Biosafety
Chapter 22
BIOSAFETY
CATHERINE L. WILHELMSEN, DVM, PHD, CBSP*; AND ROBERT J. HAWLEY, PHD, RBP, CBSP
INTRODUCTION
Biosafety
Evolution of Biosafety
RISK GROUPS AND BIOSAFETY LEVELS
Risk Groups
How Agents Are Placed in Risk Groups
Biosafety Levels
LABORATORIES IN THE LABORATORY RESPONSE NETWORK
Clinical Laboratories
Sentinel Laboratories
Reference Laboratories
National Laboratories
BIOSAFETY PROGRAM ELEMENTS REQUIRED FOR CONTAINMENT AND
MAXIMUM CONTAINMENT LABORATORIES
Measures Taken in Research to Protect Laboratory Workers
Documenting Safety Procedures
Assessing Individual Risk
Physical Barriers
Personal Protective Equipment
Medical Surveillance
Vaccinations
Protecting the Community and the Environment
Solid and Liquid Waste Inactivation and Disposal
Standard and Special Microbiological Practices
ROLE OF MANAGEMENT IN A BIOSAFETY PROGRAM
Laboratory Safety Audits
SELECT AGENT PROGRAM
Biological Defense Research Program Laboratories
Laboratory Animal Care and Use Program
THE BIOSAFETY PROFESSION
SUMMARY
*Lieutenant Colonel, Veterinary Corps, US Army (Ret); Biosafety Officer, Office of Safety, Radiation Protection, and Environmental Health, US Army
Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702; formerly, Chief, Division of Toxicology, US Army
Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

Senior Advisor, Science, Midwest Research Institute, 365 West Patrick Street, Suite 223, Frederick, Maryland 21701; formerly, Chief, Safety and Radia-
tion Protection, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland
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Medical Aspects of Biological Warfare
INTRODUCTION
Biosafety (also known as the NIH Guidelines).2 However, Appen-
dix G of the NIH Guidelines focuses primarily on physi-
Biological safety, or biosafety, is the application of cal containment involving work with recombinant de-
concepts pertaining to risk assessment, engineering oxyribonucleic acid (DNA) molecules and organisms
technology, personal protective equipment (PPE), and viruses containing recombinant DNA molecules.
policies, and preventive medicine to promote safe There are four levels of biosafety (designated 1
laboratory practices, procedures, and the proper through 4) that define the parameters of containment
use of containment equipment and facilities. In necessary to protect personnel and the environment.1
biomedicine, laboratory workers apply these tenets BSL-1 is the least restrictive, whereas BSL-4 requires a
to prevent laboratory-acquired infections and the special containment or maximum containment labora-
release of pathogenic organisms into the environ- tory facility. Positive-pressure protective suits (space
ment. A biohazard is defined as any microorganism suit or blue suit) are used solely in a maximum con-
(including, but not limited to, bacteria, viruses, fungi, tainment, or BSL-4, laboratory. Biosafety is not possible
rickettsiae, or protozoa); parasite; vector; biological without proper and extensive training. The principal
toxin; infectious substance; or any naturally occur- investigator or laboratory supervisor is responsible
ring, bioengineered, or synthesized component of for providing or arranging for appropriate training of
any such microorganism or infectious substance that all personnel within the laboratory to maintain and
is capable of causing the following: sustain a safe working environment.
" death, disease, or other biological malfunction Evolution of Biosafety
in humans, animals, plants, or other living
organisms; Steps to limit the spread of infection were prac-
" deleterious alteration of the environment; or ticed in the field of biomedicine since human illness
" an adverse impact on commerce or trade was associated with infectious microorganisms and
agreements. biologically derived toxins. However, Fort Detrick (in
Frederick, Md) is considered the birthplace (beginning
The goal of handling these hazardous agents safely in the 1940s) of modern biosafety as a discrete disci-
can be accomplished through careful integration of ac- pline. During the early years of biosafety, development
cepted microbiological practices, and the primary and of safer working practices, principles, and engineer-
secondary containments of potential biohazards. ing controls was needed.3,4 Individuals conducting
Primary containment involves placing a barrier at biomedical research commonly became infected with
the level of the hazard, confining the material to protect the organism being studied. As the hazard of work-
laboratory personnel and the immediate laboratory ing with organisms increased, so did the need to
environment through adherence to good laboratory protect laboratory personnel conducting the research.
practices and appropriate use of engineering controls. Contributions to the field of biosafety were a direct
Examples of primary containment include biological result of the innovations and extensive experiences
safety cabinets (BSCs), ventilated animal cages, and as- of Fort Detrick personnel who worked with a variety
sociated equipment. Secondary containment involves of infectious microorganisms and biological toxins.
protection of the environment external to the labora- Dr Arnold Wedum, director of industrial health and
tory from exposure to infectious or biohazardous mate- safety at Fort Detrick and regarded by many as the
rials through facility design and operational practices. father of the US biosafety profession promoted the
Combinations of laboratory practices, containment attitude that biosafety should be an integral part of
equipment, and special laboratory design are used to biomedical research.5
achieve different levels of physical containment. (His- To enhance worker safety and environmental pro-
torically, the designation  P was used to indicate the tection, Wedum4 promoted use of the following:
level of physical containment, such as P-1 through P-4.)
The current terminology is biosafety level or BSL.1 The " class III gas-tight BSC;
designation BSL is used in the Biosafety in Microbiologi- " noninfectious microorganisms in recombinant
cal and Biomedical Laboratories (BMBL),1 which focuses DNA research;
on protecting laboratory employees. BL is another des- " P-4 (today s BSL-4) principles, practices, and
ignation for biosafety level, used in Appendix G of the positive-pressure protective suit facilities
National Institutes of Health (NIH) publication Guide- when working with potential aerosol-trans-
lines for Research Involving Recombinant DNA Molecules mitted zoonotic microorganisms (eg, those
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Biosafety
causing tularemia and Q fever if a class III fixed to a frame.10 A BSC, first developed in 1964 for a
cabinet system was not available); and pharmaceutical company, used HEPA filter technology
" vaccinations of laboratory workers. to provide clean air in the work area and containment
as the primary barrier placed at the source of hazard-
Another safety enhancement was demonstrating and ous powders. Subsequent research led to the develop-
publicizing the importance of prohibiting mouth ment of a class II, type A BSC that was delivered to
pipetting for fluid transfers involving hazardous ma- the National Cancer Institute by the Baker Company
terial.6,7 Dr Emmett Barkley8 reiterated the hazard of (Sanford, Me).11 The National Cancer Institute also
oral pipetting, which should not be practiced in the developed a specification for the first class II, type B
laboratory. Barkley was chief of the Safety Division console BSC. HEPA filters have been proven to be ef-
of the National Cancer Institute (Bethesda, Md) and fective, economical, and reliable devices for removing
subsequently director of research safety at NIH when radioactive and nonradioactive particulate aerosols at
the NIH Guidelines were developed and adopted. He a high rate of collection frequency.10
was instrumental in developing physical containment Operation and retention efficiency of HEPA filters
parameters for recombinant DNA research.9 have been documented during the past years. Three
Critical to the advancement of modern biosafety mechanisms account for the collection (retention) of
was the development of air filtration technology. Dur- particles within HEPA filters:
ing the early 1940s, the US Army Chemical Warfare
Service Laboratories (Edgewood, Md) studied the 1. Small particles ranging from 0.01 to 0.2 µm
composition of filter paper captured from German gas in diameter are collected in a HEPA filter by
mask canisters in search of better smoke filters. These diffusion and are retained at an efficiency
early studies resulted in the design of collective protec- approaching 100%.
tion filter units for use at the particulate-removal stage 2. Particles in the respirable range (those of a size
by a combined chemical, biological, and radiological that may be inhaled and retained in the lungs,
purification unit of the US armed services. In the late 0.5 5.0 µm in diameter) are retained in a HEPA
1940s, the Atomic Energy Commission (precursor of filter by a combination of impaction and in-
the Nuclear Regulatory Commission) adopted this terception at an efficiency approaching 100%.
type of filter to confine airborne radioactive particles 3. Particles with an intermediate size range
in the exhaust ventilation systems of experimental (between 0.2 and 0.5 µm in diameter) are
reactors and in other areas of nuclear research. Subse- retained by a combination of diffusion and
quently, Arthur D Little Company, Inc (Boston, Mass), impaction.
and the US Naval Research Laboratory (Washington,
DC) developed a prototype glass-fiber filter paper. The HEPA filter is least efficient at retaining particles
Eventually, thin, corrugated, aluminum-alloy separa- with a diameter of 0.3 µm, with a minimum collection
tors replaced the original asbestos, thermoplastics, and efficiency of 99.97%. Hence, a standard test of HEPA
resin-treated papers. Throughout this development filter efficiency uses a generated aerosol of particles
period, military specifications were developed and that are 0.3 µm in diameter; to pass the test, the HEPA
implemented to ensure the safe operating and opti- filter must retain 99.97% of the particles.12
mal conditions of filters,10 ultimately leading to the All the air exhausted from BSCs, within which
production of high-efficiency particulate air (HEPA) infectious materials must be manipulated, is directed
filters, which are used today in a variety of engineering through a HEPA filter before recirculation to a labora-
controls, as well as in laboratory heating, ventilation, tory room or discharge to the outside environment
and air conditioning systems. through the building exhaust system. Therefore, in ad-
HEPA filters are constructed of paper-thin sheets of dition to adherence to rigorous work practice controls,
borosilicate medium that are pleated to increase their HEPA filtration of laboratory exhaust air provides an
surface area. The borosilicate sheets are tightly pleated extra margin of safety for workers, the laboratory areas,
over aluminum separators for added stability and af- and the outside environment.
RISK GROUPS AND BIOSAFETY LEVELS
Risk Groups signment helps guide the researcher in determining
the containment condition (or BSL) appropriate for
Agents infectious to humans, including agents used handling any particular agent.
in research, are placed into risk groups based on the Multiple schemes for assigning risk groups have
danger they pose to human health. The risk group as- been developed. The NIH Guidelines; the American
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Medical Aspects of Biological Warfare
Biological Safety Association (Mundelein, Ill); Health ted from one individual to another, either directly or
Canada (Ottawa, Ontario, Canada)13; other nations; indirectly. Effective treatment and preventive measures
and the World Health Organization (Geneva, Swit- are not normally available. Examples include Variola
zerland)14 all have risk group paradigms. The World virus, Ebola virus, Lassa fever virus, and Marburg
Health Organization has categorized infectious agents fever virus. The relationship of risk groups and BSLs,
and biological toxins into four risk groups. These risk practices, and equipment is illustrated in Table 22-1.
groups relate to, but do not equate to, the BSLs of
laboratories designed to work with organisms in each How Agents Are Placed in Risk Groups
risk group.14 Risk group 1 (no or low individual and
community risk) comprises microorganisms unlikely To assess the risk while working in a laboratory or
to cause human or animal disease. Risk group 2 (mod- animal environment with a specific microorganism, the
erate individual risk, low community risk) includes following criteria must be considered: number of past
pathogens that can cause human or animal disease, laboratory infections, natural mortality rate, human
but are unlikely to be serious hazards to laboratory infectious dose, efficacy of vaccination and treatment,
workers, the community, livestock, or the environ- extent to which infected animals transmit the disease,
ment. Laboratory exposures may cause serious infec- stability of the agent, and potential for exposure of
tion, but effective treatment and preventive measures the investigator.
