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OCR for page 141
Appendix B
1988 Agent Summary Statement
for HIVs, Including HTEV-IU,
LAV, HIV-l, and HIV-2
CONTENTS
Introduction
HIV Agent Summary Statement:
Agent: HIVs Including HTLV-III,
LAV, H]V-1, and H]V-2 142
Laboratory Hazards 143
Recommended Precautions .. 143
........... 142
Addendum 1 . e ee e e e e e e e e e e e e e e e e ee e e e e e 145
Laboratory Biosafety Level Criteria . . . 145
Biosafety Level 2 . .
Biosafety Level 3 . .
Vertebrate Animal Biosafety
LevelCriterm
Animal Biosafen,~ Level 2
Animal Biosafen,r Level 3
Addendum 2 .....
References
... 145
... 147
.. 149
. 149
.. 150
............. 152
... 152
Reprinted from Morbidity and Mortality Weeldy Report, 1988; 37(no. S4):1-17. The information and r=endations contained in this
appendix were developed and compiled by (1) the Division of Safety, National Institute of Allergy and Infectious Diseases, the National
Cancer Institute, and the Clinical Center of the National Institutes of Health; (2) the Food and Drug Administration; and (3) the following
units of the Centers for Disease Control: AIDS Program, Hospital Infections Program, and Office of the Director, Center for Infectious
Diseases; the Training and Laboratory Program Office; and the Office of Biosafety, Office of the Centers Director. Representatives of the
following organizations also collaborated in the effort: American Academy of Microbiology, American Biological Safety Association,
American Society for Microbiology, American Society for Clinical Pathology, Association of State and Territorial Public Health
Laboratory Directors, College of American Pathologists, Pharmaceutical Manufacturers Association, and Walter Reed Army Institute for
Research.
141
OCR for page 142
142
1988 AGENT SUMMARY STATEMENT
FOR HIVs, INCLUDING HTLV-m, LAY,
HIV-1, AND HIV-2
INTRODUCTION
In l9X4, the Centers for Disease Control (CDC)
and the National Institutes of Health ~H), in con-
sultation with experts from academic institutions,
industry, and government, published the book Bio-
safety in Microbiological and Biomedical Laborato-
ries ("Guidelines"~* (1~.
These Guidelines are based on combinations of
standard and special practices, equipment, and facili-
ties recommended for use in working with infectious
agents in various laboratory settings. The recommen-
dations are advisory; they provide a general code for
operating microbiologic and biomedical laboratories.
One section of the Guidelines is devoted to stan-
dard and special microbiologic practices, safety equip-
ment, and facilities for biosafety levels (BSL) 1
through 4. Another section contains specific agent
summary statements, each consisting of a brief de-
scription of laboratory-associated infections, the na-
ture of laboratory hazards, and recommended pre-
cautions for working with the causative agent. The
authors realized that the discovery of the availability
of information about these agents would necessitate
updating the agent summary. Such a statement for
human immunodeficiency virus ~V) (called HTLV-
III~AV when the Guidelines were published) was
published in Morbidity and Mortality Weekly Report
(MMW R) in 1986 (2~. The HIV agent summary state-
ment printed in this Supplement updates the 1986
statement.
Attached to He updated HIV agent summary state-
ment are the essential elements for BSL 2 and 3
laboratories, reproduced from the Guidelines (1) (see
Addendum 1, p. 145~. BSL 2 and 3 laboratory de-
scriptions are included because they are recommended
*Available from:
Superintendent of Documents
U.S. Govenunent Printing Office
Washington, DC 20402
Stock ft01702300167-1
APPENDIX B
for laboratory personnel working with HIV, depend-
ing on the concentration or quantity of virus or the
type of laboratory procedures used.
Ibe HIV agent summary statement does not
specifically address safety measures for collecting
and handling clinical specimens. Nonetheless, it has
been recommended Hat blood and body-fluid pre-
cautions consistently be used for ALL specimens
from ALL patients. This approach, referred to as
"universal blood and body-fluid precautions" or
"universal precautions," eliminates the need to iden-
tify all patients infected with HIV (or other blood-
borne pathogens) (3~. This subject is also covered in
other publications (3-~.
Laboratory directors, supervisors, and others are
asked to attach a copy of this revised "1988 Agent
Summary Statement for HIVs" to each copy of the
Guidelines and to all copies of their laboratory bio-
safety manual; they should review the recommended
precautions with laboratory personnel, provide ap-
propriate training in practices and operation of facili-
ties, and ensure that all personnel demonstrate profi-
ciency BEFORE being allowed to work with HIV.
The laboratory director (or the designated laboratory
supervisor) is responsible for biosafety in the labora-
tory and must establish and implement practices,
facilities, equipment, training, and work assignments
as appropriate (9~.
