STANDARD METHOD PERFORMANCE REQUIREMENTS Standard … · (SMPRs®) 2015.011: for Detection of Coxiella burnetii of Defense trained operators 1 Applicability Specific detection of

AOAC JOURNAL SPECIAL SECTION: Performance Standards for Biological Threat Agent Assays for Department of Defense Applications. 17

STANDARD METHOD PERFORMANCE REQUIREMENTS

Stakeholder Panel on agent detection aSSayS

Working group for Coxiella burnetti

James Samuel (Co-Chair), Texas A&MLinda Beck (Co-Chair), CBR Defense Concepts and Experimentation Branch, Naval Surface Warfare CenterJessica Appler, DHS/BARDAJeff Ballin, ECBCDon Bushner, JS J8, JRO-CBRNDRyan Cahall, Censeo InsightMatthew Davenport, Johns Hopkins UniversityChristina Egan, NYSDOHJoan Gebhardt, Naval Medical Research CenterTed Hadfield, HADECO, LLCMartha Hale, US ARMY MEDCOM USAMRIIDKia Hopkins, ECBCCecilia Kato, CDCAlexander Kayatani, PFPAKaren Kesterson, “CBRNE Directorate, Lab Directorate Pentagon Force Protection Agency”Saleem Khan, University Of Pittsburgh School Of MedicineKatalin Kiss, American Type Culture CollectionJohn Lednicky, University Of FloridaPejman Naraghi-Arani, LLNLSean O’Brien, DoD DUSA T-EKate Ong, JPEO-CBDRoberto Rebeil, ECBCKristian Roth, DDA/CDRH/OIR/DMDMark Scheckelhoff, DHS/OHAEmily Yost, ATECScott Coates (Staff Liaison), AOAC INTERNATIONAL

Standard Method Performance Requirements (SMPRs®) 2015.011: for Detection of Coxiella burnetii

Intended Use: Laboratory or field use by Department of Defense trained operators

1 Applicability

Specific detection of Coxiella burnetii in collection buffers from aerosol collection devices. Field-deployable assays are preferred.2 Analytical Technique

Molecular detection of nucleic acid.3 Definitions

Acceptable minimum detection level (AMDL).—Predetermined minimum level of an analyte, as specified by an expert committee, which must be detected by the candidate method at a specified probability of detection (POD).

Coxiella burnetii.—Naturally obligate intracellular bacterial pathogen of the Legionellales family.

Exclusivity.—Study involving pure nontarget strains, which are potentially cross-reactive, that shall not be detected or enumerated by the tested method.

Inclusivity.—Study involving pure target strains that shall be detected or enumerated by the alternative method.

Maximum time-to-result.—Maximum time to complete an analysis starting from the test portion preparation to assay result.

Probability of detection (POD).—Proportion of positive analytical outcomes for a qualitative method for a given matrix at a specified analyte level or concentration with a ≥0.95 confidence interval.

System false-negative rate.—Proportion of test results that are negative contained within a population of known positives.

System false-positive rate.—Proportion of test results that are positive contained within a population of known negatives.4 Method Performance Requirements

See Table 1.5 System Suitability Tests and/or Analytical Quality Control

The controls listed in Table 2 shall be embedded in assays as appropriate. Manufacturer must provide written justification if controls are not embedded in the assay.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 (2012) 19th Ed., Appendix I].

Submitted for publication September 2015.Developed by the Working Group for Coxiella burnetti and

approved by the Stakeholder Panel on Agent Detection Assays (SPADA).

Final Version Date:September 1, 2015. DOI: 10.5740/jaoacint.SMPR2015_011

16 AOAC JOURNAL SPECIAL SECTION: Performance Standards for Biological Threat Agent Assays for Department of Defense Applications.

AOAC JOURNAL SPECIAL SECTION: Performance Standards for Biological Threat Agent Assays for Department of Defense Applications. 1918 AOAC JOURNAL SPECIAL SECTION: Performance Standards for Biological Threat Agent Assays for Department of Defense Applications.

Inclusivity and exclusivity panel members must be characterized and documented to truly be the species and strains they are purported to be.7 Maximum Time-to-Results

Within 4 h.Table 2. Controls

Control Description Implementation

Positive Designed to demonstrate an appropriate test response. The

positive control should be included at a low but easily detectable

concentration, and should monitor the performance of the entire assay. The purpose of using a low concentration of positive

control is to demonstrate that the assay sensitivity is performing at a previously determined level of

sensitivity.

Single use per sample

(or sample set) run

Negative Designed to demonstrate that the assay itself does not produce a detection in the absence of the

target organism. The purpose of this control is to rule out causes

of false positives, such as contamination in the assay or test.


(or sample set) run

Inhibition

Designed to specifically address the impact of a sample or sample

matrix on the assay’s ability to detect the target organism.


run

Table 4. Exclusivity panel (near-neighbor)

Species Strain

Legionella pneumophila Philadelphia 1

Legionella pneumophila Wadsworth 1

Legionella pneumophila Sg6

Legionella longbeachae ATCC No. 33462

Rickettsiella spp. If obtainable

Table 3. Inclusivity panel

Phylogenetic group Isolate (example)

Group 1 Nine Mile RSA493 Nine Mile RSA439

Group 2 Henzerling

Group 3 Idaho Goat

Group 4 K

Group 5 G

Group 6 Dugway

Table 1. Method performance requirements

ParameterMinimum performance

requirement

AMDL 2000 genomic equivalents/mL of Coxiella burnetii

target DNA in the candidate method sample collection

buffer

Probability of detection at AMDL within sample collection buffer using Nine Mile RSA439 isolate, Clone 4

≥0.95

Probability of detection at AMDL in environmental matrix materials using Nine Mile RSA439 isolate, Clone 4

≥0.95

System false-negative rate using spiked environmental matrix materials

≤5%

System false-positive rate using environmental matrix materials

≤5%

Inclusivity All inclusivity strains (Table 3) must test positive

at 2x the AMDLa

Exclusivity

All exclusivity strains [Table 4] and all

environmental organisms [section 1.2 of AOAC Methods of Analysis

Appendix O] must test negative at 10x the AMDLa

a 100% correct analyses are expected. All discrepancies are to be retested following the AOAC Guidelines for Validation of Biological Threat Agent Methods and/or Procedures [Official Methods of Analysis of AOAC INTERNATIONAL (2012) 19th Ed., AOAC INTERNATIONAL, Rockville, MD, USA, Appendix I, http://www.eoma.aoac.org/app_i.pdf].

Standard Method Performance Requirements (SMPRs®) 2015.12: for Identification of Venezuelan Equine Encephalitis Virus (VEEV)



1 Applicability

Identification of VEEV in liquid samples from aerosol collectors. The preferential method would be a field-deployable assay.

2 Analytical Technique

Molecular methods of detecting target-specific viral component(s).

3 Definitions

Acceptable minimum identification level (AMIL).—Predetermined minimum level of an analyte, as specified by an expert committee, which must be detected and identified by the candidate method with a specified probability of identification (POI).

Exclusivity.—Study involving pure nontarget strains and species, which are potentially cross-reactive, that shall not be detected or identified by the test method.

Inclusivity.—Study involving pure target strains or species that shall be detected and identified by the alternative method.

Maximum time-to-result.—Maximum time to complete an analysis starting from the test portion preparation to assay result.

Probability of identification (POI).—Proportion of positive analytical outcomes for an identification method for a given matrix at a given analyte level or concentration.


System false-positive rate.—Proportion of test results that are positive contained within a population of known negatives.

Venezuelan Equine Encephalitis (VEE) Virus (VEEV).—VEEV encompasses several viruses all of which are within the Alphavirus genus of the Togaviridae family. For the purpose of this SMPR, VEEV includes the human pathogenic virus variants VEEV-1AB, VEEV-1C, VEEV-1D, and VEEV-1E.

4 Method Performance Requirements

See Table 1.

5 System Suitability Tests and/or Analytical Quality Control

The controls listed in Table 2 shall be made available in assays as appropriate. Manufacturer must provide written justification if controls are not available with the assay.

Submitted for publication September 2015.Developed by the Working Group for Venezuelan Equine

Encephalitis Virus and approved by the Stakeholder Panel on Agent Detection Assays (SPADA).

