AOAC Stakeholder Panel on Agent Detection Assays: Variola ......to the panel. Olson and Hadfield suggested this can be considered and decided on at the next working group meeting

Draft, Pre-Decisional

AOAC Stakeholder Panel on Agent Detection Assays: Variola Working Group Meeting Minutes Tuesday, March 19, 2014, 9:00 a.m. – 4:30 p.m.

Attendees

Meeting Participants (Present during all or part of the meeting):

AOAC Staff:

Ted Hadfield, Hadeco (WG Co-Chair) Victoria Olson, CDC (WG Co-Chair) Douglas Anders, FBI Maureen Beenan, NIH Linda Beck, DHS Kenneth Damer, Northrop Grumman Inger Damon, CDC Mark Gunnell, Dugway Proving Ground James Harnly, USDA Steven Hinrichs, University of Nebraska Penny Hitchcock, Tauri Group Anthony Hitchins, FDA (Retired) Ron Johnson, bioMérieux

Liz Kerrigan, ATCC Katalin Kiss, ATCC Hermann Meyer, The Bundeswehr, Germany Denise Pettit, NC Department of Health and Human Services Mark Scheckelhoff, DHS Frank Schaefer, EPA Brad Stawick, Microbac Elizabeth Vitalis, Lawrence Livermore National Laboratory

Jim Bradford Scott Coates Christopher Dent Nora Marshall Deborah McKenzie

Meeting Minutes

I. Presentation: Standard Method Performance Requirements and the AOAC Process

Coates provided a presentation on AOAC Standard Method Performance Requirements (SMPRs) and how they are developed. (Appendix A)

II. Presentation: Smallpox – Considerations for Testing

Olson provided an overview on the Variola virus including an introduction to and history of smallpox, the development of clinical diagnostics, the potential requirements for environmental testing. (Appendix B)

III. Presentation: Biological Detectors Review

Hadfield provided a presentation on the various types of collectors and extraction efficiency. (Appendix C)

IV. Presentation: BioWatch Presentation

Beck and Scheckelhoff provided a presentation on BioWatch testing and the transition from testing of Vaccinia to the testing of Variola.

V. Discussion: Strawman Variola Standard Method Performance Requirements

The group reviewed the document Variola SMPR v5.1, a draft SMPR for Variola which has been compiled by the core members of the working group.

A. Section 4, Definitions: (1) Under AMDL, an “alternative method” is mentioned but not identified. Action for Dent to clarify this with

AOAC. (2) Under LPOD, collaborative study data is mentioned. Since a collaborative study with Variola is not likely

to happen, the group recommended this be removed.

B. Section 6, Reference Materials: (1) Reference materials should be added to this section where they are available (e.g. Exclusivity).

C. Maximum Time to Determination:

1

March 19, 2014 Variola Working Group

Meeting Minutes v1.0 – 04/03/2014

(1) The group agreed to move this from its own item to the Method Performance Requirements table in

Section 9.

D. Section 9, Method Performance Requirements: (1) The group discussed changing the requirement from copies per filter to copies per mL (based on the core

group’s previous determination of 50 copier per microliter, 50,000 copies per mL,500,000 copies per filter). The group agreed that it is better to define this by mL rather than by filter.

(2) There was a discussion on whether or not to use the term “purified DNA” for Inclusivity and Exclusivity. The group agreed this was an appropriate term and the change was made.

E. Annex 1, Controls: (1) The group had a lengthy discussion on if all three controls (positive, negative and inhibition) were

necessary. Would including positive and negative controls in the standard change the response after an assay is run? It was agreed that the controls should remain, but the wording should be simplified to controls “must perform as designed.”

F. Annex 2, Inclusivity Panel: (1) Olson explained that the core group had discussed that the panel be designed based on bioinformatics,

and that strains should not be dictated. The group agreed to this approach. The group expressed concern that the SMPR did not list any safety precautions for dealing with Variola. Damon and Olson advised that they will add appropriate text to the SMPR and a link to the World Health Organization (WHO) regulations.

G. Annex 3, Exclusivity Panel: (1) Olson explained that the core group had selected one representative from each other species of Orthopox

and one Parapox. Bioinformatic analysis would be used to determine the other panel members.

H. Annex 4, Environmental Panel: (1) Hadfield explained that this panel was originally developed for the B. anthracis SMPR. Hadfield provided

a short history of the development of this panel. (2) The group suggested adding B. mallei to the panel. Olson and Hadfield suggested this can be considered

and decided on at the next working group meeting. (3) BioWatch representatives present confirmed that they can provide used filters for this panel, including

seasonal filters. (4) There was a lengthy discussion on the necessity of soil samples and dusts. The general consensus of the

working group was to eliminate soils and dusts and expand on the filters section of the environmental panels; stating that there should be testing with established filters to demonstrate that there is no inhibition. BioWatch representatives confirmed that they can provide pooled filters as liquid samples which would be tested at the Naval Warfare Center before they are sent out.

VI. Adjourn

a) Actions to modify the SMPR were assigned and the meeting adjourned at approximately 4:30 p.m.

MARCH 19, 2014 SPADA VARIOLA WORKING GROUP: ACTION ITEMS Action Owner Incorporate Working Group changes made on 3/19 into SMPR 5.1 and distribute a new version to group members.

Hadfield/Olson/Dent

Organize a teleconference of the full Variola Working Group to conduct a final review of the SMPR document before it is send out for wider comment.

Dent

2



Appendices: Appendix A: Coates Presentaton - Standard Method Performance Requirements and the AOAC Process Appendix B: Olson Presentation - Variola Genetic Diversity and Sequence Variation Appendix C: Hadfield Presentation –Biological Detectors Review Appendix D: Variola SMPR v5.1

3



AOAC INTERNATIONAL

Standard Method Performance Requirements

(SMPRs) for SPADA

2014 Mid-Year Meeting

Gaithersburg, Maryland, USA

4



SMPRs • Introduction

• Background

• Format

• Process

• Guideline for Development of SMPRs

• Performance Parameters

5



Standard Methods Performance Requirements

• Commonly referred to as:

• SMPRs

• “Smipper”s

• Updated format

6



SMPRs • documents a community’s analytical method

needs.

• very detailed description of the analytical requirements.

• includes method acceptance requirements.

• published as a standard.

• used to qualify methods for AOAC approval.

7



Uses of SMPRs

• Basis for method acceptance and approval. • Guidance to method developers for the

development of new methods. • Advance the state-of-the-art in a particular

direction. • Address specific analytical needs. • Allow AOAC to reach a broader community of

method developers and users.

8



AOAC has adopted 30+ SMPRs

9



SMPR Format

• Intended use

• Applicability

• Analytical technique

• Definitions

10



SMPR Format

• System suitability

• Reference materials

• Validation guidance

• Maximum time-to-determination

• Method performance requirements table

11



SMPRs are published in the OMA. SMPR ID numbers use the year and 3 numerals. OMA ID numbers use the year and 2 numerals.

12



SMPRs can be developed for all types of methods: Quantitative methods

• Trace components

• Main components

Qualitative methods

• Trace components

• Main components

Identification methods

13



SMPR Process

14



15



16



17



Fitness-for-Purpose • Very early in process

• General statement of method performance

• No or few acceptance criteria

• 1 or 2 paragraphs

• No formal format

• Not a standard

SMPR • A deliverable

• Very detailed specification of method performance requirements

• Acceptance criteria

• 2 to 3 pages

• Standard format

• Formal AOAC standard

• Published in the OMA

18



19



20



21



22



Appendix F: Guideline to SMPRs

• Complete guidance

describing SMPRs and general validation requirements.

• 19th ed. of OMA

• On-line at: http://www.eoma.aoac.org/app_f.pdf

23



Performance parameters

24



• Acceptable Minimum Detection Level (AMDL)

• Recommended Probability of Detection at the AMDL

• Inclusivity / Exclusivity Requirements

• Collaborative Study System False Negative Rate

• Collaborative Study System False Positive Rate

Performance requirements parameters for biological threat agent methods:

25



Appendix I: Guidelines for Validation of Biological Threat Agent Methods (BTAM)

• Validation requirements

for biological threat agent methods.

• 19th ed. of OMA

• On-line at: http://www.eoma.aoac.org/app_i.pdf

26



Both guidelines can be found on the AOAC website. 1. Find and mouse over on the Standards

Development dropdown menu on the homepage. 2. Click on Guidelines and References. 3. You’ll find both guidelines in the listings.

27



Summary

• SMPRs provide a logical way to define what we need in a method.

• SMPRs provide a way to standardize inclusivity/exclusivity panels.

• The process allows a community to agree on and set the minimum performance requirements for a class of methods.

28



Summary

• SMPRs provide an objective standard to judge candidate methods.

