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Biosafety

Chapter 22

BIOSAFETY

CATHERINE L. WILHELMSEN, DVM, P h D, CBSP * ; and ROBERT J. HAWLEY, P h D, 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

515

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-

cal containment involving work with recombinant de-

oxyribonucleic acid (DNA) molecules and organisms

and viruses containing recombinant DNA molecules.

There are four levels of biosafety (designated 1

through 4) that define the parameters of containment

necessary to protect personnel and the environment. 1

BSL-1 is the least restrictive, whereas BSL-4 requires a

special containment or maximum containment labora-

tory facility. Positive-pressure protective suits (space

suit or blue suit) are used solely in a maximum con-

tainment, or BSL-4, laboratory. Biosafety is not possible

without proper and extensive training. The principal

investigator or laboratory supervisor is responsible

for providing or arranging for appropriate training of

all personnel within the laboratory to maintain and

sustain a safe working environment.

Biological safety, or biosafety, is the application of

concepts pertaining to risk assessment, engineering

technology, personal protective equipment (PPE),

policies, and preventive medicine to promote safe

laboratory practices, procedures, and the proper

use of containment equipment and facilities. In

biomedicine, laboratory workers apply these tenets

to prevent laboratory-acquired infections and the

release of pathogenic organisms into the environ-

ment. A biohazard is defined as any microorganism

(including, but not limited to, bacteria, viruses, fungi,

rickettsiae, or protozoa); parasite; vector; biological

toxin; infectious substance; or any naturally occur-

ring, bioengineered, or synthesized component of

any such microorganism or infectious substance that

is capable of causing the following:

• death, disease, or other biological malfunction

in humans, animals, plants, or other living

organisms;

• deleterious alteration of the environment; or

• an adverse impact on commerce or trade

agreements.

Evolution of Biosafety

Steps to limit the spread of infection were prac-

ticed in the field of biomedicine since human illness

was associated with infectious microorganisms and

biologically derived toxins. However, Fort Detrick (in

Frederick, Md) is considered the birthplace (beginning

in the 1940s) of modern biosafety as a discrete disci-

pline. During the early years of biosafety, development

of safer working practices, principles, and engineer-

ing controls was needed. 3,4 Individuals conducting

biomedical research commonly became infected with

the organism being studied. As the hazard of work-

ing with organisms increased, so did the need to

protect laboratory personnel conducting the research.

Contributions to the field of biosafety were a direct

result of the innovations and extensive experiences

of Fort Detrick personnel who worked with a variety

of infectious microorganisms and biological toxins.

Dr Arnold Wedum, director of industrial health and

safety at Fort Detrick—and regarded by many as the

father of the US biosafety profession—promoted the

attitude that biosafety should be an integral part of

biomedical research. 5

To enhance worker safety and environmental pro-

tection, Wedum 4 promoted use of the following:

The goal of handling these hazardous agents safely

can be accomplished through careful integration of ac-

cepted microbiological practices, and the primary and

secondary containments of potential biohazards.

Primary containment involves placing a barrier at

the level of the hazard, confining the material to protect

laboratory personnel and the immediate laboratory

environment through adherence to good laboratory

practices and appropriate use of engineering controls.

Examples of primary containment include biological

safety cabinets (BSCs), ventilated animal cages, and as-

sociated equipment. Secondary containment involves

protection of the environment external to the labora-

tory from exposure to infectious or biohazardous mate-

rials through facility design and operational practices.

Combinations of laboratory practices, containment

equipment, and special laboratory design are used to

achieve different levels of physical containment. (His-

torically, the designation “P” was used to indicate the

level of physical containment, such as P-1 through P-4.)

