methods for testing  with soils  • Identify a repository for the soil samples 3 Background Soil testing has always generated the most questions as seen during: 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)

Background

Cont… 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)

Background

Then…

Soil Testing Working Group launched in order to attempt to address  concerns using soil in assay development.  Working group an interagency effort in order to include experiences  from verity of scientists and to address as many concerns about soil as  a matrix during Biothreat testing as possible.

Soil Testing Working Group Work to Date

• Working Group Launch (October 16‐17, 2018) • Five teleconferences (October 2018 – April 2019) • Drafted   “ Voluntary Consensus Standard for Collection     and  Use of Soils for Biothreat Agent Method Validation and Site  Assessments” • Public comment period (June 4, 2019 – July 12, 2019) • Comments reviewed and document prepared for  SPADA review and approval 

Soil Document Key Points

• Provide Background  Information on Soil – Terms and Definitions – Soil as a matrix • How to select Soil to  use for Testing • Processing and Using  Soil • Additional Resources • Experimental Set‐up  recommendations

9 Guidance for Soil Collection, Characterization and Application  for Biothreat Agent Detection Method and Site Evaluations • Provides guidance on the standardization of practices for  collection, application‐driven processing, characterization  and use of soil as a sample matrix or potentially interfering  substance in biothreat agent detection assays (validation  and evaluation) • Provides guidance on the standards for soils used in site  assessments, evaluation of biothreat agent  decontamination and remediation procedures • Considerations for the development of standard reference  materials • Special Considerations: Clay content/soil texture, pH,  microorganism content and characterization, moisture, soil  horizons, organic matter (carbon content)

Major Comments Submitted

• Title not as descriptive as document – Changed to "Guidance for Soil Collection, 

Characterization and Application for Biothreat Agent  Detection Method and Site Evaluations“

• Line 46 specifies laboratory, while line 26 includes  field‐deployable. Please clarify if scope includes  both. Suspicious powder collection also? – Re‐wrote the text with the intent to clarify the main  use of the guidance documents.

Major Comments Submitted

Cont… • Add reference to EPA surface sampling document. – The EPA document is excellent but did not necessarily  fit in the section indicated. Included reference in new  paragraph in section 8.4 on sampling practices . • The scenarios in 10.1 and 10.2 could use some  context – where/when is this particular  experimental set‐up needed, what is actually  being sampled? (is it a soil, or is it a sample  collection where the sample (eg aerosol, etc)  might be contaminated with soil?) – An introduction to this section was added to help with  context. 

Major Comments Submitted

Cont… • A lot of information regarding the characteristics soil, but  not much on actual sample collection. These two  documents need to be studied and referenced: 1. Guidance on Obtaining Defensible Samples:  GOODSamples. 2015. AAFCO, Champaign,  IL. https://www.aafco.org/Publications/GOODSamples 2. Guidance on Obtaining Defensible Test Portions:  GOOD Test Portions. 2018 AAFCO, Champaign, IL.  https://www.aafco.org/Publications/GoodTestPortions – Text in section 8.4 was expanded to highlight the importance of  the comments. Guidance on considering the importance of  planning for experimental error was stressed but not fully  described. The links provided in the comments were provided  and encouraged.

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Ballot: http://bit.ly/SPADA_2

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Guidance for Soil Collection, Characterization and Application 1 for Biothreat Agent Detection Method and Site Evaluations 2 3 AOAC Stakeholder Panel on Agent Detection Assays (SPADA) 1.0 Objective 8 characterization, and use of soil as a sample matrix or potentially interfering substance in 9 biothreat agent detection applications. 13 for the testing and evaluation or validation of biothreat agent detection methods and 14 systems, and 2) soils used as a sample matrix in site assessments and in evaluation of 15 biothreat agent decontamination and remediation procedures. 19 inhibition, interference, and cross-reactivity from soil to determine the reliability and/or 20 To provide guidance on standardization of practices for the collection, 10 11 12 16 17 18 2.0 Scope This standard applies to 1) soils used as a source of potentially interfering substances 3.0 Purpose It is essential to evaluate a candidate biothreat agent detection method or system for 4 5 6 7