HIV AGENT SUMMARY STATEMENT
AGENT: HIVS INCLUDING HTLV-m, LAY,
HIV-1, AND HIV-2
In the period 1984-1986, several health-care
workers (HCWs) who had no recognized risk behav-
ior for acquired immunodeficiency syndrome (AIDS)
were reported to have HIV infection (10-15~. Only
one of these HCWs was identified as a laboratory
worker. These and other reports assessed the risk of
work-related HIV infection for all HCWs as being
very low (3,6,10-12,14-18~.
National Technical Infonnation SeIvice
U.S. Department of Commerce
5282 Port Royal Road
Springfield, VA 22161
Stock #PB84-206879
OCR for page 143
APPENDIX B
In 1985, anecdotal reports were received indi-
cating that workers in two different HIV-reagent-
production laboratories had been exposed to droplets
and splashed liquid from a vessel containing concen-
trated virus. One of several workers had been cut by
glass from a broken carboy that contained HIV-in-
fected cells and medium. None of the persons ex-
posed in these episodes had developed antibody to
HIV or had clinical signs of infection 18 and 20
months, respectively, after the reported exposure.
In 1987, CDC received reports that three HCWs
had HIV infection; none of the infections were asso-
ciated with needlesticks or cuts. Two of these HCWs
were clinical laboratory workers (11~. One was a
phlebotomist whose face and mouth were splattered
with a patient's blood when the rubber stopper was
suddenly expelled from a blood-collection tube. The
second was a medical technologist who inadvertently
spilled blood on her arms and forearms while using
an apheresis apparatus to process blood from an HIV
· · -
seroposltlve patient.
In September 1987, a production-laboratory
worker was reported to have HIV infection (18~.
This person worked with large concentrations of HIV
in a BSL 3 facility. HIV was isolated from the
worker's blood; the isolate was genetically indistin
guishable from the strain of virus being cultivated in
the laboratory. No risk factors were identified, and
the worker recalled no specific incident that might
have led to infection. However, there were instances
of leakage of virus-positive culture fluid from equip
ment and contamination of the work area and centri
fuge rotors. The report concluded that the most plau
sible source of exposure was contact of the worker's
gloved hand with virus-culture supernatant, followed
by inapparent exposure to skin.
In October 1987, a second person who worked
in another HIV production facility was reported to
have HIV infection (18~. This laboratory was a well
equipped BSL 3 facility, and BSL 3 practices were
being followed. This worker reported having sus
tained a puncture wound to a finger while cleaning
equipment used to concentrate HIV.
LABORATORY HAZARDS
.
143
mentally infected nonhuman primates. In the labora-
tory, virus should be presumed to be present in all
HIV cultures, in all materials derived from HIV cul-
tures, and in/on all equipment and devices coming
into direct contact with any of these materials.
In the laboratory, the skin (especially when
scratches, cuts, abrasions, dermatitis, or other lesions
are present) and mucous membranes of the eye, nose,
mouth, and possibly the respiratory tract should be
considered as potential pathways for entry of virus.
Needles, sharp instruments, broken glass, and other
sharp objects must be carefully handled and properly
discarded. Care must be taken to avoid spilling and
splashing infected cell-culture liquid and other virus-
containing materials.
RECOMMENDED PRECAUTIONS
1. BSL 2 standards and special practices, con-
tainment equipment, and facilities, as described in
the CDC-NIH publication Bioso~fety in Microbiologi-
cal and Biomedical Laboratories (`'Guidelines"), are
recommended for activities involving all clinical
_
specimens, body fluids, and tissues from humans or
from infected or inoculated laboratory animals. These
are the same standards and practices recommended
for handling all clinical specimens. For example, and
for emphasis:
a. Use of syringes, needles, and other sharp
instruments should be avoided if possible. Used
needles and disposable cutting instruments should
be discarded into a puncture-resistant container
with a lid. Needles should not be re-sheathed,
bent, broken, removed from disposable syringes,
or otherwise manipulated by hand.
b. Protective gloves should be worn by all
personnel engaged in activities that may involve
direct contact of skin with potentially infectious
specimens, cultures, or tissues. Gloves should
be carefully removed and changed when they
are visibly contaminated. Personnel who have
dermatitis or other lesions on the hands and who
may have indirect contact with potentially infec-
tious material should also wear protective gloves.
Hand washing with soap and water immediately
after infectious materials are handled and after
work is completed-EVEN WHEN GLOVES
HAVE BEEN WORN as described above-
should be a routine practice.