are available, and the risk of infection spreading is
limited. An example is the causative agent of anthrax, " Number of past laboratory infections: The
Bacillus anthracis, in humans and animals. Risk group most frequent cause of laboratory-associated
3 (high individual risk, low community risk) includes infections in humans is the Brucella species. Ex-
pathogens that usually cause serious human or animal tra caution must be taken when working with
disease, but do not ordinarily spread from one in- this agent because of its low infectious dose for
fected individual to another. Effective treatment and humans. About 10 to 100 organisms can cause
preventive measures are available. An example is the an infection in a susceptible human host.15
causative agent of tularemia, Francisella tularensis, in " Natural mortality rate: The natural mortality
humans and animals. Risk group 4 (high individual or case-fatality rate of diseases varies widely15
and community risk) pathogens usually cause serious (Table 22-2).
human or animal disease and can be readily transmit- " Human infectious dose: Working with an
TABLE 22-1
RELATIONSHIP OF RISK GROUPS, BIOSAFETY LEVELS, PRACTICES, AND EQUIPMENT
Risk Group Biosafety Level Laboratory Type Laboratory Practices Safety Equipment
1 Basic: BSL-1 Basic teaching; research Good microbiological None; open bench work
techniques
2 Basic: BSL-2 Primary health services; Good microbiological Open bench plus BSC for
diagnostic services; techniques plus protective potential aerosols
research clothing; biohazard sign
3 Containment: Special diagnostic As level 2 plus special clothing, BSC and/or other primary
BSL-3 services; research controlled access, and devices for all activities
directional airflow
4 Maximum Dangerous pathogens; As level 3 plus airlock entry, Class III BSC, or positive-
containment: research shower exit, and special pressure protective suits in
BSL-4 waste disposal conjunction with class II
BSCs, double-door auto
clave (through the wall),
and filtered air
BSC: biological safety cabinet
BSL: biosafety level
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Biosafety
TABLE 22-2 pattern (antibiogram) of the agent under
investigation. The rationale is that treatment
CASE-FATALITY RATE BY DISEASE
will be known in advance if an inadvertent
laboratory exposure occurs. Treatment for
Disease (Untreated) Organism [Case-Fatality Rate]
exposure to a virus might be problematic,
because only symptomatic treatment may be
Plague, bubonic Yersinia pestis [50% 60%]
available. There are few available antiviral
Cholera Vibrio cholerae [50% or more]
agents that may be effective for postexposure
Tularemia, Francisella [30% 60%]
prophylaxis. Specific antiviral agents include
pulmonary tularensis
the following:
Anthrax, cutaneous Bacillus anthracis [5% 20%]
o rabies rabies immune globulin for pas-
Tularemia, typhoidal Francisella [5% 15%]
sive therapy, followed by the human dip-
tularensis
loid cell rabies vaccine or rabies vaccine,
Brucellosis Brucella species [2% or less]
adsorbed for active vaccination;
(melitensis)
o cercopithecine herpesvirus 1 (B virus) valacy-
Q fever Coxiella burnetii [1% 2.4%]
clovir hydrochloride (VALTREX; GlaxoSmith-
Kline, Research Triangle Park, NC); and
o arenaviridae and bunyaviridae (including the
viruses that cause Lassa fever, Argentine
organism having a low infectious dose for hemorrhagic fever, and Crimean-Congo
humans will place the laboratory worker at a hemorrhagic fever) ribavirin. This mate-
greater risk than working with an organism rial can be used under an Investigational
having a higher infectious dose. The infectious New Drug (IND) protocol (in the United
dose of organisms for humans varies and is States) only for empirical treatment of hem-
also dependent on the immunological com- orrhagic fever virus patients while await-
petency of the host (Table 22-3). Although ing identification of the etiological agent.
the literature contains information about the " Extent to which infected animals transmit the
potential infectious dose for humans as ex- disease: This discussion involves the zoonotic
trapolated from animal data (see Table 22-3),
an attempt to provide quantitative human
TABLE 22-3
infectious doses is not possible.16
" Efficacy of vaccination and treatment (if either
HUMAN INFECTIOUS DOSE BY ORGANISM
of these is available): Vaccines are available for
some of the agents studied within the labora- Organism Infectious Dose Route of Exposure
tory. Receiving a vaccination must be based
on a risk assessment. Only those individuals
Vibrio cholerae 108 Ingestion1
who are considered at risk should be offered
Yersinia pestis 100 20,000 Inhalation2
the vaccination. However, the potential risk of
Bacillus anthracis ~ 1,300 Inhalation3
the adverse effects from the vaccination might
Brucella species 10 500 Inhalation2
10 500 Inhalation
outweigh the risk of acquiring an infection.
(melitensis)
In addition, a vaccination might not provide
Francisella 10 Inhalation4
100% protection. An overwhelming infectious
tularensis
dose can overcome the protective capacity of
Coxiella burnetii 1 Inhalation5
a vaccination. Therefore, a vaccination should
be considered only as an adjunct to safety, not Data sources: (1) Sack DA, Sack RB, Nair GB, Siddique AK. Cholera.
Lancet. 2004;363:223 233. (2) Franz DR, Jahrling PB, Friedlander AM,
as a substitute for safety and prudent practices.
et al. Clinical recognition and management of patients exposed to
Clinical recognition and management of patients exposed to
Treatment (chemoprophylaxis) in the form
biological warfare agents. JAMA. 1997;278:399 411. (3) Dull PM,
of antibiotic therapy may also be available
Wilson KE, Kournikakis B, et al. Bacillus anthracis aerosolization as-
to treat illnesses caused by many of the sociated with a contaminated mail sorting machine. Emerg Infect Dis.
2002;8:1044 1047. (4) Jones RM, Nicas M, Hubbard A, Sylvester MD,
microorganisms being manipulated in the
Reingold A. The infectious dose of Francisella tularensis (tularemia).
laboratory, specifically by the bacterial and
Appl Biosafety. 2005;10:227 239. (5) Jones RM, Nicas N, Hubbard A,
rickettsial agents. It is necessary to deter-
Reingold A. The infectious dose of Coxiella burnetti (Q-fever). Appl
mine the antibiotic sensitivity and resistance Biosafety. 2006;11:32 41.
519
Medical Aspects of Biological Warfare
diseases or diseases that can be transmitted tants, is a result of its internal and external
from animals to humans. These diseases in- chemical compositions. For instance, spores
clude the following: of the genus Bacillus are resistant to adverse
environmental conditions and disinfectants
o those transmitted directly from animals to because of the presence of dipicolinic acid
humans (eg, rabies); (DPA [pyridine-2,6-dicarboxylic acid]) in their
o diseases that can be acquired indirectly by spore coat. DPA plays a significant role in the
humans through ingestion, inhalation, or survival of Bacillus spores exposed to wet heat
contact with infected animal products, soil, and ultraviolet radiation.17 Many viruses and
water, or other environmental surfaces bacteria are sensitive to environmental condi-
that have been contaminated with animal tions and disinfectants because of the high
waste or a dead animal (eg, anthrax); and lipid content in their outermost layer.
o a disease that has an animal reservoir, but " Potential for exposure of the investigator: The
requires a mosquito or other arthropod particular activity of an investigator, labora-
to transmit the disease to humans (eg, St tory technician, or animal handler must be
Louis encephalitis virus and Rocky Moun- considered when estimating risk. If the worker
tain spotted fever). is using a needle and syringe to inoculate
animals, the potential for autoinjection is pos-
There are exposure risks in laboratories in sible. An animal bite or scratch is another risk
which infectious disease research involving that must be considered.
use of animals may differ from the exposure
risks encountered in microbiology labora- Biosafety Levels
tories. Within the microbiology laboratory,
potentially hazardous conditions arise from BSLs are guidelines that have evolved to protect
the activities of the humans or from use of laboratory workers. These guidelines are based on data
equipment within the laboratory. In the ani- from laboratory-acquired infections and on an under-
mal facility, the animals themselves may create standing of the risks associated with various manipula-
hazards for the laboratory workers through tions of many agents transmissible by different routes.
the following means: These guidelines operate on the premise that safe work
sites result from a combination of engineering controls,
o generation of infectious aerosols; management policies, work practices and procedures,
o animal bites or scratches to the person and, occasionally, medical interventions. The different
handling the animal; and BSLs developed for microbiological and biomedical
o shedding of infectious known or unknown laboratories provide increasing levels of personnel and
zoonotic agents in animal secretions and environmental protection.1 BSL descriptions comprise
excretions, with contamination of the ani- a combination of facilities, equipment, and procedures
mal holding room, cage, bedding, equip- used to handle infectious agents to protect the labora-
ment, or other fomites. tory worker, the environment, and the community. This
combination is proportional to the potential hazard
In a controlled laboratory environment, labo- level (risk group) of a given infectious agent. Equip-
ratory workers and animal handlers can also ment serving as primary barriers consists of BSCs,
be infected by diseased or infected animals centrifuge safety cups, and containment animal cag-
via animal bites; by handling contaminated ing. Facilities also consist of secondary barriers, such
animal waste and bedding; and during ani- as self-closing/locking doors, hand-washing sinks,
mal manipulation, surgery, or necropsy. For and unidirectional airflow from the least hazardous
example, in addition to usual activities in the areas to the potentially most hazardous areas. Proce-
laboratory, handling materials contaminated dures consist of standard and special microbiological
with hantaviruses is a concern because viruses practices. Finally, PPE includes dedicated laboratory
are spread as aerosols or dusts from rodent clothing and respiratory protection.
urine, droppings, or by direct contact with There are four BSLs described in the BMBL.1 These
saliva through cuts or mucous membranes. levels range from a basic level (BSL-1) through maxi-
" Stability of the agent: The stability of an agent mum containment (BSL-4). BSL-1 consists of facilities,
(microorganism) to environmental conditions, equipment, and procedures suitable for work, with
and susceptibility or resistance to disinfec- infectious agents of no known or of minimal potential
520
Biosafety
hazard to healthy laboratory personnel. BSL-1 repre- or penetrations capable of being sealed, and
sents a basic level of containment that relies on stan- directional airflow. (Airflow is from areas of
dard microbiological practices, with no special primary low-hazard potential to areas of high-hazard
or secondary barriers recommended, other than a sink potential.) Laboratory personnel are trained
for hand washing. to understand these special design features.
BSL-2 consists of facilities, equipment, and proce- 4. Only the laboratory director can approve a
dures applicable to clinical, diagnostic, or teaching modification of these BSL-3 recommendations.
laboratories; suitable for work involving indigenous
moderate-risk infectious agents present in the com- BSL-4 comprises facilities, equipment, and proce-
munity; and associated with human disease of varying dures required for work with dangerous and exotic
severity.1 agents that pose a high individual risk of life-threaten-
Primary hazards to personnel working with these ing disease transmitted by the inhalation route and for
agents are accidental percutaneous or mucous mem- which a vaccine or therapy may not be available. Haz-
brane exposures and ingestion of infectious materials. ards to personnel working with these agents include
BSL-2 differs from BSL-1 in five ways: autoinoculation, mucous membrane or broken skin
exposure to infectious droplets, and exposure to infec-
1. Laboratory personnel receive specific training tious aerosols. BSL-4 differs from BSL-3 in six ways:
in handling pathogenic agents.
