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INDEX
FOR
ADDITIONAL REFERENCE MATERIALS FROM SLIDES
Slide 18: Emergency Plan
CDC Interim* Recommendations for Protecting Workers from Exposure to Bacillus
anthracis in Work Sites Where Mail Is Handled or Processed
(*Updated from CDC Health Advisory 45 issued 10/24/01)
Slide 21: Personal Protective Equipment
Interim CDC recommendations for personal protective equipment for responding to
biological weapons
Slide 22: OSHA Respirator Standards
CDC, OSHA respirator recommendations for potential exposures to biological agents,
such as Bacillus anthracis, in their facilities
Slide 23: OSHA Regulations: Summary of Employer Responsibilities
Slide 24: Respirator Maintenance
Slide 25: Summary of the CDC Health Advisory on the Interim Recommendations for
Protecting Workers from Exposure to Bacillus anthracis in Work Sites Where Mail Is
Handled or Processed. (Distributed via the Health Alert Network, October 31, 2001)
Slide 41: MMWR. 2002;51:482, April 5, 2002 - Suspected Cutaneous Anthrax in a
Laboratory Worker --- Texas, 2002
Laboratory Response Network
Slide 81: Cidofovir is Active Against DNA Viruses
Slide 83: Viral Hemorrhagic Fevers, Microbiology
Slide 85: Viral Hemorrhagic Fever, Clinical Features
Slide 87: Viral Hemorrhagic Fever Treatment
Slide 88: Isolation and Containment CDC: Management of patients with suspected viral
hemorrhagic fever. MMWR 37(Supplement 3):1-16, 1988.
Slide 91: Viral Encephalitides
Slide 94: Botulinum toxins.
AOEC Bioterrorism Module, Additional Reference Materials
Slide 18 Emergency Plan
CDC Interim* Recommendations for Protecting Workers from Exposure to Bacillus
anthracis in Work Sites Where Mail Is Handled or Processed
(*Updated from CDC Health Advisory 45 issued 10/24/01)
These interim recommendations are intended to assist personnel responsible for
occupational health and safety in developing a comprehensive program to reduce
potential cutaneous or inhalational exposures to Bacillus anthracis spores among workers,
including maintenance and custodial workers, in work sites where mail is handled or
processed. Such work sites include post offices, mail distribution/handling centers, bulk
mail centers, air mail facilities, priority mail processing centers, public and private
mailrooms, and other settings in which workers are responsible for the handling and
processing of mail. These interim recommendations are based on the limited information
available on ways to avoid infection and the
effectiveness of various prevention strategies and will be updated as new information
becomes available. These recommendations do not address instances where a known or
suspected exposure has occurred. Workers should be trained in how to recognize and
handle a suspicious piece of mail (<http://www.bt.cdc.gov>). In addition, each work site
should develop an emergency plan describing appropriate actions to be taken when a
known or suspected exposure to B. anthracis occurs. These recommendations are divided
into the following hierarchical categories describing measures that should be
implemented in mail-handling/processing sites to prevent potential exposures to B.
anthracis spores:
1. Engineering controls
2. Administrative controls
3. Housekeeping controls
4. Personal protective equipment for workers
These measures should be selected on the basis of an initial evaluation of the work site.
This evaluation should focus on determining which processes, operations, jobs, or tasks
would be most likely to result in an exposure should a contaminated envelope or package
enter the work site. Many of these measures (e.g., administrative controls, use of HEPA
filter-equipped vacuums, wet-cleaning, use of protective gloves) can be implemented
immediately; implementation of others will require additional time and efforts.
1. Engineering Controls in Mail-handling/processing Sites
B. anthracis spores can be aerosolized during the operation and maintenance of highspeed, mailsorting machines, potentially exposing workers and possibly entering heating,
ventilation, or airconditioning (HVAC) systems. Engineering controls can provide the
best means of preventing worker exposure to potential aerosolized particles, thereby
reducing the risk for inhalational anthrax, the most severe form of the disease. In settings
where such machinery is in use, the following engineering controls should be considered:
• An industrial vacuum cleaner equipped with a high-efficiency particulate air (HEPA)
filter for cleaning high-speed, mail-sorting machinery
• Local exhaust ventilation at pinch roller areas
• HEPA-filtered exhaust hoods installed in areas where dust is generated (e.g., areas with
highspeed, mail-sorting machinery)
• Air curtains (using laminar air flow) installed in areas where large amounts of mail are
processed
• HEPA filters installed in the building’s HVAC systems (if feasible) to capture
aerosolized spores
Note: Machinery should NOT be cleaned using compressed air (i.e.,
“blowdown/blowoff”).
2. Administrative Controls in Mail-handling/processing Sites
Strategies should be developed to limit the number of persons working at or near sites
where aerosolized particles may be generated (e.g., mail-sorting machinery, places where
mailbags are unloaded or emptied). In addition, restrictions should be in place to limit the
number of persons (including support staff and non-employees, e.g., contractors, business
visitors) entering areas where aerosolized particles may be generated. This includes
contractors, business visitors, and support staff.
3. Housekeeping Controls in Mail-handling/processing Sites
Dry sweeping and dusting should be avoided. Instead, areas should be wet-cleaned and
vacuumed with HEPA-equipped vacuum cleaners.
4. Personal Protective Equipment for Workers in Mail-handling/processing Sites
Personal protective equipment for workers in mail-handling/processing work sites must
be selected on the basis of the potential for cutaneous or inhalational exposure to B.
anthracis spores. Handling packages or envelopes may result in cutaneous exposure. In
addition, because certain machinery (e.g., electronic mail sorters) can generate
aerosolized particles, persons who operate, maintain, or work near such machinery may
be exposed through inhalation. Persons who hand sort mail or work at other sites where
airborne particles may be generated (e.g., where mailbags are unloaded or emptied) may
also be exposed through inhalation.
Recommendations for Workers Who Handle Mail
• Protective, impermeable gloves should be worn by all workers who handle mail. In
some cases, workers may need to wear cotton gloves under their protective gloves for
comfort and to prevent dermatitis. Skin rashes and other dermatological conditions are a
potential hazard of wearing gloves. Latex gloves should be avoided because of the risk of
developing skin sensitivity or allergy.
• Gloves should be provided in a range of sizes to ensure proper fit.
• The choice of glove material (e.g., nitrile, vinyl) should be based on safety, fit,
durability, and comfort. Sterile gloves (e.g., surgical gloves) are not necessary.
• Different gloves or layers of gloves may be needed depending on the task, the dexterity
required, and the type of protection needed. Protective gloves can be worn under heavier
gloves (e.g., leather, heavy cotton) for operations where gloves can easily be torn or if
more protection against hand injury is needed.
• For workers involved in situations where a gloved hand presents a hazard (e.g., close to
moving machine parts), the risk for potential injury resulting from glove use should be
measured against the risk for potential exposure to B. anthracis.
• Workers should avoid touching their skin, eyes, or other mucous membranes since
contaminated gloves may transfer B. anthracis spores to other body sites.
• Workers should consider wearing long-sleeved clothing and long pants to protect
exposed skin.
• Gloves and other personal protective clothing and equipment can be discarded in
regular trash once they are removed or if they are visibly torn, unless a suspicious piece
of mail is
recognized and handled.
If a suspicious piece of mail is recognized and handled, the worker’s protective gear
should be handled as potentially contaminated material (See “Guideline For Hand
washing And Hospital Environmental Control,” 1985, available at
<http://www.cdc.gov/ncidod/hip/guide/handwash.htm>
Hands should be thoroughly washed with soap and water when gloves are removed,
before eating, and when replacing torn or worn gloves. Soap and water will wash away
most spores that may have contacted the skin; disinfectant solutions are not needed.
Additional Recommendations for Workers Who May Be Exposed through Inhalation
• Persons working with or near machinery capable of generating aerosolized particles
(e.g., electronic mail sorters) or at other work sites where such particles may be
generated should be fitted with NIOSH-approved respirators that are at least as protective
as an N95 respirator.
• Persons working in areas where oil mist from machinery is present should be fitted with
respirators equipped with P-type filters.
• Because facial hair interferes with the fit of protective respirators, workers with facial
hair (beards and or large moustaches) may require alternative respirators (such as
powered air-purifying respirators [PAPRS] with loose-fitting hoods).
• Workers who cannot be fitted properly with a half-mask respirator based on a fit test
may require the use of alternative respirators, such as full facepiece, negative pressure
respirators, PAPRs equipped with HEPA filters, or supplied-air respirators. If a worker is
medically unable to wear a respirator, the employer should consider reassigning that
worker to a job that does not require respiratory protection.
• In addition, the use of disposable aprons or goggles by persons working with or near
machinery capable of generating aerosolized particles may provide an extra margin
of protection. In work sites where respirators are worn, a respiratory-protection program
that complies with the provisions of OSHA [29 CFR 1910.134] should be in place. Such
a program includes provisions for obtaining medical clearance for wearing a respirator
and conducting a respirator fit-test to ensure that the respirator fits properly. Without fit
testing, persons unknowingly may have poor face seals, allowing aerosols to leak around
the mask and be inhaled.