Human immunodeficiency virus has been iso-
lated from blood, semen, saliva, tears, urine, cerebro-
spinal fluid, amniotic fluid, breast milk, cervical
secretions, and tissue of infected persons and experi
OCR for page 144
144
c. Generation of aerosols, droplets,
splashes, and spills should be avoided. A bio
logical safety cabinet should be used for all pro
cedures that might generate aerosols or droplets
and for all infected cell-culture manipulations.
The Guidelines (pp. 11-13) contain additional
precautions for operating at BSL 2.
2. Activities such as producing research-labo-
ratory-scale amounts of HIV, manipulating concen-
trated virus preparations, and conducting procedures
that may produce aerosols or droplets should be per-
formed in a BSL 2 facility with the additional prac-
tices and containment equipment recommended for
BSL 3 (19) (Guidelines, pp. 14-17~.
3. Activities involving industrial-scale, large-
volume production or high concentration and ma-
nipulation of concentrated HIV should be conducted
in a BSL 3 facility using BSL 3 practices and equip-
ment (19~.
4. BSL 2 practices, containment equipment, and
facilities for animals are recommended for activities
involving nonhuman primates and any animals ex-
perimentally infected or inoculated with HIV. Be-
cause laboratory animals may bite, throw feces or
urine, or expectorate at humans, animal-care person-
nel, investigators, technical staff, and other persons
who enter the animal rooms should wear coats, pro-
tective gloves, coveralls or uniforms, and-as appro-
priate-face shields or surgical masks and eye shields
to protect the skin and mucous membranes of the
eyes, nose, and mouth.
5. All laboratory glassware, disposable mate-
rial, and waste material suspected or known to con-
tain HIV should be decontaminated, preferably in an
autoclave, before it is washed, discarded, etc. An
alternate method of disposing of solid wastes is in-
cineration.
6. Laboratory workers should wear laboratory
coats, gowns, or uniforms when working with HIV
or with material known or suspected to contain HIV.
There is no evidence that laboratory clothing poses a
risk for HIV transmission; however, clothing that
becomes contaminated with HIV preparations should
be decontaminated before being laundered or dis-
carded. Laboratory personnel must remove labora-
tory clothing before going to nonlaboratory areas.
7. Work surfaces should be decontaminated
with an appropriate chemical germicide after proce-
dures are completed, when surfaces are overdy con
APPENDIX B
laminated, and at the end of each work day. Many
commercially available chemical disinfectants (5,20-
23) can be used for decontaminating laboratory work
surfaces, for some laboratory instruments, for spot
cleaning of contaminated laboratory clothing, and
for spills of infectious materials. Prompt decontami-
nation of spills should be standard practice.
8. Universal precautions are recommended for
handling all human blood specimens for hematol-
ogic, microbiologic, chemical, and serologic testing;
these are the same precautions for preventing trans-
mission of all bloodborne infections, including hepa-
titis B (17,21,24,25~. It is not certain how effective
56°C-60°C heat is in destroying HIV in serum
(22,23,26), but heating small volumes of serum for
30 minutes at 56°C before serologic testing reduces
residual infectivity to below detectable levels. Such
treatment causes some false-positi~re results in HIV
enzyme immunoassays (27-30) and may also affect
some biochemical assays performed on serum
(27,31,32~.
9. Human serum from any source that is used
as a control or reagent in a test procedure should be
handled at BSL 2 (Guidelines, pp. 11-13~. Adden-
dum 2 (p. 152) to this report is a statement issued by
CDC on the use of all human control and reagent
serum specimens shipped to other laboratories. The
Food and Drug Administration requires that manu-
facturers of human serum reagents use a similarly
worded statement.
10. Medical surveillance programs should be in
place in all laboratories that test specimens, do re-
search, or produce reagents involving HIV. The na-
ture and scope of a surveillance program will vary
according to institutional policy and applicable local,
state, and Federal regulations (9~.
11. If a laboratory worker has a parenteral or
mucous-membrane exposure to blood, body fluid, or
viral-culture material, the source material should be
identified and, if possible, tested for the presence of
virus. If the source material is positive for HIV anti-
body, virus, or antigen, or is not available for exami-
nation, the worker should be counseled regarding the
risk of infection and should be evaluated clinically
and serologically for evidence of HIV infection. The
worker should be advised to report on and to seek
medical evaluation of any acute febrile illness that
occurs within 12 weeks after the exposure (3~. Such
an illness-particularly one characterized by fever,
rash, or lymphadenopathy~nay indicate recent HIV
OCR for page 145
APPENDIX B
infection. If seronegative, the worker should be re-
tested 6 weeks after the exposure and periodically
thereafter (e.g., at 12 weeks and 6 months after expo-
sure). During this follow-up periokespecially dur-
ing the fast 6-12 weeks after exposure, when most
infected persons are expected to show serologic evi-
dence of infection-exposed workers should be coun-
seled to follow Public Health Service recommenda-
tions for preventing transmission of HIV (3,14,25,33~.