2. Scientists experienced in handling specific 1. Laboratory personnel receive specific and
agents direct the laboratory. thorough training to handle extremely haz-
3. Access to the laboratory is limited when work ardous infectious agents. Their supervisors
is in progress. are competent scientists who are trained and
4. A laboratory-specific biosafety manual is experienced in working with these agents.
prepared or adopted. 2. Laboratory personnel understand the func-
5. Procedures capable of generating potentially tion of primary and secondary barriers and
infectious aerosols are conducted within class laboratory design features. They are trained
I or class II BSCs or other primary contain- in standard and special microbiological prac-
ment equipment. Personnel receive specific tices and the proper use of primary contain-
training in the proper use of primary con- ment equipment.
tainment equipment and adhere strictly to 3. The laboratory director strictly controls access
recommended microbiological practices. to the laboratory.
4. The laboratory is in a controlled area within a
BSL-3 includes facilities, equipment, and proce- building, completely isolated from all other ar-
dures applicable to clinical, diagnostic, research, eas of the building, or is in a separate building.
or production facilities in which work is done with 5. All activities involving agent manipulation
indigenous or exotic agents that may cause serious within the work areas of the laboratory are
or potentially lethal disease, especially after inhala- conducted within a class III BSC, or within a
tion exposure. Hazards to personnel working with class I or class II BSC used in conjunction with
these agents include autoinoculation, ingestion, and a one-piece, positive-pressure protective suit
exposure to infectious aerosols. BSL-3 differs from that is ventilated by a life-support system.
BSL-2 in four ways: 6. The BSL-4 laboratory, or maximum contain-
ment laboratory, has special engineering and
1. At BSL-3, laboratory personnel receive more design features to prevent dissemination of
extensive training in handling potentially microorganisms to the environment.
lethal pathogenic agents than the degree of
training received at BSL-2. It is important to understand how microorganisms
2. All manipulations of infectious or toxin-con- are placed in risk groups and how that knowledge is
taining materials are conducted within class used to develop procedures and physical infrastructure
II or class III BSCs or other primary contain- design to contain these agents. Next, the functions
ment equipment. Personnel are trained to use of various laboratories in response to a bioterrorist
this safety equipment properly. threat, the BSLs that these laboratories generally use,
3. The laboratory has special engineering and and the organization of these assets into a network
design features that include access zones known as the laboratory response network (LRN) will
with two locking doors, sealed penetrations be explored.
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Medical Aspects of Biological Warfare
LABORATORIES IN THE LABORATORY RESPONSE NETWORK
Clinical Laboratories laboratory, such as a state public health laboratory,
state agency laboratory, certain private sector labora-
Clinical laboratories are located in community tories, or a large local public health laboratory. These
hospitals, diagnostic centers, public health research laboratories receive specimens from sentinel labora-
institutes, and at the state government level for referral. tories in the LRN and use more specific methods to
Clinical laboratories operate at a minimum using BSL-2 confirm the preliminary identification of an organ-
principles and practices in a facility designed to support ism. Reference laboratories are the primary response
their operations. As appropriate, all of these laborato- laboratories for an overt bioterrorism event, and they
ries follow procedures for work with (a) human blood assist in a laboratory response and recovery to a co-
or blood products or with other potentially infectious vert event. The laboratories are configured as BSL-2
material,18 (b) materials with a potential for generating laboratories, but follow BSL-3 laboratory practices
an aerosol,19 (c) chemicals in the laboratory20 and sup- and equipment criteria. In addition, they conform
port areas,21 and (d) tuberculosis-causing agents.22 The to the Association of Public Health Laboratories
procedures followed are based on degree of risk. (APHL) and CDC approval process. As reference
In response to the bioterrorism incidents of 2001, laboratories, they offer state-of-the-art confirmatory
the National Laboratory Response Network for Bio- testing. They provide bioterrorism information and
terrorism was created. Coordination for this effort training for the sentinel laboratories, laboratory
was assigned to the Centers for Disease Control and support to first responders, and environmental
Prevention (CDC). The LRN consists of public and sample testing in an overt event. State public health,
private laboratories functioning together to provide federal, and academic laboratories with a capacity
timely and accurate diagnostic testing using CDC-ap- for advanced diagnostic testing (molecular assays)
proved methods. These laboratories are in compliance and the capability of toxicity testing and evaluating
with requirements of the National Electronic Disease new tests/reagents are also included as reference
Surveillance System (CDC/Public Health Information laboratories.
Network, Atlanta, Ga)23 and the Health Insurance
Portability and Accountability Act of 1996.24 The LRN National Laboratories
links local, state, and federal agencies in a three-tiered
structure (sentinel laboratories, reference laboratories, National laboratories (formerly level D labora-
and national laboratories) with a central role for the tories) use the most sensitive and specific methods
public health laboratory. for characterizing microorganisms, and include the
CDC and the US Army Medical Research Institute
Sentinel Laboratories of Infectious Diseases (USAMRIID) at Fort Detrick.
These laboratories have highly specialized facilities
Sentinel laboratories (formerly level A laboratories) for isolating and identifying, confirming, validating,
include hospital and community-based clinical laborato- and manipulating rare and extremely lethal organ-
ries. They test patient specimens using highly sensitive isms (eg, Ebola virus, Lassa fever virus, and smallpox
methods to rule out or refer microorganism isolates to a virus) in maximum containment (BSL-4) laboratories.
reference laboratory. The laboratories are configured as The missions of national laboratories differ from those
BSL-2 laboratories and follow BSL-2 laboratory practices of other laboratories, because their primary role is not
and safety equipment criteria. Work with infectious or patient care, but research. These CDC research efforts
potentially infectious material is conducted in a class provide tools to combat the infectious diseases that
II BSC. Sentinel laboratories provide a presumptive are of risk to the general population. USAMRIID s
diagnosis. They do not have a testing role for environ- research efforts are focused on protecting military
mental specimens in an overt event, nor do they conduct personnel on the battlefield by providing a means
postattack recovery sample analysis. Their role is to rule of prevention, detection, and intervention to infec-
out suspected bioterrorism agents and to recognize and tious diseases that are known to be  weaponized
report any bioterrorism suspicion, incident, or inquiry or have the potential to be used as weapons. Despite
to a LRN reference laboratory. the differences in mission for these two national
laboratories, the methods they use to protect the re-
Reference Laboratories searcher and the community from infection are very
similar. National laboratories may continue analysis
The reference laboratory (formerly included level B of environmental samples during an overt attack or
and level C laboratories) is the confirmatory (rule-in) in support of recovery operations.
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Biosafety
BIOSAFETY PROGRAM ELEMENTS REQUIRED FOR CONTAINMENT
AND MAXIMUM CONTAINMENT LABORATORIES
Measures Taken in Research to Protect Laboratory the specific training requirement, trainers should
Workers provide documentation for standard safety and labora-
tory essential training, with specific additions for the
Although BSL-3 practices, safety equipment, and laboratory that cover orientation for workers new to
facility design and construction are applicable to the laboratory and laboratory-unique procedures and
clinical, diagnostic, teaching, research, and produc- operations. Trainers should consider including in the
tion (large-scale) facilities where work is done with manual material safety data sheets (MSDSs) for the
indigenous or exotic agents with the potential for chemicals used in the laboratory. MSDSs for chemicals
respiratory transmission and lethal infection this can be obtained from vendors Web sites or from the
section will emphasize BSL-3 research laboratories. institutional chemical hygiene officer.
BSL-4 practices, safety equipment, and facility design
and construction are applicable to work in a reference Assessing Individual Risk
diagnostic or research setting with dangerous and
exotic agents that pose a high individual risk of life- For each person working in a BSL-3 and BSL-4
threatening disease. These agents may be transmitted research laboratory, that individual s supervisor
by aerosol, and there may be no available vaccine or conducts a detailed, thorough, individually tailored
therapy. BSL-4 research facilities, both class III BSC job hazard analysis or workplace hazard analysis
laboratories and protective-suit laboratories, will be (risk assessment). During this analysis, each task the
covered in this section. Due to the Biological and Toxin individual intends to perform within containment is
Weapons Convention of 1972, legitimate production evaluated in terms of its inherent risk, as described
(large-scale) BSL-4 facilities do not currently exist. in the earlier section on risk assessment and risk
management. Each task is considered in terms of a
Documenting Safety Procedures potential laboratory exposure to the infectious agent
(and its associated toxins for toxin-producing [toxi-
The Biological Safety Program Manual25 is a labora- genic] agents). Considerations include use of sharp
tory-specific manual that should include specific safety instruments and animals that could potentially result
standard/standing operating procedures (SOPs), in puncture injuries, operations that may generate
guidelines, and documents for the containment labora- infectious aerosols, and direct handling of infectious
tory. These safety SOPs identify the special hazards of agent versus observing (auditing) others working with
the laboratory and the procedures to abate or mitigate biological materials. The hazards, once identified, are
the associated risk. The SOPs or documents specify mitigated, preferably by isolating operations that pose
the following: a risk within primary and secondary containment
devices (barriers), by substituting unbreakable plastic
" laboratory entry and exit in detail; laboratory vessels for glassware and blunt instruments
" proper use of laboratory-specific safety equip- for sharp instruments, and by chemically or physically
ment (eg, BSCs, sterilizers, passboxes, and immobilizing animals to prevent or reduce the risk
dunk tanks); of sudden or unpredictable behavior leading to bites
" decontamination procedures for the specific and scratches. Once the risk assessment is written, this
laboratory; document is approved by the second-line supervisor
" maintenance of laboratory safety and mainte- and reviewed by both the biological safety officer and
nance-related records (access logs, drain flush the occupational health physician for accuracy and
logs, emergency deluge shower, and eyewash completeness.