Final Version Date: September 2, 2015. DOI: 10.5740/jaoacint.SMPR2015_012

Stakeholder Panel on Agent Detection Assays

Working group for Venezuelan equine enCephalitis Virus

Eileen Ostlund (Chair), USDA, APHIS, Veterinary ServicesJessica Appler, HHS BARDAJeff Ballin, ECBCLinda Beck, CBR Defense Concepts And Experimentation Branch, Naval Surface Warfare CenterDon Bushner, JS J8, JRO-CBRNDRyan Cahall, Censeo InsightMatthew Davenport, Department of Homeland SecurityJoan S. Gebhardt, Naval Medical Research CenterKia Hopkins, ECBCPaul J. Jackson, Lawrence Livermore National Lab (Retired)Pejman Naraghi-Arani, InSilixa Corp.Christopher Niblick, JPM NBC CA, PD CCAT&TISean P. O’Brien, DoD DUSA T&ETom Phillips, MD Department of AgricultureAnn Powers, CDCJon Rayner, Southern Research InstituteMark Scheckelhoff, DHS/OHADarci Smith, Southern Research InstituteScott Weaver, University of Texas Medical BranchEmily Yost, ATEC, Life Sciences Division, Dugway Proving GroundScott Coates (Staff Liaison), AOAC INTERNATIONAL


6 Validation Guidance


Inclusivity and exclusivity panel members must be characterized and documented to truly be the species and strains they are purported to be.

7 Maximum Time-to-Results

4 h.

Table 2. Controls


Positive Designed to demonstrate an appropriate test response.

The positive control should be included at a low but easily

detectable concentration, and should monitor the performance of the entire assay. The purpose of using a low concentration of

positive control is to demonstrate that the assay sensitivity is performing at a previously

determined level of sensitivity.

Single use per sample (or

sample set) run

Negative Designed to demonstrate that the assay itself does not produce a detection in the absence of the

target organism. The purpose of this control is to rule out causes


Single use per sample (or

sample set) run

Inhibition

Designed to specifically address the impact of a sample or sample matrix

on the assay’s ability to detect the target organism.

Single use per sample run

Table 3. Inclusivity panela

Virus Serotype/variantRepresentative

strain(s)Human illness?

VEEV VEEV-IAB Trinidad Donkey MF-8 Yes

VEEV-IC ICVE93, ICVE95 Yes

VEEV-ID 1DPA61, 1DPE98, IDPE06 Yes

VEEV-IE IEMX63, IEPA62 Yesa Method developers/evaluators must select at least one strain per

serotype/variant in this table for their inclusivity panel evaluation.

Table 1. Minimum performance requirements

ParameterMinimum performance

requirement

AMIL 5000 genome copies/mL

POI at AMIL within sample collection buffer

≥0.95

POI at AMIL in an aerosol environmental matrix

≥0.95 (Table 5; Part 1)

System false-negative rate using spiked aerosol environmental matrix

≤5% (Table 5; Part 1)

System false-positive rate using aerosol environmental matrix

≤5% (Table 5; Part 1)

Inclusivity panel purified DNA All inclusivity strains in Table 3 must be correctly identified as

VEEV at 2x the AMILa

Exclusivity panel purified DNA

All exclusivity strains [Table 4] and all environmental

organisms [section 1.2 of AOAC Methods of Analysis Appendix O]

must test negative at 10x the AMDLa

a 100% correct analyses are expected. All discrepancies are to be retested following the AOAC Guidelines for Validation of Biological Threat Agent Methods and/or Procedures [Official Methods of Analysis of AOAC INTERNATIONAL (2012) 19th Ed., AOAC INTERNATIONAL, Rockville, MD, USA, Appendix I, http://www.eoma.aoac.org/app_i.pdf]. Table 4. Exclusivity panel (near-neighbor)a

Virus Representative strain(s)

Mosso das Pedrasb 78V 3531

Evergladesb,c Fe-3-7c

Mucambob A

C (strain 71D-1252)

D

Tonateb Tonate

Pixunab Pixuna

Cabassoub Cabassou

Rio Negrob AG 80-663

EEEV PE6

WEEV CBA87a Method developers/evaluators must select at least one strain per virus

in this table for their exclusivity panel evaluation.

b Virus is related to VEEV and is in the same antigenic complex.

c Due to close genetic relationships, assays that detect Everglades virus may be considered, however this detection must be noted.

Standard Method Performance Requirements (SMPRs®) 2015.013: for Detection of Staphylococcal Enterotoxin B



1 Applicability

Detection of SEB in liquid samples. The preferential method would be a field-deployable assay.

2 Analytical Technique

Any analytical method that can detect the protein and meets the requirements of this SMPR.

3 Definitions

Acceptable minimum detection level (AMDL).—Predetermined minimum level of an analyte, as specified by an expert committee which must be detected by the candidate method at a specified probability of detection (POD).

Maximum time-to-result.—Maximum time to complete an analysis starting with recovery of toxins from the collection matrix and ending with the assay result.


Selectivity study.—A study designed to demonstrate a candidate method’s ability to detect SEB and, at the same time, demonstrate that a candidate method does not detect nontarget compounds and nontarget related toxins.

Staphylococcus enterotoxin.—A pyrogenic protein implicated in toxic shock and respiratory disorders and superantigenic response due to inhalation. Staphylococcal enterotoxin A (SEA), Staphylococcal enterotoxin B (SEB), and Staphylococcal enterotoxin C (SEC) are a part of a set of exotoxins produced by S. aureus, which comprise about 23 serologically distinct proteins that include SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, SEH, SEG, SEI, SEJ, SEK. and SEU.




See Table 1.


Working group for staphyloCoCCal enterotoxin bSandra M. Tallent (Chair), FDA - ORS/DMJessica Appler, HHS BARDAPatrice Arbault, BioAdvantage ConsultingJeff Ballin, ECBCLinda Beck, CBR Defense Concepts And Experimentation Branch, Naval Surface Warfare CenterDon Bushner, JS J8, JRO-CBRNDRyan Cahall, Censeo InsightMatthew Davenport, Department of Homeland SecurityMartha Hale, U.S. ARMY MEDCOM USAMRIIDKia Hopkins, ECBCMalcolm Johns, DHSLiz Kerrigan, ATCCKaren Kesterson, CBRNE Directorate, Lab Directorate Pentagon Force Protection AgencySaleem Khan, University of Pittsburgh School of MedicineKatalin Kiss, ATCCMarkus Lacorn, R-Biopharm AGMatthew Lesho, Luminex Stephen A. Morse, Centers for Disease Control and Prevention (Retired)Christopher Niblick, JPM NBC CA, PD CCAT&TISean P. O’Brien, DoD DUSA T&EKate Ong, JPEO-CBDTom Phillips, Maryland Department of AgricultureRoberto Rebeil, ECBCReinhardt Witzenberger, R-Biopharm AGEmily Yost, ATEC, Life Sciences Division, Dugway Proving GroundScott G. Coates (AOAC Liaison), AOAC INTERNATIONAL

Submitted for publication September 2015.Developed by the Working Group

for Staphylococcal Enterotoxin B and approved by the Stakeholder Panel on Agent Detection Assays (SPADA).

Final Version Date:September 2, 2015. DOI: 10.5740/jaoacint.SMPR2015_013



The controls listed in Table 2 shall be made available in assays as appropriate. Manufacturer or method developer must provide written justification if controls are not available in the assay.



Use pristine collection buffer solution. Sample containers with target and nontarget compounds must be: (1) blind coded, (2) randomized, (3) evaluated at the same time, and (4) masked, so that the sample identity remains unknown to the analysts. Batches are permissible.


Within 2 h.

Table 2. Controls


Positive Designed to demonstrate an appropriate test response. The

positive control should be included at a low but easily detectable

concentration, and should monitor the performance of the entire assay. The purpose of using a low concentration of positive control is to demonstrate

that the assay sensitivity is performing at a previously determined level of

sensitivity.


(or sample set) run

Negative

Designed to demonstrate that the assay itself does not produce

detection in the absence of the target. The purpose of this control is to rule

out causes of false positives, such as contamination in the assay or test.