It is recommended that all institutions establish writ-
ten policies regarding the management of laboratory
exposure to HIV; such policies should deal with con-
O~dentiality, counseling, and other related issues.
12. Other primary and opportunistic pathogenic
agents may be present in the body fluids and tissues
of persons infected with HIV. Laboratory workers
should follow accepted biosafety practices to ensure
maximum protection against inadvertent laboratory
exposure to agents that may also be present in clini-
cal specimens (34-36~.
13. Unless otherwise dictated by institutional
policy, the laboratory director (or designated labora-
tory supervisor) is responsible for carrying out the
biosafety program in the laboratory. In this regard,
the laboratory director or designated supervisor should
establish the biosafety level for each component of
the work to be done and should ensure that facilities
and equipment are adequate and in good working
order, that appropriate initial and periodic training is
provided to the laboratory staff, and that recom-
mended practices and procedures are strictly followed
(9~.
14. Attention is directed to a "Joint Advisory
Notice" of the Departments of Labor and Health and
Human Services (9) that describes the responsibility
of employers to provide "safe and healthful working
conditions" to protect employees against occupational
infection with HIV. The notice defines three expo-
sure categories of generic job-related tasks and
describes the protective measures required for em-
ployees involved in each exposure category. These
measures are: administrative measures, training and
education programs for employees, engineering
controls, work practices, medical and health-care
practices, and record-keeping. The recommendations
in this report are consistent with the "Joint Advisory
Notice"; managers/directors of all biomedical labora-
tories are urged to read this notice.
}45
ADDENDUM 1
LABORATORY BIOSAFETY LEVEL
CRITERIA
Biosafeb Level 2
Biosafety Level 2 is similar to Level 1 and is
suitable for work involving agents that represent a
moderate hazard for personnel and the environment.
It differs in that
a. laboratory personnel have specific training
in handling pathogenic agents and are di-
rected by competent scientists,
b. access to the laboratory is limited when work
is being conducted, and
c. certain procedures in which infectious aero-
sols are created are conducted in biological
safety cabinets or other physical containment
equipment.
The following standard and special practices,
safety equipment, and facilities apply to agents as-
signed to Biosafety Level 2:
A. Standard microbiologicalpractices
1. Access to the laboratory is limited or restricted
by the laboratory director when work with infectious
agents is in progress.
2. Work surfaces are decontaminated at least
once a day and after any spill of viable material.
3. All infectious liquid or solid waste is decon-
taminated before being disposed of.
4. Mechanical pipetting devices are used; mouth
pipetting is prohibited.
5. Eating, drinking, smoking, and applying cos-
metics are not permitted in the work area. Food must
be stored in cabinets or refrigerators designed and
used for this purpose only. Food storage cabinets or
refrigerators should be located outside the work area.
6. Persons are to wash their hands when they
leave the laboratory after handling infectious mate-
rial or animals.
7. All procedures are performed carefully to
minimize the creation of aerosols.
OCR for page 146
146
B. Specialpractices
1. Contaminated materials that are to be decon-
taminated away from the laboratory are placed in a
durable, leakproof container that is closed before
being removed from the laboratory.
2. The laboratory director limits access to the
laboratory. In general, persons who are at increased
risk of acquiring infection or for whom infection
may be unusually hazardous are not allowed in the
laboratory or animal rooms. The director has the
final responsibility for assessing each circumstance
and determining who may enter or work in the labo-
ratory.
3. The laboratory director establishes policies
or procedures whereby only persons who have been
advised of the potential hazard and who meet any
specific entry requirements (e.g., vaccination) enter
the laboratory or animal rooms.
4. When an infectious agent teeing worked with
in the laboratory requires special provisions for entry
(e.g., vaccination), a hazard warning sign that incor-
porates the universal biohazard symbol is posted on
the access door to the laboratory work area. The
hazard warning sign identifies the infectious agent,
lists the name and telephone number of the labora-
tory director or other responsible personas), and indi-
cates the special requirements for entering the labo-
ratory.
5. An insect and rodent control program is in
effect.
6. Laboratory coats, gowns, smocks, or uni-
forms are worn while in the laboratory. Before leav-
ing the laboratory for nonlaboratory areas (e.g., cafe-
teria, library, administrative offices), this protective
clothing is removed and left in the laboratory or
covered with a clean coat not used in the laboratory.
7. Animals not involved in the work being per-
formed are not permitted in the laboratory.
S. Special care is taken to avoid having skin be
contaminated with infectious material; gloves should
be worn when handling infected animals and when
skin contact with infectious material is unavoidable.
9. All waste from laboratories and animal rooms
is appropriately decontaminated before disposal.