The current terminology is biosafety level or BSL. 1 The

designation BSL is used in the Biosafety in Microbiologi-

cal and Biomedical Laboratories (BMBL), 1 which focuses

on protecting laboratory employees. BL is another des-

ignation for biosafety level, used in Appendix G of the

National Institutes of Health (NIH) publication Guide-

lines for Research Involving Recombinant DNA Molecules

• class III gas-tight BSC;

• noninfectious microorganisms in recombinant

DNA research;

• P-4 (today’s BSL-4) principles, practices, and

positive-pressure protective suit facilities

when working with potential aerosol-trans-

mitted zoonotic microorganisms (eg, those

516

Biosafety

causing tularemia and Q fever if a class III

cabinet system was not available); and

• vaccinations of laboratory workers.

fixed to a frame. 10 A BSC, first developed in 1964 for a

pharmaceutical company, used HEPA filter technology

to provide clean air in the work area and containment

as the primary barrier placed at the source of hazard-

ous powders. Subsequent research led to the develop-

ment of a class II, type A BSC that was delivered to

the National Cancer Institute by the Baker Company

(Sanford, Me). 11 The National Cancer Institute also

developed a specification for the first class II, type B

console BSC. HEPA filters have been proven to be ef-

fective, economical, and reliable devices for removing

radioactive and nonradioactive particulate aerosols at

a high rate of collection frequency. 10

Operation and retention efficiency of HEPA filters

have been documented during the past years. Three

mechanisms account for the collection (retention) of

particles within HEPA filters:

Another safety enhancement was demonstrating and

publicizing the importance of prohibiting mouth

pipetting for fluid transfers involving hazardous ma-

terial. 6,7 Dr Emmett Barkley 8 reiterated the hazard of

oral pipetting, which should not be practiced in the

laboratory. Barkley was chief of the Safety Division

of the National Cancer Institute (Bethesda, Md) and

subsequently director of research safety at NIH when

the NIH Guidelines were developed and adopted. He

was instrumental in developing physical containment

parameters for recombinant DNA research. 9

Critical to the advancement of modern biosafety

was the development of air filtration technology. Dur-

ing the early 1940s, the US Army Chemical Warfare

Service Laboratories (Edgewood, Md) studied the

composition of filter paper captured from German gas

mask canisters in search of better smoke filters. These

early studies resulted in the design of collective protec-

tion filter units for use at the particulate-removal stage

by a combined chemical, biological, and radiological

purification unit of the US armed services. In the late

1940s, the Atomic Energy Commission (precursor of

the Nuclear Regulatory Commission) adopted this

type of filter to confine airborne radioactive particles

in the exhaust ventilation systems of experimental

reactors and in other areas of nuclear research. Subse-

quently, Arthur D Little Company, Inc (Boston, Mass),

and the US Naval Research Laboratory (Washington,

DC) developed a prototype glass-fiber filter paper.

Eventually, thin, corrugated, aluminum-alloy separa-

tors replaced the original asbestos, thermoplastics, and

resin-treated papers. Throughout this development

period, military specifications were developed and

implemented to ensure the safe operating and opti-

mal conditions of filters, 10 ultimately leading to the

production of high-efficiency particulate air (HEPA)

filters, which are used today in a variety of engineering

controls, as well as in laboratory heating, ventilation,

and air conditioning systems.

HEPA filters are constructed of paper-thin sheets of

borosilicate medium that are pleated to increase their

surface area. The borosilicate sheets are tightly pleated

over aluminum separators for added stability and af-

1. Small particles ranging from 0.01 to 0.2 µm

in diameter are collected in a HEPA filter by

diffusion and are retained at an efficiency

approaching 100%.

2. Particles in the respirable range (those of a size

that may be inhaled and retained in the lungs,

0.5–5.0 µm in diameter) are retained in a HEPA

filter by a combination of impaction and in-

terception at an efficiency approaching 100%.

3. Particles with an intermediate size range

(between 0.2 and 0.5 µm in diameter) are

retained by a combination of diffusion and

impaction.