• SMPRs are unique in the analytical community.

• SMPRs are the future for AOAC.

29



• Questions?

30



Victoria A. Olson Ph.D. Microbiologist, Poxvirus Team Poxvirus and Rabies Branch

March 19, 2014

Adapted from presentation by Inger Damon, M.D., Ph.D.

Smallpox:

Considerat ions for test ing

Division of High Consequence Pathogens and Pathology National Center for Emerging and Zoonotic Infectious Diseases

31



Poxvirus 101 Family of large, double stranded

DNA viruses Within genera, antigenic similarity:

cross protection

Complex viruses, cytoplasmic lifecycle

Genus orthopoxvirus: 90-98% nucleotide identity across

species variola, vaccinia, cowpox, monkeypox

– all can cause human disease • Variola evolved to be a sole human

pathogen: SMALLPOX camelpox, ectromelia, taterapox are

NOT known to cause human disease

32



Object ives

• Smallpox introduction

• Development of smallpox clinical diagnostics

• Developing a framework for considering environmental smallpox detection/testing

33



SMALLPOX HISTORY AND BACKGROUND

Smallpox disease elimination and eradication variola virus elimination and (?) eradication

34



Smallpox

35



Disease Sole human pathogen

No known non-human animal reservoir

Transmit ted by respiratory route (largely airborne droplets) Rare, but notable occurrences of airborne transmission in

some hospitals

Transmit ted by percutaneous exposure

Fomites – rare cause of t ransmission

Not foodborne or waterborne

36



Source: Breman JD, Henderson DA. N Engl J Med 2002;346:1300-8.

37



Progression of smallpox

• Incubat ion Period • Pre-erupt ive Stage:

– abrupt onset high fever/const itut ional symptoms

• Macules • Papules • Vesicles • Pustules • Scabs • Scars

38



Smallpox - disease eliminat ion/eradicat ion Disease – viral exanthem

Major (“ave” 30% CFR) and minor disease (<1% CFR)/viruses

Disease prevent ion Childhood vaccination – variable rates; +/-variolation

Global Smallpox Eradicat ion program - 1958 Intensification of Smallpox eradication program 1967

• Surveillance -> contact tracing, vaccination of contacts (and contacts) • Isolation of cases, observation of contacts • Lyophilized vaccine, semi-standardized vaccine production

o Vaccinia virus o “Take” ~ protection

No non-human animal reservoir

Eliminat ion in all countries by 1977 Commission to Certify Smallpox eradication activities

• Certified as eradicated in 1979 WHA : Declaration of smallpox eradication 1980

* Smallpox and its Eradication WHO 1988

39



History- variola virus eliminat ion and eradicat ion

Consolidat ion of laboratory-held virus materials* 1975 survey by WHO, post lab exposure in 1973 (LSTMH)

• 74 labs report variola materials 1976 voluntary consolidation

• 1978 – Birmingham, England smallpox “lab”: 1 death, illness 1979 – WHO Committee of Experts recommends to preserve

variola virus stocks in a few collaborating center (CC) labs, review in 1982:->19 recommendations by the Global Commission

• 1979 – 7 labs report variola virus stocks • 1981 – 4 CC labs with variola virus • Periodic inspections for safe and secure use virus

1984 – consolidation of stocks to 2 WHO Collaborating Centers – BSL-4 facilities (WHA 33.4)

* Smallpox and its Eradication 1988

40



Virus eradicat ion – considerat ions to 1999

Addit ional Global Commission sanct ioned research – reflect (new) technologies of the t ime Cloning of variola virus genomes – in representative segments Hybrid viruses* (1981): proof of recombination/“transfection”

• Scientific Advisory Group of Experts (1984) o Vaccine research using vaccinia vector

Sequencing of virus genomes – • 1993 -2 complete variola virus “major” genomes available

Bioterrorism threat once vaccinat ion program ceases Decision to prohibit genet ic manipulat ion of variola

virus, restrict access to genomic elements and genome Discovery that Russia had attempted to “weaponize”

variola virus * Sam and Dumbell Expression of poxvirus DNA in coinfected cells and marker rescue of thermosensitive mutants by subgenomic

fragments of DNA Annales de virologie, 1981

41



SMALLPOX RESEARCH AGENDA: DIAGNOSTICS

WHO/WHA process IOM 1999, 2009 research 2000 to today

42



Smallpox Research Agenda: focused on preparedness needs

• Institute of Medicine Report recommendations for “Assessment of Future Scientific Needs for Live Variola Virus” (1999) have helped to frame the research agenda.

• Protocols approved by WHO technical subcommittee • Research updates provided annually to the WHO Advisory Committee for

Variola Virus Research • Collaborative HHS (largely CDC) and DoD (largely USAMRIID)

• All U.S. work with live variola virus occurs within the BSL-4 containment

laboratory at the CDC • Inspected regularly by U.S. security and biosafety authorities and WHO

biosafety teams • Genetic manipulation of variola virus not authorized by WHO

– 1994 Ad hoc Orthopoxvirus Advisory Committee recommendation • Full genomes of variola virus can only be maintained at the 2 WHO CCs • No lab can have more than 20% of the variola virus genome, except a WHO

Collaborat ing Center • All research findings to be made available to the international scientific

community

43



IOM recommendations* 1999-> WHO sanct ioned research agenda

• Molecular characterization of variola virus for more sensitive and specific diagnostic development – Sequencing entire genomes and specific genes

• Antiviral

• Less reactogenic vaccine development

• Animal model – pathogenesis, model system for antiviral & novel vaccine evaluation

• Fundamental research – host pathogen interaction

* Assessment of Future Scientific Needs for Live Variola Virus; N.A.Press (1999)

44



WHA resolut ions and WHO Protocol approval process

• 1999 WHA resolution to postpone decision on destruction until 2002

• 2002 WHA resolution to postpone decision

• 2005 WHA – Increased focus on “essential” public health research

• Interpreted by WHO committee to preclude fundamental research – Major review of the research to the WHA in 2011

• Advisory Group of Independent Experts (AGIES) review variola virus research in 2010

• 2011 WHA – Resolution to revisit in 3 years

• AGIES conduct second review of variola virus research in 2013

45



Virus characterizat ion

“Genomic sequencing and limited study of variola virus surface proteins derived from geographically dispersed specimens is an essential foundation for important future work. Such research could be carried out now, and could require a delay in the destruction of known stocks, but would not necessitate their indefinite retention.*”


46



Maximum likelihood analysis of single nucleot ide matrices (variola virus)

NON-WEST AFRICA

BANGLADESH NEPAL

INDIA SUMATRA MIDDLE EAST

EAST ASIA ORIENT

W AFRICA BRAZIL

48 isolates passaged 2-3 times to high titer master seed stocks DNA extracted Sequenced using primer walking methodology

Li Y. et.al. PNAS 2007;104:15787-15792 Esposito et. al Science 2006

Asian variola major

Alastrim minor

African minor AND African major

47



Diversity of variola virus ~200 kb dsDNA, ~200 ORFs

• Diversity of variola virus strains is associated with geographic distance • Alastrim minor (South America) / variola major (Asia): ~600 SNPs, ~80 Indels • Alastrim minor / variola intermediate (West Africa): ~350 SNPs, ~45 Indels • Variola African minor/major / variola major (Asia): ~150 SNPs, ~30 Indels

• Central region: virion structural proteins, enzymes - 30 gene sequences are perfectly conserved or have only synonymous SNPs, highly conserved, essential function.

• Left and Right end regions: Host range and immunomodulatory genes - majority of Indels/frameshift mutations, fragmented sequences, additional/absent of ORFs, - likely reflecting selection pressures.

• Versus other Orthopoxviruses: • Variola / camelpox-taterapox viruses: ~3200 SNPs, ~380 Indels • Variola / monkeypox virus: ~7500 SNPs, ~600 Indels

48



Diagnost ics/environmental detect ion

“If further development of procedures for the environmental detection of variola virus or for diagnostic purposes were to be pursued, more extensive knowledge of the genome variability, predicted protein sequences, virion surface structure, and functionality of variola virus from widely dispersed geographic sources would be needed.*”


49



Diagnost ics

• Why “if?”