(or sample set) run

Table 3. Nontarget toxins

SED

SEE

SEH

SEI

SEJ

SEK

SEA

SEC 1, SEC 2, SEC 3


Parameter Minimum performance requirement

AMDL 0.25 ng/mL recovered SEB in liquid

POD ≥0.95 at AMDL for SEB

Selectivity study SEB at the AMDL

All nontarget compounds [Table 3 and Appendix O, Part 1]

must test negative at 10x the AMDLa

System false-negative rate using spiked aerosol environmental matrix at the AMDL

≤5% (Table 4, Part 1)


≤5% (Appendix O, Part 2)


Standard Method Performance Requirements (SMPRs®) 2016.006: for DNA-Based Methods of Detecting Bacillus anthracis in Field-Deployable, Department of Defense Aerosol Collection Devices


Intended Use: Field-deployed use for analysis of aerosol collection filters and/or liquids

1 Applicability

Detection of Bacillus anthracis in collection buffers from aerosol collection devices. Field-deployable assays are preferred.2 Analytical Technique


Acceptable minimum detection level (AMDL).—The predetermined minimum level of an analyte, as specified by an expert committee that must be detected by the candidate method at a specified probability of detection (POD).

Environmental factors.—For the purposes of this SMPR: Any factor in the operating environment of an analytical method, whether abiotic or biotic, that might influence the results of the method.

Exclusivity.—Study involving pure non-target strains, which are potentially cross-reactive, that shall not be detected or enumerated by the candidate method.

Inclusivity.—Study involving pure target strains that shall be detected or enumerated by the candidate method.

Interferents.—A . . . substance in analytical procedures . . . that, at a (the) given concentration, causes a systematic error in the analytical result (International Union of Pure and Applied Chemistry Analytical Chemistry Division Commission on Analytical Reactions and Reagents Definition and Classification of Interferences in Analytical Procedures Prepared for Publication by W.E. Van Der Linden, Pure Appl. Chem. 61(1), 91–95(1989). Printed in Great Britain, 1989, IUPAC). Sometimes also known as interferants.

Maximum time-to-result.—Maximum time to complete an analysis starting from the collection buffer to assay result.

Probability of detection (POD).—The proportion of positive analytical outcomes for a qualitative method for a given matrix at a specified analyte level or concentration with a ≥0.95 confidence interval.




Working group for Bacillus anthracis

Ted L. Hadfield (Chair), HADECO, LLCPaul J. Jackson (Chair) Lawrence Livermore National Lab (Retired)Jessica Appler, HHS BARDALes Baillie, Cardiff UniversityEd Bailor, IABJeff Ballin, ECBCLinda Beck, Naval Surface Warfare CenterSteven Robert Blanke, University of IllinoisRyan Cahall, Censeo InsightKenneth Damer, Northrop Grumman Electronic SystemsDan Dragon, University Of AlbertaMats Forsman, FOI SwedenCrystal Jaing, LLNLMalcolm Johns, DHSNancy Lin, NISTLaura Maple, NSWCStephen A. Morse, Centers For Disease Control and Prevention (Retired)Dallas New Michael Retford, JBTDS JPM NBCCASanjiv Shah, U.S. EPAShanmuga Sozhamannan, Critical Reagents ProgramDavid Trudil, New Horizons Diagnostics Corp.Susan Welkos, USAMRIIDScott G. Coates (Staff Liaison), AOAC INTERNATIONAL

Submitted for publication April 2016.Developed by the Working Group for Bacillus anthracis and


Final Version Date: March 22, 2016. DOI: 10.5740/jaoacint.SMPR2016_006



See Table 1.


The controls listed in Table 2 shall be embedded in assays as appropriate. Manufacturer must provide written justification if controls are not embedded in the assay.


Official Methods of Analysis (2016) Appendix I: AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Biological Threat Agent Methods and/or Procedures, AOAC INTERNATIONAL, Rockville, MD, USA.

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.

7 Maximum Time-to-Result

Within 4 h.

8 Guidance on Combining DNA for Exclusivity Evaluation

DNA from exclusivity panel organisms 1–9 in Table 4

may be tested as isolated DNA, or combined to form a pool

of exclusivity panel organisms, with each panel organism

represented at 10 times the AMDL. If an unexpected result

occurs, each of the exclusivity organisms from a failed pool

must be individually retested at 10 times the AMDL.

DNA from exclusivity panel organisms 10–15 in Table 4

cannot be combined for exclusivity evaluation.



AMDL 2000 standardized BA Ames spores per mL liquid in the candidate method sample collection buffer

Probability of detection at AMDL within sample collection buffer ≥0.95

Probability of detection at AMDL in environmental matrix materials ≥0.95


≤5%

System false-positive rate using environmental matrix materials ≤5%

Inclusivity All inclusivity strains (Table 3) must test positive at 2x the AMDLa

Exclusivity All exclusivity strains (Table 4 and Appendix O, Part 1) must test negative at 10x the AMDLa


Table 2. Controls


Positive Designed to demonstrate an appropriate test response. The positive control should be included at a low but easily detectable concentration,

and should monitor the performance of the entire assay. The purpose of using a low concentration of positive control is to demonstrate that the assay sensitivity is

performing at a previously determined level of sensitivity.

Single use per sample (or sample set) run

Negative Designed to demonstrate that the assay itself does not produce a detection in the absence of the target organism. The purpose of this control is to rule out causes of

false positives, such as contamination in the assay or test.


Inhibition Designed to specifically address the impact of a sample or sample matrix on the assay’s ability to detect the target organism.



No. Cluster Genotype Strain Origin Characteristics

1 A1a 7 Canadian bison Wood bison pXO1+, pXO2+, VNTRa genotype group A1a

2 A3a 45b V770-NP-1R Vaccine (United States) pXO1+, pXO2–, VNTR genotype group A3A

3 A2 29 PAK-1 Sheep (Pakistan) pXO1+, pXO2+, VNTR genotype group A2

4 A3a 51 BA1015 Bovine (MD) pXO1+, pXO2+, VNTR genotype group A3a

5 A3b 62 Ames Bovine (Texas) pXO1+, pXO2+, VNTR genotype group A3b

6 A3c 67 K3 South Africa pXO1+, pXO2+, VNTR genotype group A3c

7 A3d 68 Ohio ACB Pig pXO1+, pXO2+, VNTR genotype group A3d

8 A4 69 SK-102 (Pakistan) Imported wool pXO1+, pXO2+, VNTR genotype group A4

9 A4 77 Vollum 1B USAMRIIDc pXO1+, pXO2+, VNTR genotype group A4

10 B1 82 BA1035 Human (South Africa) pXO1+, pXO2+, VNTR genotype group B1

11 B2 80 RA3 Bovine (France) pXO1+, pXO2+, VNTR genotype group B2

12 A1a 8 Pasteur USAMRIID pXO1–, pXO2+, VNTR genotype group A1a

13 A3b 59, 61b Sterne USAMRIID pXO1+, pXO2–, VNTR genotype group A3b

14 A1b 23 Turkey No. 32 Human (Turkey) pXO1+, pXO2+, VNTR genotype group A1b

a VNTR = Variable number tandem repeat.

b Organism contains only seven of eight multiple locus variable number tandem repeat analysis (MLVA) markers due to the absence of pXO2. Genotypes listed are consistent with seven of the eight markers.

c USAMRIID = United States Army Medical Research Institute for Infectious Diseases.


No. Species Strain Plasmid status

1 B. cereus S2-8 pXO1–, pXO2–

2 B. cereus 3A pXO1–, pXO2–

3 B. thuringiensis HD1011 pXO1–, pXO2–


5 B. cereus D17 pXO1–, pXO2–


7 B. cereus Al Hakam pXO1–, pXO2–

8 B. cereus ATCC 4342 pXO1–, pXO2–

9 B. cereus FM1 pXO1–, pXO2–

10 B. cereus E33L pXO1–, pXO2–

11 B. thuringiensis 97-27 pXO1–, pXO2–

12 B. cereus G9241 pBCXO1+a, pXO2–

13 B. cereus 03BB102 pXO1+, capA+, capB+, capC+b

14 B. cereus 03BB108 pX01+, capA+, capB+, capC+b

a pBCXO1 is pX01-like, but not identical.

b capA, capB, and capC are contained within the Bacillus anthracis pXO2 plasmid; however, the capA, capB, and capC sequences are found in strains 03BB102 and 03BB108 in the absence of the pxO2 plasmid.


