Microbial Forensics

Microbial Forensics:
A Primer for Armed Forces
Medical Laboratory Scientists
CPT Dana Perkins, PhD
SAFMLS Annual Meeting, 28 - 31 March 2011
UNCLASSIFIED
Disclaimer
The views expressed in this presentation
are those of the author and do not reflect
the official policy or position of the
Department of the Army, Department of
Defense, or the U.S. Government.
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Briefing Agenda

Microbial (Bio-) Forensics: definition &
goals

Overview of bioforensics methods

Illustrative case: Amerithrax

Bioforensics in Army operations:
application, implications, and challenges
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Why does this matter to you
“The infantry Soldier’s weapon is his rifle;
and Medical Service
the Chemical Soldier’s weapons are his
knowledge of and access to accurate
CBRN information
and his ability to advise his Commander”
-The Chemical Corps of the Future- From Apprentice to CBRN ExpertMAJ John Shank, Army Chemical Review, Jan-June 2007, p.27
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Comprehensive Bio-Weapons (BW) / Terrorism (BT)
Preparedness Strategy
POST-EVENT
PRE-EVENT
PREVENTION PROTECTION
SURVEILLANCE
& DETECTION

National Strategy to Combat Weapons of Mass Destruction
(2002)

National Strategy for Combating Terrorism (2006)

National Military Strategy to Combat Weapons of Mass
Destruction (2006)

National Strategy for Countering Biological Threats (2009)

National Health Security Strategy (2009)

National Security Strategy (2010)

National Response Framework (2008 revision)

National Disaster Recovery Framework (2010 draft)
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RESPONSE
& RECOVERY
“Attribution is the investigative
process by which the United States
Government (USG) links the identity
of a perpetrator or perpetrators of
illicit activity and the pathway leading
to criminal activity”.
-- National Strategy to Support Research in
Microbial Forensics Attribution Investigations and
National Security, 2009 –
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Federal Response Trigger Points

Credible Intelligence of a Plan to Conduct a Bio Attack

Notification of an Actionable Result from BioWatch

Confirmed Cases of Bioterrorism Agent Disease in U.S.
• Declaration a Public Health Emergency
• Establish the ESF #8 command structure
• Lead the public health & medical
emergency response actions
• Declaration of an Incident of National Significance
• Initiate the NRF
• Raise the Terror Threat Advisory Level
• Coordinate overall nonmedical support and
response actions
Investigation of criminal activities
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Outbreak Detection

Determination of a Disease Outbreak

Laboratory Confirmation

Identification (Analysis and Confirmation)

Instances of disease that raise the “index of suspicion”
of terrorist or criminal activities are reported to FBI.

If warranted, the FBI, HHS and/or USDA and respective
State/local health officials will conduct a
joint law enforcement and epidemiological investigation
Attribution
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The National Bioforensic Analysis Center

NBFAC was designated in
HSPD-10 (Biodefense for
the 21st Century) as the
lead Federal facility to
conduct and facilitate the
technical forensic analysis
and interpretation of
materials recovered
following a biological attack

To meet this mission,
NBFAC is developing
forensic tools, methods,
and strain repositories for
pathogens of concern
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http://www.whitehouse.gov/sites/default/files/National_Strategy_for_Countering_BioThreats.pdf
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2009
http://www.whitehouse.gov/files/documents/ostp/NSTC%20Reports/National%20MicroForensics%20R&DStrategy%202009%20UNLIMITED%20DISTRIBUTION.pdf
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Microbial Forensics / Bioforensics /
Forensic Microbiology

Forensic science integrates
information obtained from
intelligence, field investigation,
the crime scene, and the
laboratory

Bioforensics: Examination of
traces of a biological agent from a
bioterrorism act, bio-crime or
investigation, as well as a naturally
occurring biological agent release

Integrated data analysis is used
for attribution purposes


Scientific evidence is only one
piece of the attribution puzzle
Characterization of specific
information about an agent that
will help to determine whether it
was released intentionally,
accidentally, or appears naturally
in the environment

Providing answers to crucial
questions, such as what kind of
biological agent was used, where
and when it was made and how
it was prepared
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Bioforensics Methods

Genotyping tools for identifying
molecular signatures of
biological agents:
– PCR-based fingerprinting
– Pulsed-Field Gel
Electrophoresis (PFGE)
– Ribotyping
– Whole genome shotgun
sequencing
– Multi Locus Sequence Typing
(MLST)
– MLVA (Multi Locus VNTR
Analysis)
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Rapid Multi-Locus Sequence Typing Using Microfluidic Chips
Read et al, PLoS ONE, 5(5), 2010
Databases:
Plant Associated and Environmental
Microbes Database (PAMDB):
http://genome.ppws.vt.edu/cgi-bin/MLST
MLVA-NET: http://www.pasteur.fr/mlva
Multi Locus Sequence Typing:
http://www.mlst.net
PubMLST: http://pubmlst.org
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Bioforensics Methods