periodic test logs); The preferred means to mitigate risk is by using
" floor plan with hand-wash sinks and all other engineering controls (eg, BSCs, chemical fume hoods,
safety features annotated; sealed centrifuge rotors, and safety cups) and partial
" emergency and routine communication pro- containment caging for animals (eg, microisolator
cedures for the specific laboratory; and cages; ventilated cage racks; and ventilated, nega-
" laboratory-specific training. tive-pressure, HEPA-filtered rigid cubicles or flexible
isolators). Where the hazard cannot be eliminated
A compilation of existing SOPs, specifying how a by physical means, the hazard can be managed by
laboratory worker would access the SOPs (on-line, administrative controls that provide specific training
paper copy in a binder, or both) is suggested. To meet on procedures. Examples of such procedures include
523
Medical Aspects of Biological Warfare
disposal of used injection needles without recapping Cabinetry - Class II (laminar flow) biosafety cabinetry
them or use of an approved, one-handed practice to Standard 4926 on initial installation, at least annually
recap needles, either the one-handed scoop technique thereafter, or after every major repair or relocation of
or a one-handed technique using a recapping device the cabinet. It is recommended that accredited certifiers
(an engineering control that holds the cap in place). be engaged for provision of class II BSC certification
Specific training is provided to encourage workers to and repair service. Class II cabinets may be used in
use safe methods and operations to prevent aerosol BSL-3 laboratories, when supplemented by use of PPE
generation, skin and mucosal contact with infectious (gloves, gowns, and respiratory protection), and may
agents, and handling of sharps where they cannot be be used in BSL-4 laboratories in conjunction with wear-
eliminated. ing a one-piece, positive-pressure, ventilated suit with
If the hazard cannot be eliminated by engineering a life-support system, an in-line HEPA or high-purity
or administrative controls, it may be mitigated by filter, and supplied with grade D breathing air. When
the use of PPE to protect against contact, mucosal, class II cabinets that recirculate air to the laboratory
and respiratory exposure. Vaccinations, when avail- are used in BSL-4 facilities operated by US Depart-
able and where medically indicated, may serve as an ment of Defense (DoD) organizations or contractors
adjunct to PPE, but never as a substitution for PPE. in support of biological research, development, test,
Once all the tasks an individual will perform have and evaluation operations, they must be field certified
been assessed and all the infectious and toxic agents every 6 months.27
the individual will work with have been identified, When working within a class II BSC, the equipment
the tasks and agents are recorded in a document that and materials are arranged in a clean-to-dirty layout,
the worker and the supervisor prepare together. The with clean materials (uncontaminated materials) in the
mitigating controls are then chosen with input from center of the work space and contaminated materials
safety professionals and occupational health and at one end of the work space within the cabinet and
medical staff to form a collection of primary barri- contaminated waste materials at the other end of the
ers, approved practices, PPE, and vaccinations. Based work space.28 Class III cabinets are totally enclosed,
on an individual worker s current educational and ventilated, gas-tight cabinets. They provide the high-
experience levels and state of health, certain controls est level of product, personal, and environmental
may not be feasible. High-risk tasks may have to be protection, and are most suitable for work in BSL-3
avoided, on a spectrum that may range from observing and BSL-4 laboratories. They also provide absolute
high-risk tasks (in-vivo work, such as manipulations protection against respiratory exposure to infectious
of exposed animals) and performing low-risk tasks or toxic aerosols. Operations are conducted using
(in-vitro work with infected cell cultures in a BSC), to shoulder-length gloves or half-suits connected to the
the extreme that the individual may not be granted cabinets. Air is supplied to the class III cabinet through
access to the containment laboratory. a HEPA filter, and air exhausted from the cabinet to the
atmosphere passes through two HEPA filters in series
Physical Barriers (or one HEPA filter and an exhaust air incinerator).
Materials are removed from the cabinet by passing
Primary barriers include class II and class III BSCs, them through an interlocked, double-door sterilizer or
protective suits, and containment animal housing. through a chemical dunk tank filled with an appropri-
Class II BSCs are open-fronted cabinets with HEPA- ate disinfectant for the infectious agents or toxins in
filtered laminar airflow. Class II type A1 and type A2 use. Several class III cabinets housing a refrigerator,
cabinets may exhaust HEPA-filtered air back into the cell culture incubator, centrifuge, or aerosol-generating
laboratory or may exhaust the air to the environment equipment may be connected in a cabinet line as an
through an exhaust canopy. Class II type B1 cabinets integrated system for use in a BSL-3 laboratory or in a
have HEPA-filtered down-flow air composed of uncon- BSL-4 cabinet laboratory. A complete change of cloth-
taminated, recirculated in-flow air (30%) and exhaust ing is required, with wearing of a dedicated laboratory
most (70%) of the contaminated air through a dedi- scrub suit, jumpsuit or gown, shoes, and examination
cated duct with a HEPA filter to the atmosphere. Class gloves for hand protection in case of a puncture or
II type B2 (total exhaust) cabinets exhaust all in-flow if a pinhole develops in the cabinet shoulder-length
and down-flow air to the atmosphere after passing gloves, or half-suits.
through a HEPA filter located in a dedicated exhaust Primary barriers for animal housing include the
duct. To verify proper operation, all class II BSCs must following: (a) microisolator cages for rodents that have
be field certified in accordance with NSF International filter tops; (b) ventilated rodent cage racks; (c) venti-
Standard/American National Standard for Biosafety lated, negative-pressure, HEPA-filtered cubicles; (d)
524
Biosafety
ventilated, negative-pressure, HEPA-filtered flexible Elastomers LLC]) may be indicated to protect against
film isolators; and (e) rigid, ventilated, negative-pres- exposure to other contaminated materials, such as
sure, HEPA-filtered isolation cages.29 Rigid, venti- toxins, organic solvents, and caustics. Gloves should
lated, negative-pressure, HEPA-filtered, mobile animal be changed frequently, followed by thorough hand
transport carts have been developed at USAMRIID to washing. Disposable gloves should not be reworn.
isolate animals during transfer between containment To ensure protection when working with highly haz-
animal facilities.30 Other primary containment devices ardous materials, double gloving (wearing two pairs
include ventilated, filtered enclosures for continuous of gloves) should be practiced. If the outer glove is
flow centrifuges and use of sealed rotors and centri- punctured or torn, the protective skin barrier should
fuge safety cups in conventional centrifuges. Primary still be maintained by the inner glove if it, too, was not
containment devices used in necropsy rooms include breached (provision of redundant protection). If work-
downdraft necropsy tables, specially designed class II ing with contaminated sharps (eg, needles, scalpels,
cabinets for conducting necropsies, and HEPA-filtered glass slides, capillary tubes, pipettes) or with infected
vacuum shrouds for oscillating bone saws. animals that may bite or scratch, laboratory workers
should consider wearing cut-resistant overgloves
Personal Protective Equipment (eg, Kevlar [EI Du Pont de Nemours and Company,
Wilmington, Del]; armored, stainless-steel mesh; or
In BSL-3 containment, laboratory workers wear leather gloves) for additional protection.33 If working
protective clothing, such as solid-front or wraparound with materials where there is a splash hazard, the use
gowns, scrub suits, or coveralls. This protective cloth- of safety goggles or face shields and head covers (bon-
ing is not to be worn outside the laboratory. To aid nets, caps, hood) may be indicated.
in enforcement of this rule, laboratory clothing may When entering rooms housing infected animals,
be color-coded, so that it can be readily identified use of additional PPE (wraparound gowns or Tyvek
if worn outside the laboratory. Scrub suits are typi- [DuPont Tyvek, Richmond, Va] coveralls, foot covers or
cally two-piece ensembles composed of trousers and boots, head covers, eye and respiratory protection, etc)
tunics. Tunics with long sleeves that terminate in knit is required. These PPE requirements will be indicated
wrist cuffs aid in donning protective gloves. Gloves on the warning sign posted on the door of the animal s
are drawn over the cuffs and may be secured in place cage. Respiratory protection is provided by using
using tape. Long-sleeved tunics are favored over short- properly fitted respirators approved by the National
sleeved tunics because long sleeves with gloves taped Institute of Occupational Safety and Health (NIOSH).
to the sleeves can provide a physical barrier to protect Surgical masks or nuisance dust masks do not meet
the skin of the wrists and arms from potential expo- the NIOSH definition of a respirator. NIOSH-approved
sure to infectious agents, including bacterial spores.31 respiratory protection systems are commonly used
Disposable clothing should not be reused. Reusable in BSL-3 laboratories and animal rooms when the
clothing is decontaminated, usually by autoclaving, respiratory hazard cannot be completely engineered
before being laundered to prevent an exposure haz- out through the use of primary containment devices.
ard to laundry workers.32 Clothing is changed when Useful and comfortable negative-pressure respirators
overtly contaminated or after every work session, include disposable N-100 filtering face pieces with
depending on facility policy. The wearing of dedicated integral exhalation valves and tight-fitting, half-face,
laboratory shoes or safety shoes may be required in negative-pressure respirators fitted with N-100 par-
BSL-3 facilities. Otherwise, disposable shoe covers ticulate filters. These respirators have an assigned
should be worn. Wearing dedicated laboratory socks protection factor of 10, meaning there are 10-fold fewer
provides comfort to the feet and extra skin protection particulates at the breathing zone inside the respirator
to exposed ankles, if trousers are not long enough to than outside the respirator, providing the respirator is
cover the legs fully. properly fitted and worn. A properly fitted and worn
Protective gloves must be worn when handling full-face piece, negative-pressure respirator has an as-
infectious materials, animals, and contaminated mate- signed protection factor of 50 to 100 and also provides
rial. Gloves are selected to meet the needs of the risk eye protection. All users of respirators must be enrolled
assessment. Nitrile or latex gloves may be appropri- in a respiratory protection program in accordance with
ate if they provide the worker with protection from the Occupational Safety and Health Administration
the infectious agent being handled. However, gloves (OSHA) Respiratory Protection Standard.19 Users of
manufactured from other materials (eg, neoprene tight-fitting respirators must be fit tested annually
[DuPont Performance Elastomers LLC, Wilmington, using an approved qualitative or quantitative fit test.
Del], butyl rubber, and Hypalon [DuPont Performance Wearers of tight-fitting respirators must not have facial
525
Medical Aspects of Biological Warfare
hair that could interfere with the fit of the respirator, In a class III BSC operation (BSL-4 cabinet labora-
nor should eyeglasses interfere with the tight seal. tory), personnel must remove all personal clothing
Users of full-face, tight-fitting respirators who wear and undergarments and shoes. Complete laboratory
eyeglasses will need special optical inserts that may clothing including undergarments, pants, shirts,
be worn inside the respirator face piece. jumpsuits, shoes, and gloves is provided and worn
When working in a BSL-3 environment, such as by laboratory workers.1 Workers wear nitrile or latex
a room housing infected animals in open cages or a examination gloves for extra protection when working
necropsy room equipped with a downdraft table and in class III BSCs, just in case the shoulder-length box
an oscillating bone saw, greater respiratory protec- gloves develop pinholes, punctures, or tears.
tion might be needed. A NIOSH-approved powered In BSL-4 suit laboratories and BSL-4 animal facilities,
air-purifying respirator (PAPR) with a loose-fitting personnel must remove all personal clothing, including
hood or a tight-fitting full face piece is often used and undergarments, socks, shoes, and jewelry. Complete
provides an assigned protection factor of 1,000. Benefits laboratory clothing including undergarments, pants,
of wearing a loose-fitting hood include comfort, no shirts, jumpsuits, socks, and gloves is provided for,
requirement for fit testing, and amenability to use by and used by, laboratory workers. Some institutes opt to
individuals with facial hair. Reusable turbo blowers omit wearing undergarments in containment. Workers
for PAPRs are powered by rechargeable batteries. The don a fully encapsulating positive-pressure protective
blowers may be equipped with N-100 particulate filters suit supported by an umbilical-supplied air system.
or with combination cartridges that incorporate a par- The suit can be fitted with integral protective over-
ticulate filter with activated charcoal or other chemical boots or with legs terminating in soft booties. If a suit
absorbent for use in atmospheres of greater than 19.5% of the latter design is used, the worker dons protective
oxygen that have contaminated particulates and low overboots inside the BSL-4 suit facility, after passing
levels of organic or other specified chemical vapors. through an airlock equipped with a decontaminating
The airflow in cubic feet per minute, with cartridges chemical suit shower. When not in use, protective
installed, must be checked with a flow gauge before overboots are stored inside the BSL-4 facility. As of this
each work session. Because there are no OSHA stan- writing, positive-pressure encapsulating suits for use
dards or end-of-service life indicators for particulate in a BSL-4 environment are not federally regulated by
filters when used with infectious agents, institutes OSHA as level A chemical suits or as respirators, and
have to develop local criteria for determining when such suits are not currently NIOSH approved. How-
to replace particulate filters. For example, USAMRIID ever, the compressor and filter system must provide
has established an empirically based policy to replace minimum grade D breathing air to the positive-pres-
particulate filters after 80 hours of use. As a complete sure encapsulating suits.19,27
protective ensemble, PAPRs with loose-fitting hoods
may be worn in conjunction with Tyvek suits or long- Medical Surveillance
sleeved scrub suits, gloves, laboratory socks, and shoes
with shoe covers or overboots. All NIOSH-approved Medical surveillance comprises baseline and period-
respirators are approved as a complete system, so ic (usually annual) studies, including the following:
components cannot be switched between different
manufacturers products without negating the ap- " complete medical history,
proval. For example, a NIOSH-approved PAPR system " urinalysis,
consists of the turbo blower unit, battery, belt, hose, " hematology,
filters or cartridges, and loose-fitting hood or tight- " serum chemistry panel,
fitting face piece, all assembled and marketed by the " serum protective antibody titers for specific
manufacturer as a complete system. Only approved, disease agents,
compatible replacement components from the same " physical examinations, and
manufacturer may be used with a given respiratory " ancillary studies.
protection system.