(See December 11, 1998, MMWR, available at
<http://www.cdc.gov/mmwr/preview/mmwrhtml/00055954.htm>
Slide 21
Interim CDC recommendations for personal protective equipment for responding to
biological weapons
The interim CDC recommendations for personal protective equipment, including
respiratory protection and protective clothing, are based upon the anticipated level of
exposure risk
associated with different response situations, as follows:
1.Responders should use a NIOSH-approved, pressure-demand SCBA in conjunction
with a Level A protective suit in responding to a suspected biological
incident where any of the following information is unknown or the event is uncontrolled:
- the type(s) of airborne agent(s);
- the dissemination method;
- if dissemination via an aerosol-generating device is still occurring or it has stopped
but there is no information on the duration of dissemination, or what the exposure
concentration might be.
2. Responders may use a Level B protective suit with an exposed or enclosed NIOSHapproved pressure-demand SCBA if the situation can be defined in which:
- the suspected biological aerosol is no longer being generated;
- other conditions may present a splash hazard.
3. Responders may use a full facepiece respirator with a P100 filter or powered airpurifying respirator (PAPR) with high efficiency particulate air (HEPA) filters when it
can be determined that:
- an aerosol-generating device was not used to create high airborne
concentration,
- dissemination was by a letter or package that can be easily bagged.
These types of respirators reduce the user’s exposure by a factor of 50 if the user has
been properly fit tested.
Slide 22: OSHA Respirator Standards
CDC, OSHA respirator recommendations for potential exposures to biological agents,
such as Bacillus anthracis, in their facilities:
Summary
The goal of using a respirator is to reduce the exposure to the contaminant of concern to
an acceptable level that will not adversely affect the wearer. According to the Centers for
Disease Control and Prevention (CDC) and Occupational Safety and Health
Administration (OSHA) all National Institute for Occupational Safety and Health
(NIOSH) approved particulate respirators will help reduce exposures to biological
aerosols such as B. anthracis, the bacteria that causes anthrax. Recently the CDC and
OSHA have published respirator selection guidance, based on the expected risk of
exposure, for individuals who may be potentially exposed to B. anthracis while engaged
in mail handling, first responder, or investigative activities. However, no safe exposure
levels (i.e. the amount you can inhale without adverse health effects) have been set for
biological aerosols, including B. anthracis. Therefore, it must be recognized that
respirators can reduce inhalation exposures but cannot eliminate the risk of contracting
infection, illness, or disease. Each facility or individual must use the best available
information in determining the appropriate respiratory protection for the level of
exposure reduction that they feel is appropriate for potential occupational exposures to B.
anthracis in their facility.
Respirator Use Limitations
A properly fitted respirator can only help reduce exposures when used immediately prior
to and during the release of B. anthracis spores. Unfortunately, in the case of terrorist
activity it is unlikely that you would have warning or knowledge of your exposure until
symptoms started to appear in infected people. Once anthrax symptoms appear, a
respirator will not be effective in helping to prevent the disease.
Before selecting respiratory products for biological agents, such as B. anthracis, there are
important considerations you must be aware of. The airborne concentration of these
agents will be unknown; therefore it may not be possible to select the most appropriate
respirator. In addition,
NIOSH is the government agency responsible for testing and certifying respirators.
NIOSH tests and certifies respirators for use against particles, gases, and vapors. NIOSH
does not certify respirators for use against specific particles or biological agents, such as
B. anthracis spores. Therefore, their efficacy against biological warfare agents is not
known.
Respirators may help protect your lungs, however, they will not prevent entry through
other routes such as the skin (cutaneous), which would require additional personal
protective equipment (PPE).
Without proper decontamination, materials could create a hazard by bringing the spores
into areas thought to be uncontaminated.
Proper fit of the respirator to the face is extremely important. If it does not fit properly,
you will increase your likelihood of exposure to the B. anthracis you are trying to filter.
Individuals wearing tight fitting face pieces must be clean-shaven at all times when
wearing respirators.
Respirators are designed for occupational/professional use by adults who are properly
trained in their use and limitations.
Individuals with a compromised respiratory system should consult with a physician prior
to use.
In the event of a known or suspected biological warfare agent release; respirators should
be used for escape only; leave the area immediately; do not remove respirator until going
through decontamination and are in a clean environment; seek medical advice; and
dispose of respirator immediately in accordance to your employers directions.
Filtering Bacillus anthracis
Biological agents such as B. anthracis are particles and can be removed by particulate
filters with the same efficiency as non-biological particles having the same physical
characteristics (size, shape, etc.), although their efficacy against biological agents is not
known. According to the CDC the typical size of B. anthracis spores is between 1 - 5
microns.
Both OSHA and the CDC recommend a NIOSH certified class 95 or higher filter for use
against B. anthracis spores. However, the type of respirator facepiece and filter class
required does vary depending activities and risk of exposure. Consult the OSHA and
CDC requirements before selecting a respirator for potential occupational exposures to B.
anthracis.
NIOSH class 95 filters are certified to be at least 95% efficient against a particle of 0.3
microns. Therefore, the filter will be 95% efficient or greater for particles in the 1 to 5
micron size range. A NIOSH certified class 100 or HEPA filter is 99.97% efficient
against this most penetrating particle size of 0.3 microns.
Importance of Proper Fit
The fit of a respirator is equally as important as filter efficiency. While a respirator may
be equipped with filter media to effectively capture a high percentage of airborne
particles, excessive particles may enter the respirator through leaks around the facepiece
of an improperly fitted facepiece.
A tight sealing respirator, one where the sealing surface contacts the face, will not
provide an adequate seal when placed over facial hair.
A bearded worker will typically require a respirator where the wearer’s facial hair does
not interfere with the face seal. In many instances this will consist of a powered airpurifying respirator (PAPR) with a hood or helmet.
Assigned Protection Factors
It is important to understand that since the safe level of exposure to B. anthracis spores
has not been established, there is no assurance that any respirator will mitigate or prevent
anthrax infection or disease. Respirators are traditionally selected after determining the
airborne concentration of the contaminant, the exposure limit of the contaminant and the
assigned protection factor of the respirator. Since the exposure limit and concentration
are unknown for biological agents the traditional respirator selection method cannot be
uniformly applied.
All NIOSH certified respirators have an assigned protection factor (APF), which predicts
how much the respirator may reduce a wearer’s exposure.
The assigned protection factor is only applicable when the respirator is correctly selected,
properly used by a trained and fit tested wearer and the respirator is maintained in good
working order.
A respirator with a higher protection factor will provide greater exposure reduction when
the respirator is used properly and fitted to the individual.
Here is an example of how to use assigned protection factors when choosing an
appropriate respirator: Assume the contaminant concentration in the air is 10,000
particles. A person has passed a fit test and is wearing a half mask respirator with an
assigned protection factor of 10. This means the person could expect to reduce their
exposure by 10 times, resulting in a possible inhalation of 1000 particles. A full-face
respirator would reduce the exposure by 50 times resulting in a possible inhalation of 200
particles.
When a facility decides to make respiratory products a part of its emergency management
or response plan, it is essential they follow all aspects of the OSHA respiratory protection
standard, 29 CFR 1910.134.
CDC and OSHA Respirator Recommendations
The CDC has published three documents with respirator recommendations for mail
handlers, first responders, and investigators who may be potentially exposed to B.
anthracis. CDC recommends that a combination of controls be used to help reduce mail
handlers potential exposure to B. anthracis. These include engineering controls (e.g.,
local exhaust ventilation), administrative controls (e.g., limiting number of people who
could be exposed), and good work practices (e.g., wet cleaning). These recommendations
for mail handlers do not address instances where known or a suspected exposure has
occurred. For instances where a known or suspected exposure has occurred CDC
recommends respirators with a higher assigned protection factor for the first responders
and investigators who must enter into these environments to perform their duties. In
addition, CDC states that in work sites where respirators are worn, a respiratory
protection program that complies with the provisions of the OSHA Respiratory
Protection Standard (29 CFR 1910.134) should be in place. CDC emphasizes the need for
users to be fit tested to ensure the respirator fits properly. CDC states “Without fit testing,
persons unknowingly may have poor face seals, allowing aerosols to leak around the
mask and be inhaled”. The following table lists 3M respirators that satisfy the CDC
recommendations.
OSHA has published a workplace anthrax exposure guidance document titled the
Anthrax Matrix. This document is intended to assist employers and employees in dealing
with possible workplace exposure to B. anthracis in mail handling operations today. The
matrix helps guide employers in assessing risk to their workers, providing appropriate
protective equipment and specifying safe work practices for low, medium and high risk
levels in the workplace. Following is a summary of the respiratory protection
requirements outlined in this document. Note, the document also specifies engineering
controls, work practices, and other personal protective equipment (PPE). Please consult
the Anthrax Matrix for complete details of this guidance document. The following table
lists 3M respirators that satisfy the OSHA recommendations.