10. Hypodermic needles and syringes are used
only for parenteral injection and aspiration of fluids
from laboratory animals and diaphragm bottles. Only
APPENDIX B
needle-locking syringes or disposable syringe-needle
units (i.e., the needle is integral to the syringe) are
used for the injection or aspiration of infectious fluid.
Extreme caution should be used when handling
needles and syringes to avoid autoinoculation and
the generation of aerosols during use and disposal. A
needle should not be bent, sheared, replaced in the
sheath or guard, or removed from the syringe follow-
ing use. The needle and syringe should be promptly
placed in a puncture-resistant container and decon-
taminated, preferably by autoclaving, before discard
or reuse.
11. Spills and accidents that result in overt expo-
sures to infectious material are immediately reported
to the laboratory director. Medical evaluation, sur-
veillance, and treatment are provided as appropriate,
and written records are maintained.
12. When appropriate, considering the agents
handled, baseline serum samples for laboratory and
other at-risk personnel are collected and stored. Ad-
ditional serum specimens may be collected periodi-
cally, depending on the agents handled or on the
function of the facility.
13. A biosafety manual is prepared or adopted.
Personnel are advised of special hazards and are re-
quired to read instructions on practices and proce-
dures and to follow them.
C. Containment equipment
Biological safety cabinets (Class I or II) or other
appropriate personal-protection or physical-contain-
ment devices are used when:
1. Procedures with a high potential for creating
infectious aerosols are conducted. These may in-
clude centrifuging, grinding, blending, vigorous shak-
ing or mixing, sonic disruption, opening containers
of infectious materials whose internal pressures may
be different from ambient pressures, inoculating ani-
mals intranasally, and harvesting infected tissues from
animals or eggs.
2. High concentrations or large volumes of in-
fectious agents are used. Some types of materials
may be centrifuged in the open laboratory if sealed
heads or centrifuge safety cups are used and if the
containers are opened only in a biological safety
cabinet.
OCR for page 147
APPENDIX B
D. Laboratoryfaciliiies
1. The laboratory is designed so that it can be
easily cleaned.
2. Bench tops are impervious to water and resis-
tant to acids, alkalis, organic solvents, and moderate
heat.
3. Laboratory furniture is sturdy, and spaces
between benches, cabinets, and equipment are acces-
sible for cleaning.
4. Each laboratory contains a sink for hand-
washing.
5. If the laboratory has windows that open, they
are fitted with fly screens.
6. An autoclave for decontaminating infectious
laboratory wastes is available.
Biosafety Level 3
Biosafety Level 3 is applicable to clinical, diag-
nostic, teaching, research, or production facilities in
which work is done with indigenous or exotic agents
that may cause serious or potentially lethal disease as
a result of exposure by inhalation. Laboratory per-
sonnel have specific training in handling pathogenic
and/or potentially lethal agents and are supervised by
competent scientists who are experienced in working
with these agents. All procedures involving the ma-
nipulation of infectious material are conducted within
biological safety cabinets or other physical contain-
ment devices or by personnel wearing appropriate
personal-protection clothing and devices. The labo-
ratory has special engineering and design features. It
is recognized, however, that many existing facilities
may not have all the facility safeguards recommended
for Biosafety Level 3 (e.g., access zone, sealed pene-
trations, and directional airflow). In these circum-
stances, acceptable safety may be achieved for
routine or repetitive operations (e.g., diagnostic pro-
cedures involving the propagation of an agent for
identification, typing, and susceptibility testing) in
laboratories in which facility features satisfy Bio-
safety Level 2 recommendations if the recommended
"Standard Microbiological Practices," "Special Prac-
tices," and "Containment Equipment" for Biosafety
Level 3 are rigorously followed. The decision to
implement this modification of Biosafety Level 3
recommendations should be made only by the labo-
ratory director.
|47
The following standard and special practices,
safety equipment, and facilities apply to agents as-
signed to Biosafety Level 3:
A. Stand`~rd~rucrobiologicalpractices
1. Work surfaces are decontaminated at least
once a day and after any spill of viable material.
2. All infectious liquid or solid waste is decon-
taminated before being disposed of.
3. Mechanical pipetting devices are used; mouth
pipetting is prohibited.
4. Eating, drinking, smoking, storing food, and
applying cosmetics are not permitted in the work
area
5. Persons wash their hands after handling in-
fectious materials and animals and every time they
leave the laboratory.
6. All procedures are performed carefully to
minimize the creation of aerosols.
B. Specialpractices
1. Laboratory doors are kept closed when ex-
periments are in progress.
2. Contaminated materials that are to be decon-
taminated at a site away from the laboratory are
placed in a durable, leakproof container that is closed
before being removed from the laboratory.