• Proponents, in 1999, that EM and standard PCR techniques were sufficient for smallpox diagnostics – As of 2002 – survey of EM capacity in state health

departments reveals only 3-8 with skilled capability to any capability

– Newer technology: real time PCR

50



Diagnost ics: Nucleic acid test ing Real t ime PCR Assays

• Platform supported at Laboratory Response Network (LRN)

• High throughput • Sensitive/specific

– Sensitive to 1-50 genome copies – Historically lesion samples contained 104-107

infectious virions – Assays validated against authentic variola virus

genomic material • Limitations:

– time to get samples to reference labs

51



Diagnost ics developed • CDC developed/evaluated (real time) PCR assays targeting Orthopoxvirus

genus and various species (variola, monkeypox, vaccinia, etc. ) – Provide reagents/facilities for others to evaluate assays

• ~13 peer-reviewed publications evaluate PCR assays against authentic variola virus genomic material

• Subset used in LRN (variola, monkeypox, cowpox and vaccinia virus detection) – 2002 onward: vaccine AE identification – 2003: response to monkeypox outbreak

• Regulatory agency approval

– De novo 510K submitted on orthopoxvirus non-variola assay • Approved September 2012

– Discussion initiated with FDA (2002) on variola virus assay

• Initiating, technology transfer to other countries – Monkeypox – Smallpox laboratory diagnostics network (WHO sponsored)

52



Clinical diagnost ic approaches used at the WHO CC at CDC

• Nucleic acid testing

• Viral isolation

• Serologic assays

• Protein based/virus detection – In development – Commercial assay available

• Only one Orthopoxvirus diagnostic assay has achieved regulatory approval – LRN Orthopoxvirus non-variola real-time PCR assay

• FDA de novo 510(k) approved September 2012 • Dependent upon LRN algorithm

53



Clinical laboratory algorithm development and successes

• Focuses clinical attention to most serious look-a-likes

• Focuses lab attention to most serious contenders

• Helps define/remind what conditions are most frequently confused with possible smallpox

• Frames logic for approaching diagnostics

• Minimizes false positives

• Use of the algorithm in 2002*

Seward et. al. CID 2004

54



LRN

55



CLINICAL DIAGNOSTIC PERFORMANCE RELATED TO DISEASE PREVALENCE

Predictive value vs. sensitivity and specificity

56



Test parameters

• Sensit ivity: – the ability to identify as positive all those with the disease

• Specificity: – the ability to identify as negative all those without the

disease • Predict ive value posit ive (PV+):

– the proportion of true positives among those testing positive

• Predict ive value negat ive (PV-): – the proportion of true negatives among those testing

negative

57



Disease

present absent

a=true positives

b=false positives

c=false negatives

d=true negatives

Test result

POS

NEG

total

a+b

c+d

TOT a+c b+d

Sensitivity = a/ (a+c)

Specificity = d/ (b+d)

PV+ = a/ (a+b)

PV- = d/ (c+d)

58



Pre-event/Post-event

• Test > 90% sensitivity • Pre-event prevalence of smallpox is zero:

– If test has 95% specificity, 10 tests done per month, in 6 to 8 sites, every month there will be 3 or 4 false positives

• Post-event prevalence of smallpox is finite: – If test has 95% specificity, 1000 tests done per week,

every week 50 results will be false positives – If test has 99% sensitivity, 1000 tests done per week,

10 results will be false negatives

59



Test parameters

• Sensitivity and specificity are independent of the prevalence of disease

• Predictive value positive and negative vary with disease prevalence (Bayes’ Theorem)

• Implications for smallpox testing “pre-event” and “post-event”: use of tests for decision making

60



Test parameters - examples

• Sensitivity 95% • Specificity 95%

PREV PV+ PV-50% 95% 95%10% 67.80% 99%1% 16% 99.95%

0.10% 1.80% 99.99%0.01% 0.20% 99.99%

61



Test parameters - examples

• Sensitivity 99% • Specificity 99%

PREV PV+ PV-10.00% 92.80% 99.80%

1% 50% 99.99%0.10% 9% 100.00%

62



Example: “Pre-event” Use more than 1 test to increase PV+ Individual with clinical scenario with fever, followed by centifugal rash:

Test 1: sensitivity 99%,

specificity 99%

PREV PV+ PV-10.00% 92.80% 99.80%

1% 50% 99.99%0.10% 9% 100.00%

Test 2: sensitivity 95%, specificity 95%

PREV PV+ PV-50.00% 95.00% 95.00%

10% 67% 99.00%1.00% 16% 99.50%

63



Pre event: prevalence of smallpox is zero

Peri event: prevalence of smallpox is low

• Clinical scenario should be consistent – Use febrile rash algorithm; validate algorithm

• Wide availability of other key diagnostic tests especially rapid VZV testing to rule in VZV

• Limit the number of laboratories performing variola virus testing – Establish confirmatory testing protocols

64



Pre event: prevalence of smallpox is zero

Peri event: prevalence of smallpox is low

• Approaches to improving predictive value positive : implications for result use (rule in, and institution of vaccination campaign vs. rule out) – Use more than 1 test (different targets) – Use tests without common sources of false

positives

65



Post event

• Need to assess what needs for testing will be: – One scenario: greatest needs in beginning,

and near end of smallpox re-eradication

• May need to test more “low suspicion samples” – Sensitivity vs. specificity

66



Issues relevant to implementat ion of smallpox diagnost ics

• Biosafety: – containment; – Standard vs. Universal vs. Airborne precautions; – vaccination

• Biosecurity • Reagents

– WHO, CDC, select agent recommendations on use of variola virus DNA • Centralized/regionalized testing

– Transportation of specimens • QA/QC, Proficiency testing: how to • Communication between clinician/epidemiologists/laboratory

– Clinical history, case patient photos • Development of a disease confirmatory algorithm

– Screening tests, confirmatory tests: regional vs. centralized – Presumed positive, Confirmed Positive – Communication of results, and public health response – Role of viral isolation by culture

67



LRN

68



NA TEST VALIDATION Iterative approaches to test validation

69



Previous validations:

Sensitivity analysis: Serial 100-fold dilutions (1ng/µl to 1 fg/µL) of DNA Inclusivity panel – variola virus DNA: 2 purified viral stocks 33 crude viral stocks 2 Human scab samples

Specificity analysis: Serial 100-fold dilutions (1ng/µl to 1 fg/µL) of DNA Exclusivity panel – near neighbor (Orthopoxvirus): Eurasian: 1 ectromelia 2 monkeypox viruses 2 camelpox viruses 1 cowpoxvirus 1 taterapoxvirus 5 vaccinia viruses North American: 1 skunkpoxvirus Exclusivity panel – other rash-causing illnesses: 1 Varicella Zoster virus 1 Herpes simplex virus (type 1) 1 Rickettsia strain Exclusivity panel – negatives: Myxoma & tissue culture (2)

70



Complementary set:

Inclusivity Panel Species Strain Sample

Variola virus 102 Crude Variola virus 103 Crude Variola virus 66-39 Crude Variola virus 7124 Crude Variola virus 7125 Crude Variola virus 72-119 Crude Variola virus 73-175 Crude Variola virus 77-1605 Crude Variola virus Bombay Crust Variola virus Brazil-Garcia Crude Variola virus Congo Crude Variola virus Eth-17 Crude Variola virus Harper Crude Variola virus Harvey Crude Variola virus Heidelberg Crude Variola virus Higgins Crude Variola virus Hinton Crude Variola virus Horn Crude Variola virus Horn Pure Variola virus K1629 Crude Variola virus Kali Mathu Crude Variola virus Kembula Crude Variola virus Minnesota 124 Crude Variola virus MS Lee Crude Variola virus Nepal Pure Variola virus New Dehli Crude Variola virus Nigeria Kuclano Crust Variola virus Nur Islam Crude Variola virus Rumbec Crude Variola virus Shahzamon Crude Variola virus Solaiman Crude Variola virus Stillwell Crude Variola virus V68-59 Crude Variola virus V70-222 Crude Variola virus V70-228 Crude Variola virus Variolator-4 Crude Variola virus Yamada Crude

Exclusivity Panel - near neighbor (Orthopoxviruses) Species Strain

Ectromelia virus Moscow Monkeypox virus 79-0266 Monkeypox virus 79-0005 Camelpoxvirus LLC Camelpoxvirus V78-I-903 Cowpoxvirus Brighton

Taterapoxvirus (Gerbilpox) Vaccinia virus Lister Vaccinia virus VTH Vaccinia virus Wyeth Vaccinia virus WYH pGS62-9-v1-1-1 Vaccinia virus Rabbitpoxvirus Skunkpovirus

Exclusivity Panel - other rash-causing illnesses Species Strain

Varicella Zoster Virus Webster Herpes Simplex Virus-1 HFEM

Rickettsia conorii

Exclusivity Panel - other negatives

Species Strain

Myxoma

Human tissue culture cells Sup-T

Monkey kidney tissue culture cells BSC-40

71



Validated diagnostic real-time PCR assays Target multiple regions of

the genome Target sequences specific

to variola virus

Recently acquired Cowpox virus sequences Exhibit extensive

phylogenetic diversity Contain certain regions

previously thought to be specific to variola virus

• Now assays predicted to cross-react with cowpox virus

Variola virus : diagnostic creation

and validation

D. Carroll, et. al

72



Variola virus signature (eroded specificity): assay cross-reacts with cowpox virus