To be approved to use a respirator, a user must be Ancillary studies can include the following:
medically cleared, be enrolled in an employer-provid-
ed OSHA-compliant respiratory protection program,19 " periodic chest radiograph;
receive initial and annual training on the use of the as- " periodic electrocardiogram;
signed respirator or additional training when a different " annual audiogram;
type of respirator is assigned, and undergo annual fit " annual visual acuity testing;
testing for negative-pressure, tight-fitting respirators. " annual evaluation of respiratory capacity; and
526
Biosafety
" mental fitness, neurological examinations,
mentor before a laboratory worker is considered profi-
and random testing for illicit substance use
cient to work independently in BSL-4 containment. After
(as needed).
demonstrating proficiency, the laboratory worker can
begin independent work in the BSL-4 containment suite.
An effective occupational health program benefits both
During normal operations in the BSL-4 containment
the employee and the employer. This program may
suite, workers may disconnect briefly from the breath-
reduce time lost to injuries. This occupational health
ing air supply to move about and then couple to an air
program will comply with OSHA and other applicable
line in a new location within the suite. One manufac-
federal and state laws and regulations.
turer advises that up to a 5-minute residual air supply
Medical surveillance is a critical part of a compre- may remain in the suit if there is an unanticipated loss
hensive occupational health and safety program. An
or interruption of the breathing air supply.36 In regular
occupational health and safety program has the fol- operations, it is prudent not to remain disconnected
lowing objectives34:
from the air supply for more than 2 or 3 minutes, be-
cause the carbon dioxide concentration and humidity
" protection of workers against health and
level will quickly rise within the suit space. Generally,
safety hazards in the work environment;
the visor fogs ups before the carbon dioxide concentra-
" proper placement of workers according to
tion builds up to a hazardous level, thus prompting
their physical, mental, and emotional abilities;
the user to connect to the air supply expeditiously.
" maintenance of a pleasant, healthy work
It is important that personnel are fit for the physical
environment;
challenges of working in a BSL-4 suit laboratory. An
" establishment of preplacement health
ongoing medical surveillance program ensures that,
examinations;
in the event of occupational exposure to an infectious
" establishment of regular, periodic health ex- agent or toxin, the medical needs of the worker will be
aminations (medical surveillance);
met immediately. If a laboratory worker should become
" diagnosis and treatment of occupational inju- ill without obvious exposure to an agent, the individual
ries, exposures, and diseases;
will be assessed to determine whether the illness is
" consultation with the worker s personal phy- related to an unknown laboratory exposure.
sician, with the worker s consent, of other
related health problems;
Vaccinations
" health education and counseling for workers;
" safety education for workers;
The decision to vaccinate is based on a benefit-to-
" identification of hazardous situations or find- risk analysis or a risk-reduction analysis.37 To justify
ing the means to prevent or mitigate hazard- use of a vaccine, the benefit from vaccination must
ous situations; and
outweigh any potential untoward effects of the vaccine.
" establishment of surveys and studies of the
Benefits of vaccination include induction of specific
industrial environment for protection of work- humoral (antibody-based) and cellular immunity to a
ers, their families, and the community.
given infectious agent or toxin. Risks of vaccinations
consist of local or systemic reactions.
Laboratory workers employed in a BSL-4 suit facil- Even the safest vaccine product has a likelihood of
ity are enrolled in a medical surveillance program, and
producing unwanted or unexpected side effects or an
they should be medically evaluated for fitness to use
adverse event in a small percentage of the population
an encapsulating, positive-pressure protective suit. At
receiving the vaccine.37 For at-risk personnel in-
USAMRIID, workers in the BSL-4 suit laboratories are
cluding laboratory workers use of appropriate pro-
enrolled in a hearing protection program. When the 8- phylactic vaccines can provide an additional level of
hour, time-weighted average level is 85 dB or greater,
protection.1 Each institute should have a written policy
workers must be enrolled in an employer-provided
that defines at-risk personnel, specifies the risks and
hearing protection program to comply with OSHA
benefits of specific vaccinations, and identifies appro-
regulations.35 The program requires employees to
priate prophylactic vaccine products. The requirements
undergo initial baseline and annual surveillance audi- and recommendations should address the infectious
ometry, fitting, and training to use hearing protectors
agents known to be present or likely to be encountered
(ear plugs or muffs).
in a given institute.
It is required that personnel receive initial familiar- For all clearly identified at-risk personnel, licensed
ization training to wear the suit, as well as extensive,
vaccines for which the benefits clearly exceed the risks
documented, tailored training provided by an assigned
should be offered.1 Examples of licensed vaccines for
527
Medical Aspects of Biological Warfare
identified at-risk personnel at USAMRIID include protect persons and animals in the community from
those for protection against hepatitis B, yellow fever, infectious agents in the event of an accidental release
Japanese encephalitis, rabies, anthrax, smallpox, and within the laboratory.1 Secondary barriers in BSL-3
other orthopox virus infections. containment facilities include entry vestibules or
Recommendations must be carefully considered for personnel airlocks that feature two self-closing and
the following situations1: lockable doors, clothes change rooms and shower
facilities, and a hand-washing sink in each individual
" giving less efficacious vaccines (eg, those with laboratory room. The sink is located near the room exit
diminished immunogenicity or loss of potency); door and has hands-free operation (using foot pedals,
" giving vaccines associated with high rates of or knee/elbow paddles) or is automatically activated
local or systemic reactions (vaccines with safe- by an infrared sensor. Other secondary barriers include
ty concerns, such as excessive reactogenicity); floor, wall, and ceiling finishes constructed for easy
" giving vaccines that induce increasingly cleaning and decontamination; sealed penetrations
severe reactions with repeated use (vaccines in floors, walls, and ceilings; and sealable openings to
that induce hypersensitivity reactions); and facilitate decontamination. Laboratory furniture has
" giving unlicensed vaccines under IND protocols. waterproof and chemical-resistant bench tops, and any
chairs are covered with nonfabric material to permit
IND vaccines used under a US Food and Drug Ad- easy decontamination. An autoclave is available in the
minstration exemption for research and vaccination of facility. The facility is equipped with a ducted exhaust
laboratory personnel include the following38: ventilation system that creates inward directional air-
flow from areas of lower potential hazard to areas of
" two Venezuelan equine encephalitis vaccines, higher potential hazard (negative-pressure gradient)
" Eastern equine encephalitis vaccine, without recirculation of air. To confirm inward airflow,
" Western equine encephalitis vaccine, a visual monitoring device (eg, a Magnehelic differ-
" pentavalent botulinum toxoid, ential pressure gauge [Dwyer Instruments, Michigan
" Rift Valley fever inactivated vaccine, City, IN], Photohelic gauge [Dwyer Instruments,
" Q-fever vaccine, and Michigan City, IN], rodimeter,  tell-tail ) should be
" tularemia vaccine. available at the laboratory entry.
In animal BSL (ABSL)-3 facilities, room fittings and
Possible contraindications for subject participation ventilation should be in accordance with the Guide for
in vaccination programs or for work within biocontain- Care and Use of Laboratory Animals (The Guide)40 and
ment laboratories39 include the following medical issues: the BMBL.1 If the ABSL-3 facility has floor drains,
the drain traps are always filled with an appropriate
" chronic, serious, or uncontrolled medical disinfectant. Additional environmental protection
problems; design features (enhancements) in BSL-3 laboratories
" acute or temporary medical conditions; and animal-holding spaces (including provision of
" autoimmune disorders; personnel showers and effluent decontamination,
" impaired immunity; HEPA filtration of exhaust air, and containment of
" conditions that may obscure recognition of ad- piped services) may be indicated, depending on the
verse events from investigational vaccines; nature of the infectious agents to be used (eg, arbovi-
" conditions that could lead to unpredictable ruses and high-consequence animal pathogens); the
behavior or collapse, leading to increased risk risk assessment (or maximum credible event analysis)
of an individual or coworker to exposure or of the site (eg, laboratory to be located in a highly
medical emergency within a laboratory; populated urban center or in a remote region having a
" untoward reactions to multiple vaccinations; low-density population); and applicable federal, state,
and and local regulations.
" vaccine-specific contraindications. Secondary barriers required in BSL-4 laboratories
and ABSL-4 animal-holding spaces are all those
Protecting the Community and the Environment specified for BSL-3 laboratories and ABSL-3 animal-
holding spaces, with additional provisions. Other
Secondary barriers are the elements of laboratory required secondary barriers include a dedicated,
facility design and construction that (a) contribute to nonrecirculating ventilation system with supply and
protection of laboratory personnel, (b) provide a barrier exhaust components balanced to ensure directional
to protect persons outside of the laboratory, and (c) airflow from areas of lower potential hazard to areas
528
Biosafety
of higher potential hazard. Also required is HEPA Solid and Liquid Waste Inactivation and Disposal
filtration of supply air and double HEPA filtration
of exhaust air, with redundancy (backup exhaust The US Environmental Protection Agency (EPA) de-
duct with fan and in-line double HEPA filters), and fines antimicrobial pesticides as substances or mixtures
an alarm and daily monitoring to prevent positive of substances used to destroy or suppress the growth of
pressurization of the laboratory or animal-holding harmful microorganisms (eg, bacteria, viruses, or fungi)
space. In large, complex operations, a supervisory on inanimate objects and surfaces. Public health antimi-
control and data acquisition system (also known as crobial products are intended to control microorganisms
a building automation system) may be installed to infectious to humans in any inanimate environment.
monitor and control room pressures automatically. These products include sterilizers (sporicides) and
An automatically starting emergency power source disinfectants (see http://www.epa.gov/pesticides/
(usually a diesel-powered generator) is required as a factsheets/antimic). Sterilizers (sporicides) are used to
minimum for the redundant exhaust ventilation sys- destroy or eliminate all forms of microbial life, including
tems, redundant life-support (breathing air) systems, fungi, viruses, and all forms of bacteria and their spores.
alarms, lighting, entry and exit controls, and BSCs. In Sterilization is widely used in hospitals for infection
practice, the freezers and other laboratory equipment control. Types of sterilizers include steam under pres-
(incubators and refrigerators) are generally also on sure (autoclaves), dry-heat ovens, low-temperature gas
circuits that can switch to emergency backup power. (ethylene oxide), and liquid chemical sterilants. All types
Other infrastructure elements that contribute to the of sterilizers are also applicable for use in microbiological
secondary barrier include change rooms, person- and biomedical laboratories. In laboratories, autoclaving
nel showers, effluent decontamination by a proven is used to prepare sterile instruments, equipment, and
method (preferably heat treatment), and containment microbiological nutrient media and to render micro-
of piped services. Floor and sink drain traps must biologically contaminated liquid and solid waste sterile
be kept filled with an appropriate disinfectant (one before it enters the waste-disposal stream. Laboratory
with proven efficacy for the microorganisms handled glassware is dried, sterilized, and depyrogenated (ren-
within the BSL-4 facility). Required at the contain- dered free of endogenous pyrogens) in dry-heat ovens.