Standard Method Performance Requirements (SMPRs®) 2016.007: for Detection of Francisella tularensis in Aerosol Collection Devices



Working group for francisella tularensis

Paul S. Keim (Chair), Northern Arizona UniversityDavid Wagner (Chair), Northern Arizona UniversityJessica Appler, HHS BARDATimothy Bauer, Naval Surface Warfare Center DahlgrenLinda Beck, CBR Defense Concepts and Experimentation Branch, Naval Surface Warfare CenterJulie Boylan, Defense Threat Reduction AgencyRyan Cahall, Censeo InsightKenneth Damer, Northrop Grumman Electronic SystemsMatthew Davenport, Department of Homeland SecurityMats Forsman, FOI SwedenSteven H. Hinrichs, University of Nebraska Medical CenterKatalin Kiss, ATCCLaura Maple, NSWCStephen A. Morse, Centers for Disease Control and Prevention (Retired)Pejman Naraghi-Arani, InSilixa Corp.Jeannine Petersen, Centers for Disease Control and Prevention (CDC)Denise Pettit, N.C. Department of Health and Human ServicesMichael Retford, JBTDS JPM NBCCAMark Scheckelhoff, DHS/OHASanjiv Shah, U.S. EPAShanmuga Sozhamannan, DoD ECBCScott G. Coates (Staff Liaison), AOAC INTERNATIONAL


1 Applicability

Detection of Francisella tularensis in collection buffers from aerosol collection devices. Field-deployable assays are preferred.2 Analytical Technique


Acceptable minimum detection level (AMDL).—The predetermined minimum level of an analyte, as specified by an expert committee which must be detected by the candidate method at a specified probability of detection (POD).




Interferents.—A . . . substance in analytical procedures . . . that, at a (the) given concentration, causes a systematic error in the analytical result (International Union of Pure and Applied Chemistry Analytical Chemistry Division Commission on Analytical Reactions and Reagents Definition and Classification of Interferences in Analytical Procedures Prepared for Publication by W.E. Van Der Linden, Pure & Appl. Chem. 61(1), 91–95(1989). Printed in Great Britain, 1989, IUPAC). Sometimes also known as interferants.





See Table 1.

Submitted for publication April 2016.Developed by the Working Group for Francisella tularensis and





AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Biological Threat Agent Methods and/or Procedures [Official Methods of Analysis AOAC INTERNATIONAL (2016) Appendix I].


In silico analysis.—In silico screening shall be performed on signature sequences (e.g., oligo primers/probes/amplicons) to predict specificity and inclusivity across available sequenced Francisella strains. In silico results are suggestive of potential performance issues. Basic Local Alignment Search Tool (BLAST) (or a comparable tool)

should be used to examine potential hybridization events

between signature components and available Francisella

genomic sequence data in GenBank®. Results of in silico

analyses shall be included in method/assay performance

evaluation reports.


Within 4 h.

8 Guidance on Combining DNA for Exclusivity Evaluation

Organisms may be tested as isolated DNA, or combined

to form a pool of isolated DNA. Isolated DNA may be

combined into pools of up to 10 exclusivity panel organisms,

with each panel organism represented at 10 times the AMDL,

where possible. If an unexpected result occurs, each of the

exclusivity organisms from a failed pool must be individually

retested at 10 times the AMDL.



AMDL 2000 standardized cells per mL liquid in the candidate method sample collection buffer


Probability of detection at AMDL in environmental matrix materials

≥0.95


≤5%




a 100% correct analyses are expected. All discrepancies are to be retested following the AOAC Guidelines for Validation of Biological Threat Agent Methods and/or Procedures [Official Methods of Analysis of AOAC INTERNATIONAL (2016) 20th Ed., AOAC INTERNATIONAL, Rockville, MD, USA, Appendix I; http://www.eoma.aoac.org/app_i.pdf].

Table 2. Controls


Positive Designed to demonstrate an appropriate test response. The positive control should be included at a low but easily detectable concentration, and should

monitor the performance of the entire assay. The purpose of using a low concentration of positive control is to demonstrate that the assay sensitivity is

performing at a previously determined level of sensitivity.


Negative Designed to demonstrate that the assay itself does not produce a detection in the absence of the target organism. The purpose of this control is to rule out causes







No. UCCa ID Genus and species Strain Characteristics

1 FRAN001 Francisella tularensis subsp. tularensis Type A2 (Type strain)

2 FRAN004 Francisella tularensis subsp. holarctica (LVS) Type B (Russian)

3 FRAN012 Francisella tularensis subsp. holarctica Type B (United States)

4 FRAN016 Francisella tularensis subsp. tularensis (SCHU S4) Type A1 (United States)

5 FRAN024 Francisella tularemia subsp. holarctica JAP (Cincinnati) Type B (Japanese)

6 FRAN025 Francisella tularensis subsp. tularensis (VT68) Type A1 (United States)

7 FRAN029 Francisella tularensis subsp. holarctica (425) Type B (United States)

8 FRAN031 Francisella tularensis subsp. tularensis (Scherm) Type A1 (United States)

9 FRAN072 Francisella tularensis subsp. tularensis (WY96) Type A2 (United States)

10 N/A Francisella tularensis subsp. mediasiaticaa UCC = Department of Defense Unified Culture Collection; components available through Biodefense and Emerging Infections Research Resources

Repository.


No. Species Strain

1 Francisella philomiragia Jensen O#319L ATCC 25015

2 Francisella philomiragia Jensen O#319-029 ATCC 25016



5 Francisella philomiragia D7533, GA012794

6 Francisella philomiragia E9923, GA012801

7 Francisella novicida D9876, GA993548

8 Francisella novicida F6168, GA993549

9 Francisella novicida U112, GA993550

10 Francisella hispaniensis DSM 22475

Standard Method Performance Requirements (SMPRs®) 2016.008: for DNA-Based Methods of Detecting Yersinia pestis in Field-Deployable, Department of Defense Aerosol Collection Devices



Working group for Yersinia pestis

Luther Lindler (Chair), DHSJessica Appler, HHS BARDAJeff Ballin, ECBCTimothy Bauer, Naval Surface Warfare Center DahlgrenLinda Beck, CBR Defense Concepts and Experimentation Branch, Naval Surface Warfare CenterJulie Boylan, Defense Threat Reduction AgencyRobert Bull, Department of Homeland SecurityRyan Cahall, Censeo InsightKenneth Damer, Northrop Grumman Electronic SystemsMatthew Davenport, Department of Homeland SecurityChristina Egan, NYSDOHKen Gage, CDCJennifer Gibbons, ECBC/ExcetTed L. Hadfield, HADECO, LLCMalcolm Johns, DHSMatthew Lesho, LuminexLaura Maple, NSWCTraci Pals, DTRARobert Perry, University of KentuckyMichael Retford, JBTDS JPM NBCCAMark Scheckelhoff, DHS/OHASanjiv Shah, U.S. EPAShanmuga Sozhamannan, Critical Reagents ProgramDavid Wagner, Northern Arizona UniversityDavid Watson, DTRAPatricia Worsham, USAMRIIDScott G. Coates (Staff Liaison), AOAC INTERNATIONAL


1 Applicability

Detection of Yersinia pestis in collection buffers from aerosol collection devices. Field-deployable assays are preferred.2 Analytical Technique


Acceptable minimum detection level (AMDL).—The predetermined minimum level of an analyte, as specified by an expert committee which must be detected by the candidate method at a specified probability of detection (POD).




Interferents.—A . . . substance in analytical procedures . . . that, at a (the) given concentration, causes a systematic error in the analytical result (International Union of Pure and Applied Chemistry Analytical Chemistry Division Commission on Analytical Reactions and Reagents Definition and Classification of Interferences in Analytical Procedures Prepared for Publication by W.E. Van Der Linden, Pure Appl. Chem. 61(1), 91–95(1989). Printed in Great Britain, 1989, IUPAC). Sometimes also known as interferants.





Submitted for publication April 2016.Developed by the Working Group for Yersinia pestis and





See Table 1.