Exclusivity panel Species Strain Name Assay 1

ct for 5 ng DNA Assay 2

ct for 5 ng DNA Vaccinia Copenhagen Negative Negative Vaccinia WR Negative Negative Vaccinia ACAM 2000 Negative Negative Vaccinia BRZ SERRO Negative Negative Cowpox CPXV-NOR1995-MAN Negative Negative Cowpox CPXV GER1980-EP4 19 Negative Cowpox CPXV GER1991-3 18 Negative Cowpox CPXV_GER1998_2 17 Negative Cowpox CPXV FIN 2000 Negative

Ectromelia ECTV Moscow Negative Negative Monkeypox MPXV RCG 2003 358 Negative Negative Monkeypox MPXV USA 2003 044 Negative Negative Raccoonpox RACV V71-I-84 Negative Negative Skunkpox SKPV 1991 Negative Negative Volepox VPXV 2004-CA-007 Negative Negative

Camelpox CMLV-78-I-2379 17 Negative Taterapox (gerbilpox) TATV-71-I-016 16 Negative

Parapoxvirus Orf Vaccine for sheep Negative Negative

73



What we have learned about variola virus diagnostic assay development/validation

Bioinformatic analysis should lead design of validation panels Inclusivity panel include all variola virus strains with

differences in assay target region Exclusivity panel (near neighbor Orthopoxviruses)

contain viruses with assay target regions most similar to variola virus

Exceedingly difficult to construct uniform panels for all assays due to high similarity between Orthopoxviruses

Simultaneous identification of multiple variola virus signatures will increase confidence in initial identification/verification of the pathogen with real-time PCR

74



Sensitivity analysis: Two low dilutions (100 and 10 fg/µL) of DNA Inclusivity panel – variola virus: ≥ 2 purified viral stocks (at least one from each primary clade) (encompass differences in target region)

Specificity analysis: One high dilution (100 pg/µl) of nucleic acid Exclusivity panel – near neighbor (Orthopoxvirus): Eurasian: 1 ectromelia 2 monkeypox viruses (one from each clade) 1 camelpox virus 5 cowpoxviruses (one from each predicted clade) 1 taterapoxvirus 4 vaccinia viruses (one from each clade) North American: 1 raccoonpoxvirus 1 skunkpoxvirus 1 volepoxvirus

Current recommendations: tailor panel to assay based on bioinformatics

75



Specificity analysis (cont.): One high dilution (100 pg/µl) of DNA

Exclusivity panel (cont.) – other rash-causing illnesses: 2 Varicella Zoster viruses (J clade and E1 clade) 2 Herpes simplex viruses (type 1 and 2) 2 Rickettsia strains Chordopoxviruses: 1 Parapoxvirus (Orf) Negatives: 13 bacteria strains (skin flora) water

Current recommendations: tailor panel to assay based on bioinformatics

76



Current Recommendations: Exclusivity panels

100 pg/µl Exclusivity panel - near neighbors

(Orthopoxviruses) Species Strain Name

Ectromelia ECTV Moscow Monkeypox MPXV RCG 2003 358 Monkeypox MPXV USA 2003 044 Camelpox CMLV-78-I-2379 Cowpox CPXV-NOR1995-MAN Cowpox CPXV GER1980-EP4 Cowpox CPXV GER1991-3 Cowpox CPXV FIN-2000-MAN Cowpox CPXV_GER1998_2

Taterapox (gerbilpox) TATV-71-I-016 Vaccinia Copenhagen Vaccinia WR Vaccinia ACAM 2000 Vaccinia BRZ SERRO

Raccoonpox RACV V71-I-84 Skunkpox SKPV 1991 Volepox VPXV 2004-CA-007

Exclusivity panel - other rash-causing illnesses Species Strain Name

Varicella-zoster virus pOKA (J clade)

Varicella-zoster virus Webster (E1 clade)

Herpes simplex virus type 1 F

Herpes simplex virus type 2 G

Rickettsia conorii CDC

Rickettsia akari CDC

Parapoxvirus Orf Vaccine for sheep

Exclusivity panel - Negatives Species ID number

Enterococcus faecalis ATCC 29212 Eschericia coli ATCC 25922

Klebsiella pneumoniae ATCC 33495 Peptostreptococcus anaerobius ATCC 27337

Propionibacterium acnes ATCC 6919 Pseudomonas aeruginosa ATCC 27853

Staphylococcus aureus (strain1) ATCC 12600 Staphylococcus aureus (strain 2) ATCC 25923

Staphylococcus epidermidis (strain 1) ATCC 49134 Staphylococcus epidermidis (strain 2) ATCC 12228 Staphylococcus epidermidis (strain 3) ATCC 14990

Streptococcus gallylyticus ATCC 49147 Streptococcus pyogenes ATCC 49117

Water

77



THINKING ABOUT ENVIRONMENTAL DETECTION

78



Considerat ions in addit ion to those of clinical diagnost ics….

How to verify authent ic agent? Sufficient vs. necessary Culture? Nucleic acid tests

• How much of the genome?

How to verify infect ious risk? Absent a clinically ill human, what is sufficient, what is necessary?

How and where to evaluate validate WHO considerations what is sufficient, necessary to have a public

safety/health actionable assay result; What should that action be?

79



Environmental variola screening test posit ives (26) >808,000 samples tested

confirmatory testing: no test result was confirmed positive

Test Date Assay Sample ID Parameter Result CT Value 2/10/2008 08:09:00 PM CDC Screen Panel P066079 VRL3 reactive 40.54 2/10/2008 08:07:00 PM CDC Screen Panel P066060 VRL3 reactive 40.54 2/10/2008 10:18:00 PM Verification panel for Variola

virus P066060 VRL1 reactive 27.89

2/10/2008 08:11:00 PM CDC Screen Panel P065784 VRL3 reactive 40.54 2008 4

5/6/2009 02:43:01 PM CDC Screen Panel 050609-654 VRL3 reactive 28.93 5/6/2009 02:43:00 PM CDC Screen Panel 050609-192 VRL3 reactive 28.93 5/6/2009 02:43:01 PM CDC Screen Panel 050609-666 VRL3 reactive 28.93

11/23/2009 01:16:00 PM CDC Screen Panel P069407 VRL3 reactive 41.69 11/23/2009 01:16:00 PM CDC Screen Panel P069411 VRL3 reactive 41.69 12/17/2009 03:34:00 PM CDC Screen Panel P070763 VRL3 reactive 43.28 12/17/2009 03:34:00 PM CDC Screen Panel P070670 VRL3 reactive 43.28 10/9/2009 03:21:00 PM CDC Screen Panel P133565 VRL3 reactive 20.66

2009 8 4/17/2010 08:37:00 PM CDC Screen Panel P298637 VRL3 reactive 28.91 4/17/2010 08:37:00 PM CDC Screen Panel P298640 VRL3 reactive 28.36 4/17/2010 08:37:00 PM CDC Screen Panel P298645 VRL3 reactive 28.03 4/26/2010 02:13:00 PM CDC Screen Panel P222219 VRL3 reactive 42.28 4/3/2010 03:40:00 PM CDC Screen Panel P113811 VRL3 reactive 36.92 4/3/2010 03:40:00 PM CDC Screen Panel P113819 VRL3 reactive 36.92

2/23/2010 01:03:00 PM CDC Screen Panel D549214 VRL3 reactive 44.07 3/29/2010 09:48:00 AM CDC Screen Panel P554250 VRL3 reactive 41.02 3/29/2010 09:48:00 AM CDC Screen Panel P554251 VRL3 reactive 41.02 3/29/2010 09:48:00 AM CDC Screen Panel P554252 VRL3 reactive 41.02 3/17/2010 02:50:00 PM CDC Screen Panel P076217 VRL3 reactive 26.53 3/17/2010 02:50:00 PM CDC Screen Panel P076225 VRL3 reactive 26.53 8/5/2010 07:33:00 PM CDC Screen Panel P409223 VRL3 reactive 42.87

2010 13 12/29/2011 07:47:00 PM CRP Screen Panel P307499 POX2 reactive 32.74 12/29/2011 07:43:00 PM CRP Screen Panel P307493 POX2 reactive 32.74 7/21/2011 07:39:00 PM CDC Screen Panel P066838 VRL3 reactive 1/7/2011 02:20:00 PM CDC Screen Panel P194059 VRL3 reactive 44.01

2011 4 29

80



For more information please contact Centers for Disease Control and Prevention

1600 Clifton Road NE, Atlanta, GA 30333 Telephone, 1-800-CDC-INFO (232-4636)/TTY: 1-888-232-6348 E-mail: [email protected] Web: www.cdc.gov

Acknowledgements Poxvirus Team members 1999-present

“ TNTC” CDC and external partners

National Center for Emerging and Zoonotic Infectious Diseases Division of High Consequence Pathogens and Pathology

The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.