ment barrier is an autoclave with two interlocked Ethylene oxide sterilization is used to sterilize materials
doors with the outer door sealed to the outer wall such as delicate instruments and laboratory notebooks,
(a so-called bioseal). The autoclave is automatically which cannot withstand steam sterilization, but is
controlled so that the outer door cannot be opened seldom used to sterilize solid waste. Liquid sterilants,
until a sterilization cycle has been completed. Also used to sterilize delicate instruments by immersion and
provided is a dunk tank, fumigation chamber, or a to sterilize impervious surfaces by surface application,
ventilated equipment airlock for passage of materials can be added to suspensions of infectious materials to
into the containment area and safe decontamination chemically inactivate them. Disinfectants, according
and removal of materials that cannot be steam ster- to the EPA, are used on hard inanimate surfaces and
ilized from the containment area. The walls, floors, objects to destroy or irreversibly inactivate infectious
and ceilings are constructed as a sealed internal fungi and bacteria, but not necessarily their spores. The
shell (the containment envelope) capable of being EPA divides disinfectant products into two major types:
decontaminated using a fumigant. Bench tops have (1) hospital and (2) general use. Hospital disinfectants
seamless surfaces impervious to water, resistant are most critical to infection control in hospitals and
to chemicals, and free of sharp edges. Appropriate are used on medical and dental instruments and on
electronic communications are provided between the hospital environmental surfaces. General disinfectants
BSL-4 containment area and the noncontainment area, are products used in households, swimming pools, and
which may include a telephone, facsimile, two-way water purifiers.
radio, intercom, and a computer system on a local An example of a liquid sterilant-disinfectant is
area network or wireless network. BSL-4 protective Alcide EXSPORE (Alcide Corporation, Redmond,
suit laboratories also have a dedicated area for stor- Wash) 4:1:1 base concentrate (1.52% sodium chlorite;
ing suits and boots, and a double-door personnel EPA Registration No. 45631-3), which comes with a
airlock equipped with a chemical shower for surface separate activator concentrate (9.5% lactic acid) as a set.
decontamination of protective suits. Animal-holding This sterilant-disinfectant must be freshly prepared by
rooms need to meet the standards specified in The diluting the base with water per the manufacturer s in-
Guide.40 Containment operational parameters are structions before adding activator to generate chlorine
inspected and verified daily before work is initiated dioxide.41 The prepared sterilant-disinfectant should be
in the BSL-4 facility. used immediately and must be freshly prepared daily.
529
Medical Aspects of Biological Warfare
An example of a hospital disinfectant is MICRO- and local regulations. In the United States, disposal
CHEM PLUS (National Chemical Laboratories, Inc, of several categories of solid waste (regulated medi-
Philadelphia, Pa; EPA Registration No. 1839-95- cal waste, perceived medical waste, and pathological
2296) a proprietary mixture of two quaternary am- waste) is regulated at the state level. Many states have
monium compounds and inert ingredients which is strict regulations that require that such waste be steril-
labeled to kill listed microorganisms (specified viruses, ized and rendered unrecognizable (by processes such
fungi, and nonspore-forming bacteria) when mixed at as incineration, shredding, or grinding with steam
the rate of 2 ounces of the concentrated product per sterilizing or irradiating) before final disposal in a
gallon of water.42 sanitary landfill.
An example of a general (household) disinfectant is
Clorox Regular Bleach (The Clorox Company, Oakland, Standard and Special Microbiological Practices
Calif; 6.00% sodium hypochlorite; EPA Registration
No. 5813-50). When mixed at the rate of 1/4 cup per Standard and special microbiological practices
gallon of water, it is labeled to kill listed microorgan- universal to all BSLs are as follows:
isms (specified viruses, fungi, and nonspore-form-
ing bacteria).43 Bleach is not registered by the EPA " The laboratory director limits or restricts ac-
as a sterilant. During the subsequent cleaning and cess to the laboratory when experiments are
decontamination of spore-contaminated postal facili- in progress.
ties after the 2001 anthrax-by-mail incidents, the EPA " A biohazard sign may be posted at the en-
issued crisis exemptions on a case-by-case basis to trance of the BSL-1 laboratory if infectious
use bleach for emergency decontamination subject to agents are present. A biohazard sign is posted
adherence with specified conditions of application (see at the entrance of BSL-2, BSL-3, and BSL-4
http://www.epa.gov/pesticides/factsheets/chemi- laboratories and animal rooms when infec-
cals/bleachfactsheet). tious agents are present.
In BSL-4 laboratories and in BSL-3 and ABSL-3 facil- " Policies for the safe handling of sharps are
ities, if indicated by the risk assessment, liquid effluent instituted.
(laboratory sewage) must be inactivated by a proven " All procedures are performed carefully to
process, generally heat treatment under pressure. minimize the creation of aerosols.
Steam sterilization of laboratory sewage may be either " Work surfaces are decontaminated at least once
a continuous flow or a batch process. Solids suspended daily and after any spill of viable material.
in the liquid waste are comminuted (finely ground). " All infectious waste is decontaminated by
The effluent is heated to specified temperature and an approved process (eg, autoclaving before
held at that temperature for a certain period of time. disposal).
Then, it is cooled, sampled for sterility testing, and " A pest (insect and rodent) control program
released to a municipal or nonpublic sewer system. The must be in effect.
time temperature relationship for the selected process
depends on the inactivation profile of the infectious Additional standard practices common to BSL-1
microorganisms that could potentially be present in the through BSL-3 facilities are as follows:
liquid waste. The current process at Fort Detrick holds
the heated effluent at 132°C (270°F) for a minimum of " Personnel wash their hands after handling
12 minutes, sufficient to inactivate fungal and bacterial viable materials, after removing gloves, and
spores. The standard liquid biowaste process used at before leaving the laboratory.
the Canadian Science Centre for Human and Animal " Eating, drinking, smoking, handling contact
Health (Winnipeg, Manitoba, Canada) heats the efflu- lenses, taking medication, and storing food
ent to 121°C (250°F) for a 30-minute holding time, but for human consumption in the laboratory or
has the capability of achieving a temperature as high animal-holding facility are not permitted. If
as 141°C (286°F).44 The standard process is sufficient contact lenses are worn in the laboratory or
to inactivate fungal and bacterial spores. The higher animal-holding area, goggles or a face shield
temperature is available, if needed, to inactivate prions should also be worn. Personnel should refrain
(heat-resistant infectious proteins).45 from applying cosmetics or lip balm, chewing
After infectious materials have been inactivated by gum, and taking oral medications while in the
an appropriate method of sterilization or disinfection, laboratory or animal-holding facility.
they may be removed from the laboratory and dis- " Mouth pipetting is prohibited. Only mechani-
posed of in accordance with applicable federal, state, cal pipetting devices are to be used.
530
Biosafety
There are no special practices for the BSL-1 labora- appropriate initial training, and annual
tory. The following special practices apply to BSL-2, training, and additional training on po-
BSL-3, and BSL-4 laboratories, as well as to ABSL-2, tential hazards in the laboratory or animal
ABSL-3, and ABSL-4 animal-holding areas: facility; precautions to take to prevent
exposures; and procedures on evaluating
" Secure all laboratories registered for select potential exposures. The laboratory director
agents and toxins.46 Keep BSL-2 and BSL-3 is also responsible for ensuring that the pre-
laboratory room doors closed when work- viously described training is appropriately
ing with infectious agents. Keep doors in documented.
BSL-4 laboratories and in ABSL-2, ABSL-3, " Use caution with needles and syringes. In
and ABSL-4 animal-holding areas closed and BSL-3 and BSL-4 laboratories and in ABSL-3
locked at all times. and ABSL-4 animal-holding facilities, use
" Do not allow people who are at a heightened only needle-locking syringes or disposable
risk of becoming infected (eg, immunocom- syringe needle systems in which the needle
promised individuals) access to the labora- is integral to the syringe. Use syringes that
tory or animal room when work with infec- resheath the needle and systems without nee-
tious agents is in progress. Only individuals dles. Dispose of used sharps in conveniently
advised of the potential hazards who meet located puncture-resistant containers.
specific entry requirements may enter the " Place all potentially infectious materials in
laboratory or animal-holding room. covered, leakproof containers during col-
" In ABSL-2, ABSL-3, and ABSL-4 animal-hold- lection, manipulation, storage, transport, or
ing facilities, the Institutional Animal Care shipping. Place viable material to be removed
and Use Committee and the Institutional from a class III BSC or a BSL-4 facility in an
Biosafety Committee approve special policies unbreakable, sealed primary container that
and procedures. is enclosed in a unbreakable, sealed second-
" Along with the biohazard sign, post the follow- ary container. Pass this enclosed material
ing information at the entrance to the laboratory through a chemical disinfectant dunk tank,
or animal-holding room: the agents in use, the fumigation chamber, or airlock having a
BSL, required vaccinations, any PPE required, chemical suit shower (in the case of a BSL-4
the name and phone number of the principal suit facility).
investigator, and any procedures required to " Decontaminate work surfaces and laboratory
exit the laboratory or animal-holding room. equipment with an effective disinfectant rou-
" At-risk individuals entering the laboratory or tinely, after work with infectious materials is
animal-holding room are to receive appropri- completed, and after any spills. Contaminated
ate vaccinations and skin tests, if available for equipment must be appropriately decontami-
the agents being handled or agents potentially nated before repair or maintenance or packag-
present in the room. ing for transport.
" Store baseline and periodic serum samples col- " Immediately report to the laboratory direc-
lected from at-risk personnel. At intervals, col- tor (supervisor) any spill or accident that
lect and analyze serum samples from at-risk results in exposure to infectious materials.
personnel working in ABSL-4 containment Institute medical evaluation, surveillance,
and communicate the results to those at-risk and treatment as appropriate and document
personnel. this medical care in writing. In BSL-3 and
" Describe biosafety procedures for BSL-2 and BSL-4 containment facilities, develop and
ABSL-2 facilities in SOPs. Describe biosafety post spill procedures. Professional staff or
procedures for BSL-3 and BSL-4 laboratories other appropriately trained personnel must
and ABSL-3 and ABSL-4 animal-holding facili- decontaminate, contain, and clean up any spill
ties in a biological safety manual specific to the of infectious material. In BSL-4 containment,
laboratory or animal-holding facility. Advise establish practical and effective protocols for
personnel of the specific hazards, require them emergency situations, including the evacua-
to read/understand the manual, and make tion of incapacitated staff.
certain that they comply with it. " Animals, plants, and clothing unrelated to
" The laboratory director must ensure that the work conducted are not permitted in the
laboratory and support personnel receive laboratory.
