Testing For and Assessment
of Agents of Bioterrorism
Viruses in May – John Bates
Overview
• Classification of biological agents
• What you need to be a good bioterrorist
• Features of biological agents that make them attractive
• Epidemiological clues for a bioterrorist attack
• Scenarios for the use of biological agents
• Possible modes of distribution
• The agents and capabilities for detection
• Laboratory response protocols
• Forensic microbiology
• The RCPA Biosecurity Quality Assurance Programme
Tier 1 Biological Agents
• High-priority agents that include organisms that pose a threat to national
security because they:
o Can be easily disseminated or transmitted person-to-person
o Cause high mortality, with potential for major public health impact
o Might cause public panic and social disruption; and
o Require special action for public health preparedness.
• Variola Major (smallpox)
• Bacillus anthracis (anthrax)
• Yersinia pestis (plague)
• Clostridium botulinum toxin & organism (botulism)
• Francisella tularensis (tularensis)
• Burkholderia mallei and B. pseudomallei
• SARS Coronavirus
• Foot & Mouth Disease virus
• Filoviruses,
o Ebola haemorrhagic fever
o Marburg haemorrhagic fever
• Arenaviruses,
o Lassa (Lassa fever)
o Junin (Argentine haemorrhagic fever) and related viruses (Chapare & Lujo)
Tier 2 Biological Agents
• The second highest priority agents includes those that
o are moderately easy to disseminate
o cause moderate morbidity and low mortality: and
o require specific enhancements of CDC’s diagnostic capacity
and enhanced disease surveillance
• Coxiella burnetti (Q Fever) & Typhus
• Brucella species (Brucellosis)
• alphaviruses
o Venezuelan encephalomyelitis
o eastern and western equine encephalomyelitis
• Ricin toxin from Ricinus communis (Castor bean)
• Epsilon toxin of Clostridium perfringens; and
• Staphylococcal enterotoxin B
Tier 2 Biological Agents
Food and Water Agents
•A subset of Tier 2 includes agents that are
food and/or waterborne.
•These pathogens include but are not
limited to:
•Salmonella species
•Shigella dysenteriae
•Escherichia coli O157:H7
•Vibrio cholerae; and
•Cryptosporidium parvum
Tier 1 Agents
Tier 2 Agents
Abrin (reportable quantity 5 mg)
African swine fever virus
Bacillus anthracis (Anthrax—virulent strains)
Capripoxvirus
(Sheep pox virus and Goat pox virus)
Botulinum toxin (reportable quantity 0.5 mg)
Classical swine fever virus
Ebolavirus
Clostridium botulinum
(Botulism; toxin-producing strains)
Foot-and-mouth disease virus
Francisella tularensis (Tularaemia)
Highly pathogenic influenza virus, infecting humans
Lumpy skin disease virus
Marburgvirus
Peste-des-petits-ruminants virus
Ricin (reportable quantity 5 mg)
Yellow fever virus (non-vaccine strains)
Rinderpest virus
SARS coronavirus
Variola virus (Smallpox)
Yersinia pestis (Plague)
Routes of Entry
Dermal
Respiratory
Gastrointestinal
Questions for a Budding
Terrorist
• Do we have access to the agents we want
to use? Where do we get them?
• Do we have the facilities to safely handle
these agents?
• Do we have the facilities to produce large
quantities of these agents?
• Having grown them, do we have the
technology and knowledge to stabilise these
agents for long term storage?
Questions for a Budding
Terrorist
• Do we have the technology and knowledge
to weaponise the agent?
• Do we want to use an agent that is not
contagious, or one that will spread rapidly
throughout the population?
Bioterrorist’s Shopping List
• Production - easy to produce in large amounts
• Storage - easy to store for long periods in a stable form
• Immunity - should be low level herd immunity to the agent
• Virulence - agent should be virulent & effective in low doses
• Incubation Period - should be short
• Controllable - agent may or may not result in an uncontrollable epidemic
Setting the Scene
• Although hundreds of potential pathogens and toxins exist,
not all of them present the same threat as weapons
• While one organism may have a high virulence, it may be
very difficult to culture, disperse or handle.
• Conversely, an organism with limited destructive potential
may be readily available and easy to disseminate
• How do we evaluate the potential threats posed by
biological agents?
Criteria to Evaluate Potential
Threats - Summary
• Availability
• Ease of Culture
• Survivability in the environment
• Ease of dispersion
• Infectivity
• Morbidity
Criteria to Evaluate Potential
Threats- Summary
• Impact on target population
• Immunity of target
• Therapeutic control measures
• Ability to be transmitted subject to subject
• Ability to be bioengineered to increase virulence or to make the agent resistant
to vaccines or therapeutics
Epidemiological Clues to
Bioterrorism
• Higher morbidity or mortality than expected for a common disease or syndrome
• A common syndrome with an unusual geographic or seasonal distribution
• The presence of a large epidemic or multiple simultaneous outbreaks in discrete
population(s)
• Illness in persons suggesting exposure at a common venue or unusual setting
(e.g., workplace, major public event)
• Unusual, atypical, or antiquated agent strain
• Unexplained illness and death in animal populations
Scenarios for the Use of
Biological Agents (1)
•1. A Hoax Attack
oStill has to be treated as a real event
oHoaxer achieves all the aims of a real scenario
oThreat capability of the hoaxer must be determined
oCOMPLACENCY IS A REAL LONG-TERM
PROBLEM!