“Collateral” forensics indicators by:
A: Distribution of chlorine within a spore (by SIMS)
B: Image of spore surface (by AFM)
– Microscopy (EM, AFM)
– 14C Accelerator Mass Spectrometry
(AMS)
– Matrix-Assisted Laser Desorption
Ionization (MALDI) MS
– 13C/12C and 15N/ 14N isotope ratios
– Secondary-Ion MS (SIMS)
– Electro-Spray Ionization (ESI) MS
and Derivatization Gas
Chromatography MS
– Bayesian network as a DST
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A
B
S.P. Velsko, Physical and Chemical Analysis:
A Key Component of Bioforensics,
AAAS Annual Conference, 2005
Morphology of spores coated with silica by two
different methods
http://www.microbialrosettastone.com
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Amerithrax timeline

17 Sep 2008: FBI Director Robert
Mueller tells the Senate Judiciary
Committee that the FBI will seek an
independent review of the scientific
evidence of the anthrax case "because
of the importance of the science to
this particular case and perhaps
cases in the future“

18 Sep 2001: The first letters containing
anthrax are mailed

04 Oct 2001- 21 Nov 2001: five people died
from inhaling anthrax and 17 others were
infected after exposure

09 Oct 2001- 19 Feb 2010: FBI investigation

29 July 2008: Bruce Ivins, a USAMRIID
scientist suspected in the 2001 anthrax attacks,
 19 Feb 2010: The Department of
commits suicide
Justice formally concludes its
investigation of the anthrax mailings:
"Evidence developed from [the]
investigation established that Dr.
[Bruce] Ivins, alone, mailed the
anthrax letters"

http://cohort11.americanobserver.net/adambehsudi/BehsudiFlashFinal/BehsudiFinal.html
15 Feb 2011: National Research
Council (NRC) of the National Academy
of Sciences (NAS) publishes the
“Review of the Scientific Approaches
Used During the FBI’s Investigation
of the 2001 Anthrax Letters”
http://www.fbi.gov/about-us/history/famous-cases/anthrax-amerithrax/amerithrax-investigation
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http://www.justice.gov/amerithrax/docs/amx-investigative-summary.pdf
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B. anthracis in letters was Ames, not genetically engineered.

Multiple distinct colony morphological types, or morphotypes, of B. anthracis Ames
were present in the letters. Molecular assays of specific genetic sequences
associated with these morphotypes provided an approach to determining
relationships among evidentiary samples.
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
The FBI created a repository of Ames strain
B. anthracis samples and performed
experiments to determine relationships
among the letter materials and the
repository samples. The scientific link
between the letter material and flask
number RMR-1029 is not as conclusive as
stated in the DOJ Investigative Summary.

Silicon was present in the letter powders
but there was no evidence of intentional
addition of silicon-based dispersants.

Physico-chemical and radiological
experiments were properly conducted to
evaluate the samples for potential
signatures connecting them to a source but
proved to be of limited forensic value.
http://www.nap.edu/catalog.php?record_id=13098
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Biological Threats and Military Operations

The deliberate or accidental
employment or threat of BW and
attacks with biological materials

Dual-use nature of biotechnology
impedes situational awareness

Unique challenges to military
operations (from blackmail or acts of
terrorism during peace, to limited use
during MOOTW; during a conflict/war
the probability of use may increase)

Microbial forensics, sample
management, chain of evidence,
and other TTPs related to
attribution, are important elements
of military response to BW/BT
(in particular in DSCA & WMD-E)
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Defense Support of Civil Authorities (DSCA)

Support provided by US
Federal military forces,
National Guard forces
(performing duty in accordance
with Title 32 U.S.C.), DOD
Civilians and contract
personnel, and DOD agencies
and components, in response to
requests for assistance during
domestic incidents

In addition to supporting
Federal agencies in
consequence management
operations, DOD, as authorized
by law, plays a supporting role
in assisting FBI in its response
to terrorist incidents (e.g. threat
assessment, forensic testing in
DOD labs, etc)
Refs.:DODD 5111.113, FEB 09, GAO MAR 2010
“…Requests for DOD assistance for law enforcement and criminal
investigation during the incident come from the Attorney General to
the Secretary of Defense through the DOD Executive Secretary.
Once the Secretary approves the request, the order is transmitted
either directly to the unit involved or through the Chairman of the
Joint Chiefs of Staff. The FBI SAC informs the Principal Federal
Official (PFO), if one has been designated, when requesting this
additional assistance…”
-- NRF Terrorism Incident Law Enforcement & Investigation Annex --
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WMD - Elimination (WMD-E)