3. The laboratory director controls access to
the laboratory and limits access only to persons whose
presence is required for program or support pur-
poses. Persons who are at increased risk of acquiring
infection or for whom infection may be unusually
hazardous are not allowed in the laboratory or animal
rooms. The director has the final responsibility for
assessing each circumstance and determining who
may enter or work in the laboratory.
4. The laboratory director establishes policies
and procedures whereby only persons who have been
advised of the potential biohazard, who meet any
specific entry requirements (e.g., vaccination), and
who comply with all entry and exit procedures enter
the laboratory or animal rooms.
5. When infectious materials or infected ani-
mals are present in the laboratory or containment
module, a hazard warning sign (incorporating the
universal biohazard symbol) is posted on all labora-
tory and animal-room access doors. The hazard warn
OCR for page 148
148
ing sign identifies the agent, lists the name and tele-
phone number of the laboratory director or other re-
sponsible personas), and indicates any special re-
quirements for entering the laboratory, such as the
need for vaccinations, respirators, or other personal-
protection measures.
6. All activities involving infectious materials
are conducted in biological safety cabinets or other
physical-containment devices within the containment
module. No work is conducted in open vessels on the
open bench.
7. The work surfaces of biological safety cabi-
nets and other containment equipment are decon-
taminated when work with infectious materials is
finished. Plastic-backed paper toweling used on non-
perforated work surfaces within biological safety
cabinets facilitates clean-up.
8. An insect and rodent control program is in
effect.
9. Laboratory clothing that protects street cloth-
ing (e.g., solid-front or wrap-around gowns, scrub
suits, coveralls) is worn in the laboratory. Laboratory
clothing is not worn outside the laboratory, and it is
decontaminated before being laundered.
10. Special care is taken to avoid skin contami-
nation with infectious materials; gloves are worn
when handling infected animals and when skin con-
tact with infectious materials is unavoidable.
11. Molded surgical masks or respirators are
worn in rooms containing infected animals.
12. Animals and plants not related to the work
being conducted are not permitted in the laboratory.
13. All waste from laboratories and animal rooms
is appropriately decontaminated before being dis-
posed of.
14. Vacuum lines are protected with high-effi-
ciency particulate air (HEPA) filters and liquid disin-
fectant traps.
15. Hypodermic needles and syringes are used
only for parenteral injection and aspiration of fluids
from laboratory animals and diaphragm bottles. Only
needle-locking syringes or disposable syringe-needle
units (i.e., the needle is integral to the syringe) are
used for the injection or aspiration of infectious fluids.
Extreme caution is used when handling needles and
syringes to avoid autoinoculation and the generation
of aerosols during use and disposal. A needle should
not be bent, sheared, replaced in the sheath or guard,
or removed from the syringe following use. The
needle and syringe should be promptly placed in a
APPENDIX B
puncture-resistant container and decontaminated,
preferably by autoclaving, before being discarded or
reused.
16. Spills and accidents that result in overt or
potential exposures to infectious material are imme-
diately reported to the laboratory director. Appropri-
ate medical evaluation, surveillance, and treatment
are provided, and written records are maintained.
17. Baseline serum samples for all laboratory
and other at-risk personnel are collected and stored.
Additional serum specimens may be collected peri-
odically, depending on the agents handled or the
function of the laboratory.
18. A biosafety manual is prepared or adopted.
Personnel are advised of special hazards and are re-
quired to read instructions on practices and proce-
dures and to follow them.
C. Containment equipment
Biological safety cabinets (Class I, II, or III) or
other appropriate combinations of personal-protec-
tion or physical-containment devices (e.g., special
protective clothing, masks, gloves, respirators, cen-
trifuge safety cups, sealed centrifuge rotors, and con-
tainment caging for animals) are used for all activi-
ties with infectious materials that pose a threat of
aerosol exposure. These include: manipulation of
cultures and of clinical or environmental material
that may be a source of infectious aerosols; the aero-
sol challenge of experimental animals; harvesting of
tissues or fluids from infected animals and embryon-
ated eggs; and necropsy of infected animals.
D. Laboratoryfacilities
1. The laboratory is separated from areas that
are open to unrestricted traffic flow within the build-
ing. Passage through two sets of doors is the basic re-
quirement for entry into the laboratory from access
corridors or other contiguous areas. Physical separa-
tion of the high-containment laboratory from access
corridors or other laboratories or activities may also
be provided by a double-doored clothes-change room
(showers may be included), airlock, or other access
facility that requires passing through two sets of doors
before entering the laboratory.
2. The interior surfaces of walls, floors, and
ceilings are water resistant so that they can be easily
cleaned. Penetrations in these surfaces are sealed or
OCR for page 149
APPENDIX B
capable of being sealed to facilitate decontaminating
the area.