The controls listed in Table 2 shall be embedded in assays as appropriate. Manufacturer must provide written justification if controls are not embedded in the assay.


Official Methods of Analysis (2016) Appendix I: AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Biological Threat Agent Methods and/or Procedures, AOAC INTERNATIONAL, Rockville, MD, USA.

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.7 Maximum Time-to-Results

Within 4 h.8 Guidance on Combining DNA for Exclusivity Evaluation

Organisms may be tested as isolated DNA, or combined to form a pool of isolated DNA. Isolated DNA may be combined into pools of up to 10 exclusivity panel organisms, with each panel organism represented at 10 times the AMDL, where possible. If an unexpected result occurs, each of the exclusivity organisms from a failed pool must be individually retested at 10 times the AMDL.



AMDL 2000 standardized cells of Yersinia pestis strain CO92 per mL liquid in the candidate method sample collection buffer

Probability of detection at AMDL within sample collection buffer

≥0.95

Probability of detection at AMDL in environmental matrix materials

≥0.95


≤5%

System false-positive rate using environmental matrix materials

≤5%



a 100% correct analyses are expected. All discrepancies are to be retested following the AOAC Guidelines for Validation of Biological Threat Agent Methods and/or Procedures [Official Methods of Analysis of AOAC INTERNATIONAL (2016) 20th Ed., AOAC INTERNATIONAL, Rockville, MD, USA, Appendix I; http://www.eoma.aoac.org/app_i.pdf].

Table 2. Controls


Positive Designed to demonstrate an appropriate test response. The positive control should be included at a low but easily detectable concentration, and should monitor the

performance of the entire assay. The purpose of using a low concentration of positive control is to demonstrate that the assay sensitivity is performing at a previously

determined level of sensitivity.


Negative Designed to demonstrate that the assay itself does not produce detection in the absence of the target organism. The purpose of this control is to rule out causes of






No. StrainAchtman Genotype Comments Availabilitya

1 CO92 1.ORI.c Well-studied example of epidemic strain of pestis, recent isolate CDC, USAMRIID

2 KIM 2.Med Well-studied strain in academic circles, virulence data extensive CDC, USAMRIID

3 Antiqua 1.Ant b Ancient strain near root of tree CDC, USAMRIID

4 Pestoides B 0.PE1 CDC, USAMRIID

5 Pestoides F 0.PE2.a pPst negative, old strain in terms of phylogeny CDC, USAMRIID

6 Pestoides G 0.PE2.b pPst negative CDC, USAMRIID

7 Angola 0.PE3 A “pestoides” in everything except name CDC, USAMRIID

8 Nairobi 1.Ant a CDC, USAMRIID

9 Harbin35 2 Ant Rumored to be used or resulted from infection during experiments by Japanese BW Unit 731

CDC, USAMRIID

10 PBM19 1.ORI.a CDC, USAMRIID

11 Java9 1.ORI pFra negative CDC, USAMRIID

12 A1122 1.ORI.a Well-characterized U.S. isolate that is pgm- and pCD-; also has 2X large pPst plasmid

CDC, USAMRIID

13 Nicholisk 41 2.ANT CDC, USAMRIID

14 Shasta 1.ORI YE0387; Shasta (20 Oct 54); Shasta; human case; USA: Ca; 1960 6LY; UCC YERS074

CDC, USAMRIID

15 Dodson 1.ORI Dodson (Aug 70); human case: male age 4.5 years; USA: Arizona (Tuba City); 27 Jun 67; UCC YERS073

CDC, USAMRIID

16 El Dorado

Note on plasmid nomenclature: pMT1 = pFRA; pPCP1 = pPST = pPLA; pCD1 = pYB = pCAD

a CDC = Centers for Disease Control and Prevention; USAMRIID = U.S. Army Medical Research Institute of Infectious Diseases.


Species Strain Comments Availabilitya

YPNN1 Yersinia ruckeri YERS063 USAMRIID

YPNN2 Yersinia rohdei YERS062 USAMRIID

YPNN3 Yersinia pseudotuberculosis PB1/+ 1 Sequenced WRAIR

YPNN4 Yersinia pseudotuberculosis IP32953 1 Sequenced WRAIR

YPNN5 Yersinia pseudotuberculosis YPIII 3 Sequenced WRAIR

YPNN6 Yersinia pseudotuberculosis Pa3606 1b WRAIR

YPNN7 Yersinia pseudotuberculosis IB 1b WRAIR

YPNN8 Yersinia pseudotuberculosis EP2/+ 1 WRAIR

YPNN9 Yersinia pseudotuberculosis MD67 1 WRAIR

YPNN10 Yersinia pseudotuberculosis 1 1a WRAIR

YPNN11 Yersinia enterocolitica WA O:8 WRAIR

YPNN12 Yersinia enterocolitica 8081 O:8 Sequenced WRAIR

YPNN13 Yersinia enterocolitica 2516-87 O:9 WRAIR

YPNN14 Yersinia kirstensenii Y231 Non-pathogenic WRAIR

YPNN15 Yersinia frederiksenii Y225 Non-pathogenic WRAIR

YPNN16 Yersinia intermedia Y228 Non-pathogenic WRAIR

YPNN17 Yersinia aldovae 670-83 Non-pathogenic WRAIR

a USAMRIID = U.S. Army Medical Research Institute of Infectious Diseases; WRAIR = Walter Reed Army Institute of Infectious Diseases.


Standard Method Performance Requirements (SMPRs®) 2016.009: for DNA-Based Methods of Detecting Brucella suis in Field-Deployable, Department of Defense Aerosol Collection Devices

Submitted for publication August 2016.Developed by the Working Group for Brucella suis and approved

by the Stakeholder Panel on Agent Detection Assays (SPADA). Final Version Date: September 1, 2016. DOI: 10.5740/jaoacint.SMPR2016_009



1 Applicability

Detection of Brucella suis in collection buffers from aerosol collection devices. Field-deployable assays are preferred.2 Analytical Technique



Exclusivity.—Study involving pure nontarget strains, which are potentially cross-reactive, that shall not be detected or enumerated by the candidate method.






See Table 1.5 System Suitability Tests and/or Analytical Quality control


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).


If a specified inclusivity or exclusivity isolate is not commercially available in the United States at this time, use


Working Group for Brucella suis

Frank Roberto, Chair, Idaho National LaboratoryJennifer Arce, PNNLLinda C. Beck, CBR Defense Concepts and Experimentation Branch, Naval Surface Warfare CenterThomas R. Blank, NBFACRyan Cahall, Censeo InsightKenneth Damer, Northrop Grumman Electronic SystemsThomas Ficht, TAMUJeffrey Foster, University of New HampshireKatalin Kiss, ATCCMikeljon Nikolich, Walter Reed Army Institute of ResearchSteven Olsen, DVM USDA/ARSRich Ozanich, Pacific Northwest National LaboratoryDavid Rozak, USAMRIIDFrank Schaefer, U.S. EPA (retired)Shanmuga Sozhamannan, DoD ECBCRebekah Tiller, CDCScott G. Coates, AOAC INTERNATIONAL

the GenBank accession number to test the genomic sequence with in silico analysis.


Within 4 h.

8 Guidance

Organisms may be tested as isolated DNA, or combined to form pooled isolated DNA. Isolated DNA may be combined

into pools of up to 10 exclusivity panel organisms, with each panel organism represented at 10 times the AMDL. If an unexpected result occurs, each of the exclusivity organisms from a failed pool must be individually retested at 10 times the AMDL.

If the isolate is not commercially available in the United States at this time, use the GenBank accession number to test the genomic sequence with in silico analysis.



AMDL 2000 genomic equivalents of Brucella suis (biovar 1, type strain 1330) per mL liquid in the

candidate method sample collection buffer



System false-negative rate using spiked environmental matrix materials ≤5%


Inclusivity All inclusivity strains (Table 3) must test positive at 2× the AMDLa

Exclusivity All exclusivity strains (Table 4 and Appendix O, Part 1) must test negative at 10× the AMDLa

a 100% correct analyses are expected. All discrepancies are to be retested following the 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., AOAC INTERNATIONAL, Rockville, MD, USA, Appendix I, http://www.eoma.aoac.org/app_i.pdf].