The HEPA filter is least efficient at retaining particles

with a diameter of 0.3 µm, with a minimum collection

efficiency of 99.97%. Hence, a standard test of HEPA

filter efficiency uses a generated aerosol of particles

that are 0.3 µm in diameter; to pass the test, the HEPA

filter must retain 99.97% of the particles. 12

All the air exhausted from BSCs, within which

infectious materials must be manipulated, is directed

through a HEPA filter before recirculation to a labora-

tory room or discharge to the outside environment

through the building exhaust system. Therefore, in ad-

dition to adherence to rigorous work practice controls,

HEPA filtration of laboratory exhaust air provides an

extra margin of safety for workers, the laboratory areas,

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

handling any particular agent.

Multiple schemes for assigning risk groups have

been developed. The NIH Guidelines ; the American

Agents infectious to humans, including agents used

in research, are placed into risk groups based on the

danger they pose to human health. The risk group as-

517

Medical Aspects of Biological Warfare

Biological Safety Association (Mundelein, Ill); Health

Canada (Ottawa, Ontario, Canada) 13 ; other nations;

and the World Health Organization (Geneva, Swit-

zerland) 14 all have risk group paradigms. The World

Health Organization has categorized infectious agents

and biological toxins into four risk groups. These risk

groups relate to, but do not equate to, the BSLs of

laboratories designed to work with organisms in each

risk group. 14 Risk group 1 (no or low individual and

community risk) comprises microorganisms unlikely

to cause human or animal disease. Risk group 2 (mod-

erate individual risk, low community risk) includes

pathogens that can cause human or animal disease,

but are unlikely to be serious hazards to laboratory

workers, the community, livestock, or the environ-

ment. Laboratory exposures may cause serious infec-

tion, but effective treatment and preventive measures

are available, and the risk of infection spreading is

limited. An example is the causative agent of anthrax,

Bacillus anthracis , in humans and animals. Risk group

3 (high individual risk, low community risk) includes

pathogens that usually cause serious human or animal

disease, but do not ordinarily spread from one in-

fected individual to another. Effective treatment and

preventive measures are available. An example is the

causative agent of tularemia, Francisella tularensis, in

humans and animals. Risk group 4 (high individual

and community risk) pathogens usually cause serious

human or animal disease and can be readily transmit-

ted from one individual to another, either directly or

indirectly. Effective treatment and preventive measures

are not normally available. Examples include Variola

virus, Ebola virus, Lassa fever virus, and Marburg

fever virus. The relationship of risk groups and BSLs,

practices, and equipment is illustrated in Table 22-1.

How Agents Are Placed in Risk Groups

To assess the risk while working in a laboratory or

animal environment with a specific microorganism, the

following criteria must be considered: number of past

laboratory infections, natural mortality rate, human

infectious dose, efficacy of vaccination and treatment,

extent to which infected animals transmit the disease,

stability of the agent, and potential for exposure of

the investigator.

• Number of past laboratory infections: The

most frequent cause of laboratory-associated

infections in humans is the Brucella species. Ex-

tra caution must be taken when working with

this agent because of its low infectious dose for

humans. About 10 to 100 organisms can cause

an infection in a susceptible human host. 15

• Natural mortality rate: The natural mortality

or case-fatality rate of diseases varies widely 15

(Table 22-2).

• 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

Basic: BSL-1

Basic teaching; research Good microbiological

None; open bench work

techniques

Basic: BSL-2

Primary health services; Good microbiological

Open bench plus BSC for

diagnostic services;

techniques plus protective

potential aerosols

research

clothing; biohazard sign

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

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

518

Biosafety

TABLE 22-2

pattern (antibiogram) of the agent under

investigation. The rationale is that treatment

will be known in advance if an inadvertent

laboratory exposure occurs. Treatment for

exposure to a virus might be problematic,

because only symptomatic treatment may be

available. There are few available antiviral

agents that may be effective for postexposure

prophylaxis. Specific antiviral agents include

the following:

o rabies—rabies immune globulin for pas-

sive therapy, followed by the human dip-

loid cell rabies vaccine or rabies vaccine,

adsorbed for active vaccination;

o cercopithecine herpesvirus 1 (B virus)—valacy-

clovir hydrochloride (VALTREX; GlaxoSmith-

Kline, Research Triangle Park, NC); and

o a renaviridae and bunyaviridae (including the

viruses that cause Lassa fever, Argentine

hemorrhagic fever, and Crimean-Congo

hemorrhagic fever)—ribavirin. This mate-

rial can be used under an Investigational

New Drug (IND) protocol (in the United

States) only for empirical treatment of hem-

orrhagic fever virus patients while await-

ing identification of the etiological agent.