Scenarios for the Use of
Biological Agents (2)
•2. A Real attack where the Agent is
Identified
oTerrorist reveals nature of the agent and where &
how it has been released
oEnormous implications for public health
oInevitable casualties amongst first response
personnel and medical staff, especially if agent is
infectious
Scenarios for the Use of
Biological Agents (3)
•3. A Real Attack where the Agent is
not Identified
oTerrorist aims for maximum destruction to the fabric
of society
oGreatest effect on its target because of the fear
factor
oLarge scale panic in population
Possible Modes of Distribution
•Light plane - airborne dispersal
Sentinel Cows
Photo courtesy of Lee Taylor, Department of Primary Industries, Qld., Biloela
Possible Modes of Transmission
•Mail
Possible Modes of Transmission
•Subways/Public Transport Systems
Possible Modes of Transmission
•Contaminated Food
Possible Modes of
Transmission
•Person to Person Transmission:
• Most likely from a virological perspective
• May involve someone who deliberately infects themselves and then just walks
through a large city bumping into people – a very realistic scenario for a
smallpox terrorist (see SBS doco)
• This adds new meaning to a “Lone Wolf” attack, but would require access to
very highly secured biological material. Low technological bioterrorism using
human beings as the vector is a subset of suicide terrorism.
How might we become
aware of an attack?
• Rapid death of livestock on the outskirts of populated areas
• Large influx of seriously ill people at emergency departments suffering flu-like
symptoms
• Rapid spread of unknown infectious agent throughout the population
• Serious illness in a large number of people from unknown agents that are
resistant to treatment (genetically modified to be multi-drug resistant eg. Plague)
Photo courtesy of Dr David Looke, Infection Management Service, Princess Alexandra Hospital, Brisbane
Characteristic Buboes
Intravascular coagulation
Chimeras
• With the rapid advances in molecular genomics, the threat from chimeras is
becoming very real.
• Large numbers of self styled scientists are doing gene splicing experiments in
their labs in garages at home.
• There are already controls on the publication of experiments deemed to be dualuse, which has caused some consternation in the scientific community, eg.
experiments which increase the virulence of influenza strains.
• The former USSR had an active program (Vector) designed to increase virulence
and antibiotic resistance in dangerous bacteria – this would be much easier to
achieve with today’s technology
• What’s to stop someone putting the genes for an Ebola virus in an E. coli, an
organism common to all warm-blooded species?
• Stephen Hawking spoke recently about the threat posed by
genetically engineered viruses
Testing for Agents
• There are five labs in Australia with affiliation with the Laboratory Response
Network (LRN), which is run out of the CDC
• Membership of the LRN provides access to procedures for testing a full range of
agents in different specimen matrices (clinical samples, powders, liquids,
environmental swabs, etc.), as well as specific quality controlled reagents and
positive control material (if you can get it into the country!).
• Whilst PCR provides the fastest means of detecting a particular agent, the LRN
protocols stress the need for other supplementary testing to confirm an agent,
where possible (this probably applies to the bacteria more than the viruses).
• Other possible tests include DFA, tenacity, phage-lysis and TRF.
• Generally, the LRN prefers at least two positive assays before confirming an
agent, but reporting a presumptive positive based on a PCR result in an
appropriate epidemiological setting is warranted.
Anthrax
Sample Types
• Animal samples
o Biosecurity Queensland, UQ Vet School
• Clinical samples
o Hospitals, private and public pathology laboratories, Public Health
• Environmental Samples
o Government clients, Public Health
• White Powders
o Police and Queensland Fire and Emergency Service
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Capabilties
• B. anthracis, F. tularensis, Y. pestis
o Isolation, Culture, microscopy (capsule staining), DFA, RAMP, phage, real time PCR (inhouse and LRN), MLVA, WGS
• Burkholderia sp
o Culture, Real-time PCR (in-house and LRN)
• Brucella sp
o Culture, Real-time PCR (in house speciation by HRM)
• C. botulinum
o Mice bioassay, culture, real-time PCR (in-house and LRN) for toxin typing
• Ricin
o R. communis DNA detection by real-time PCR, RAMP
• Abrin
o A. precatorius DNA detection by real-time PCR, lateral flow device
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Capabilties
• Extensive experience in molecular typing for use in outbreak
investigations
• Microbial Source Tracking
• Cluster analysis
• Phylogenetic studies
• Molecular typing methods ranging from simple PCR through to Whole Genome
Sequencing
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New Technologies
• The (Australian (counter) Bioterrorism Laboratory Network (ABLN) is constantly
scanning the published literature for new testing capabilities:
• There have been some notable problems with identification systems
o MALDI-TOF – may be problems with mis-identification because the database does not
contain select agents. Also, the issue of contamination of automated equipment arose in
the management of Ebola cases in the US.
o Film-array systems – the Bio-threat-E system can detect 27 targets and 17 pathogens.