81



For more information please contact Centers for Disease Control and Prevention

1600 Clifton Road NE, Atlanta, GA 30333 Telephone, 1-800-CDC-INFO (232-4636)/TTY: 1-888-232-6348 E-mail: [email protected] Web: www.cdc.gov

Extra slides

82



Ectromelia

Cowpox Camelpox

Taterapox Variola

Vaccinia Brazilian Isolates

Cowpox Vaccinia

Monkeypox Volepox

Skunkpox Raccoonpox

0.01 substitutions/site

Clethrionomys glareolus

Orthopoxvirus Host Systems

Tatera kempii

Microtus californicus

Apodemus sylvaticus

Oryzomys sp Hemagluttinin (HA)

neighbor-joining

?

Courtesy of Darin Carroll, Ph.D.

83



http://wadict.soas.ac.uk/wadict/dictdb_guest/wadict_search.php?searchfield=usg_cn&searchitem=dou%20sad

Mandate to respond to threats of bioterrorism has provided further

‘guidance’

• Development/deployment of Algorithm for Evaluating Patients for Smallpox (‘Rash Illness Algorithm’)

• Developing enhanced orthopoxvirus diagnostics for deployment beyond CDC

• Respond to large-scale vaccination

84



Sensitivity analysis: Serial 10-fold dilutions (1ng/µl to 1 fg/µL) of DNA Inclusivity panel - Variola DNA: 5 purified viral stocks 11 crude viral stocks 3 Human scab samples

Specificity analysis: Serial 10-fold dilutions (1ng/µl to 1 fg/µL) of DNA Exclusivity panel – near neighbor (Orthopoxvirus): Eurasian: 1 ectromelia 1 monkeypox virus 1 camelpox virus 1 cowpoxvirus 1 taterapoxvirus 1 vaccinia virus North American: 1 raccoonpoxvirus Exclusivity panel – other rash-causing illnesses: 1 Varicella Zoster virus 2 Herpes simplex viruses (type 1 and 2) 1 Rickettsia strain Water

Small set:

85



Small set: Inclusivity Panel

Species Strain Sample Variola virus 68-258 Crude Variola virus 7124 Pure Variola virus 72-143 Crude Variola virus 73-225 Crude Variola virus 77-1252 Crude Variola virus Afghanistan Pure Variola virus Bangledesh Crude Variola virus Bangledesh Pure Variola virus Butler Crude Variola virus Iran Crude Variola virus Juba Crude Variola virus Kembula Crust Variola virus Mannon Crude Variola virus Parvin Crust Variola virus Rafig Lahore Crude Variola virus Solomain Crust Variola virus Somalia Pure Variola virus Sumatra Pure Variola virus v74-227 Congo 9 Crude

Exclusivity Panel - Near Neighbor (Orthopoxvirus)

Species Strain Ectromelia virus Moscow

Monkeypox virus V96-I-16

Camelpoxvirus E2379

Cowpoxvirus Brighton

Taterapoxvirus (Gerbilpox)

Vaccinia virus IHDW

Raccoonpoxvirus

Exclusivity Panel - other rash-causing illnesses

Species Strain

Varicella Zoster virus OKA

Herpes Simplex Virus-1 Justin

Herpes Simplex Virus-2 patient 920

Rickettsia akari

water

86



Complementary set:

Sensitivity analysis: Serial 100-fold dilutions (1ng/µl to 1 fg/µL) of DNA Inclusivity panel – variola virus DNA: 2 purified viral stocks 33 crude viral stocks 2 Human scab samples

Specificity analysis: Serial 100-fold dilutions (1ng/µl to 1 fg/µL) of DNA Exclusivity panel – near neighbor (Orthopoxvirus): Eurasian: 1 ectromelia 2 monkeypox viruses 2 camelpox viruses 1 cowpoxvirus 1 taterapoxvirus 5 vaccinia viruses North American: 1 skunkpoxvirus Exclusivity panel – other rash-causing illnesses: 1 Varicella Zoster virus 1 Herpes simplex virus (type 1) 1 Rickettsia strain Exclusivity panel – negatives: Myxoma & tissue culture (2)

87



Complementary set:


Variola virus 102 Crude Variola virus 103 Crude Variola virus 66-39 Crude Variola virus 7124 Crude Variola virus 7125 Crude Variola virus 72-119 Crude Variola virus 73-175 Crude Variola virus 77-1605 Crude Variola virus Bombay Crust Variola virus Brazil-Garcia Crude Variola virus Congo Crude Variola virus Eth-17 Crude Variola virus Harper Crude Variola virus Harvey Crude Variola virus Heidelberg Crude Variola virus Higgins Crude Variola virus Hinton Crude Variola virus Horn Crude Variola virus Horn Pure Variola virus K1629 Crude Variola virus Kali Mathu Crude Variola virus Kembula Crude Variola virus Minnesota 124 Crude Variola virus MS Lee Crude Variola virus Nepal Pure Variola virus New Dehli Crude Variola virus Nigeria Kuclano Crust Variola virus Nur Islam Crude Variola virus Rumbec Crude Variola virus Shahzamon Crude Variola virus Solaiman Crude Variola virus Stillwell Crude Variola virus V68-59 Crude Variola virus V70-222 Crude Variola virus V70-228 Crude Variola virus Variolator-4 Crude Variola virus Yamada Crude

Exclusivity Panel - near neighbor (Orthopoxviruses) Species Strain

Ectromelia virus Moscow Monkeypox virus 79-0266 Monkeypox virus 79-0005 Camelpoxvirus LLC Camelpoxvirus V78-I-903 Cowpoxvirus Brighton

Taterapoxvirus (Gerbilpox) Vaccinia virus Lister Vaccinia virus VTH Vaccinia virus Wyeth Vaccinia virus WYH pGS62-9-v1-1-1 Vaccinia virus Rabbitpoxvirus Skunkpovirus

Exclusivity Panel - other rash-causing illnesses Species Strain


Rickettsia conorii

Exclusivity Panel - other negatives

Species Strain

Myxoma

Human tissue culture cells Sup-T

Monkey kidney tissue culture cells BSC-40

88



Sensitivity analysis:

Serial 100-fold dilutions (1ng/µl to 1 fg/µL) of DNA Inclusivity panel - Variola DNA: 9 different strains Specificity analysis: Serial 100-fold dilutions (1ng/µl to 1 fg/µL) of DNA Exclusivity panel – near neighbor (Orthopoxvirus): Eurasian: 1 monkeypox virus 1 camelpox viruses 2 vaccinia viruses North American: 1 skunkpoxvirus Exclusivity panel – other rash-causing illnesses: 2 Varicella Zoster virus 1 Herpes simplex virus (type 1) Water

IWG set:

89



IWG set:

Inclusivity Panel

Species Strain

Variola virus 7124

Variola virus Afghanistan

Variola virus Bangledesh

Variola virus Congo

Variola virus Horn

Variola virus Nepal V73-175

Variola virus Parvin

Variola virus V73-225

Variola virus V78-2379

Exclusivity Panel - near neighbor (Orthopoxvirus)

Species Strain


Camelpox virus CP58

Vaccinia virus Dryvax

Vaccinia virus IHDJ

Exclusivity Panel - other rash-causing illnesses

Species Strain

Varicella Zoster Virus OKA

Varicella Zoster Virus Webster


water

90



Sensitivity analysis: Serial 10-fold dilutions (1ng/µl to 1 fg/µL) or three select concentrations (1ng/µl, 100 fg/µL, 10 fg/µL) of DNA

Inclusivity panel - Variola DNA:

2 purified viral stocks 10 crude viral stocks Specificity analysis: Select concentrations [(1ng/µl, 100 fg/µL, 10 fg/µL) or (1ng/µl, 10 fg/µL, 1 fg/µL)] of DNA

Exclusivity panel – near neighbor (Orthopoxvirus): Eurasian: 3 monkeypox viruses 2 camelpox viruses 2 cowpoxviruses 1 taterapoxvirus 6 vaccinia viruses North American: 1 raccoonpoxvirus 1 skunk poxvirus 1 volepoxvirus

FDA Panel:

91



Specificity analysis (cont.): One high dilution (1 ng/µl) of DNA

Exclusivity panel (cont.) – other rash-causing illnesses: 2 Varicella Zoster viruses 3 Herpes simplex viruses (type 1 and 2) 2 Rickettsia strains Negatives: 16 bacteria strains (skin flora) 2 tissue culture cellular DNAs water

FDA Panel:

92



FDA Panel:

