Anthrax and Other Suspicious Powders

Anthrax and Other Suspicious Powders
John Bates
Chair, Public Health Laboratory Network
Co-Chair, Australian (counter) Bioterrorism Laboratory Network
Anthrax for Beginners
A Guide for the Uninitiated
Historical Aspects (1)
Anthrax is mentioned in Homer’s Iliad,
where Apollo’s “burning wind of plague”
struck down the pack animals first,
followed by the soldiers.
Historical Aspects (2)
Similarly, Virgil describes how the plague
spread from animals to humans……..
“For neither might the hides be used, nor could one
cleanse the flesh by water or master it by fire. They could
not even shear the fleeces, eaten up with sores and filth,
nor touch the rotten web. Nay, if any man donned the
loathsome garb, feverish blisters and foul sweat would run
along his foetid limbs, and not long had he to wait ere the
accursed fire was feeding on his stricken limbs”
Historical Aspects (3)
The father of microbiology, Louis Pasteur,
in conjunction with Robert Koch,
demonstrated that Bacillus anthracis was
the cause of the disease anthrax
This provided essential proof for the germ
theory of disease.
In 1881, Pasteur produced a live
attenuated vaccine that offered protection
to animals
Historical Aspects (4)
As if they didn’t have enough troubles,
many soldiers in the trenches in World
War 1 died of anthrax after cutting
themselves shaving
The organism was introduced from goathair shaving brushes contaminated with
spores.
Historical Aspects (5)
In Australia, anthrax is predominantly a
disease of ruminant animals (livestock).
First outbreak reported in Cumberland,
NSW
As the disease spread over time, it led to
the creation of the “anthrax belt”
This extends from north east Victoria to
the Darling Downs in Queensland
Clinical Syndromes Veterinary
Animals may become infected either through the
pulmonary route or through an exposed lesion
Once the organism gains entry to the body, it is
carried to the lymphatics where it starts to
multiply rapidly, creating a “continuous culture”type scenario.
The action of the lethal toxin on the endothelial
lining of blood vessels results in internal bleeding
that eventually produces the characteristic
bleeding discharges from external orifices
Clinical Syndromes - Human
Human infections are rare and usually result
from contact with infected animals or hides, with
resultant skin lesions.
The most recent human case was in 1998, when
there was an infection recorded in a worker in a
Brisbane factory who handled hessian bags
imported from India.
This case subsequently developed a very large
lesion on his thigh before successful treatment.
Intentional release of spores => pulmonary
anthrax
Cutaneous Anthrax
This is the most common route of
infection and is an occupational hazard in
those who handle hides, wool and hair or
are involved in the butchering, skinning
and dissection of infected carcasses, an
environment where cuts on the hands are
common.
The recent anthrax attacks in the United
States resulted in a number of skin
infections amongst mail workers and
office staff who handled infected mail.
Cutaneous Anthrax
Within a couple of days, this develops into a fluid-filled
vesicle that subsequently breaks down to form a painless
ulcer known as an eschar.
This ulcer is 1-3 cm in diameter and has a characteristic
black necrotic centre. There is also pronounced oedema in
the surrounding area due to the release of oedema factor
by the anthrax bacilli.
There may also be swelling of adjacent lymph nodes. It is
unusual for death to occur (only 20% of cases) unless the
bacilli either gain access to the bloodstream or there is
respiratory distress due to the associated oedema in the
cervical and upper thoracic areas.
Antibiotic treatment is usually effective
Anthrax Clinical Lesions
Gastrointestinal Anthrax
Results from the consumption of anthraxinfected meat
Initial symptoms include nausea, loss of
appetite, vomiting and fever. This is
followed by abdominal pain, vomiting of
blood (haematemesis) and severe
diarrhoea.
There are no accurate statistics on
mortality but it is thought to be as high as
60%
Inhalational Anthrax
Inhalational anthrax now suggests the intentional
release of spores although in the past it also
resulted from exposure of textile or tannery
workers to hides or animal fibres contaminated
with B anthracis spores (wool sorter’s disease).
The incubation period varies from 1 to 6 days,
depending on the dose inhaled (and therefore the
degree of milling of the powder).
The disease onset is fairly non-specific.
Inhalational Anthrax
Fever, malaise and fatigue may be present
initially with a possible non-productive cough.
Following this, the patient’s condition may
improve for a few days.
However, the onset of severe respiratory distress
marks the terminal stages of illness and death
usually follows some 24-36 hours later
Routes of Entry
Dermal
Respiratory
Gastrointestinal
Signs & Symptoms
Basic Bacteriology
A Gram positive, spore forming bacillus
Virulent strains produce a capsule
Look like “train carriages” stuck end to
end in a Gram stain.
Non-motile, penicillin sensitive and nonhaemolytic and colonies exhibit tenacity
Produces three potent factors - Protective
antigen, Lethal factor and Oedema factor.