3. Bench tops are impervious to water and re-
sistant to acids, alkalis, organic solvents, and moder-
ate heat.
4. Laboratory furniture is sturdy, and spaces
between benches, cabinets, and equipment are acces-
sible for cleaning.
5. Each laboratory contains a sink for washing
hands. The sink is foot-, elbow-, or automatically
operated and is located near the laboratory exit door.
6. Windows in the laboratory are closed and
sealed.
7. Access doors to the laboratory or contain-
ment module are self-closing.
8. An autoclave for decontaminating laboratory
wastes is available, preferably within the laboratory.
9. A ducted exhaust-air ventilation system is
provided. This system creates directional airflow that
draws air into the laboratory through the entry area.
The exhaust air is not recirculated to any other area of
the building, is discharged to the outside, and is dis-
persed away from occupied areas and air intakes.
Personnel must verify that the direction of the airflow
is proper (i.e., into the laboratory). The exhaust air
from the laboratory room can be discharged to the
outside without being filtered or otherwise treated.
10. The HEPA-filtered exhaust air from Class I
or Class II biological safety cabinets is discharged di-
rectly to the outside or through the building exhaust
system. Exhaust air from Class I or II biological
safety cabinets may be recirculated within the labora-
tory if the cabinet is tested and certified at least every
12 months. If the HEPA-f~ltered exhaust air from
Class I or II biological safety cabinets is to be dis-
charged to the outside through the building exhaust
system, it is connected to this system in a manner
(e.g., thimble-unit connection) that avoids any inter-
ference with the air balance of the cabinets or build-
ing exhaust system.
VERTEBRATE ANIMAL BIOSAFETY LEVEL
CRITERIA
Animal Biosafety Level 2
A. Standard practices
1. Doors to animal rooms open inward, are self-
closing, and are kept closed when infected animals
are present.
|49
2. Work surfaces are decontaminated after use
or spills of viable materials.
3. Eating, drinking, smoking, and storing of
food for human use are not permitted in animal rooms.
4. Personnel wash their hands after handling
cultures and animals and before leaving the animal
room.
5. All procedures are carefully performed to
minimize the creation of aerosols.
6. An insect and rodent control program is in
effect.
B. Specialpractices
1. Cages are decontaminated, preferably by
autoclaving, before being cleaned and washed.
2. Surgical-type masks are worn by all person-
nel entering animal rooms housing nonhuman pri-
mates.
3. Laboratory coats, gowns, or uniforms are
worn while in the animal room. This protective cloth-
ing is removed before leaving the animal facility.
4. The laboratory or animal-facility director
limits access to the animal room only to personnel
who have been advised of the potential hazard and
who need to enter the room for program or service
purposes when work is in progress. In general, per-
sons who may be at increased risk of acquiring infec-
tion or for whom infection might be unusually haz-
ardous are not allowed in the animal room.
5. The laboratory or animal-facility director es-
tablishes policies and procedures whereby only per-
sons who have been advised of the potential hazard
and who meet any specific requirements (e.g., vacci-
nation) may enter the animal room.
6. When an infectious agent in use In the an~-
mal room requires special-entry provisions (e.g., vac-
cination), a hazard warning sign (incorporating the
universal biohazard symbol) is posted on the access
door to the animal room. The hazard warning sign
identifies the infectious agent, lists the name and
telephone number of the animal-facility supervisor
or other responsible personas), and indicates the spe-
cial requirements for entering the animal room.
7. Special care is taken to avoid contaminating
skin with infectious material; gloves should be worn
when handling infected animals and when skin con-
tact with infectious materials is unavoidable.
8. All waste from the animal room is appropri-
ately decontaminated preferably by autoclaving
OCR for page 150
lS0
before being disposed of. Infected animal carcasses
are incinerated after being transported from the ani-
mal room in leakproof, covered containers.
9. Hypodermic needles and syringes are used
only for the parenteral injection or aspiration of fluids
from laboratory animals and diaphragm bottles. Only
needle-locking syringes or disposable syringe-needle
units (i.e., the needle is integral to the syringe) are
used for the injection or aspiration of infectious fluids.
A needle should not be bent, sheared, replaced in the
sheath or guard, or removed from the syringe follow-
ing use. The needle and syringe should be promptly
placed in a puncture-resistant container and decon-
taminated, preferably by autoclaving, before being
discarded or reused.
10. If floor drains are provided, the drain taps
are always fUled with water or a suitable disinfec-
tant.
11. When appropriate, considering the agents
handled, baseline serum samples from animal-care
and other at-risk personnel are collected and stored.
Additional serum samples may be collected periodi-
cally, depending on the agents handled or the func-
tion of the facility.