*Red zone (Workplaces Where Authorities Have Informed You That Contamination with
Anthrax Spores Has Been Confirmed or Is Strongly Suspected). The employer is notified
by law enforcement or public health authorities that a facility is strongly suspected of or
confirmed as having been contaminated with anthrax spores. The employer is engaged in
emergency response to and clean up of bio-terrorist releases of anthrax spores.
*Yellow Zone (Workplaces Where Contamination with Anthrax Spores Is Possible). This
zone is where workplace contamination is possible. Risk factors that should be
considered in this zone include handling bulk mail, handling mail from facilities that are
known to be contaminated, working near equipment such as high-speed
processors/sorters that could aerosolize anthrax spores; workplaces in close proximity to
other workplaces known to be contaminated; or workplaces that may be targets of bioterrorists.
*Green Zone (Workplaces Where Contamination with Anthrax Spores Is Unlikely).
OSHA states this zone covers the vast majority of workplaces in the United States. Since
October 2001, anthrax spores have only been discovered in a very limited number of
workplaces.
Additional Information
Please use the links below to access the most recent governmental information regarding
respiratory protection against Bacillus anthracis:
OSHA Homepage
CDC Homepage
OSHA Guidance Document: The Anthrax Matrix (November 16, 2001)
CDC Advisory: CDC Interim* Recommendations for Protecting Workers from Exposure
to Bacillus anthracis in Work Sites Where Mail Is Handled or Processed (October 31,
2001)
CDC Advisory: Interim Recommendations for the Selection and Use of Protective
Clothing and Respirators Against Biological Agents (October 24, 2001)
CDC Advisory: Protecting Investigators Performing Environmental Sampling for
Bacillus anthracis: Personal Protective Equipment (November 6, 2001)
Last updated: 11/21/01
Slide 23: OSHA Regulations: Summary of Employer Responsibilities
In summary, employers must:
Maintain a written respiratory protection program with worksite specific procedures for
fit testing and training.
Provide instruction on the respiratory hazards to which the workers are potentially
exposed during routine and emergency situations.
Provide instruction on the uses and limitations of all respirators worn in the work area.
Instruct and demonstrate to employees how to properly don and adjust any respirators
worn according to the manufacturers' instructions..
Allow the employees an opportunity to practice these procedures.
Provide user seal check instructions.
Fit test each employee to be assigned a respirator.
Instruct the employees in the procedures for the maintenance and storage of the
respirators being used.
Inform the employees how to recognize medical signs and symptoms that may limit or
prevent the effective use of the respirators.
Document the successful completion of training and fit testing for all employees wearing
respirators.
Slide 24: CHANGING YOUR RESPIRATOR/ FILTERS/ CARTRIDGES
If you are using a dust mask (also called a filtering facepiece or "N95" respirator) OR a
rubber or plastic facepiece respirator wih replaceable particulate filters (e.g. N95, P100),
replace the dust mask or change the filters when you notice any of the following:
Increased breathing resistance, OR
Physical damage to any part of the face piece or filters, OR
Inside of the dust mask becomes unsanitary, OR
Time use limitations on the package require replacement.
If you are using a half facepiece with replaceable chemical cartridges (e.g. organic vpors,
ammonia), the cartridges must be replaced:
In accordance with a change schedule, OR
Earlier if smell, taste, or irritation from the contaminant(s) is detected.
Slide 25
Summary of the CDC Health Advisory on the Interim Recommendations for Protecting
Workers from Exposure to Bacillus anthracis in Work Sites Where Mail Is Handled or
Processed. (Distributed via the Health Alert Network, October 31, 2001)
On October 31, 2001, the CDC issued a "revised" advisory titled Official CDC Health
Advisory: CDC Interim Recommendations for Protecting Workers from Exposure to
Bacillus anthracis in Work Sites Where Mail is handled or Processed.* This document is
intended to assist personnel responsible for occupational health and safety in developing
a comprehensive program to reduce potential cutaneous or inhalational exposures to
Bacillus anthracis spores among workers, including maintenance and custodial workers,
in work sites where mail is handled or processed.
These recommendations do not address instances where known or a suspected exposure
has occurred.
The document stresses the need for engineering controls (e.g., local exhaust ventilation),
administrative controls (e.g., limiting number of people who could be exposed), and good
work practices (e.g., wet cleaning) to minimize worker exposure to Bacillus anthracis.
Personal protective equipment recommendations including gloves, eye protection, and
other personal protective clothing are also discussed.
According to the CDC, in the event an employer chooses to provide respirators for
employees handling or processing mail the advisory provides the following guidance for
selecting respiratory protection products to help reduce potential exposures to Bacillus
anthracis:
Persons working with or near machinery capable of generating aerosolized particles (e.g.,
electronic mail sorters) or at other work sites where such particles may be generated
should be fitted with NIOSH-approved respirators that are at least as protective as an N95
respirator.
Persons working in areas where oil mist from machinery is present should be fitted with
respirators equipped with P-type filters.
Because facial hair interferes with the fit of protective respirators, workers with facial
hair (beards and or large mustaches) may require alternative respirators (such as powered
air-purifying respirators [PAPRs] with loose-fitting hoods).
Workers who cannot be fitted properly with a half-mask respirator based on a fit test may
require the use of alternative respirators, such as full facepiece, negative pressure
respirators, PAPRs equipped with HEPA filters, or supplied-air respirators. If a worker is
medically unable to wear a respirator, the employer should consider reassigning that
worker to a job that does not require respiratory protection.
In addition, CDC states that in work sites where respirators are worn, a respiratoryprotection program that complies with the provisions of the OSHA Respiratory
Protection Standard (29 CFR 1910.134) should be in place. CDC emphasizes the need for
users to be fit tested to ensure the respirator fits properly. CDC states "Without fit testing,
persons unknowingly may have poor face seals, allowing aerosols to leak around the
mask and be inhaled".
Slide 41:
Additional Reference: MMWR. 2002;51:482, April 5, 2002 - Suspected Cutaneous
Anthrax in a Laboratory Worker --- Texas, 2002
On April 5, 2002, CDC reported a case of suspected cutaneous anthrax in a worker at
laboratory A who had been processing environmental samples for Bacillus anthracis
in support of CDC investigations of the 2001 bioterrorist attacks in the United
States.1 Since the initial report, the worker had serial serology performed at the CDC
laboratory. A greater than fourfold rise from baseline in the concentration of
immunoglobulin G to protective antigen was demonstrated. The peak antibody level
was observed 7-8 weeks after the onset of symptoms, and the time course and levels
of detectable antibodies were consistent with those seen in other cases of cutaneous
anthrax. On the basis of case definitions developed during the recent investigation,
these additional findings confirm this as a case of cutaneous anthrax.2 This case
brings the number of anthrax cases identified in the United States since October 3,
2001, to 23, including 11 inhalation and 12 cutaneous (eight confirmed and four
suspected). This is the first laboratory-acquired case of anthrax associated with the
recent investigation.
The epidemiologic and environmental investigation of this case indicated that the
probable source of exposure was the surface of vials containing B. anthracis isolates
that the worker had placed in a freezer. The storage vials had been sprayed with 70%
isopropyl alcohol, which is not sporicidal, instead of a bleach solution because bleach
had caused labels to become dislodged. The worker did not wear gloves when
handling the vials. A culture of the vial tops performed at laboratory A tested positive
for B. anthracis. The vial top specimen was confirmed positive for B. anthracis at
CDC. Multiple-locus variable-number tandem repeat analysis found this isolate to be
indistinguishable from the culture of the worker's clinical specimen. This case
underscores the importance of safe laboratory procedures and anthrax vaccination for
workers routinely handling B. anthracis isolates.3
A number of events during the past several years have served to focus attention on the
threat of terrorism and the use of biological or chemical weapons against civilian
populations.1 A single or sentinel act of bioterrorism, whether announced or
unannounced, would initially be recognized at the local or state level, as would the
initial public health response. The Centers for Disease Control and Prevention (CDC)
was designated by the Department of Health and Human Services to prepare the
nation's public health system to respond to such a bioterrorism event.2 An effective
public health response would need to be timely because there is only a short window
to provide prophylaxis or implement other control measures that are designed to
minimize the number of casualties.3 To enhance local and state preparedness, the
CDC funded cooperative agreements with states and several large cities that focused
on preparedness activities.4 Five areas were emphasized during the first 3 years of
this program (1999-2001): preparedness planning and readiness assessment,
surveillance and epidemiology capacity, biological laboratory capacity, chemical
laboratory capacity, and health alert network and training.
The recent mail-borne attacks using spores of Bacillus anthracis have made bioterrorism
a reality in the United States.5,6 These attacks demonstrated the 2 types of scenarios
characteristic of bioterrorism events: covert (unannounced) and overt (announced).