It’s ease of use, single specimen pouch and wide array of detection targets have the
potential to bring routine detection of bioterrorist agents to the level of the clinical
laboratory. The FDA has approved the EZ rRT-PCR assay, the BioFire FilmArray
biothreat panel, the Roche LightMix Ebola Zaire rRT-PCR system and the Cepheid Xpert
test.
o In the past, the Commonwealth Dept of Health has been very generous is providing
equipment and kits to the ABLN labs doing this work.
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Preparation with First
Responders
• G20 planning team created flowcharts outlining ideal sampling
procedures to distribute to QPS/QFES/Defence
• G20 planning team gave a Powerpoint presentation to the
QPS/QFES describing minimum requirements for in-field
testing prior to submission
• Appropriate tubes
• Volumes
• One contact number given out, which was to be answered 24
hours a day. Responders encouraged to call number BEFORE
sample is dispatched
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Sampling – White Powders
Biological samples
Swab (for
difficult to
reach or small
samples).
Rayon or
Decron (no
wooden
handled
swabs) and
place in a
sterile screwcap
polyethylene
plastic tube
Approx 1 g
(small shovel
scoop) in
approx 1 mL
of sterile
water in a
sterile screwcap
polyethylene
plastic tube
2 samples required –
one for Biological
testing and one for
Chemical testing
Chemical samples
Approx 1 g (small shovel
scoop) placed in a sterile
screw-cap polyethylene plastic
tube
Outside surfaces of each screw-cap plastic tube wiped down with 10% sodium hypochlorite solution or
equivalent decon agent. Each screw-cap plastic tube then placed into individual clean clip sealed plastic bags
Preparation as laboratory
• FSS Public Health Microbiology:
o Developed processing flowcharts depending on the sample type ie white powder, liquid
o Quick Reference Protocol book, molecular calculator spreadsheets etc
o Ensured all molecular capabilities included an in-house assay as back-up
o Designed clear simple report templates – interim and final
o Ordering – stockpiled all necessary reagents, PPE
o Expanded capabilities
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Gathering of Evidence for
Biocrimes - Forensic
Microbiology
• “The application of forensic procedures
to the generation of epidemiological
typing data that can be used to link
bacterial isolates or viral agents to each
other, particularly when biocrime is
involved.”
Basic Requirements
• Forensics has additional and more stringent
requirements than standard microbiology.
• For example, maintaining a chain of custody on
evidentiary samples.
• The intent of microbial forensics is to identify a
bioattack organism in the greatest detail possible.
• There is also a requirement for high level quality
assurance systems that will stand up in a court of law.
• Increasingly this will apply to all legal investigations
Scope
• The list of bioattack organisms needs to be expanded
to include microorganisms relevant to food and water
threats.
• The purposeful “doctoring” of organisms also needs to
be considered eg.
o Enhanced antibiotic resistance,
o Addition of genes from other species, especially in “harmless”
bacteria,
o Modification of known pathogens in such a way as to avoid
known existing detection mechanisms
o Combinations of pathogens e.g. Ebola Pox genetic
combination of Ebola and smallpox, said to have been
developed by Russian biological weapons experts.
What are the Likely Uses?
• In the absence of a bioattack on Australia, there are still a number
of justifications for adopting a microbial forensics approach:
o 1. Investigation of foodborne and waterborne outbreaks
o 2. Investigation of outbreaks of infectious disease with a suspicious
signature
o 3. Investigation of outbreaks involving bioattack organisms in
animals, eg anthrax in cattle.
o 4. Cases of medical negligence involving hospital-acquired infection
due to inadequate hygiene
A New Science
• At the PHML, we have unit dedicated to Molecular
epidemiology
• What is the difference between molecular epidemiology
and microbial forensics?
• Microbial forensic data must hold up not only to the
scrutiny of scientists in the health care community, but
also to the scrutiny of judges and juries
• Increasingly, the use of DNA sequencing is being seen
as the only definitive way to establish a link between
two epidemiologically related isolates
Proficiency Testing SSBA
Rationale for the program
• Biological weapons and bioterrorism, biowarfare - increasingly
secular
• Natural infectious disease outbreaks
• Laboratory safety and security
• Impacts of agent on economy, society and governments
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Proficiency Testing SSBA
Selecting a suitable agent
•
Availability
•
Production capability
•
Ease of dissemination
• Treatment available
Other factors
•
Robustness
•
Detection/Traceability
•
Contagiousness
• Population protection
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Proficiency Testing SSBA
Defence against biological terrorism
• Intelligence gathering
• Counter-acquisition strategies
• Epidemiological surveillance systems
• Syndromic
• Laboratory
• Environmental
• Consequence mitigation
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Proficiency Testing SSBA
• Suspicious white powder found at Parliament House Canberra
(August 2011)
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