Inclusivity panel (1 ng/ul, 100fg/ul, 10 fg/ul) Species Strain

Variola virus 66-39 Variola virus 68-258 Variola virus 7124, purified Variola virus Garcia Variola virus Heidelberg Variola virus Kembula Variola virus Minn 124 Variola virus MS Lee Variola virus Nigeria Kudano Variola virus Shahzamon Variola virus v74-227 Congo 9

Inclusivity panel 10-fold dilutions (1 ng/ul - 1 fg/ul) Species Strain

Variola virus Afghanistan - purified

Exclusivity panel - near neighbor (1 ng/ul, 100fg/ul, 10 fg/ul)

Species Strain Monkeypox virus Sierra Leone V70-I-260 Monkeypox virus V96-I-16

Cowpoxvirus V00-I-20 (Swedish) Vaccinia virus Dryvax

Exclusivity panel - near neighbor (1 ng/ul, 10fg/ul, 1 fg/ul)

Species Strain Monkeypox virus V79-I-005 Camelpoxvirus V78-I-2379 Camelpoxvirus V78-I-903 Cowpoxvirus Brighton

Taterapoxvirus (Gerbilpoxvirus) Vaccinia virus Dryvax Vaccinia virus IHDJ Vaccinia virus IHDW Vaccinia virus Lister Vaccinia virus WR Vaccinia virus Wyeth

Raccoonpoxvirus Skunkpoxvirus Volepoxvirus

93



FDA Panel:

Exclusivity panel - other rash causing illnesses (1 ng/ul)

Species Strain

Varicella Zoster Virus OKA

Varicella Zoster Virus Webster

Herpes Simplex Virus-1 HFEM


Herpes Simplex Virus-2 patient 920

Rickettsia akari

Rickettsia conorii

Exclusivity panel - negatives (1 ng/ul) Sample

Candida albicans

Diphtheroid

E. coli: 25922

Enterococcus faecalis

Klebsiella pneumoniae

Peptostreptococcus anaerobius

Propionibacterium acnes

Pseudomonas aeruginosa

Staph aureus (strain 1)



Staph epidermidis (strain 1)



Strep bovis (alpha-Strep)

strep pyogenes

BSC40 cells

Human SupT

Water

94



Sensitivity analysis: Serial 10-fold dilutions (1ng/µl to 10 fg/µL) of DNA Inclusivity panel - Variola DNA: 3 purified viral stocks 9 crude viral stocks Specificity analysis: Exclusivity panel – near neighbor (Orthopoxvirus): Two concentrations (1ng/µl and 100 pg/µL) of DNA Eurasian: 1 ectromelia 3 monkeypox virus 1 camelpox virus 2 cowpoxvirus 2 vaccinia virus

Exclusivity panel – other rash-causing illnesses/negatives: High concentration (1 ng/µl) 1 varicella Zoster virus 2 herpes simplex viruses (type 1) 1 staph strain 2 tissue culture cell lines and water

GHSAG:

95



GHSAG:


Variola virus 66-39 Crude Variola virus 68-258 Crude Variola virus 7124 Pure Variola virus Afghanistan Pure Variola virus Garcia Crude Variola virus Heidelberg Crude Variola virus Higgins Crude Variola virus Kembula Crude Variola virus MS Lee Crude Variola virus Shahzamon Crude Variola virus Somalia Pure Variola virus Congo 9 V74-227 Crude

Exclusivity Panel - near neighbors (Orthopoxvirus)

Species Strain

Ectromelia Moscow

Monkeypox virus Sierra Leone V70-I-260



Camelpoxvirus V78-I-903

Cowpoxvirus Brighton

Cowpoxvirus V00-I-20 (Swedish)

Vaccinia virus Dryvax

Vaccinia virus Lister

Exclusivity Panel - other rash-causing illness/negatives Species Strain


Staph aureus strain 2 Tissue culture cells (Monkey) BSC-40 Tissue culture cells (Human) SupT

Water

96



Biological Detectors Review

Ted Hadfield HADECO, LLC 321 474-3153

97



References

• Methods for sampling of airborne viruses. D. Verreault S, Moineau S, Duchaine C. Microbiol Molecular Biology Reviews. 2008;72:413-444

• BIAD Bacillus anthracis Assay Development Project

• MRIGlobal internal support data

98



Types of Biological Aerosol Samplers

• Liquid Impingers wet collection • Cyclone Samplers wet collection • Slit Samplers dry collection • Electrostatic precipitators dry collection • Filter units (DFU) dry collection • Other samplers

99



Aerosol Partical Size

• The means (type of generator) of aerosolization has a critical impact on the aerodynamic size and on the behavior of the airborne particles

• The size of the virus particle itself does not rule the airborne particle size.

• Particle size distribution of artificially produced submicrometer and ultrafine aerosols of culture media is not affected by the presence of virus.

100



Liquid Impinger Collector Most commonly used collectors for comparison studies Used at Dugway Proving Grounds for Aerosol “truth” data during indoor and outdoor agent release exercises Particles are collected in the liquid Survival of bacterial organisms is good Restriction on how much air flow can be pushed through the liquid

101



Cyclone Collector Particles impact on a layer of water that is recycled to concentrate particles. Collection efficiency is good with large to medium sized particles Organisms survival seems to be good in cyclone collectors if the water isn’t evaporated to dryness

102



http://www.google.com/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&docid=wycczlmakcgWuM&tbnid=851IOSfEfILh4M:&ved=0CAYQjRw&url=http://www.ebay.com/itm/Burkard-Multi-Vial-Cyclone-Sampler-for-Detection-of-Airborne-Spores-Particles-/380781920566&ei=wfgmU7-0Nqmh2QWM84GoCg&bvm=bv.62922401,d.b2I&psig=AFQjCNEHVBhCdE6afDE7tF798i0iGNLVMQ&ust=1395149262583394

Impact Collectors, Slit Sampler

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http://www.google.com/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&docid=-8bwY3hmiJv6WM&tbnid=W0H2qgJCjhUgvM:&ved=0CAYQjRw&url=http://inspectapedia.com/mold/Cassette_Study_DJF.htm&ei=YfomU-LaF4rC2wXpvIGgCQ&bvm=bv.62922401,d.b2I&psig=AFQjCNFM4moFmnXNQv3KjDRWrsPp2zSXBQ&ust=1395149529707435

Examples of Impact Collectors

Impact detectors are more efficient at capturing large particles – Anderson Sampler – Slit Samplers – Cyclone Samplers – Liquid impingers (AGIs)

104



Electrostatic Collector

High voltage corona charges particles that precipitate onto a grounded surface Fluid is used to collect and concentrate the particles

105



Dry Filter Unit Collector Filters are very destructive for viruses but are most efficient at collecting submicrometer particles Filter efficiency is based on five mechanisms

Interception Inertial impaction Diffusion Gravitational settling Electrostatic attraction

Filters can cause viral structural damage resulting in loss of infectivity Desiccation of samples during collection interferes with viral infectivity Gelatin filters can be used and do not appear to significantly affect viral infectivity.

Use is limited by environmental condition Low humidity causes drying High humidity causes the filter to dissolve

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http://www.google.com/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&docid=TqoRS6Bu4Fce2M&tbnid=d7nFfsC03imtWM:&ved=0CAYQjRw&url=http://www.automatedbuildings.com/news/may05/articles/meso/meso.htm&ei=Q_wmU-W2Lqmv2QWJu4HwAg&psig=AFQjCNHkZxGiIHOXazEQ_ZKLSZMAwjq3xw&ust=1395150261449752

Aerosol Particle Size

• Small particles are highly governed by the diffusion phenomenon

• Large particles tend to impact and to be intercepted.

• Both types of behaviors are at their lowest at 0.3uM; thus filters are least efficient at removing .3uM particles.

• Filter efficiency improves with increasing and decreasing particle size

107




• Single viral particles do exist in the air but tend to aggregate quickly to form larger particles

• Aggregation depends on size distribution of the particles the concentration of the aerosol, and the thermodynamic conditions and occurs by diffusion, impaction, interception or electrostatic attraction

• When a droplet evaporates its final size depends on the relative humidity in the chamber

108




• The smaller size limit of a viral aerosol is limited to the virus diameter.