An unannounced release of anthrax spores would likely go unnoticed, as happened
with the index case, Mr Stevens.7 Others coexposed in the Stevens attack left the area
long before the act of terrorism became evident. The first signs that anthrax spores
had been released did not become apparent until days later, when 2 individuals
became ill and sought medical care. An astute clinician and laboratorian provided
public health officials with initial clues that an unannounced attack had occurred.
However, increased physician awareness of unusual signs and symptoms that may be
associated with a bioterrorist attack is needed because 7 individuals with cutaneous or
inhalational anthrax sought medical treatment in New York, New Jersey, and Florida
before the index case was recognized.8
Because of their terrorism response training, traditional "first responders" (eg, firefighters
or law enforcement) are the most likely to respond to an announced attack, such as
the letter received by Senator Tom Daschle's office,6 or to the numerous hoaxes that
occurred during the same period. Therefore, the initial recognition of bioterrorism,
whether unannounced or announced, would be at the local level and state level and
would result in a comprehensive public health response involving epidemiological
investigation, medical treatment and prophylaxis for affected persons, and initiation
of disease prevention activities. The success of this response depends on, to a large
extent, physician recognition and rapid and accurate identification of the threat agent.
In this issue of Mayo Clinic Proceedings, articles by Varkey et al,9 Espy et al,10 and
Uhl et al 11 highlight the need for physician awareness and laboratory preparedness.
Many biological agents can cause illness in humans, but not all are capable of affecting
public health and medical infrastructure on a large scale. To focus on public health
preparedness activities, the CDC convened a meeting of national experts to review
potential criteria for selecting the biological agents that posed the greatest threat to
civilian populations.12 The list of "Critical Agents"2 was prioritized based on
considerations such as (1) ability of the agent to cause mass casualties, (2) ability of
the agent to be disseminated widely, (3) ability of the agent to be transmitted from
person to person, (4) public perception associated with the intentional release of the
agent, and (5) special public health preparedness needs based on stockpile
requirements, enhanced surveillance, or diagnostic needs. As currently defined,
Category A agents are those most likely to cause mass casualties if deliberately
disseminated and require broad-based public health preparedness efforts. Such agents
are responsible for anthrax, smallpox, tularemia, plague, botulism, and viral
hemorrhagic fevers. In the United States, both clinical and laboratory experience are
limited for Category A agents. Physician education is of paramount importance for
early recognition of covert releases of biothreat agents. Several Web sites (eg,
www.idsociety.org/bt/toc.htm ) and articles such as that by Varkey et al 9 provide
practicing physicians with knowledge of the clinical presentation, diagnosis, and
management of diseases caused by Category A agents. Physicians also need to be
aware that biological agents can be introduced into a civilian population in several
ways (eg, aerosol, food contamination, water, animal vectors) and that clinical
presentation can vary depending on the route of entry.
Rapid laboratory identification of the biothreat agent is necessary to minimize the impact
of a bioterrorist attack. However, as previously mentioned, laboratory experience
with Category A agents is limited. Before October 2001, the low number of infections
caused by these agents in the United States was given as a major reason why the
commercial sector did not develop and manufacture specific Food and Drug
Administration-approved diagnostic tests in this country (ie, a small market). In
addition, many of these agents pose risks to laboratory workers and must be handled
at Biosafety Level 3 or 4.12 This situation has created the need for development and
restricted distribution of biodetection assays and specialized reagents, regardless of
the size of the market. Such market-independent actions are necessary to support the
emergency public health response infrastructure and to meet the security interests of
the United States. These assays and reagents are employed within the Laboratory
Response Network (LRN) for Bioterrorism, which was developed by the CDC in
concert with the Association of Public Health Laboratories and with collaboration
from the Federal Bureau of Investigation, US Army Medical Research Institute of
Infectious Diseases, Naval Medical Research Center, and Lawrence Livermore
National Laboratory to detect and respond to agents that are released by a bioterrorist
and those that occur naturally. This is particularly important because the dispersal
mechanism (ie, intentional vs natural) will generally not be known at the time of
initial detection. The LRN is a multilevel system that will ultimately link state and
some local public health laboratories with military, veterinary, agricultural, water, and
food-testing laboratories. Currently, the LRN operates as a network of laboratories
(laboratory levels designated A through D) with progressively stringent levels of
safety, containment, and technical proficiency necessary to perform the essential ruleout, rule-in, and referral functions required for agent identification.
Level A laboratories are, for the most part, the hospital and other clinical laboratories
with certified biological safety cabinets that participate in the LRN by ruling out or
referring the critical agents that they encounter in their routine work to the nearest
Level B or C laboratory. However, before the clinical laboratory can fulfill this role it
must address several issues: (1) knowledge of the current biosafety level within the
laboratory; (2) familiarity with Level A protocols, which are available on the Internet
at either www.asmusa.org or www.bt.cdc.gov ; (3) knowledge of current guidelines to
ensure safe handling and shipment of biological agents 13; (4) familiarity with
protocols related to chain of custody, collection, preservation, and shipment of
specimens; and (5) location of the nearest LRN Level B or C laboratory. Until rapid
and reliable detection systems and diagnostic tests are available for the Level A
laboratory, it must rely on conventional methods to obtain as much information in the
least amount of time to rule out specific agents effectively. The article by Uhl et al 11
describes the use and advantages of real-time polymerase chain reaction (PCR) assays
and their potential use in clinical laboratories, in an attempt to rapidly rule in or rule
out infection by a likely bioterrorism infectious agent.
Most clinical laboratories are incapable of processing specimens for the diagnosis of
smallpox. The article by Espy et al 10 describes the observation that proper
autoclaving of specimens can eliminate hazards associated with infectivity while the
DNA of the microorganism remains detectable by PCR. Nucleic acid amplification
assays, such as those described by Espy et al, have high analytic sensitivity and do
not distinguish between viable and nonviable agents. Thus, care should be taken to
avoid cross-contamination of negative, nonviable specimens during autoclaving.
Laboratory Response Network Level B, C, and D laboratories have access to the
biodetection assays and specialized reagents that are used in validated protocols for
the confirmation of critical agents. Level B laboratories are primarily state and local
public health laboratories that have Biosafety Level 2 facilities where Biosafety Level
3 practices are observed; Level C laboratories are primarily public health laboratories
with Biosafety Level 3 facilities or certified animal facilities, which are necessary for
performing the mouse toxicity assay for botulinum toxin. Level C laboratories can
perform all Level B tests and additional tests requiring Biosafety Level 3
containment, such as those that involve the handling of powders suspected of
containing anthrax spores. Level D laboratories are federal laboratories (ie, CDC and
US Army Medical Research Institute of Infectious Diseases) with the Biosafety Level
4 capacity to handle agents (eg, Ebola and variola major) that other laboratories
cannot handle. Level D laboratories have the capacity to perform all the Level B and
C procedures. In addition, they can identify agents in specimens that have been
referred by Level B or C laboratories, identify recombinant microorganisms that may
not be recognizable with conventional isolation and identification methods, and
maintain extensive culture collection of critical agents against which the isolate(s)
from a bioterrorist event may be compared to determine its origin.
The LRN became operational in August 1999. Since then, considerable effort has been
expended to develop and validate rapid assays. Robust real-time PCR assays using
TaqMan (Roche Diagnostics Corporation, Indianapolis, Ind) chemistry have been
developed and optimized for several commercially available platforms including the
LightCycler (Roche Applied Science, Indianapolis, Ind), SmartCycler (Cepheid,
Sunnyvale, Calif), and the GeneAmp 5700 and PRISM 7700 (Applied Biosystems,
Foster City, Calif). The effort expended in the development of these assays ensures
that they will have the accuracy, reproducibility, sensitivity, and specificity that are
required for use in the LRN. High-confidence real-time PCR assay development starts
with the identification of primer pairs or signatures that are able to recognize unique
regions of the target microorganism. These primer pairs should detect nucleic acid
derived from any strain of the target organism but not react with nucleic acid from
phylogenetically related organisms or organisms that constitute the culture or sample
background and could be copurified from the sample. The selected primer pair-probe
combinations, referred to as specific signatures, are used as a panel to identify the
target agent. For agent identification, a specific algorithm must be used based on
positive or negative reactions of the signature panel. The robustness of these assays
has been evaluated through a multicenter validation study involving an average of 10
laboratories. Having confidence in the test results also requires well-trained
laboratorians. The CDC recently completed six 1-week courses during which 1
person from each state public health laboratory and other selected LRN laboratories
received training in realtime PCR assays.
In summary, an effective public health response to a bioterrorism event will depend on a
few key factors: the ability of medical professionals to rapidly recognize the clinical
signs and symptoms of disease caused by a biothreat agent; the capability of
laboratory professionals to rapidly detect and confirm the identity of the agent; an
epidemiological investigation to determine the source of the infection; and, most
importantly, communication and coordination among all the responders involved in
the event.
Richard F. Meyer, PhD
Rapid Response and Advanced Technology Laboratory; Bioterrorism Preparedness and
Response Program; National Center for Infectious Diseases
Stephen A. Morse, MSPH, PhD
Bioterrorism Preparedness and Response Program; National Center for Infectious
Diseases
References
Tucker JB. Historical trends related to bioterrorism: an empirical analysis. Emerg Infect
Dis. 1999;5:498-504.