531
Medical Aspects of Biological Warfare
" In BSL-3 and BSL-4 containment facilities, remove and leave all laboratory clothing in
the laboratory director must ensure that all the inner change room. Take a decontaminat-
personnel are proficient in standard microbio- ing (soap and water) personal wet shower
logical practices, laboratory-specific practices, on exit from the laboratory. Autoclave soiled
and operations before they begin work with laboratory clothing before laundering. Use the
microorganisms. equipment airlock to enter or exit the labora-
" In BSL-3 and BSL-4 containment facilities, con- tory only in an emergency.
duct open manipulations of infectious agents " Bring supplies and materials into the BSL-4
in BSCs or other primary containment devices. facility through the double-door autoclave,
Conducting work in open vessels on the open fumigation chamber, or equipment airlock,
bench is prohibited. Vessels with tight-fitting which is decontaminated before and after
covers (gasketed caps, O-ring seals) should each use. Secure the airlock outer door before
be used to hold viable cultures within water the inner door is opened. Secure the airlock
baths and shaking incubators. Use sealed ro- inner door after materials are brought into the
tors or centrifuge safety containers fitted with facility.
O-ring seals to contain centrifuge tubes. Use " Autoclave or decontaminate all materials
plastic-backed paper towels on nonperforated other than materials to be retained in a viable
surfaces to facilitate cleanup. Use plastic ves- state before removing them from the BSL-4
sels in place of glass vessels. facility.
" At BSL-4, maintain a physical or electronic " In BSL-4 containment, establish a system to
log of all personnel, with the time of each report laboratory accidents and exposures,
person s laboratory entry and exit recorded. employee absenteeism, and medical surveil-
This requirement also applies to all person- lance of a potential laboratory-acquired illness.
nel who have access to areas in which select " Make available a facility for quarantine, isola-
agents and toxins are used or stored.46 tion, and medical care of personnel who work
" In BSL-4 containment (and in BSL-3 contain- in BSL-4 containment and who are affected
ment, if indicated by risk assessment, site- with a potential or known laboratory-acquired
specific conditions, or applicable regulations), illness.
enter and exit the laboratory only through
the clothing change and shower rooms. In ABSL-4 containment, personnel assigned to work
Remove and leave personal clothing in the with infected animals should work in pairs. Appro-
outer change room. Change completely into priate procedures should be used to reduce possible
laboratory clothing. On exiting the laboratory, exposure to infectious agents.
ROLE OF MANAGEMENT IN A BIOSAFETY PROGRAM
Management must consider safety a top priority personnel (each spoke of the wheel) in an institute
and, on a daily basis, work closely with and support must be considered important, regardless of their
safety personnel. While management must provide perception of their contributions.
a biosafety program, as well as engineering features The goals of a biosafety program include the fol-
and equipment designed to reduce the risks associ- lowing: (a) prevention of injury, infection, and death
ated with the research conducted at the institute, of employees and the public; (b) prevention of envi-
safety is also an individual responsibility. To illus- ronmental contamination; (c) conformance to prudent
trate this point (Figure 22-1), consider the mission biosafety practices; and (d) compliance with federal,
or purpose of an institute as the hub of a wheel. All state, and local regulations/guidelines. The ultimate
personnel regardless of education, experience, or objective of these goals is to keep everyone healthy
job description are the spokes of the wheel and while supporting productive research. Personnel train-
must be reminded regularly of the importance of ing is paramount. Both initial and refresher training
their contributions to an institute. If one (or more) of personnel must address the institutional biological
of the spokes is (are) not functioning as designed, safety program and the elements of biosafety. Train-
the wheel does not operate smoothly. Consequently, ing can be conducted as a discussion rather than as
it takes longer to meet not only personal goals and a formal lecture to promote audience participation.
objectives, but also institute goals and objectives. All This technique allows individuals to have ownership
532
Biosafety
policies through an integrated program of safety engi-
neering, vaccination, health surveillance, and medical
management of illness.
Risk encompasses an awareness of the risk, an as-
sessment (or evaluation) of the risk, and management
of the risk. Communication is a fundamental part of
risk assessment and training.
The US Government developed a five-step risk
management process (Figure 22-2).47 The five sequen-
Custodial staff;
tial steps of the risk management process include the
Security staff
MISSION
housekeeping
following:
1. Identify hazards What is the hazard?
2. Assess hazards What is the danger of this
hazard?
3. Develop controls and make risk decision
What controls can be used to remove this haz-
ard, or make a decision to accept some risk?
4. Implement controls Controls developed for
the risk are implemented (or put into opera-
tion or practice).
Fig. 22-1. Institute personnel are depicted as the spokes of a
5. Supervise and evaluate After a period of
wheel that work together to accomplish a common mission.
evaluation, the controls implemented are re-
viewed to determine whether they were ade-
quate, or if additional controls must be added.
in the dialogue, which, for the most part, will result
in better adherence to compliance of institute and Laboratory Safety Audits
regulatory policies.
The philosophy of a biosafety program is based on An audit is a methodical examination and review.
an early estimation of risk, followed by application In the present context, it is a systematic, critical review
of appropriate containment and protective measures. of laboratory safety features. The terms survey (com-
It is very important to investigate and review safety prehensive view) and inspection (a critical appraisal,
incidents at the institute because presentation of this
data will heighten the awareness of individuals that
accidents do happen despite safeguards. Concluding
Develop
remarks for each training session should reiterate the
controls and make
Assess
description of some obvious hazards and how safety
risk decision
hazards
personnel try to minimize the risk of these hazards.
Safety personnel must emphasize that their role is
to try to identify hazards, conduct risk assessments,
develop risk management strategies, and evaluate
the effectiveness of those strategies over time. Safety
personnel must actively engage with and seek the
help of all administrative and laboratory personnel
Identify
in hazard identification. It must be understood that a
hazards
Implement
safety department cannot provide absolute safety, but
controls
strives to provide reasonable safety. Safety personnel
advise, guide, provide limited training, and imple-
Supervise
and evaluate
ment institute and regulatory policies (in conjunction
with the institutional biosafety committee). The safety
department, with continued support from manage-
ment and all facility personnel, can minimize the risk
Fig. 22-2. Five steps of the risk management process.
of hazards by implementing institute and regulatory Adapted from: US Army Safety Center, Fort Detrick, Maryland.
533
Administration
Safety staff
Laboratory
technicians
Facility
ineers
g
en
Animal
caretake
s
Principal
rs
investigator
Medical Aspects of Biological Warfare
an official examination, or checking or testing against the magnehelic and photohelic gauges; (e) checking
established standards) are often used interchangeably documentation that emergency communication de-
with the term audit. During the laboratory safety audit, vices have been tested at least monthly; (f) testing and
safety practices and equipment are evaluated. General recording during the audit operating status of alarms,
safety, life safety, biological safety, chemical hygiene, emergency lights, and emergency exit lights; and (g)
and radiation safety are topics covered in a typical spot checking laboratory SOPs, laboratory biosafety
laboratory safety audit. Laboratory audits should be manuals, and laboratory personnel training records.
scheduled on a regular basis and may be announced Four events that warrant conducting a formal, un-
or unannounced. scheduled audit of a laboratory include the following50:
Self-audits of required safety practices provide a
measure for achieving compliance with safety rules 1. accident or injury in the workplace,
and regulations.48 Designated safety specialists can 2. follow-up to implementation of new biosafety
conduct regular safety audits at quarterly intervals, regulations or procedures,
accompanied by the laboratory supervisor and a facili- 3. a new funding source requesting documenta-
ties management representative. Deficiencies can be tion of workplace safety, and
pointed out during the audit. Later, a written report 4. new infectious agents proposed for use in the
with suggestions for corrective action may be sent laboratory.
to the laboratory supervisor. The supervisor reports
progress on remediation to the safety specialist within An urgent time for evaluation of biosafety SOPs
a mutually agreed on, fixed-time period. Support from may be before a major outside organization or agency
higher management is essential for an audit to have the conducts a site visit.50 Two examples of organizations
desired effect of improving employee safety, as well as conducting site visits are the Joint Commission on Ac-
instituting compliance with applicable regulations.48 creditation of Healthcare Organizations and the Asso-
Use of a checklist ensures a systematic, standard- ciation for Assessment and Accreditation of Laboratory
ized audit, thus reducing the chance of missing critical Animal Care (AAALAC) International. Examples of
items. Citing the pertinent requirement or applicable agencies that conduct inspections of laboratories regis-
regulation on the checklist provides a ready reference tered for select agents are the CDC and USDA-Animal
and justification for each item listed on the checklist. and Plant Health Inspection Service (APHIS) Select
Within the overall laboratory safety audit, the fol- Agent Program Laboratory Inspection Programs. For
lowing list of biosafety elements should be covered49: subordinate laboratories of the US Army Medical Re-
search and Materiel Command, safety office personnel
" autoclave repair and operational records, conduct periodic safety site assistance visits.27 For DoD
" proper use of PPE, research, development, test, and evaluation (RDTE)
" appropriate laboratory clothing, laboratories, the director of Army safety conducts
" no food or drink in the laboratory, biological defense safety evaluation site visits.27
" proper use of sharps and sharps disposal In DoD RDTE facilities, health and safety profes-
containers, sionals must conduct internal inspections (audits)
" decontamination of infectious materials before of BSL-1 and BSL-2 laboratories at least quarterly
disposal, and must conduct internal inspections of BSL-3 and
" proper disposal of laboratory waste, BSL-4 laboratories at least monthly.27 Inspections
" proper laboratory signage, must be documented, deviations from safe practices
" current certification of BSCs, and recorded, and recommended corrective actions taken.
" use of in-line HEPA filters on laboratory If deviations are life-threatening, access to the labora-
vacuum outlets. tory area is restricted until corrective actions have
been taken. New RDTE efforts involving biological
Additional biosafety elements audited at USAMRIID agents must be evaluated and inspected before startup.
include (a) weekly flushing floor and sink drains and Any Department of the Army headquarters agency
recording the action in a drain flush log; (b) flushing can recommend special studies or reviews when (a)
the eyewash weekly and recording the action in an conditions or practices that may affect safety have
eyewash flush log; (c) testing (flushing and measuring changed; (b) major system modifications to facility
the flow rate) the emergency deluge shower at least design and physical configuration are made; and (c)
weekly and recording the action in an emergency safety, health, and environmental protection standards
shower test log; (d) recording during the audit differ- and requirements have changed significantly.27 Safety
ential pressures for laboratory rooms as displayed on officials maintain safety inspection records for 3 years,
534
Biosafety
and they review records annually to note trends that BSL-3, and BSL-4 laboratories to identify potential
require corrective actions.27 Laboratory supervisors problems. These quarterly inspections augment the
review their work areas at least weekly and take any monthly inspections conducted by laboratory suite
needed corrective actions promptly. supervisors or their designees. Inspections, which may
At USAMRIID, safety professionals assigned to be announced or unannounced, include coverage of
the Office of Safety and Radiation Protection conduct general safety practices and safety practices specific
quarterly comprehensive inspections of BSL-1, BSL-2, to a particular BSL.51
SELECT AGENT PROGRAM
Legislation for the CDC Select Agent Program Services, CDC,46 or APHIS (Agricultural Bioterrorism
was initially enacted in 1996 to document transfers Protection Act of 2002).58,59 Within each registered
between microbial culture and toxin repositories and entity, a designated  responsible official (entity
laboratory facilities of certain pathogens infectious to owner, director, commander, or other designee within
humans and biological toxins injurious to humans, management), alternate responsible official(s) (to act
termed select agents and toxins.52,53 The objective in the absence of the responsible official), principal
of the legislation was to prevent transfer of these investigator(s), and staff member(s) having direct,
restricted biological materials to unauthorized indi- unescorted access to the restricted biological ma-
viduals and facilities having the intent to use them terials must be named individually in the entity s
for potentially nefarious purposes. The legislation registration application to the CDC or to APHIS.