• The larger limit depends on the4 size of the particle with which it is associated

• With a liquid impinger sampler, 65-70% of virus laden particles were ≥6uM in diameter; 19-24% were 3 uM in diameter

• ~10% were ≤3uM

109



Factors affecting Recovery of Airborne Viruses

• Relative humidity • Temperature • Radiation • Aerosolization medium • Exposure period • chemical composition of the air • The sampling method • The infectivity of the airborne assay

110



Effects of RH on Infectivity of Airborne Viruses

Virus Optima

l RH Family Nucleic Acid Size Envelope

Influenza Low Orthomyxoviridae

ssRNA (-) 80-120 Yes

Newcastle Disease

Low Paramyhxoviridae

ssRNA (-) 150 Yes

Vesicular Stomatitis

Low Rhabdoviridae ssRNA(-) 60 x 200 Yes

JEV Low Flaviviridae ssRNA(+) 40-60 Yes

Porcine R&R Low Ateriviridae

ssRNA(+) 45-60 Yes

Coronavirus Mid-range

Coronaviridae ssRNA(+) 120-160 Yes

Polio High Picornaviridae ssRNA(+) 25-30 No

Picornavirus High Picornaviridae ssRNA(+) 25-30 No

Rhinotracheitis High Herpesviridae dsDNA 200 yes

111



Virus Factors influencing Detection

• Enveloped viruses are more stable at low RH • Nonenveloped viruses are more stable at high RH • Some viruses are not influenced by RH

– (St Louis encephalitis virus) • Some viruses are more stable at low temperature

and are not influenced by RH • Vaccinia is more susceptible to UV radiation at

low RH than high RH • Detection Assay-growth, immunoassay, PCR,

other molecular assays

112



Virus Capture and Recovery • Efficiency of viral recovery is an indicator of the

integrity and infectivity of the captured virus – May capture but lose viral infectivity

• Viral detection may be done by – Culture – Immunoassay – Molecular biology assays (PCR) – Metagenomic sequencing – Animal inoculation

• Few studies evaluate virus capture followed by PCR or

other ultrasensitive detection methods

113



Things influencing viral collection and detection

• Viral concentration in the aerosol • Viability of the virus in the aerosol • Efficiency of viral capture • Viability in the capture matrix • Time and insult to the virus on the matrix • Efficiency of virus extraction from the sampler

matrix • Efficiency of test method

114



Summary • Many features influence the extraction efficiency of samples from a

particular collection apparatus • Virus type, enveloped or nonenveloped, humidity, UV intensity, time of

collection, area of collection, type of collector, type of detection assay, concentration of the aerosol, volume of air being sampled, time attributes of the collection, presence of other particulates are a few of the variables influencing extraction efficiency

• There are many collector types, the vendor selected collector should be evaluated for extraction efficiency with standardized reference suspensions of the target organism(s) on a standardized reference “dirty” filter

• Lack of standardized methods make extraction efficiency difficult to compare

• Reported efficiencies range from 10%-55% for most (bacterial) organisms, elution/extraction efficiencies are rarely reported in publications

115



Summary

• Vendor should include testing of “negative” filters collected throughout the year and from multiple geographic locations to assess false positive reactions.

• The extraction efficiency of the chosen collector samples and extraction method should meet AMDL requirements

116



•Questions Comments and Discussion

117



Not a Standard EXAMPLE Do Not Distribute

1

AOAC SMPR 2014.XXX; Version 5.1 Method Name: Detection and Identification of Variola Virus Approved Body: AOAC Stakeholder Panel on Agent Detection Assays Approval Date: Final version date: 1. Intended Use: Laboratory use by trained technicians. 2. Applicability: Detection and identification of Variola virus in aerosol collection filters

and/or liquids. 3. Analytical Technique: Polymerase Chain Reaction (PCR) Methods.

4. Definitions:

Acceptable Minimum Detection Level (AMDL)

The predetermined minimum level of an analyte, as specified by an expert committee which must be detected by the alternative method with an estimated 5% lower confidence limit on the probability of detection (POD) of 0.95 or higher. The AMDL is dependent on the intended use. (Draft ISO 16140) 1

Exclusivity

Study involving pure non-target strains, which are potentially cross-reactive, that shall be not detected or enumerated by the tested method. (Draft ISO 16140)

Environmental Interference Ability of the assay to be unaffected by environmental substances. Substances will be tested for cross reactivity and inhibition.

Inclusivity

Study involving pure target strains that shall be detected or enumerated by the alternative method. (Draft ISO 16140)

Laboratory Probability of Detection (LPOD) The POD value obtained from combining all valid collaborator data sets for a method for

a given matrix at a given analyte level or concentration. LPOD(0) = the LPOD at 0 concen-trations of the target microorganism. (AOAC)

1

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

118




2

Probability of Detection (POD)2 The proportion of positive analytical outcomes for a qualitative method for a given matrix at a given analyte level or concentration. (AOAC and Draft ISO).

System False-Negative Rate

Rate of negative system results in a population of known positive test portions.

System False-Positive Rate Rate of positive system results in a population of known negative test portions.

Variola Virus The poxvirus of the genus Orthopoxvirus that is the causative agent of smallpox.

5. System suitability tests and/or analytical quality control: The controls listed in Annex 1 shall be embedded in assays as appropriate. Manufacturer must provide written justification if controls are not appropriate to an assay.

6. Reference Material(s): 7. Validation Guidance: AOAC INTERNATIONAL Methods Committee Guidelines for Validation

of Biological Threat Agent Methods and/or Procedures (AOAC INTERNATIONAL Official Methods of Analysis, 2012, Appendix I).

8. Maximum Time-To-Determination: Maximum time to complete an analysis starting from

the test portion preparation to final determination must be 24 hours.

2 Appendix H: Probability of Detection (POD) as a Statistical Model for the Validation of Qualitative

Methods, Official Methods of Analysis of AOAC INTERNATIONAL, 19th

edition, 2012.

119




3

9. Method Performance Requirements:

Parameter Minimum Performance Requirement

Acceptable Minimal Detection Level (AMDL)

500,000 genome copies/filter (assume 10% filter extraction efficiency) of Variola virus in the candidate method sample collection buffer.

Probability of Detection @ AMDL

0.95 or higher.

Inclusivity panel 100% positive results†

Exclusivity panel 100% negative results†

Collaborative Study System False-Negative Rate

≤ 5%

Collaborative Study System False-Positive Rate

≤5%

Notes: † 100% correct analyses are expected. All aberrations are to be re-tested following the

AOAC Guidelines for Validation of Biological Threat Agent Methods and/or Procedures3.

Some aberrations may be acceptable if the aberrations are investigated, and acceptable explanations can be determined and communicated to method users.

• 3

Official Methods of Analysis of AOAC INTERNATIONAL (2012) 19th Ed., AOAC INTERNATIONAL, Gaithersburg, MD, USA, APPENDIX I; also on-line at http://www.eoma.aoac.org/app_i.pdf.

Comment [CU1]: Make sure limitations on filter and processing are pointed out to the working group.

120




4

ANNEX I: Controls

Positive Control

This control is designed to demonstrate an appropriate test response. This 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 avoid contamination of the test sample and/or instrument.

Single use per sample (or sample

set) run

Success - Control detected at expected levels. Failure - Control not detected or at levels below expected.

Negative Control

This control is 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 contamination in the assay or test.

Single use per sample (or sample

set) run

Success - No detections made. Failure - Detections made.

Inhibition Control

This control is 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

Success - Control detected at expected levels. Failure - Control not detected or at levels below expected.

121




5

Annex II: Inclusivity Panel The inclusivity panel should be designed based on bioinformatics analysis of the sequenced Variola virus strains in the assay target region. At least two representative strains from each major clade of Variola virus should be included within the inclusivity panel as well as any other strain with differences within the assay primer and/or probe target sequences.

122




6

Annex III: Exclusivity Panel The exclusivity panel should be designed based on bioinformatics analysis of the assay target region. All poxvirus strains listed below should be included. In addition, any other strain of the poxvirus species below with greater similarity to your target region than the core exclusivity panel strain should be added to the exclusivity panel.

CORE EXCLUSIVITY PANEL

Species Strain Commercial availability

Vaccinia Elstree (Lister vaccine) ATCC VR-1549

Cowpox Brighton ATCC VR-302

Ectromelia Moscow ATCC VR-1374

Monkeypox WRAIR 7-61 BEI NR-27

Monkeypox USA-2003 BEI NR-2500

Raccoonpox Herman ATCC VR-838

Skunkpox SKP1991 ATCC (initial sample to be provided by CDC) x040224

Volepox 2004-CA-007 ATCC (initial sample to be provided by CDC) x040215

Camelpox V78-I-2379 BEI (initial sample to be provided by CDC) x083899

Taterapox (gerbilpox) V71-I-016 BEI (initial sample to be provided by CDC) x040170

Parapoxvirus Orf vaccine Colorado Serum Company

Comment [CU2]: Discussion on whether the vendor or an independent group would conduct this

analysis. If independent group, who would fund? If

not external party review, provide detailed list of the

bioinformatics analysis (BLAST search, etc.) which

should be submitted with other validation data?