2. Centers for Disease Control and Prevention. Biological and chemical terrorism:
strategic plan for preparedness and response: recommendations of the CDC Strategic
Planning
Workgroup. MMWR Recomm Rep. 2000;49(RR-4):1-14.
3. Kaufmann AF, Meltzer MI, Schmid GP. The economic impact of a bioterrorist attack:
are prevention and postattack intervention programs justifiable?
Emerg Infect Dis. 1997;3:83-94.
4. Khan AS, Morse S, Lillibridge S. Public-health preparedness for biological terrorism
in the USA. Lancet. 2000;356:1179-1182.
5. Centers for Disease Control and Prevention. Update: investigation of anthrax
associated with intentional exposure and interim public health guidelines, October
2001. MMWR Morb Mortal Wkly Rep. 2001;50:889-893.
6. Centers for Disease Control and Prevention. Update: investigation of bioterrorismrelated anthrax and interim guidelines for exposure management and antimicrobial
therapy, October 2001 [published correction appears in MMWR Morb Mortal Wkly
Rep. 2001;50:962]. MMWR Morb Mortal Wkly Rep. 2001;50:909-919.
7. Jernigan JA, Stephens DS, Ashford DA, et al. Bioterrorism-related inhalational
anthrax: the first 10 cases reported in the United States. Emerg Infect Dis.
2001;7:933-944.
8. Lipton E, Johnson K. A nation challenged: the anthrax trail; tracking bioterror's
tangled course. New York Times. December 26, 2001; sect A:1.
9. Varkey P, Poland GA, Cockerill FR III, Smith TF, Hagen PT. Confronting
bioterrorism: physicians on the front line. Mayo Clin Proc. 2002;77:661-672.
10. Espy MJ, Uhl JR, Sloan LM, Rosenblatt JE, Cockerill FR III, Smith TF. Detection of
vaccinia virus, herpes simplex virus, varicellazoster virus, and Bacillus anthracis
DNA by LightCycler polymerase chain reaction after autoclaving: implications for
biosafety of bioterrorism agents. Mayo Clin Proc. 2002;77:624-628.
11. Uhl JR, Bell CA, Sloan LM, et al. Application of rapid-cycle real-time polymerase
chain reaction for the detection of microbial pathogens: the Mayo-Roche Rapid
Anthrax Test. Mayo Clin Proc. 2002;77:673-680.
12. Rotz LD, Khan AS, Lillibridge SR, Ostroff SM, Hughes JM. Public health
assessment of potential biological terrorism agents. Emerg Infect Dis. 2002;8:225230.
13. Centers for Disease Control and Prevention, National Institutes of Health. Biosafety
in Microbiological and Biomedical Laboratories, 4th ed. Washington, DC: US
Government Printing Office; 1999. Available at:
www.cdc.gov/od/ohs/biosfty/bmb14tochtm . Accessibility verified May 29, 2002.
Slide 81: Cidofovir
Cidofovir is Active Against DNA Viruses
•Nucleotide analogue, inhibits viral DNA polymerase by competitive inhibition of dCTP
•Phosphorylated to active diphosphateform by host cellular enzymes
•Cidofovir diphosphate-choline adduct
–T½ = 87 hours
–intracellular reservoir
–weekly administration
•FDA approved for treatment of CMV retinitis in persons with AIDS
Cidofovir Dosing and Toxicity
•Usual Dose = 5 mg/kg, intravenously, once weekly
•Cleared in kidneys
•Toxicities:
–Nephrotoxicity, with proteinuria and?creatinine
- Neutropenia
Reference: Lea AP, Bryson HM. Cidofovir. Drugs 1996;52:225-30 .
Slide 83: Viral Hemmorhagic Fevers
Additional Reference: Textbook of Military Medicine, Chapter 29, Viral Hemorrhagic
Fevers, page 594:
The Filoviridae includes the causative agents of Ebola and Marburg hemorrhagic fevers.
These filoviruses have an exotic, threadlike appearance when observed via electron
microscopy. Marburg virus was first recognized in 1967 when a lethal epidemic of VHF
occurred in Marburg, Germany, among laboratory workers exposed to the blood and
tissues of African green monkeys that had been imported from Uganda; secondary
transmission to medical personnel and family members also occurred.10 In all, 31
patients became infected, 9 of whom died. Subsequently, Marburg virus has been
associated with sporadic, isolated, usually fatal cases among residents and travelers in
southeast Africa.11
Ebola viruses are taxonomically related to Marburg viruses; they were first recognized
in association with explosive outbreaks that occurred almost simultaneously in 1976 in
small communities in Zaire12 and Sudan.13 Significant secondary transmission occurred
through reuse of unsterilized needles and syringes and nosocomial contacts. These
independent outbreaks involved serologically distinct viral strains. The Ebola–Zaire
outbreak involved 277 cases and 257 deaths (92% mortality), while the Ebola–Sudan
outbreak involved 280 cases and 148 deaths (53% mortality). Sporadic cases occurred
subsequently. In 1989, a third strain of Ebola virus appeared in Reston, Virginia, in
association with an outbreak of VHF among cynomolgus monkeys imported to the
United States from the Philippines.14 Hundreds of monkeys were infected (with high
mortality) but no human cases occurred, although four animal caretakers seroconverted
without overt disease. Recently, small outbreaks involving new strains of Ebola virus
occurred in human populations in Côte d’Ivorie in 1994 and Gabon in 1995; a larger
outbreak involving the Ebola-Zaire strain involved more than 300 people, with 75%
mortality, in Zaire in 1995.15
Very little is known about the natural history of any of the filoviruses. Animal reservoirs
and arthropod vectors have been aggressively sought without success.
Slide 85: Viral Hemorrhagic Fever
Additional Reference: Textbook of Military Medicine, Chapter 29, Viral Hemorrhagic
Fevers, page 594-597:
CLINICAL FEATURES OF THE VIRAL HEMORRHAGIC FEVER SYNDROME
The VHF syndrome develops to varying degrees in patients infected with these
viruses. The exact nature of the disease depends on viral virulence and strain
characteristics, routes of exposure, dose, and host factors. For example, dengue
hemorrhagic fever is typically seen only in patients previously exposed to heterologous
dengue serotypes.19 The target organ in the VHF syndrome is the vascular bed;
correspondingly, the dominant clinical features are usually a consequence of
microvascular damage and changes in vascular permeability.20 Common presenting
complaints are fever, myalgia, and prostration; clinical examination may reveal only
conjunctival injection, mild hypotension, flushing, and petechial hemorrhages. Fullblown VHF typically evolves to shock and generalized bleeding from the mucous
membranes, and often is accompanied by evidence of neurological, hematopoietic, or
pulmonary involvement. Hepatic involvement is common, but a clinical picture
dominated by jaundice and other evidence of hepatic failure is seen in only a small
percentage patients with Rift Valley fever, Crimean-Congo hemorrhagic fever, Marburg
hemorrhagic fever, Ebola hemorrhagic fever, and yellow fever. Renal failure is
proportional to cardiovascular compromise, except in HFRS caused by hantaviruses,
where it is an integral part of the disease process; oliguria is a prominent feature of the
acutely ill patient.8 VHF mortality may be substantial, ranging from 5% to 20% or higher
in recognized cases. Ebola outbreaks in Africa have had particularly high case fatality
rates, from 50% up to 90%.12,13
The clinical characteristics of the various VHFs are somewhat variable. For Lassa
fever patients, hemorrhagic manifestations are not pronounced, and neurological
complications are infrequent, occurring only late and in only the most severely ill group.
Deafness is a frequent sequela of severe Lassa fever. For the South American
arenaviruses, (Argentine and Bolivian hemorrhagic fevers), neurological and
hemorrhagic manifestations are much more prominent. RVF virus is primarily
hepatotropic; hemorrhagic disease is seen in only a small proportion of cases. In recent
outbreaks in Egypt, retinitis was a frequently reported component of Rift Valley fever.21
Unlike Rift Valley fever, where hemorrhage is not prominent, Crimean-Congo
hemorrhagic fever infection is usually associated with profound disseminated
intravascular coagulation (DIC) (Figure 29-1). Patients with Crimean-Congo
hemorrhagic fever may bleed profusely; and since this occurs during the acute, viremic
phase, contact with the blood of an infected patient is a special concern: a number of
nosocomial outbreaks have been associated with C-CHV virus.
The picture for diseases caused by hantaviruses is evolving, especially now in the
context of HPS syndrome. The pathogenesis of HFRS may be somewhat different;
immunopathological events seem to be a major factor. When patients present with HFRS,
they are typically oliguric. Surprisingly, the oliguria occurs while the patient’s viremia is
resolving and they are mounting a demonstrable antibody response. This has practical
significance in that renal dialysis can be started with relative safety.
For the diseases caused by filoviruses, little clinical data from human outbreaks exist.