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suitability of the method or system in the presence of this environmental factor. It is also 21 imperative that methods used for site assessments detect reliably the target agent in the 22 soil matrix. Currently, however, there are no generally agreed-upon standards for the 23 preparation and characterization of soils, nor for the use of soil samples in biothreat agent 24 detection applications. This voluntary consensus standard will help to establish 25 uniformity in the use of soils for evaluation of candidate biothreat agent detection 26 methods and field-deployable technologies. This will result in increased confidence in the 27 reliability of methods and systems and allow for direct comparison of data among studies. 31 Stakeholder Panel on Agent Detection Assays (SPADA) has developed 16 Standard 32 Method Performance Requirements (SMPRs) for various biothreat agent detection 33 methods (1). As part of the validation requirements, the methods are assessed for 34 environmental interferences, including testing with a variety of soil types. Chemical or 35 biochemical (e.g., nucleic acid or protein) components in soils can cause positive or 36 negative interferences in biothreat agent detection methods and systems based on 37 polymerase chain reaction (PCR) or immunoassay technologies. No guidance, however, 38 is provided in these documents regarding how to choose, collect, process, and test the 39 soils in a standard manner. Guidance is challenging due to the complexity of the 40 operational environment, the impact the various methods and systems under evaluation 41 may have on the type and amount of sample required, and the lack of ability to cover 42 every mission type/constraint that may drive sample choice, collection, and processing. 43 28 29 30 4.0 Introduction Through a voluntary consensus standard development process, the AOAC

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This standard focuses on two main experimental uses for soils: 1) soils as a positive 44 and/or negative interference in biothreat agent detection methods with a focus on field 45 deployable detectors and assays during either laboratory or field experiment set-ups; and 46 2) soils tested as part of a site survey or as part of pre- and post-decontamination and 47 remediation assessments. In the first instance, soil is not the intended matrix for the 48 method, but soil components may become airborne and be collected on filters, in liquid 49 aerosol collectors, on surfaces, and in water as contaminants. In the second instance, soil 50 is the intended sample matrix for the method. 55 matter accumulation, maximum biological activity, and/or eluviation of materials such as 56 iron and aluminum oxides and silicate clays. 57 ( b ) B horizon .—The soil horizon, usually beneath the A horizon, that is characterized 58 by one or more of the following: 1) a concentration of silicate clays, iron and aluminum 59 oxides, and humus, alone or in combination; 2) a blocky or prismatic structure; and 3) 60 coatings of iron and aluminum oxides that give darker, stronger or redder color. The B 61 horizon accumulates clay minerals that have leached from upper layers. 62 ( c ) C horizon .—A mineral horizon, generally beneath the solum, that is relatively 63 unaffected by biological activity and pedogenesis and is lacking properties diagnostic of 64 an A or B horizon. The C horizon consists of partially altered parent material and tends to 65 51 52 5.0 Terms and Definitions 53 The following terms and definitions are from Weil and Brady (2): 54 ( a ) A horizon .—The surface horizon of a mineral soil having maximum organic

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contain more characteristics of the bedrock below or the displaced material deposited at 66 the site. 67 ( d ) Cation exchange capacity (CEC) .—The sum total of exchangeable cations that a 68 soil can adsorb. Sometimes called total-exchange capacity, base-exchange capacity or 69 cation-adsorption capacity. CEC is expressed in centimoles of charge per kilogram 70 (cmolc/kg) of soil (or of other adsorbing material, such as clay). 71 ( e ) Clay .—A soil consisting of particles <0.002 mm in diameter. Clay is negatively 72 charged and has capacity for water retention. 73 ( f ) Clay mineral .—Naturally occurring inorganic material (usually crystalline) found 74 in soils and other earthy deposits, the particles being of clay size. 75 ( g ) E horizon .—Light colored mineral horizon where most of the organic matter and 76 smaller minerals have eluviated or leached out of the layer. 77 ( h ) Fine sand .—Comprised of particles in diameter range of 0.2-0.02 mm. Made of 78 weathered primary rock minerals and particles that do not pack together easily. Air 79 enters easily and water flows through fine sand rapidly. 80 ( i ) Fulvic acid.— A term of varied usage but usually referring to the mixture of 81 organic substances remaining in solution upon acidification of a dilute alkali extract from 82 the soil. 83 ( j ) Humic acid .—A mixture of variable or indefinite composition of dark organic 84 substances, precipitated upon acidification of a dilute alkali extract from soil. 85 ( k ) Humin .—The fraction of the soil organic matter that is not dissolved upon 86 extraction of the soil with dilute alkali. 87