Table 2. Controls


Positive control Designed to demonstrate an appropriate test response. The positive control should be included at a low but easily detectable concentration, and should


performing at a previously determined level of sensitivity. It is recommended that a technique (i.e., unique distinguishable signature) is used to confirm whether the positive control is the cause of a positive signal generated by a sample.


Negative control Designed to demonstrate that the assay itself does not produce a detection in the absence of the target organism. The purpose of this control is to rule out

causes of false positives, such as contamination in the assay or test.


Inhibition control Designed to specifically address the impact of a sample or sample matrix on the assay’s ability to detect the target organism.


Table 3. Inclusivity panela

No. Strain designa-tion

Biovar ATCC/BEI/GB Accession No.

Available from

Comments

1 B. suis 1330 1 ATCC 23444 BEI NR-302

BEI Resourc-es

Swine, USA

2 B. suis Thomsen 2 ATCC 23445

BEI NR-303

BEI Resourc-es

Hare, Denmark

3 B. suis 686 3 ATCC 23446

BEI NR-304

BEI Resourc-es

Swine, USA

4 B. suis 40 4 ATCC 23447

BEI NR-305

BEI Resourc-es

Reindeer, Russia

5 B. suis 513 5 ACBK00000000b GenBank Mouse, Russia

6 B. suis S2 NA ALOS00000000.1b GenBank Naturally attenuated vaccine strain used in China

a The Brucella Working Group recognizes that B. suis biovar 5 is difficult to distinguish from the other B. suis biovars. The working group concluded that B. suis biovar 5 should be included as a part of the B. suis inclusivity panel with caution that B. suis biovar 5 may be very difficult to differentiate from other B. suis biovars. However, the SMPR does not require candidate assays to differentiate biovars.

b Available in the whole genome database at GenBank.


Table 4. Exclusivity panela,b

No. Strain designation Biovar ATCC/BEI/Accession No.

Available from Comments

1 B. abortus S19 1 NR-10134 NVSL S19 vaccine strain, smooth

2 B. abortus RB51 1 BEI NR-2552 NVSL BEI Resources

RB51 vaccine strain, rough

3 B. abortus 86/8/59 2 ATCC 23449 BEI NR-231

BEI Resources Bovine, England

4 B. abortus 12 3 ATCC 17385 BEI NR-229

BEI Resources

5 B. abortus Tulya 3 ATCC 23450 BEI NR-232

BEI Resources Human, Uganda

6 B. abortus 292 (39/94) 4 ATCC 23451 BEI NR-233


7 B. abortus B3196 5 ATCC 23452 BEI NR-234



BEI Resources Bovine, Africa

9 B. abortus 63/75 7 ATCC 23454 BEI NR-262

BEI Resources Bovine, Africa

10 B. abortus C68 9 ATCC 23455 BEI NR-263




12 B. melitensis 16M 1 ATCC 23456 BEI NR-256

BEI Resources Goat, USA

13 B. melitensis 63/9 2 ATCC 23457 CP007789 CP007788

BEI NR-257

Not commercially available in the U.S. at this time

Goat, Turkey

14 B. melitensis Ether 3 ATCC 23458 BEI NR-258

BEI Resources Goat, Italy

15 B. melitensis bv. 1 str. Rev. 1

1 ACEG00000000 Not commercially available in the U.S. at this time

Elberg origin, B. melitensis vaccine strain

16 B. canis RM-666 NA ATCC 23365 NR-683

ATCC Dog

17 B. neotomae 5K33 NA ATCC 23459 BEI NR-684

ATCC BEI Resources

Desert Wood Rat

18 B. ovis 63-390 NA ATCC 25840 BEI NR-682

ATCC BEI Resources

Ram, Australia

19 B. ceti B1/94 NA AZBH02000000 Not commercially available in the U.S. at this time

Porpoise, Scotland

20 B. pinnipedialis B2/94 NA ACBN00000000 Not commercially available in the U.S. at this time

Seal, Scotland

21 Brucella spp. 83/13 NA ACBQ00000000 Not commercially available in the U.S. at this time

Rat, Australia

22 B. inopinata BO1 NA ADEZ00000000 Not commercially available in the U.S. at this time

Human, Oregon

23 Brucella sp. BO2 NA ADFA00000000 Not commercially available in the U.S. at this time

Human, Australia

24 B. papionis F8/08-60(T) NA ACXD00000000 Not commercially available in the U.S. at this time

Novel Brucella associated with primates (NVSL 07-

0026)

26 B. microti CCM 4915 NA CP001578 CP001579

Not commercially available in the U.S. at this time

Vvole, Czech Republic

27 B. vulpis NA LN997863-LN997864 Not commercially available in the U.S. at this time

Red fox, Austria

28 Agrobacterium tumefaciens NA ATCC 4452 ATCC

Table 4. (continued)

No. Strain designation Biovar ATCC/BEI/Accession No.

Available from Comments

29 Ochrobactrum anthropi NA ATCC 49188 ATCC

30 Ochrobactrum intermedium LMG 3301

NA SAMN02472089

a The Brucella Working Group is aware that B. canis can infect humans, causing approximately 100 cases of human brucellosis annually. The working group is also aware of the close relationship between B. suis and B. canis. In fact, the taxonomic classification of all Brucella spp. has undergone debate during the last few decades, with some scientists proposing that all Brucella spp. should be reclassified as B. melitensis on the basis of results of DNA-DNA hybridization, and that the current species should be reclassified as biovars. However, the classic taxonomic scheme for the Brucella spp. and existing biovars was reapproved in 2003 [Osterman, B., & Moriyon, I. (2006) International Committee on Systematics of Prokaryotes: Subcommittee on the Taxonomy of Brucella, Int. J. Syst. Evol. Microbiol. 56, 1173–1175] on the basis of host specificity, phenotypic characteristics, varying virulence, and genotyping data. For these reasons as well as directions from DoD to focus on B. suis, the working group determined to develop this SMPR for the specific detection of B. suis.

b The Brucella Working Group is aware of Russian vaccines using B. abortus SR82 and B. abortus 7579, and other strains may also be in use. These vaccine strains were not available at the time this SMPR was adopted. Consequently the working group decided not to include these vaccine strains in the exclusivity panel.


Standard Method Performance Requirements (SMPRs®) 2016.010: for DNA-Based Methods of Detecting Burkholderia pseudomallei in Field-Deployable, Department of Defense Aerosol Collection Devices

Submitted for publication August 2016.Developed by the Working Group for Burkholderia pseudomallei

and approved by the Stakeholder Panel on Agent Detection Assays (SPADA).




1 Applicability

Detection of Burkholderia pseudomallei in collection buffers from aerosol collection devices. Field-deployable assays are preferred.2 Analytical Technique



Exclusivity.—Study involving pure nontarget strains, which are potentially cross-reactive, that shall not be detected or enumerated by the candidate method.






See Table 1.5 System Suitability Tests and/or Analytical Quality Control




Working Group for Burkholderia pseudomallei

Jay Gee, Chair, Centers For Disease Control And PreventionJennifer Arce, PNNLLinda C. Beck, CBR Defense Concepts and Experimentation Branch, Naval Surface Warfare CenterThomas R. Blank, NBFACLarry Blyn, Ibis BiosciencesRyan Cahall, Censeo InsightAmanda J. Clark, Naval Surface Warfare Center Dahlgren VABart Currie, Tropical and Emerging Infectious Diseases DivisionKenneth Damer, Northrop Grumman Electronic SystemsMatthew Davenport, Department of Homeland SecurityDavid DeShazer, USAMRIIDMalcolm Johns, DHSMr. Paul S. Keim, Northern Arizona UniversityKatalin Kiss, ATCCMatthew Lesho, Luminex CorporationNancy Lin, NISTStephen A. Morse, Centers For Disease Control and Prevention (Retired)Pejman Naraghi-Arani, InSilixa Corp.Rich Ozanich, Pacific Northwest National LaboratoryFrank Roberto, Idaho National LaboratoryDavid Rozak, USAMRIIDJason Sahl, Northern Arizona UniversityFrank Schaefer, US EPA (retired)Steven Schutzer, UMDNJHerbert P Schweizer, University of FloridaShanmuga Sozhamannan, DoD ECBCApichai Tuanyok, University of FloridaScott Coates, AOAC INTERNATIONAL


If an isolate designated in the inclusivity or exclusivity panel is not commercially available in the United States at this time, use the genomic sequence for in silico analysis.