• Extent to which infected animals transmit the

disease: This discussion involves the zoonotic

CASE-FATALITY RATE BY DISEASE

Disease (Untreated) Organism

[Case-Fatality Rate]

Plague, bubonic

Yersinia pestis

[50%–60%]

Cholera

Vibrio cholerae

[50% or more]

Tularemia,

Francisella

[30%–60%]

pulmonary tularensis

Anthrax, cutaneous Bacillus anthracis

[5%–20%]

Tularemia, typhoidal Francisella

[5%–15%]

tularensis

Brucellosis

Brucella species

[2% or less]

( melitensis )

Q fever

Coxiella burnetii

[1%–2.4%]

organism having a low infectious dose for

humans will place the laboratory worker at a

greater risk than working with an organism

having a higher infectious dose. The infectious

dose of organisms for humans varies and is

also dependent on the immunological com-

petency of the host (Table 22-3). Although

the literature contains information about the

potential infectious dose for humans as ex-

trapolated from animal data (see Table 22-3),

an attempt to provide quantitative human

infectious doses is not possible. 16

• Efficacy of vaccination and treatment (if either

of these is available): Vaccines are available for

some of the agents studied within the labora-

tory. Receiving a vaccination must be based

on a risk assessment. Only those individuals

who are considered at risk should be offered

the vaccination. However, the potential risk of

the adverse effects from the vaccination might

outweigh the risk of acquiring an infection.

In addition, a vaccination might not provide

100% protection. An overwhelming infectious

dose can overcome the protective capacity of

a vaccination. Therefore, a vaccination should

be considered only as an adjunct to safety, not

as a substitute for safety and prudent practices.

Treatment (chemoprophylaxis) in the form

of antibiotic therapy may also be available

to treat illnesses caused by many of the

microorganisms being manipulated in the

laboratory, specifically by the bacterial and

rickettsial agents. It is necessary to deter-

mine the antibiotic sensitivity and resistance

TABLE 22-3

HUMAN INFECTIOUS DOSE BY ORGANISM

Organism

Infectious Dose Route of Exposure

Vibrio cholerae

10 8

Ingestion 1

Yersinia pestis

100–20,000 Inhalation 2

Bacillus anthracis

~ 1,300

Inhalation 3

Brucella species

10–500

Inhalation 2

( melitensis )

Francisella

Inhalation 4

tularensis

Coxiella burnetii

Inhalation 5

Clinical recognition and management of patients exposed to

biological warfare agents. JAMA . 1997;278:399–411. (3) Dull PM,

Wilson KE, Kournikakis B, et al. Bacillus anthracis aerosolization as-

sociated with a contaminated mail sorting machine. Emerg Infect Dis .

2002;8:1044–1047. (4) Jones RM, Nicas M, Hubbard A, Sylvester MD,

Reingold A. The infectious dose of Francisella tularensis (tularemia).

Appl Biosafety . 2005;10:227–239. (5) Jones RM, Nicas N, Hubbard A,

Reingold A. The infectious dose of Coxiella burnetti (Q-fever). Appl

Biosafety. 2006;11:32–41.

519

10–500

Inhalation

Data sources: (1) Sack DA, Sack RB, Nair GB, Siddique AK. Cholera.

Lancet . 2004;363:223–233. (2) Franz DR, Jahrling PB, Friedlander AM,

et al. Clinical recognition and management of patients exposed to

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