WMD-E OPS: actions to
systematically locate, characterize,
secure, disable, or destroy WMD
programs and related capabilities

Objective: to combat further WMD
proliferation and prevent regeneration
of a WMD capacity

Tactical WMD-E OPS are military-led
– Planned
– Targets of opportunity
Exploitation: The intent of this task is to gain an understanding of an adversary’s
WMD programs & capabilities to attribute & connect to the adversary’s network,
which may determine future targets; collect evidence of a WMD program; and
provide force protection from immediate WMD threats, if required.
Reference: Joint Publication 3-40, Combating WMDs, 10 June 2009
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BW Facility
/ Event
Ref: FM 3-11.86 Multi Service TTPs for Biological Surveillance
& FM 3-90.10 CBRNE Operational HQ
Seize / Secure
Site Assessment
Evidence Gathering
Transport to Laboratory
USG-designated laboratories for internationally
accepted definitive confirmation
Lab Testing
OR
Lab Reporting
UN-designated laboratories ?
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Russia’s allegations
http://www.mid.ru/brp_4.nsf/e78a48070f128a7b43256999005bcbb3/cc9c7d192f0ebc5ac325777a0057e1ae?OpenDocument
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Establishing a bioforensics capability in DOD

Develop, integrate, and institutionalize

Incorporate bioforensics into strategic, operational,
and tactical level planning

Integrate bioforensics into operational and tactical
level exercises

Ensure the “plug and play” augmentation and reach
back in deployments and exercises

Eliminate the possibility of ad-hoc approaches to
integration of bioforensics (as a component of
exploitation/assessment in WMD-E) in conjunction
with warfighting

Ensure the synergy between S&T; policy, plans &
doctrine; and operations

Facilitate combined operations with Allies through
exercises and help them develop similar capabilities

Foster constructive engagement and coordination
with the United Nations
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UN Secretary General’s Mechanism for Investigation of
Alleged Use of Chemical, Biological or Toxin Weapons


Triggered by a request to the Secretary General
to carry out promptly investigations in response
to reports that may be brought to his attention by
any UN Member State concerning the possible use
of CBTW (UNGA Res. A/RES/42/37C of 11/30/87)
UNSGM
TGP: Technical Guidelines and Procedures
(A/44/561 of 1989) endorsed by the UNGA in 1990

UNODA serves as a focal point within the UN
Secretariat to facilitate the administrative and
substantive support and coordination for the efficient
functioning of the investigative mechanism

TGPs
Fact-Finding Activities [at site(s) of alleged
use]:
–
–
–
–
–
–
–
Locate site(s) precisely
Examine terrain, vegetation and animal life
Degree of contamination
In situ detection and analysis
Collect physical evidence
Collect biomedical samples
Conduct interviews
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Currently:
Experts >237
Labs >42
Roster
of
Experts
& Labs
Reinvigorating the UNSGM: an opportunity for
developing a bioforensics capability at the UN level?
General Assembly Resolution “UN Global Counter-Terrorism Strategy”
(A/RES/60/288 (2006):
Member States encourage the Secretary General to update the roster
of experts and laboratories, as well as the technical guidelines and
procedures, available to him for the timely and efficient investigation
of alleged use
2006 Sixth Review Conference of the Biological Weapons Convention
(BWC):
UNSGM represents an international institutional mechanism for
investigating cases of alleged use of biological or toxin weapons
In order to become an operational mechanism able to inform the policy / international
law decision-making process, the UNSGM should update its TGPs to include
bioforensics considerations; standards/guidelines for field/lab investigations and data
validation; integration of human, animal, and plant forensic/lab data; and an effective,
credible, and defensible technical capability (i.e. established and sustainable expertise,
infrastructure, and resources)
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UN Secretary General’s Authority

Investigations can be carried out either at the request of States or of the
Security Council (under Chapter VII of the UN Charter)

Chemical investigations: OPCW can be called upon to provide inspection
services under UN direction

Biological investigations: the Secretary General has the authority to make
advance arrangements himself for investigating alleged use of BW at the
request of any Member State

Although the standing authority of the Secretary General is limited to
investigating the use of BW, the Security Council can request the Secretary
General to investigate any threat, including BW development, possession,
testing, transfer, or accidental release
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Contact information
Dana Perkins, Ph.D.
CPT Dana Perkins
Senior Science Advisor
Office of the Assistant Secretary for
Preparedness and Response
U.S. Department of Health and Human Services
Tel: 202 205 5716 (office)
E-mail: [email protected]
CBRN Officer
7th Civil Support Command HHC
USAREUR
Tel: 410 446 7256 (mobile)
E-mail: [email protected]
UNCLASSIFIED