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( l ) Humus .—The more or less stable fraction of the soil organic matter remaining 88 after the major portions of added plant and animal residues have decomposed. Usually it 89 is dark in color. 90 ( m ) Silt .—Comprised of particles in the size diameter range of 0.02-0.002 mm. 91 Smaller than sand and more difficult to drain. 92 ( n ) Soil horizon .—A layer of soil, approximately parallel to the soil surface, differing 93 in properties and characteristics from adjacent layers below or above it. 94 ( o ) Soil profile .—A vertical section of the soil through all its horizons and extending 95 into the parent material. 96 ( p ) Solum .—Comprised of surface and subsoil layers that have undergone the same 97 soil-forming conditions. 101 testing with soils difficult to scope. The study of soil is interdisciplinary involving 102 chemistry, biology, physics, genesis and taxonomy, in addition to agricultural and 103 conservation practices. Soils are an important natural resource. They are a medium for 104 plant growth, a regulator for water supply, a recycler of raw materials, a habitat for soil 105 organisms, an engineering medium and an environmental interface. Overall, soils are a 106 very complex matrix including physical and living components that lead to ever-changing 107 compositions. 108 Variability in soils can be problematic. The physical and living components of soil 109 change with depth of the soil, leading to soil horizons in a single soil profile that have 110 98 99 100 6.0 Background Information on Soil There are over 19,000 identified soils in the United States alone, making experimental

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different characteristics. With the different characteristics in mind, care must be 111 exercised when collecting samples to avoid mixing soil types. These horizons can have 112 different pH, organic content and clay minerals. Soils also vary seasonally and over time. 113 A collected soil sample is considered a catch sample and represents a snapshot in time of 114 that soil. Outside of the soil profile, soils change with distance such that 2 soil samples 115 collected only a few feet apart can have very different characteristics. When collecting 116 soil samples, reviewing soil maps and preparing to analyze the sample shortly after 117 collection is recommended in order to confirm the characteristics desired for the 118 experimental purpose. If planning on combining sub-samples of collected soil, field 119 texture methods and field soil pH kits are helpful in establishing similar characteristics 120 between the sub-samples. 124 can be divided into 3 main mineral groups: sand, silt and clay. Clay is the most active 125 component of soil, having the smallest size and therefore the largest surface area. Any 126 non-organic material >2 mm is considered gravel and is most often not included in soil 127 experiments or testing. The ratio of sand, silt and clay determines the texture of the soil 128 (Figure 1) and varies little over time. Texture is critical to soil behavior including gas 129 exchange, active fraction, nutrient retention, and water retention. 121 122 123 6.1 Soil Texture Soil consists of organic and non-organic components. The non-organic components

130 131

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132 134 135 136 140 141 142 146 147

Figure 1. Soil texture triangle. Reprinted from 133 https://soils4teachers.org/files/images/s4t/texture-triangle.jpg

6.2 Soil Organisms 137 2 mm) and macro fauna (>2 mm). One gram of soil is typically expected to harbor 10 8 - 138 10 9 live bacteria. In addition to bacteria and archaea, larger organisms like fungi, 139 protozoa, nematodes, and micro arthropods are found in large numbers. 143 ecosystem diversity. The typical pH range is 4.5 to about 8.4 but can be lower than 3.5 144 and higher than 9.0. The cation exchange capacity (CEC) is also dependent on the soil 145 pH. Soil is also an important habitat for organisms including microbes, meso fauna (0.1 - 6.3 Soil pH The pH of a soil impacts the behavior of chemicals and plays a role in the soil