Within 4 h.

8 Guidance

Organisms may be tested as isolated DNA, or combined to form pooled isolated DNA. Isolated DNA may be combined into pools of up to 10 exclusivity panel organisms, with each panel organism represented at 10 times the AMDL. If an unexpected result occurs, each of the exclusivity organisms from a failed pool must be individually retested at 10 times the AMDL.



AMDL 2000 standardized cells of Burkholderia pseudomallei 1026b per mL liquid in the candidate method sample collection

buffer




≤5%


Inclusivity All inclusivity strains (Table 3) must test positive at 2× the AMDLa

Exclusivity All exclusivity strains (Table 4 and Appendix O, Part 1) must test negative at 10× the AMDLa

a 100% correct analyses are expected. All discrepancies are to be retested following the 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., AOAC INTERNATIONAL, Rockville, MD, USA, Appendix I, http://www.eoma.aoac.org/app_i.pdf].

Table 2. Controls




performing at a previously determined level of sensitivity. It is recommended that a technique (i.e., unique distinguishable signature) is used to confirm whether the positive control is the cause of a positive signal generated by a sample.


Negative control Designed to demonstrate that the assay itself does not produce a detection in the absence of the target organism. The purpose of this control is to rule out

causes of false positives, such as contamination in the assay or test.


Inhibition control Designed to specifically address the impact of a sample or sample matrix on the assay’s ability to detect the target organism.




Species Isolate Available from Comments

B. pseudomallei MSHR668 BEI NR-9922

BEI Resources Clinical Australian isolate

B. pseudomallei MSHR1655 Clinical Australian isolate DBPAOa

B. pseudomallei K96243 BEI NR-4073

BEI Resources Clinical Thai isolate

B. pseudomallei MSHR305 BEI NR-44225

BEI Resources Clinical Australian isolate

B. pseudomallei 1026b BEI NR-9910 BEI NR-4074


B. pseudomallei 7894 DBPAO

B. pseudomallei MSHR840 Clinical Australian isolate DBPAO

B. pseudomallei 576a BEI NR-9916


B. pseudomallei HBPUB10134a BEI NR-44220


B. pseudomallei RF80 Environmental isolate from Thailanda DBPAO = Defense Biological Products Assurance Office.

Table 4. Exclusivity panel (near neighbor)a

Species Isolate

1 B. mallei Strain 6NCTC 10248

BEI NR-36126

2 B. mallei China 5BEI NR-21

3 B. thailandensis CDC3015869 (TXDOH)

4 B. thailandensis H0587

5 B. thailandensis Malaysia20

6 B. thailandensis E1

7 B. humptydooensis (proposed)

MSMB43ATCC BAA-2767

8 B. humptydooensis (proposed)

MSMB1589

9 Burkholderia species MSMB264

MSMB0265

10 B. oklahomensis 1974002358

11 B. oklahomensis-like BDU8

12 Burkholderia species MSMB175

TSV85

13 B. ubonensis MSMB2036

14 B. ubonensis MSMB1189

15 B. multivorans AU1185

16 B. stagnalis MSMB735

17 B. cepacia (B. cenocepacia)

MSMB1824

18 B. vietnamiensis FL-2-3-30-S1-D0

19 B. vietnamiensis AU1233a Strains and species from items 3 to 19 can be used as an

exclusivity panel for B. mallei assays.

Standard Method Performance Requirements (SMPRs®) 2016.011: for Detection of Botulinum Neurotoxins A1 and A2 in Field-Deployable, Department of Defense Aerosol Collection Devices


Intended Use: Laboratory or field use by trained operators within the Department of Defense

1 Applicability

Detection of botulinum neurotoxins A1 and A2 in collection buffers from aerosol collection devices. Field-deployable assays are preferred.2 Analytical Technique

Any analytical method that can detect the protein and meets the requirements of this SMPR.3 Definitions


Maximum time-to-assay result.—Maximum time to complete an analysis starting with recovery of toxins from the collection matrix and ending with the assay result.


Selectivity study.—A study designed to demonstrate a candidate method’s ability to detect the various forms of botulinum neurotoxin A, and at the same time, demonstrate that a candidate method does not detect nontarget compounds and related nontarget toxins.4 System Suitability Tests and/or Analytical Quality Control

The controls listed in Table 1 shall be made available in assays as appropriate. Manufacturer or method developer must provide written justification if controls are not available in the assay.5 Validation Guidance


Equal numbers of botulinum neurotoxin A1 and A2 and botulinum neurotoxin A1 and A2 complex samples must be represented in the selectivity study. Use pristine buffer solution. Samples with target and nontarget compounds


Working Group for Botulinum Neurotoxins A1 and A2

Shashi Sharma, Chair, FDA - CFSANJennifer Arce, PNNLLinda C. Beck, CBR Defense Concepts and Experimentation Branch, Naval Surface Warfare CenterRyan Cahall, Censeo InsightMr. Kenneth Damer, Northrop Grumman Electronic SystemsPaul J. Jackson, Lawrence Livermore National Lab (Retired)Matthew Lesho, Luminex CorporationRich Ozanich, Pacific Northwest National LaboratoryMark Poli, USAMRIIDDavid Rozak, USAMRIIDSanjiv Shah, US EPATheresa Smith, USAMRIIDShanmuga Sozhamannan, DoD ECBCNagarajan Thirunavukkarasu, USFDAScott G. Coates, AOAC INTERNATIONAL

Submitted for publication August 2016.Developed by the Working Group for Botulinum Neurotoxins A1

and A2 and approved by the Stakeholder Panel on Agent Detection Assays (SPADA).



must be: (1) blind coded; (2) randomly mixed together; (3) evaluated at the same time; and (4) masked, so that the sample identity remains unknown to the analysts. Batches are permissible provided that these four conditions are met.

Information on other subtypes is desirable but not required.


See Table 2.

7 Maximum Time-to-Assay Results

4 h.

Table 1. Controls


Positive Designed to demonstrate an appropriate test response. The positive control should be included at a low but easily detectable concentration, and should monitor the performance of the entire

assay. The purpose of using a low concentration of positive control is to demonstrate that the assay sensitivity is performing at a

previously determined level of sensitivity. It is recommended that a technique (i.e., unique distinguishable signature) is used to confirm

whether the positive control is the cause of a positive signal generated by a sample.


Negative Designed to demonstrate that the assay itself does not produce detection in the absence of the target organism. The purpose of this control is to rule out causes of false positives, such as

contamination in the assay or test.






AMDL 1.25 ng/mL recovered botulinum neurotoxin A1 and A2 complexes

in collection buffers

Selectivity study POD ≥0.95 at AMDL for botulinum neurotoxin A1 and A2 complex

Tetanus toxin must test negative at 10× the AMDLa

System false-negative rate using spiked aerosol environmental matrix at the AMDL


System false-positive rate using aerosol environmental matrix at the AMDL


a 100% correct analyses are expected. All aberrations are to be retested following the 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., AOAC INTERNATIONAL, Rockville, MD, USA, Appendix I, http://www.eoma.aoac.org/app_i.pdf]. Some aberrations may be acceptable if the aberrations are investigated, and acceptable explanations can be determined and communicated to method users.

Standard Method Performance Requirements (SMPRs®) 2016.012: for Detection and Identification of Variola Virus


Intended Use: Laboratory use by trained technicians

1 Applicability

Detection of Variola virus DNA in collection buffers from aerosol collection devices for DoD applications.

Note: Method developers are advised to check the AOAC website for the most up to date version of this SMPR before initiating a validation.2 Analytical Technique

Polymerase chain reaction (PCR) methods3 Definitions

Acceptable minimum detection level (AMDL).—Predetermined minimum level of an analyte, as specified by an expert committee that must be detected by the candidate method at a specified probability of detection (POD). The AMDL is dependent on the intended use. (Draft EN ISO/CD 16140-1: Microbiology of food and animal feeding stuffs—Method validation—Part 1: Terminology of method validation, vs 17-03-2011)

Exclusivity.—Study involving pure nontarget strains that are potentially cross-reactive that shall not be detected or enumerated by the tested method. (Draft EN ISO/CD 16140-1: Microbiology of food and animal feeding stuffs—Method validation—Part 1: Terminology of method validation, vs 17-03-2011)

Inclusivity.—Study involving pure target strains that shall be detected or enumerated by the alternative method. (Draft EN ISO/CD 16140-1: Microbiology of food and animal feeding stuffs—Method validation—Part 1: Terminology of method validation, vs 17-03-2011)

Maximum time-to-assay result.—Maximum time to complete an analysis starting from the collection buffer to assay result.