Capsulated Anthrax
Spore-forming bacilli
Mode of Action
The protective antigen is necessary for
entry into the host cell
There are two exotoxins:
– Oedema factor, responsible for the
prominent oedema at the site of infection
– Lethal factor, leads to the inhibition of
intracellular signalling.
Near Neighbours
B. anthracis is a member of the B. cereus
group, which includes B. cereus, B.
thuringiensis, B. anthracis and
B.mycoides.
The RCPA Biosecurity QAP has
challenged labs enrolled in the program
with each of these organisms to build up
familiarity with the similarities and
differences of each species.
Near Neighbours
B. anthracis has been viewed as a highly
monomorphic species
However, new molecular methods have found
genetic differences amongst natural isolates
The pathogenicity of B. anthracis is related to the
presence of the pXO1 and pXO2 plasmids
These were once thought to separate B. anthracis
from other members of the genus
Near Neighbours
Within the last 5 years, B. cereus strains
containing pXO1 and pXO2 have been
discovered
A specific mutation in B. anthracis’ global
regulator plcR gene, which controls the
transcription of virulence factors in B.
cereus and B. thuringiensis, now appears
to be the key difference
All B. anthracis strains carry a mutation
that makes the plcR gene dysfunctional
Long Laboratory Case
Definition
–Definitive criteria
1) Isolation of Bacillus anthracis from blood, wounds,
or intestinal contents
–Suggestive criteria
1) Detection of Bacillus anthracis by NAT
2) Gram positive square-ended bacilli, non-motile and
encapsulated (well shown with polychrome
methylene blue stain), from blood or other normally
sterile site.
Laboratory Capabilities
Most PHLN labs have the capability and
facilities to culture and identify or rule out
anthrax
The DoHA has resourced a number of the
major labs with the purchase of an AB
7500 DX FAST real-time PCR platform for
use with LRN assays
Bioterrorist Scenarios
A Hoax Attack - eg. White Powders
A Real Attack Where the Agent is
Identified
A Real Attack Where the Agent is Not
Identified - eg. US Mail attacks
Possible Modes of
Distribution
Light plane - sentinel cows
Mail
Subways
Contaminated food
Possible Modes of
Distribution
Light plane - sentinel cows
Possible Modes of
Transmission
Mail
Possible Modes of
Transmission
Subways
Possible Modes of
Transmission
Contaminated Food
I’m Dreaming of
a White
Christmas
A Summary of “White Powder” Incidents in
Queensland since October 15th, 2001
Summary of Incident
Numbers
October
November December January
Anthrax
Screen:
Physical
Examination
but no culture
67
16
7
5
Nasal Swabs
44
1
0
3
Culture for
anthrax
19
24
28
20
PCR & culture
for anthrax
41
14
4
2
The Incidents
Diagnostic Issues
As a “dry-run”, these provided us with an
excellent introduction to the chemistry of
white powders
Capabilities have improved considerably
as a result of this exposure
PCR is useful for screening large numbers
of culture broths.
Culture proved just as effective once
numbers dropped.
The Amerithrax Investigation
Less than a month after September 11, 2001,
letters containing spores of anthrax were sent
through the US mail.
Between October 4 and November 20, 2001, 22
individuals developed anthrax
5 of the cases were fatal.
Although this started as a public health
investigation, it was quickly taken over by the
FBI when it became obvious that this was a
deliberate act
The Amerithrax Investigation
The scientific investigation led to the
development of “Microbial forensics”
This became an essential part of the
scientific investigation in the hands of the
FBI, as well as traditional procedures
This led to the FBI naming a USAMRIID
anthrax researcher, Dr. Bruce Ivins, as the
perpetrator of the anthrax attacks
Scientific Critique of the FBI
Investigation
The B. anthracis in the letters was the Ames
strain and was 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
Scientific Critique of the FBI
Investigation
The FBI created a repository of Ames strain
samples and performed experiments to determine
relationships among the letter materials and the
repository samples. The scientific link between
the letter material and flask no. 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
It is difficult to draw conclusions about the
amount of time needed to prepare the spore
material or the skill set of the perpetrator
Scientific Critique of the FBI
Investigation
Physicochemical and radiological experiments were
properly conducted to evaluate the samples for potential
signatures connecting them to a source but this proved to
be of limited forensic value
There was inconsistent evidence of Ames DNA in
environmental samples that were collected from an
overseas site
There are other tools, methods, and approaches available
today for a scientific investigation like this one
Organisational structure and oversight are critical aspects
of a scientific investigation. The FBI generated an
organisational structure to accommodate the complexity of
this case and received advice from prominent experts
Looking to the Future
A review should be conducted of the classified
materials that are relevant to the FBI’s
investigation of the 2001 mailings, including all of
the data and material pertaining to the overseas
environmental sample collections
The goals of forensic science and realistic
expectations regarding its use in the
investigation of a biological attack must be
communicated to the public and policymakers
with as much clarity and detail as possible
before, during, and after the investigation.