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6.4 Soil Organic Matter (SOM) 149 animal decomposition, material synthesized by organisms, and cells/debris from soil 150 organisms. SOM impacts the physical and chemical properties of soils, including soil 151 quality and function. The organic matter component of soil is comprised of substances from plant and

152 153 154 155

6.5 Soil Formation and Horizons

Soils are formed by 5 main soil-forming factors: 1) Climate, 2) Parent Material 156 (topography), and 5) Time. Time as a forming factor refers to the time of active 157 weathering versus the standard linear time scale. For example, a soil in Hawaii can be 158 considered older than a soil found in North America due to active weathering. These 5 159 soil-forming factors lead to unique characteristics in each soil and the formation of soil 160 horizons along a vertical profile (Figure 2). There are 5 identified horizons, called O, A, 161 E, B, and C horizons layered above the unweathered parent material. Each horizon has 162 distinct properties as defined in the Terms and Definitions. A soil may contain all or just 163 a few of these horizons. The top horizon may be an O horizon of loose, partly decayed 164 organic matter or an A horizon consisting of mineral matter mixed with organic material. 165 For most experimental purposes related to the very upper portion of the Earth’s crust, the 166 O, A and B horizons tend to dominate sample collection and handling practices. Figure 2 167 is an actual soil profile showing the boundaries and variability of depth of horizons. The 168 soil in Figure 2 has a 4-inch A horizon above a 20-inch B horizon. The C horizon is of an 169 unknown depth. Soil horizons can vary in depth from a few inches to 40 feet or more. 170 (bedrock and deposited sediments), 3) Organisms (micro and macro), 4) Relief

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171 173 174 175 176 177

Figure 2. Soil Profile with measurements in inches. Reprinted from 172 https://www.nrcs.usda.gov/wps/portal/nrcs/detail/nj/soils/?cid=nrcs141p2_018867

7.0 Characterization and Selection of Soils

7.1 Soil Characterization Tests

7.1.1 Physico-Chemical Characteristics 178 impact the performance of an analytical method or system. Three main soil properties are 179 expected to have the most impact on an experiment or test: organic carbon content, clay 180 content/soil texture, and soil pH. In addition, moisture content can affect the viability of 181 biothreat agents as well as interactions between biothreat agents and chemical or 182 biochemical constituents in the soil. 183 Soil is a very complex medium and the physico-chemical characteristics of a soil can

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Methods for determining soil characteristics should be selected from standardized 184 procedures, preferably the ISO methods listed in the Handbook of Soil Analysis (3). Most 185 of these methods are performed at agricultural analytical laboratories across the nation 186 (Table 2). When submitting a soil for analysis, it is recommended the laboratory’s soil 187 preparation steps are utilized and the laboratory is alerted prior to sending the soil if it is 188 not from the local region. Most analytical laboratories will calibrate instruments for soil 189 characteristics specific to their region. If sending a soil from another part of the nation or 190 the world, alerting them to the possible differences will allow them to tailor their methods 191 towards the expected characteristics of the sample. For example, soil pH varies greatly 192 with more acidic soil typically found on the east coast and more basic soils in the western 193 U.S. Alerting the laboratory that a soil may have a different pH than typically found in 194 the local region will ensure they calibrate the pH probe correctly for the soil. Additional 195 tests often offered by these analytical laboratories include CEC, moisture content, and 196 water holding capacity. Samples to be submitted to a laboratory for chemical and 197 physical characterization should be sterilized by autoclaving (see Section 8.7) with the 198 understanding that this impacts culturing and assessment of heat labile materials. 199 Alternatively, there are field test kits available for determination of soil moisture and 200 other parameters. 204 content over time. Although free RNA degrades rapidly, encapsulated RNA (e.g., RNA 205 viruses) can be very stable. Additionally, many vegetative forms of bacterial pathogens 206 201 202 203 7.1.2 Microbiological Characterization The microbiological stability of soil samples can be gauged by measuring RNA

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