established the original list of agents and required All named individuals must undergo a successful
such facilities to be registered with the Department security risk assessment conducted by the Federal
of Health and Human Services before transfers of Bureau of Investigation before these individuals are
restricted biological materials could be made to other approved by the CDC or APHIS for unescorted ac-
registered facilities within the United States. As estab- cess to an entity s biological materials. As part of
lished, this Select Agent Rule, codified in Title 42 CFR the entity registration process, inspectors from the
Part 72.6,53 also required an initial, periodic inspection CDC or APHIS will visit the entity with or without
of each registered facility to ensure that safety criteria prior notice to inspect biological safety and physical
were met. After the terrorist events of September 2001 security features of the laboratory facility, records of
and the ensuing anthrax-by-mail incidents shortly training, and health surveillance for personnel who
thereafter, the Patriot Act, enacted in 2001,54,55 and the have access to the restricted biological material, and
Public Health Security and Bioterrorism Prepared- also inventory records of all registered biological ma-
ness and Response Act of 2002 (Bioterrorism Act)56,57 terials maintained by a given entity. In the inventory
extended the jurisdiction of control of the Select record, each registered biological material is listed
Agent Rule from facilities and individuals that only by name, along with its location of storage and use
transfer select agents and toxins to all facilities and and the name of the principal investigator. Inventory
individuals that store and use select agents and tox- records must document each approved transfer and
ins. The purpose of the new legislation was to protect destruction of the biological materials, and must
against misuse of select agents and toxins whether account for possession, propagation, and consump-
inadvertent or the result of terrorist acts against the tion in the course of bona fide mission work. Written
US homeland or other criminal acts. The codified regulations, protocols, and operating procedures (the
regulations developed to implement the legislation46,58 so-called Biological Safety Manual) pertaining to work
ensured appropriate availability of biological agents with the regulated biological material in the regis-
and toxins for legitimate biomedical, agricultural, or tered laboratory areas are examined. Also inspected
veterinary research; education; and other purposes are electronic security measures and the emergency
(while excluding their availability for illegitimate response plan, including steps taken to report and
applications). The original list of biological materials recover lost, stolen, or diverted biological material.
was greatly expanded. An entity registration has to be renewed every 3
All laboratories ( entities ) having listed speci- years, and an individual s security risk assessment
fied select agents and toxins, USDA select agents, has to be repeated every 5 years. Felony convictions
overlap agents (agents appearing on both of the for violations of the Select Agent Rule legislation can
preceding lists), or listed plant pathogens must, by result in substantial sentences, including heavy fines
federal law, register each of their biological material and lengthy prison terms. The Select Agent Final Rule
holdings with the Department of Health and Human was published in 2005.45,58
535
Medical Aspects of Biological Warfare
Biological Defense Research Program Laboratories Use of Animals64 regulate the care and use of labora-
tory animals used in research. Many of the applicable
All laboratories involved in DoD RDTE operations regulations and policies are summarized in the Guide
must comply with the Department of the Army Bio- for the Care and Use of Laboratory Animals40 (the Guide).
logical Defense Safety Program.27 These regulations The responsible administrative official at each institu-
specify safety policy, responsibilities, and procedures tion using laboratory research animals must appoint
for military and contract laboratories conducting op- an Institutional Animal Care and Use Committee
erations at BSL-2, BSL-3, and BSL-4 in support of the US representative to oversee and evaluate the institution s
military biological defense program. This regulation animal program, procedures, and facilities to ensure
predates the Title 42 CFR Part 73,46 but shares many that they are consistent with the AWRs, Public Health
features with the select agent program regulation and Service policy (for those institutions that receive NIH
CDC/NIH guidelines.1 The DoD Biological Surety funding), and recommendations specified in the Guide.
(Biosurety) Program is a new program implemented This guide covers many aspects of an institutional ani-
in DoD biological defense RDTE laboratories that use mal care and use program, including the following:
DoD-provided biological agents.60 This biosurety pro-
gram is patterned after existing nuclear and chemical " policies and responsibilities;
surety programs. The purpose of the biosurety pro- " monitoring care and use of animals;
gram is to ensure the safe and secure use of biological " veterinary care;
agents. The program encompasses physical security, " qualifications and training of personnel who
biological safety, biological agent accountability, and work with animals; and
personal reliability as measures to prevent unauthor- " occupational health and safety of personnel
ized access to agents of bioterrorism (select agents).60,61 working with animals, physical facilities, and
One provision implements a two-person rule when animal husbandry.
working with biological select agents and toxins
(BSATs), accomplished by having two individuals Under the heading of occupational health and safety,
physically present in the laboratory room during work critical topics in an effective animal care and use pro-
with these materials. This requirement can also be met gram include the following:
by using surveillance cameras and random observa-
tions by roving observers. Physical security measures " hazard identification and risk assessment;
include inspection of all personal belongings on entry " personnel training, hygiene, safe facilities, and
into the laboratory building and random exit inspec- procedures;
tions, as well as physical security upgrades to harden " health monitoring;
the laboratory building. Biological safety refers to the " animal experimentation involving biological
provisions of the Department of the Army Biological and other hazardous agents;
Defense Safety Program. Agent accountability consists " use of PPE;
of ensuring that documentation exists for storage " medical evaluation; and
and access to BSATs. A biological personal reliability " preventive medicine for personnel working
program (BPRP), required by the biosurety program, with animals.
exceeds the scope of the CDC Select Agent Program.46
In addition to the security risk assessment required A voluntary program exists for the assessment
by the CDC Select Agent Program, the BPRP requires and accreditation of institutional animal care and
a background security investigation conducted at use programs. At the request of a given institution,
the level of that needed for a secret clearance for all AAALAC International will send laboratory animal
individuals who work with or potentially have access technical experts to the institution to conduct a site
to BSATs. The BPRP also requires initial and periodic visit and evaluate all aspects of an institution s animal
urinalyses for illegal drug use and continuous medical care and use program. If all aspects of the program
and suitability screenings for as long as an individual meet the high standards of AAALAC International,
remains enrolled in the BPRP. the institution may be granted the coveted designation
 AAALAC accredited, which is effective for 3 years.
Laboratory Animal Care and Use Program Triennial renewals require a complete, comprehensive
reassessment of an institution s animal care and use
Federal animal welfare regulations62,63 (AWRs) program. Accreditation by AAALAC International is
from USDA and APHIS, state and local laws, and the mandatory for DoD organizations and facilities main-
Public Health Service Policy on Humane Care and taining animals for use in DoD programs.65
536
Biosafety
THE BIOSAFETY PROFESSION
Many biological safety professionals begin their ca- professionals to meet minimum set standards of exper-
reers as bench scientists in the biological sciences, par- tise and proficiency. The ABSA, the national organiza-
ticularly microbiology, or as professionals in medicine tion of biological safety professionals, has established
or the allied health sciences, and subsequently transfer two levels of credentialing: (1) the Registered Biosafety
into the biological safety field to work as biological Professional (RBP) and (2) the Certified Biological Safety
safety officers, occupational health and safety manag- Professional (CBSP). The RBP is an individual with a
ers or specialists, or in closely related positions. With documented university education or specialized train-
the quickening tempo of biological defense research ing in relevant biological safety disciplines who has sub-
and the establishment of new, high, biocontainment mitted an application and has been found to be eligible
laboratories, the demand for competent biological for registration by the ABSA RBP Evaluation Review
safety professionals is increasing. Academic institu- Panel.67 The RBP has sufficient understanding of cell bi-
tions and government agencies are beginning to ology, pathogenic microbiology, molecular genetics, host
recognize the need to establish didactic and practical immune responses, and concepts of infectious agent
training opportunities in biological safety. For example, transmission to enable the RBP to apply safeguards
the Division of Occupational Health and Safety and the when working with biohazardous materials.
National Institute of Allergy and Infectious Diseases The CBSP is an individual who has a combination
of NIH have jointly established a National Biosafety of documented university education, specialized
and Biocontainment Training Program offering 2-year training, and experience in relevant biological safety
postbaccalaureate and postdoctoral fellowships at the disciplines, and has further demonstrated knowledge
NIH campus in Bethesda, Maryland. This program and proficiency by passing the Specialist Microbiolo-
specifically trains fellows to support BSL-3 and BSL-4 gist in Biological Safety Microbiology examination ad-
research environments by acquiring the necessary ministered by the National Registry of Microbiologists
knowledge and skills to meet scientific, regulatory, of the American Society for Microbiology. Every 5
biocontainment, biosafety, engineering, communica- years, qualification as a Specialist Microbiologist may
tions, management, and public relations challenges be renewed by submitting to the National Registry
associated with conducting research in such facilities.66 of Microbiologists evidence of acceptable continuing
An example of an academic fellowship program is the education credits or by retaking and passing the ex-
biosafety fellowship program at Washington Univer- amination. The CBSP also participates in a certification
sity School of Medicine in St. Louis, Missouri. maintenance program administered by ABSA in which
Credentialing biological safety professionals is not the individual submits a certain number of accept-
currently mandated or regulated. A formal, voluntary able certification maintenance points every 5 years to
credentialing process exists to enable biological safety maintain certification.
SUMMARY
A successful biosafety program is based on an force. Presentation of concepts must be expressed in
early estimation of risk and application of appropriate understandable terms. Initial and refresher training
containment and protective measures. It is important of personnel must address elements of biosafety and
to review safety incidents that occur in the institute, the institute s biological safety program. To promote
because these data will heighten individual awareness audience attentiveness, participation, and retention
that accidents do happen despite implementing safe- of information, training is best conducted in an infor-
guards. The goals of a biosafety program are to: mal discussion format. Training success is gauged by
how well the work force collectively internalizes the
" facilitate safe, productive research; biosafety program, as evaluated within the overall
" prevent environmental contamination; context of a positive safety culture that permeates all
" conform to prudent biosafety practices; and work attitudes and operations. Elements of a positive
" comply with federal, state, and local regula- safety culture include the following68:
tions and guidelines.
" applying (regularly) safety practices and using
To achieve the goals of the biosafety program, infor- safety terms in the workplace;
mation pertaining to the program must be conveyed " including safety practices in the employee s
to the work force, along with how it benefits the work job description and performance appraisals;
537
Medical Aspects of Biological Warfare
" specifying and monitoring safe behaviors in Management must consider safety a top prior-
the workplace; ity and work closely on a daily basis with safety
" providing tangible rewards for promoting safety; professionals, who need management s support
" articulating safety concerns in interactions on policies to be implemented. Management must
with management, peers, and subordinates; provide a safety program, engineering features, and
" emphasizing safety procedures when starting equipment designed to reduce research-associated
new tasks; risks in the institute. Biosafety professionals strive
" briefing employees on safety procedures and to provide reasonable assurance of biological safety,
the consequences of ignoring safety practices but cannot guarantee absolute safety. In the end, the
or engaging in unsafe behaviors; success of the safety program depends on the em-
" observing, reporting, and correcting hazards ployees themselves. Safety is as much an individual
promptly; and responsibility as any other assigned performance
" using PPE appropriately (always). objective.
Acknowledgments
Opinions, interpretations, conclusions, and recommendations are those of the authors and are not
necessarily endorsed by the US Army and Midwest Research Institute. Research was conducted in
compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals
and experiments involving animals, and adheres to principles stated in the Guide for the Care and Use
of Laboratory Animals.40 The facility where this research was conducted is fully accredited by AAALAC
International.
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