Although mortality is high, outbreaks are rare and sporadic. Marburg and Ebola viruses
produce prominent maculopapular rashes, and DIC is a major factor in their pathogenesis.
Therefore, treatment of the DIC should be considered, if practicable, for these patients.
Among the flaviviruses, yellow fever virus is, of course, hepatotropic: black vomit
caused by hematemesis has been associated with this disease. Patients with yellow fever
develop clinical jaundice and die with something comparable to hepatorenal syndrome.
Dengue hemorrhagic fever and shock are uncommon, life-threatening complications of
dengue, and are thought—especially in children—to result from an immunopathological
mechanism triggered by sequential infections with different dengue viral serotypes.19
Although this is the general epidemiological pattern, dengue virus may also rarely cause
hemorrhagic fever in adults and in primary infections.22
DIAGNOSIS
The natural distribution and circulation of VHF agents are geographically restricted
and mechanistically linked with the ecology of the reservoir species and vectors.
Therefore, a high index of suspicion and elicitation of a detailed travel history are critical
in making the diagnosis of VHF. Patients with arenaviral or hantaviral infections often
recall having seen rodents during the presumed incubation period, but, since the viruses
are spread to humans by aerosolized excreta or environmental contamination, actual
contact with the reservoir is not necessary. Large mosquito populations are common
during the seasons when RVF virus and the flaviviruses are transmitted, but a history of
mosquito bite is sufficiently common to be of little assistance in making a diagnosis,
whereas tick bites or nosocomial exposure are of some significance when CrimeanCongo hemorrhagic fever is suspected. History of exposure to animals in slaughterhouses
should raise suspicions of Rift Valley fever and Crimean-Congo hemorrhagic fever in a
patient with VHF. When large numbers of military personnel present with VHF
manifestations in the same geographical area over a short period of time, medical
personnel should suspect either a natural outbreak (in an endemic setting) or possibly a
biowarfare attack (particularly if the virus causing the VHF is not endemic to the area).
VHF should be suspected in any patient presenting with a severe febrile illness and
evidence of vascular involvement (subnormal blood pressure, postural hypotension,
petechiae, hemorrhagic diathesis, flushing of the face and chest, nondependent edema)
who has traveled to an area where the etiologic virus is known to occur, or where
intelligence suggests a biological warfare threat. Signs and symptoms suggesting
additional organ system involvement are common (headache, photophobia, pharyngitis,
cough, nausea or vomiting, diarrhea, constipation, abdominal pain, hyperesthesia,
dizziness, confusion, tremor), but they rarely dominate the picture. A macular eruption
occurs in most patients who have Marburg and Ebola hemorrhagic fevers; this clinical
manifestation is of diagnostic importance.
Laboratory findings can be helpful, although they vary from disease to disease and
summarization is difficult. Leukopenia may be suggestive, but in some patients, white
blood cell counts may be normal or even elevated. Thrombocytopenia is a component of
most VHF diseases, but to a varying extent. In some, platelet counts may be near normal,
and platelet function tests are required to explain the bleeding diathesis. A positive
tourniquet test has been particularly useful in diagnosing dengue hemorrhagic fever, but
this sign may be associated with other hemorrhagic fevers as well. Proteinuria or
hematuria or both are common in VHF, and their absence virtually rules out Argentine
hemorrhagic fever, Bolivian hemorrhagic fever, and hantaviral infections. Hematocrits
are usually normal, and if there is sufficient loss of vascular integrity perhaps mixed with
dehydration, hematocrits may be increased. Liver enzymes such as aspartate
aminotransferase (AST) are frequently elevated. VHF viruses are not primarily
hepatotropic, but livers are involved and an elevated AST may help to distinguish VHF
from a simple febrile disease.
For much of the world, the major differential diagnosis is malaria. It must be borne in
mind that parasitemia in patients partially immune to malaria does not prove that
symptoms are due to malaria. Typhoid fever and rickettsial and leptospiral diseases are
major confounding infections; nontyphoidal salmonellosis, shigellosis, relapsing fever,
fulminant hepatitis, and meningococcemia are some of the other important diagnoses to
exclude. Ascertaining the etiology of DIC is usually surrounded by confusion. Any
condition leading to DIC could be mistaken for diseases such as acute leukemia, lupus
erythematosus, idiopathic or thrombotic thrombocytopenic purpura, and hemolytic
uremic syndrome.
Definitive diagnosis in an individual case rests on specific virological diagnosis. Most
patients have readily detectable viremia at presentation (the exception is those with
hantaviral infections). Infectious virus and viral antigens can be detected and identified
by a number of assays using fresh or frozen serum or plasma samples. Likewise, early
immunoglobulin (Ig) M antibody responses to the VHF-causing agents can be detected
by enzyme-linked immunosorbent assays (ELISA), often during the acute illness.
Diagnosis by viral cultivation and identification requires 3 to 10 days for most (longer for
the hantaviruses); and, with the exception of dengue, specialized microbiologic
containment is required for safe handling of these viruses.23 Appropriate precautions
should be observed in collection, handling, shipping, and processing of diagnostic
samples.24 Both the Centers for Disease Control and Prevention (CDC, Atlanta,
Georgia.) and the U.S. Army Medical Research Institute of Infectious Diseases
(USAMRIID, Fort Detrick, Frederick, Maryland.) have diagnostic laboratories operating
at the maximum Biosafety Level (BL-4; see Chapter 19, The U.S. Biological Warfare and
Biological Defense Programs, for further discussion of BLs). Viral isolation should not
be attempted without BL-4 containment.
In contrast, most antigen-capture and antibody-detection ELISAs for these agents can
be performed with samples that have been inactivated by treatment with b-propiolactone
(BPL).25 Likewise, diagnostic tests based on reverse transcriptase polymerase chain
reaction (RT-PCR) technology are safely performed on samples following RNA
extraction using chloroform and methanol. RT-PCR has been successfully applied to the
real-time diagnosis of most of the VHF agents.26,27 When isolation of the infectious
virus is difficult or impractical, RT-PCR has proven to be extremely valuable; for
example, with HPS, where the agent was recognized by PCR months before it was finally
isolated in culture.9
When the identity of a VHF agent is totally unknown, isolation in cell culture and
direct visualization by electron microscopy, followed by immunological identification by
immunohistochemical techniques is often successful.14 Immunohistochemical techniques
are also useful for retrospective diagnosis using formalin-fixed tissues, where viral
antigens can be detected and identified using batteries of specific immune sera and
monoclonal antibodies. Although intensive efforts are being directed toward the
development of simple, qualitative tests for rapid diagnosis in the field, definitive
diagnosis for these diseases today requires, at a minimum, an ELISA capability coupled
with specialized immunological reagents, supplemented (ideally) with an RT-PCR
capability.
Ebola and Marburg Virus Genomic Structure, Comparative and Molecular Biology
Provided by John Crowley (B.S.) and Ted Crusberg (Ph.D.) [email protected]. Dept. of
Biology & Biotechnology, Worcester Polytechnic Institue, Worcester MA 01609
Ebola is a member of the negative-stranded RNA virus family Filoviridae. These
filoviruses (Ebola, Marburg and Reston) are very similar in morphology, density and
sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) profile (Klenk,
1994). The particles are pleomorphic, meaning they can exist in many shapes.
Their basic structure is long and filamentious, essentially bacilliform, but the viruses
often takes on a "U" shape, and the particles can be up to 14,000 nm in length and
average 80 nm in diameter. The virus consists of a nucleocapsid, surrounded by a crossstriated helical capsid. There is an axial channel in the nucleocapsid, and the whole virion
is surrounded by a lipoprotein unit derived from the host cell. In addition, there are 7 nm
spikes placed 10 nm apart visible on the surface of the virion.
The genome consists of a single negative strand of RNA that is non-infectious itself, nonpolyadenylated, with a linear arrangement of genes, with some occurrence of overlap.
The order is:
3'-untranslated region
nucleoprotein
viral structural protein
VP35
VP40 glycoprotein
VP30
VP24
polymerase(L)
5'-untranslated region
Once inside the cell (mechanism not yet known) the virus transcribes its RNA and
replicates in the cytoplasm of the infected cell. Replication is mediated by the synthesis
of an antisense positive RNA strand what will serve as template for additional viral
genomes. As the infection progresses the cytoplasm of the infected cell develops
"prominent inclusion bodies" that contain the viral nucelocapsid, which will become
highly structured. The virus then assembles, and buds off the host cell, attaining its
lipoprotein coat from the infected cell's outer membrane.
The transcriptional start site was determined to be at base 54 (3'UACUCCUUCUAAUU-). The stop site was identified due to its sequence homology
with the Sendai virus polyadentlation (transcriptional signaling) site and its position after
the open reading frame for the nuceloprotein gene: (3'-UAAUUCUUUUUU). The
location of these sequences determined that there are long non-coding sequences within
the nucleoprotein gene itself, 417 bp at the 5'-end and 341 bp at the 3'-end. The coding
region begins with two AUG codons and ends with a UGA stop codon. The first protein
is predicted to have 739 amino acids, and 83.3 KDa molecular weight, lower than that
observed by PAGE.