Probability of detection (POD).—Proportion of positive analytical outcomes for a qualitative method for a given matrix at a specified analyte level or concentration with a ≥0.95 confidence interval [Appendix H: Probability of Detection (POD) as a Statistical Model for the Validation of Qualitative Methods, Official Methods of Analysis of AOAC INTERNATIONAL, 20th Ed., 2016].



Variola virus.—A member of the genus Orthopoxvirus and the causative agent of smallpox.


Working Group for Variola Virus

Victoria Olson, Chair, Centers For Disease Control and PreventionJennifer Arce, PNNLLinda C. Beck, CBR Defense Concepts and Experimentation Branch, Naval Surface Warfare CenterLarry Blyn, Ibis BiosciencesRyan Cahall, Censeo InsightAmanda J. Clark, M.S., Naval Surface Warfare Center Dahlgren VirginiaKenneth Damer, Northrop Grumman Electronic SystemsMohamed Sofi Ibrahim, ECBCPaul J. Jackson, Lawrence Livermore National Lab (Retired)Katalin Kiss, ATCCDr. Hermann Meyer, Institute of Microbiology of the BundeswehrPejman Naraghi-Arani, InSilixa Corp.Denise Pettit, N.C. Department of Health and Human ServicesFrank Schaefer, US EPA (retired)Mark Scheckelhoff, DHS/OHAShanmuga Sozhamannan, DoD ECBCElizabeth Vitalis, Lawrence Livermore National LaboratoryScott G. Coates, AOAC INTERNATIONAL

Submitted for publication August 2016.Developed by the Working Group for Variola virus and approved by

the Stakeholder Panel on Agent Detection Assays (SPADA). Final Version Date: September 1, 2016. DOI: 10.5740/jaoacint.SMPR2016_012




AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Biological Threat Agent Methods and/or Procedures (Official Methods of Analysis of AOAC INTERNATIONAL, 20th Ed., 2016, Appendix I).6 Method Performance Requirements

See Table 1.7 Maximum Time-to-Assay Result

≤4 h.

ANNEX I Inclusivity Panel

The inclusivity panel shall include:(1) Sequences from at least two representative strains,

one strain from each major clade of Variola virus [Li et. al. (October 2, 2007) On the origin of smallpox: correlating Variola phylogenics with historical smallpox records, PNAS 104(40), 15787–15792]

(2) Any other strain with differences in the assay primer and/or probe target sequences based on bioinformatic analysis. See Annex IV.

Note: The World Health Organization (WHO) restricts access to Variola virus genomic material; use of any genomic sequences greater than 500 bp requires written permission/approval from the WHO. Insertion of Variola virus DNA into other Orthopoxviruses is prohibited.

More details can be found at:WHO Advisory Committee on Variola Virus Research:

Report of the Seventeenth Meeting: Annex 5: WHO recommendations concerning the distribution, handling and synthesis of Variola virus DNA, http://apps.who.int/iris/bitstream/10665/205564/1/WHO_OHE_PED_2016.1_eng.pdf

WHO recommendations concerning the distribution, handling and synthesis of Variola virus DNA, http://www.who.int/csr/disease/smallpox/SummaryrecommendationsMay08.pdf

ANNEX II Exclusivity Panel (Near Neighbor)

The exclusivity panel shall include:(1) All poxvirus strains listed in Table 3 (Note: See AOAC

website for the most recent list.)

(2) Any additional strains determined through the bioinformatics analysis, performed in accordance with Annex III, with greater similarity to the assay’s target region(s) than the strains listed in Table 3.

ANNEX III Bioinformatics Analyses of Signature Sequences

Underlying Variola Virus Assays

In silico screening will be performed on signature

sequences (e.g., oligo primers) to demonstrate specificity to Variola virus and inclusivity across all sequenced Variola virus strains.

In silico results are suggestive of potential performance issues, so will guide necessary additions to the wet screening panels. In silico identification of potential cross-reactions (false positives) or nonverifications (false negatives) would require the affected strains be included in the exclusivity or inclusivity panels, respectively, if available.

A vendor-selected tool to carry out the bioinformatics evaluation should be able to predict hybridization events between signature components and a sequence in a database including available genomic sequence data, using public GenBank nt (http://www.ncbi.nlm.nih.gov/genbank/). The selected tool should be able to identify predicted hybridization events based on platform annealing temperatures, thus ensuring an accurate degree of allowed mismatch is incorporated in predictions. The program should detect possible amplicons from any selected database of sequence.

Potential tools for in silico screening of real-time PCR signatures include:

(1) http://sourceforge.net/projects/simulatepcr/files/ ?source=navbar. This program will find all possible amplicons and real-time fluorescing events from any selected database of sequence.

(2) NCBI toolsThe vendor submission should include:(1) Description of sequence databases used in the in silico

analysis(2) Description of conditions used for in silico analysis.

Stringency of in silico analysis must match bench hybridization conditions.

(3) Description of tool used for bioinformatics evaluation. Data demonstrating the selected tool successfully predicts specificity that has been confirmed by wet-lab testing on designated isolates. These data can be generated retrospectively using published assays

(4) List of additional strains to be added to the inclusivity (Annex I) or exclusivity (Annex II) panels based on the bioinformatics evaluation



Acceptable minimum detection level (AMDL) 50 000 copies/mL Variola virus target DNA in the candidate method sample collection buffer. Copies/mL refers to number of viral

genomes or equivalent plasmid copies containing target viral gene or gene fragment.

Probability of detection at AMDL within sample collection buffer

≥0.95

Probability of detection at AMDL in an aerosol environmental matrix

≥0.95 (Appendix O, Part 2)

Inclusivity panel purified DNA All inclusivity strains (Annex I) must test positive at 2× the AMDLa

Exclusivity panel purified DNA All exclusivity strains (Annex II and Appendix O, Part 1) must test negative at 10× the AMDLa

System false-negative rate using spiked aerosol environmental matrix




a 100% correct analyses are expected. All aberrations are to be retested following the 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., AOAC INTERNATIONAL, Rockville, MD, USA, Appendix I, http://www.eoma.aoac.org/app_i.pdf]. Some aberrations may be acceptable if the aberrations are investigated, and acceptable explanations can be determined and communicated to method users.

Table 2. Controls




performing at a previously determined level of sensitivity. It is recommended that a technique (i.e., unique distinguishable signature) is used to confirm whether the

positive control is the cause of a positive signal generated by a sample.


Negative control

Designed to demonstrate that the assay itself does not produce a detection in the absence of the target organism. The purpose of this control is to rule out causes of



Inhibition control

Designed to specifically address the impact of a sample or sample matrix on the assay’s ability to detect the target organism.

Single use per sample run

Table 3. Core exclusivity panel

Species StrainCommercial availability

Vaccinia Elstree (Lister vaccine)

ATCC VR-1549

Cowpox Brighton ATCC VR-302

Ectromelia Moscow ATCC VR-1374

Monkeypox V79-I-005 BEI NR-2324

Monkeypox USA-2003 BEI NR-2500

Raccoonpox Herman ATCC VR-838

Skunkpox SKPV-USA-1978-WA

ATCC VR-1830

Volepox VPXV-USA-1985-CA

ATCC VR-1831

Camelpox V78-I-2379 BEI NR-49736

Taterapox V71-I-016 BEI NR-49737

Parapoxvirus Orf Vaccine Colorado Serum Co.

44 AOAC JOURNAL SPECIAL SECTION: Performance Standards for Biological Threat Agent Assays for Department of Defense Applications.

STANDARD METHOD PERFORMANCE REQUIREMENTS Standard … · (SMPRs®) 2015.011: for Detection of Coxiella burnetii of Defense trained operators 1 Applicability Specific detection of

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