123




7

Annex III: ENVIRONMENTAL FACTORS PANEL FOR VALIDATING PCR DETECTORS FOR BIOTHREAT AGENTS

The Environmental Factors Panel is intended to supplement the biothreat agent near- neighbor exclusivity testing panel, and it should be applicable to all PCR biothreat agent detection assays. The panel criteria are divided into two main groups – the environmental panel of identified environmental organisms and the matrix panel of unknown environmental samples. This panel will test for potential cross-reactive amplification as well as PCR inhibitors.

Environmental Panel Organisms - This list is comprised of identified organisms from the environment. The organisms and cell lines will be tested as isolated DNA or pools of isolated DNA. It is acknowledged that some of these microorganisms are Select Agents.

Other biothreat agents Bacillus anthracis Ames Yersinia pestis Colorado-92

Francisella tularensis subsp. tularensis Schu-S4 Burholderia pseudomallei Coxiella burnetii Brucella melitensis Ricinus communis – use ricin plant leaves as source of DNA Clostridium botulinum Type A

Cultivatable bacteria identified as being present in air and soil

Acinetobacter lwoffii

Agrobacterium tumefaciens

Bacillus cohnii

Bacillus psychrosaccharolyticus Bacillus benzoevorans Bacillus megaterium Bacillus horikoshii Bacillus macroides Bacteroides fragilis Burkholderia cepacia Burkholderia gladoli Burkholderia stabilis Burkholderia plantarii Chryseobacterium indologenes Clostridium sardiniense Clostridium perfringens Deinococcus radiodurans

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Delftia acidovorans

Escherichia coli K12 Fusobacterium nucleatum Lactobacillus plantarum Moraxella nonliquefaciens Mycobacterium smegmatis Neisseria lactamica Pseudomonas aeruginosa Rhodobacter sphaeroides Riemerella anatipestifer Shewanella oneidensis Staphylococcus aureus

Stenotophomonas maltophilia

Streptococcus pneumoniae Streptomyces coelicolor Synechocystis

Vibrio cholerae

Legionella pneumophila Listeria monocytogenes

DNA Viruses Vaccinia virus (pox) Adenovirus vaccine Herpes simplex or CMV – whichever is available

Microbial eukaryotes Freshwater amoebae Acanthamoeba castellanii Naegleria fowleri Fungi Alternaria alternata Aspergillus fumagatis Aureobasidium pullulans Cladosporium cladosporioides Cladosporium sphaerospermum Epicoccum nigrum Eurotium amstelodami Mucor recemosus Paecilomyces variotii Penicillum chrysogenum Wallemia sebi

Comment [CU3]: This is already in the near

neighbor exclusivity panel

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DNA from higher eukaryotes Plants Zea mays (corn) Pollen from Pinus spp. (pine) Cotton – use leaves from cotton plant as source of DNA Arthropods Aedes aegypti (ATCC /CCL-125) mosquito cell line Mosquito (C6/36)* (Culex sp?) Dust mite (commercial source) Flea (Rocky Mountain labs) Drosophilia cell line Musca domestica (housefly) ARS, USDA, Fargo, ND Gypsy moth cell lines LED652Y cell line (baculovirus)– Invitrogen Cockroach (commercial source) Tick (Amblyoma ) Mammals Mus musculus (ATCC/HB-123) mouse Rattus norvegicus (ATCC/CRL-1896) rat Canis familiarus(ATCC/CCL-183) dog Felis catus (ATCC/CRL-8727) cat Homo sapiens (HeLa) human Avian Chicken

Biological insecticides – includes Bacillus thuringiensis subspecies that are widely used in agriculture. It is acknowledged that this organism is a near-neighbor of B. anthracis and has been included in the BA exclusivity panel. Furthermore, it is not closely related to Y. pestis and F. tularensis. However, strains of B. thuringiensis present in commercially available insecticides have been extensively used in hoaxes and are likely to be harvested in air collectors. For these reasons, it should be used to assess the specificity of these threat assays. B. thuringiensis subsp. israelensis B. thuringiensis subsp. kurstaki B. thuringiensis subsp. morrisoni Viral agents have also been used for insect control. Two representative products are: Gypcheck for gypsy moths (Lymanteria dispar nuclear polyhedrosis virus) Cyd-X for coddling moths (Coddling moth granulosis virus)

Environmental biowatch filter pooled samples – Comment [CU4]: Chris – do you have any more to add to this description??

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The Environmental Panel Organism list as presented here was approved by SPADA on 12/13/07 and amended on 9/17/08.

Environmental Substances and Matrices – environmental matrices (i.e., air and soil) can be the source of diverse and dynamic microbial populations potentially affecting assay specificity. Many environmental matrices contain substances that inhibit PCR reactions and ultimately affect assay sensitivity. The various matrices and their characteristics are:

Soil samples – represent complex mixtures of microorganisms and potential PCR inhibitors. This is a potential source of organisms found in air samples or contaminants collected by a first responder. This category includes 5 soil types – sandy, loam, clay, subsoil and silt. Soil of each type will be collected from five U.S. regions – East, West, North, South and Central. Soils will be pooled by soil type. Individual soil samples should be negative for biothreat agents as determined by prescreening using PCR prior to pooling. [Note: As of 12-28-08, we have collected and received soil types from 5 areas – El Paso, Houston, District of Columbia, Kansas and Seattle. ]

Pooled soils will be characterized by 1) type of soil; 2) biothreat agent-specific PCR (positives will be cultured); 3) standard soil analysis by USDA Natural Resources Conservation Services; and 4) PCR inhibition. MRI will use the commercially-available Mo-Bio kit for soils for characterization purposes. Eventually, the soil samples will be subjected to full metagenomic sequencing as part of another contract.

Pooled soil samples representing each soil type will be tested for inhibition of PCR and potential cross reactivity from soil microorganisms. Freshly prepared suspensions of each soil type at 0.1 g/mL are to be tested spiked with one target organism at the AMDL. Freshly prepared suspensions of each soil type at 0.1 g/mL are to be tested spiked with one specified exclusivity organism at the required exclusivity level. Freshly prepared suspensions of each soil type at 0.1 g/mL are to be tested alone. Inhibition and cross reactivity testing will be performed by testing the samples according to the manufacturer’s protocol (DNA extraction and PCR).

The recommendations for Soil Samples as presented here were approved by SPADA on 12/13/07 with the following caveats:

1. SPADA must decide who will test and characterize the soils and what standardized methods will be used.

2. SPADA must define what is meant by a large quantity to be collected. 3. Inhibition testing is for informational purposes only and is to be

documented in the package insert. 4. SPADA must decide what quantity of soil is to be used for aerosol method

testing.

On 9/17/08, the EFWG recommended testing 0.1 g of each soil type and a large quantity was defined as 40 lbs (standard USDA collection bag)

Comment [CU5]: Discussion point – should these environmental panels (soil, powders) be

removed since focus is on aerosol collected samples?

Should the inhibition PCR control be considered sufficient for identifying that there is not interference

with the reaction??

Comment [CU6]: I know that the mission of the

variola working group is to focus on the construction

of the panels, but one limitation to note is that none

of these samples will be able to be spiked with the target organism or even the whole genomic DNA of

the target organism. They will only be able to be

spiked with plasmid containing the target sequence

of the assay.

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SPADA amended and approved the recommendations for soil testing as presented here on 1/22/09.

Standardized Dust-loaded samples - represent the complex mixtures of microorganisms and potential inhibitors found in indoor and outdoor air from various regions.

Raw dust was purchased from an industrial contractor. Dusts were collected from air conditioning ducts, filters, air handling equipment and vacuum bags. Dusts were collected from government buildings, commercial buildings and private residences. Dusts were collected in five states ranging from Utah to Delaware. Dusts were sieved to a fine particle size, tested for Bacillus anthracis signature, were pooled and retested for Bacillus anthracis signature activity, and then were bottled in 100 g lots under vacuum. Dust was negative for Bacillus anthracis signature, and was not inhibitory to PCR reactions using 0.1g of dust as a sample size. Dust consists predominately of 10-30u diameter size particles and can be consistently deposited on a dry filter unit filter using the MRI D3 instrument. For filter matrices, 0.1 g of standardized dust will be applied to filters using the MRI D3 device for blowing aerosolized material onto filters. For liquid collection samples, a 0.1 g of standardized dust will be suspended in collection liquid using a cyclone sampler. Filters or liquid collection samples should be tested for inhibitory effects against a standard amount of biothreat agent using the manufacturer’s protocol (e.g., follow manufacturer’s recovery and DNA extraction procedures prior to PCR). Filters or liquid collection samples should be tested for cross reactivity by following the manufacturer’s protocol for recovery and DNA extraction prior to PCR.

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Documents

AOAC Stakeholder Panel on Agent Detection Assays: Variola ......to the panel. Olson and Hadfield suggested this can be considered and decided on at the next working group meeting