A Kyte-Doolittle analysis of the predicted amino acid sequence yields a definitive
hydrophobic N-terminal region, and a hydrophilic C-terminal end. Transcripts from the
cloned gene run against natural viral mRNA on acid-urea-agarose gels were identical.
Translated proteins from wild type and cloned genes were also identical on SDS-PAGE.
Sanchez et al (1993) has published the sequence of the complete genome of Ebola virus
and determined the gene order to be 3'-NP-VP35-VP40-GP-VP30-VP24-L.
Three areas of overlap occur in the genome, that average 18 bp in length. The first
overlap is between the VP35 and VP40 genes, the second between GP and VP30 and the
third between VP24 and L. These overlaps are limited to the conserved sequences
determined for the transcriptional signals. In addition there are three non-coding
sequences between VP30 and VP24. Except for the start site the L gene (RNA dependent
RNA polymerase) has yet to be completely sequenced. For the Ebola (+) leader RNA
sequence a potential stem-loop structure is possible, and may play a role in gene
expression (perhaps by altering ribosome binding). For example the hairpin shape of the
(+) leader strand may be conducive to nucleoprotein binding, and the subseqeuent
conformational change may either promote replication of transcription. Norther blot
analysis performed on all transcripts yielded appropriate length mRNAs except for the
glycoprotein gene (GP). It was found that the GP gene produces both a full-length
transcript and a shorter mRNA derived from an atypical transcriptional stop sequence
located in the middle coding region of the gene. The protein produced by this transcript
would appear to have no particular purpose, and may not be tolerated within infected
cells.
Aligning the gene sequences of the Ebola and Marburg viruses along side one another
and comparing protein products from the two viruses show similarities although no
immunological cross reactivitiy exists:
Ebola and Marburg genomes are both very large containing 3' and 5' non-coding regions.
Both genomes contain overlaps that consist of the transcriptional start and stop signals,
although Ebola has three and Marburg has only one. Their positions in relation to the
intergenic regions also differ.
The intergeneic regions are not conserved in either genome, and both contain one
unusually lengthy region (>94 bp)
All the polyadenylation sites contain (3'-UAAUU) excpet for the VP40 gene of the
Marburg virus
Both viruses produce mRNA that can form stem-loop structures
Ebola GP gene produces two transcripts but Marburg produces only one
The Marburg virus does not contain the polyadenylation sequence that is found in the
Ebola GP gene. The proteins of Ebola and Marburg are likewise similar. The C-terminal
end of the GP proteins of both viruses are 80.9% homologous and are comparable to the
same protein found in oncogeneic retroviruses. This may provide some insight into the
pathogenicitity of this family of viruses, as synthetic peptides of this gene yield a highly
compormised immune system, leading to an inhibition of blastogenesis of lymphhocytes,
a reduced chemotactiic ability of monocytes and macrophages and inhibition of natural
killer (NK) cells. Other similarities in the GP protein include a variable hydrophilic
central region flanked by hydrophobic regions that contain most of the cycfteins used in
disulfide bridges.
The sequences of the VP40 and VP24 proteins show that they are primarily hydrophobic,
and may be membrane-associated. VP40 may be a matrix protein because of its net
positive charge and similarity to like proteins from other viruses. VP30 is presumed to be
a second nucleoprotein, base on its amino acid sequence and proposed structure. Elliott et
al.,(1993) has reporerted on the amiono acid compositioni of VP35 and VP40. VP35 is
the second protein synthesized after the nucleoprotein so there is a distinct possibility that
it may be a non-structural protein involved in transcriptional events. Unlike regulatory
proteins VP35 is not phosophorylated. VP30 which on the other hand is phosphorylated
is often found tightly bound to nucleoprotein, providing strong evidence that it too is in
fact a nucleoprotein
The Marburg virus GP is acylated by myristic and palmitic acids (Funke, et al., 1995) in
an insect cell culture system, with cystein residues acting as the sites for acylation. In
Marburg virus acylation appears to play a role in receptor binding and fusion activities.
References
Funke, C., et al., Acylation of the Marburg Virus Glycoprotein, Virology 208, 289297(1995).
Sanchez, A., et al. (1993), Sequence analysis of the Ebola virus genome: organization,
genetic elements and comparison with the enome of Marburg virus, Virus Res. 29, 215240(1993).
Klenk, H.D., et al. (1994), Marburg and Ebola Viruses, Encyclopedia of Virology, Vol. 2,
827-832.
last modified May 18, 1995
Slide 87: VHF Treatment
Additional Reference Material From: CDC: Management of patients with suspected viral
hemorrhagic fever. MMWR 37(Supplement 3):1-16, 1988.
Treatment is largely supportive and typically requires intensive care monitoring to avoid
fluid overload (pulmonary edema) while maintaining hemodynamic stability and
providing appropriate comfort measures (sedation, pain medication). Systemic
coagulopathy should be treated in manner similar to DIC. Ribavirin is an antiviral
(available only on an IND basis) medication that has been used in therapy and
prophylaxis for Lassa fever, Hemorrhagic Fever with Renal Syndrome, and CCHF. The
only available vaccine is against yellow fever. Other vaccines are currently under
investigation.
Additional Reference: Textbook of Military Medicine, Chapter 29, Viral
Hemorrhagic Fevers, page 597-599:
MEDICAL MANAGEMENT
Patients with VHF syndrome require close supervision, and some will require
intensive care. Since the pathogenesis of VHF is not entirely understood and availability
of specific antiviral drugs is limited, treatment is largely supportive. This care is
essentially the same as the conventional care provided to patients with other causes of
multisystem failure. The challenge is to provide this support while minimizing the risk of
infection to other patients and medical personnel.
Supportive Care
Patients with VHF syndrome generally benefit from rapid, nontraumatic
hospitalization to prevent unnecessary damage to the fragile capillary bed. Transportation
of these patients, especially by air, is usually contraindicated because of the effects of
drastic changes in ambient pressure on lung water balance. Restlessness, confusion,
myalgia, and hyperesthesia occur frequently and should be managed by reassurance and
other supportive measures, including the judicious use of sedative, pain-relieving, and
amnestic medications. Aspirin and other antiplatelet or anticlotting-factor drugs should be
avoided.
Secondary infections are common and should be sought and aggressively treated.
Concomitant malaria should be treated aggressively with a regimen known to be effective
against the geographical strain of the parasite; however, the presence of malaria,
particularly in immune individuals, should not preclude management of the patient for
VHF syndrome if such is clinically indicated.
Intravenous lines, catheters, and other invasive techniques should be avoided unless
they are clearly indicated for appropriate management of the patient. Attention should be
given to pulmonary toilet, the usual measures to prevent superinfection, and the provision
of supplemental oxygen. Immunosuppression with steroids or other agents has no
empirical and little theoretical basis, and is contraindicated except possibly for HFRS.
The diffuse nature of the vascular pathological process may lead to a requirement for
support of several organ systems. Myocardial lesions detected at autopsy reflect cardiac
insufficiency antemortem. Pulmonary insufficiency may develop, and, particularly with
yellow fever, hepatorenal syndrome is prominent.16
Treatment of Bleeding
The management of bleeding is controversial. Uncontrolled clinical observations
support vigorous administration of fresh frozen plasma, clotting factor concentrates, and
platelets, as well as early use of heparin for prophylaxis of DIC. In the absence of
definitive evidence, mild bleeding manifestations should not be treated at all. Moresevere hemorrhage indicates that appropriate replacement therapy is needed. When
definite laboratory evidence of DIC becomes available, heparin therapy should be
employed if appropriate laboratory support is available.
Treatment of Hypotension and Shock
Management of hypotension and shock is difficult. Patients often are modestly
dehydrated from heat, fever, anorexia, vomiting, and diarrhea, in any combination. There
are covert losses of intravascular volume through hemorrhage and increased vascular
permeability.28 Nevertheless, these patients often respond poorly to fluid infusions and
readily develop pulmonary edema, possibly due to myocardial impairment and increased
pulmonary vascular permeability. Asanguineous fluids—either colloid or crystalloid
solutions—should be given, but cautiously. Although it has never been evaluated
critically for VHFs, dopamine would seem to be the agent of choice for patients with
shock who are unresponsive to fluid replacement. -Adrenergic vasoconstricting agents
have not been clinically helpful except when emergent intervention to treat profound
hypotension is necessary. Vasodilators have never been systematically evaluated.
Pharmacological doses of corticosteroids (eg, methylprednisolone 30 mg/kg) provide
another possible but untested therapeutic modality in treating shock.
Specific Antiviral Therapy
Ribavirin is a nonimmunosuppressive nucleoside analogue with broad antiviral
properties,31 and is of proven value for some of the VHF agents. Ribavirin reduces
mortality from Lassa fever in high-risk patients,32 and presumably decreases morbidity in
all patients with Lassa fever, for whom current recommendations are to treat initially with
ribavirin 30 mg/kg, administered intravenously, followed by 15 mg/kg every 6 hours for
4 days, and then 7.5 mg/kg every 8 hours for an additional 6 days.30 Treatment is most
effective if begun within 7 days of onset; lower intravenous doses or oral administration
of 2 g followed by 1 g/d for 10 days also may be useful.
The only significant side effects have been anemia and hyperbilirubinemia related to a
mild hemolysis and reversible block of erythropoiesis. The anemia did not require
transfusions or cessation of therapy in the published Sierra Leone study32 or in
subsequent unpublished limited trials in West Africa. Ribavirin is contraindicated in
pregnant women, but, in the case of definite Lassa fever, the predictability of fetal death
and the need to evacuate the uterus justify its use. Safety of ribavirin in infants and
children has not been established.
A similar dose of ribavirin begun within 4 days of disease is efficacious in patients
with HFRS.33 In Argentina, ribavirin has been shown to reduce virological parameters of
Junin virus infection (ie, Argentine hemorrhagic fever), and is now used routinely as an
adjunct to immune plasma. However, ribavirin does not penetrate the brain and is
expected to protect only against the visceral, not the neurological phase of Junin
infection.
Small studies investigating the use of ribavirin in the treatment of Bolivian
hemorrhagic fever and Crimean-Congo hemorrhagic fever have been promising, as have
preclinical studies for Rift Valley fever.33 Conversely, ongoing studies conducted at
USAMRMC predict that ribavirin will be ineffective against both the filoviruses and the
flaviviruses. No other antiviral compounds are currently available for the VHF agents.
Interferon alpha has no role in therapy, with the possible exception of Rift Valley
fever,34 where fatal hemorrhagic fever has been associated with low interferon responses
in experimental animals. However, as an adjunct to ribavirin, exogenous interferon
gamma holds promise in treatment of arenaviral infections.
Slide 88: Isolation and Containment CDC: Management of patients with suspected viral
hemorrhagic fever. MMWR 37(Supplement 3):1-16, 1988.
VHF may be transmitted by bodily fluids, but the exact mechanism is unknown. This
disease does not appear to be readily transmitted by the airborne route. (This is not the
case for Ebola among monkeys.) The highest risk of transmission is during the latter
stages of the illness, which are characterized by vomiting, diarrhea, shock, and
hemorrhage. Since most strains of VHF are known to spread in the hospital environment,
universal precautions are essential.
Patients suspected of having VHF should be isolated in a single room with an adjoining
anteroom that serves as the only entrance. This anteroom should be stocked with
personal protective gear (gloves, gowns, and masks) for staff. The patient’s room should
have negative air pressure compared with the anteroom and the outside hall. Strict
barrier-nursing techniques should be enforced. Patients should be cared for at the
hospital where they were first seen, since transferring patients may increase the potential
for secondary transmission. These viruses are easily inactivated with soaps, detergents,
and routine disinfectant solutions.
In previous outbreaks, simple barrier nursing was enough to reduce health care provider
infection rate to virtually zero.
Patients with VHF syndrome generally have significant quantities of virus in their
blood, and perhaps in other secretions as well (with the exceptions of dengue and classic
hantaviral disease). Well-documented secondary infections among contacts and medical
personnel not parenterally exposed have occurred. Thus, caution should be exercised in
evaluating and treating patients with suspected VHF syndrome. Over-reaction on the part
of medical personnel is inappropriate and detrimental to both patient and staff, but it is
prudent to provide isolation measures as rigorous as feasible.30 At a minimum, these
should include the following:
stringent barrier nursing;
mask, gown, glove, and needle precautions;
hazard-labeling of specimens submitted to the clinical laboratory;
restricted access to the patient; and
autoclaving or liberal disinfection of contaminated materials, using hypochlorite or
phenolic disinfectants.
For more intensive care, however, increased precautions are advisable. Members of
the patient care team should be limited to a small number of selected, trained individuals,
and special care should be directed toward eliminating all parenteral exposures. Use of
endoscopy, respirators, arterial catheters, routine blood sampling, and extensive
laboratory analysis increase opportunities for aerosol dissemination of infectious blood
and body fluids. For medical personnel, the wearing of flexible plastic hoods equipped
with battery-powered blowers provides excellent protection of the mucous membranes
and airways.
Slide 91
Additional Reference: Textbook of Military Medicine, Chapter 28, Viral
Encephalitides
The IA, IB, and IC variants of VEE virus are pathogenic for equines and have the
capacity for explosive epizootics with epidemic human disease. Epidemics of VEE
affecting 20,000 to 30,000 people, or more, have been documented in Venezuela and
Ecuador. In contrast to the other alphavirus encephalitides, EEE and WEE, epizootic
strains of VEE are mainly amplified in equines, rather than birds, so that equine disease
normally occurs prior to reports of human disease. Enzootic VEE strains (variants ID, IE,
and IF and subtypes II, III, IV, V, and VI) are not recognized as virulent for equines, but
disease has been documented with most of these variants in humans who reside in or
move into enzootic foci, or after laboratory infections. The resulting syndromes appear to
be similar, if not indistinguishable, from the syndrome produced by epizootic variants,
which ranges from undifferentiated febrile illness to fatal encephalitis.
Following an incubation period that can be as short as 28 hours but is usually 2 to 6
days, patients typically develop a prostrating syndrome of chills, high fever (38°C–
40.5°C), headache, and malaise. Photophobia, sore throat, myalgias, and vomiting are
also common symptoms. Frequent signs noted on physical examination include
conjunctival injection, erythematous pharynx, and muscle tenderness. Although
essentially all human infections with VEE virus are symptomatic, only a small percentage
manifest neurological involvement. In one epidemic, it was estimated that the ratio of
encephalitis to infections is less than 0.5% in adults, although possibly as high as 4% in
children. Mild CNS involvement is evidenced by lethargy, somnolence, or mild
confusion, with or without nuchal rigidity. Seizures, ataxia, paralysis, or coma herald
more severe CNS involvement. In children with overt encephalitis, case fatalities range as
high as 35% compared with 10% for adults. However, for those who survive encephalitic
involvement, neurological recovery is usually complete. School-age children are believed
to be more susceptible to a fulminant form of disease, in which depletion of lymphoid
tissues is prominent and which follows a lethal course over 48 to 72 hours.
In the first 3 days of illness, leukopenia and elevated serum glutamic-oxaloacetic
transaminase (SGOT) are common. For those with CNS involvement, a lymphocytic
pleocytosis of up to 500 cells per microliter will be observed in the CSF. The CSF
pleocytosis may acutely be polymorphonuclear but soon becomes predominantly
lymphocytic.
Specific diagnosis of VEE can be accomplished by virus isolation, serologic testing,
or both. During the first 1 to 3 days of symptoms of nonspecific febrile illness, VEE virus
may be recovered from either the serum or the nasopharynx. Despite the theoretical
possibility of person-to-person transmission of virus present in the nasopharynx, no
evidence of such occurrences has been reported. Identification of the VEE subtype of an
isolate involved can be accomplished by cross-neutralization tests. HI, enzyme-linked
immunosorbent assay (ELISA), or plaque reduction neutralization (PRN) antibodies
appear as viremia diminishes. Complement-fixing (CF) antibodies make their appearance
later during convalescence. VEE IgM antibodies are present in acute phase sera, and it
has been reported that the VEE IgM tests do not react with sera from patients with EEE
or WEE. Since patients with encephalitis typically come to evaluation later in the course
of clinical illness, virus is recovered less often from them, and they usually have serum
antibody by the time of clinical presentation.
Immunity after infection is probably lifelong to the homologous serotype, but crossimmunity is weak or nonexistent to heterologous serotypes. Thus, when viewed either as
an endemic disease threat or as a potential biological warfare threat, adequate
immunization will require polyvalent vaccines.
Slide 94: Botulinum Toxins
Textbook of Military Medicine, Chapter 33, Botulinum toxins.
The seven serotypes of botulinum toxin produced by Clostridium botulinum are the
most toxic substances known. They are associated with lethal food poisoning after the
consumption of canned foods. This family of toxins was evaluated by the United States
as a potential biological weapon in the 1960s and is believed to be an agent that could be
used against our troops. Unlike other threat toxins, botulinum neurotoxin appears to cause
the same disease after inhalation, oral ingestion, or injection. Death results from skeletal
muscle paralysis and resultant ventilatory failure. Because of its extreme toxicity, the
toxin typically cannot be identified in body fluids, other than nasal secretions, after
inhalation of a lethal dose. The best diagnostic sample for immunologic identification of
the toxin is from swabs taken from the nasal mucosa within 24 hours after inhalational
exposure. Because of the small quantity of toxin protein needed to kill, botulinum toxin
exposure does not typically induce an antibody response after exposure.
Prophylactic administration of a licensed pentavalent vaccine fully protects laboratory
animals from all routes of challenge. Passive immunotherapy with investigational
hyperimmune plasma also prevents illness if it is administered before the onset of clinical
intoxication.