C. Containment equipment
Biological safety cabinets, other physical-contain-
ment devices, and/or personal-protection devices (e.g.,
respirators, face shields) are used when procedures
with a high potential for creating aerosols are con-
ducted. These include necropsy of infected animals,
harvesting of infected tissues or fluids from animals
or eggs, intranasal inoculation of animals, and ma-
nipulation of high concentrations or large volumes of
infectious materials.
D. Animalfacilities
1. The animal facility is designed and con-
structed to facilitate cleaning and housekeeping.
2. A sink for washing hands is available in the
room that houses infected animals.
3. If the animal facility has windows that open,
they are fitted with fly screens.
4. It is recommended, but not required, that the
direction of airflow in the animal facility is inward
and that exhaust air is discharged to the outside with-
out being recirculated to other rooms.
S. An autoclave that can be used for decon
APPENDIX B
laminating infectious laboratory waste is available in
the same building that contains the animal facility.
Animal Biosafety Level 3
A. Standard practices
1. Doors to animal rooms open inward, are self-
closing, and are kept closed when work with infected
animals is in progress.
2. Work surfaces are decontaminated after use
or after spills of viable materials.
3. Eating, drinking, smoking, and storing of
food for human use are not permitted in the animal
room.
4. Personnel wash their hands after handling
cultures or animals and before leaving the labora-
tory.
5. All procedures are carefully performed to
minimize the creation of aerosols.
6. An insect and rodent control program is in
effect.
B. Special practices
1. Cages are autoclaved before bedding is re-
moved and before they are cleaned and washed.
2. Surgical-type masks or other respiratory pro-
tection devices (e.g., respirators) are worn by person-
nel entering rooms that house animals infected with
agents assigned to Biosafety Level 3.
3. Wrap-around or solid-front gowns or uni-
forms are worn by personnel entering the animal
room. Front-button laboratory coats are unsuitable.
Protective gowns must remain in the animal room
and must be decontaminated before being laundered.
4. The laboratory director or other responsible
person limits access to the animal room only to per-
sonnel who have been advised of the potential hazard
and who need to enter the room for program or
service purposes when infected animals are present.
In general, persons who may be at increased risk of
acquiring infection or for whom infection might be
unusually hazardous are not allowed in the animal
room.
5. The laboratory director or other responsible
person establishes policies and procedures whereby
only persons who have been advised of the potential
hazard and meet any specific requirements (e.g., vac
cination) may enter the animal room.
OCR for page 151
APPENDIX B
6. Hazard warning signs (incorporating the uni-
versal biohazard warning symbol) are posted on ac-
cess doors to animal rooms containing animals in-
fected with agents assigned to Biosafety Level 3 are
present. The hazard warning sign should identify the
agents in use, list the name and telephone number
of the animal room supervisor or other responsible
personas), and indicate any special conditions of en-
tq into the animal room (e.g., the need for vaccina-
tions or respirators).
7. Personnel wear gloves when handling in-
fected animals. Gloves are removed aseptically and
autoclaved with other animal room waste before being
disposed of or reused.
8. All wastes from the animal room are auto-
claved before being disposed of. All animal car-
casses are incinerated. Dead animals are transported
from the animal room to the incinerator in leakproof,
covered containers.
9. Hypodermic needles and syringes are used
only for Savage or parenteral injection or aspiration
of fluids from laboratory animals and diaphragm
bottles. Only needle-locking syringes or disposable
synnge-needle units (i.e., the needle is integral to the
syringe) are used. A needle should not be bent,
sheared, replaced in the sheath or guard, or removed
from the syringe following use. The needle and sy-
ringe should be promptly placed in a puncture-resis-
tant container and decontaminated, preferably by
autoclaving, before being discarded or reused. When
possible, cannulas should be used instead of sharp
needles (e.g., for Savage).
10. If floor drains are provided, the drain traps
are always filled with water or a suitable disinfec-
tant.
11. If vacuum lines are provided, they are pro-
tected with HEPA filters and liquid disinfectant traps.
12. Boots, shoe covers, or other protective foot-
wear and disinfectant footbaths are available and
used when indicated.
C. Containment equipment
1. Personal-protection clothing and equipment
andfor other physical-containment devices are used
for all procedures and manipulations of infectious
materials or infected animals.
2. The risk of infectious aerosols from infected
animals or their bedding can be reduced if animals
are housed in partial-containment caging systems,
IS!
such as open cages placed in ventilated enclosures
(e.g., laminar-flow cabinets), solid-wall and -bottom
cages covered by filter bonnets, or other equivalent
primary containment systems.
D. Animalfacilities
1. The animal facility is designed and con-
smucted to facilitate cleaning and housekeeping and
is separated from areas that are open to unrestricted
personnel traffic within the building. Passage through
two sets of doors is the basic requirement for entry
into the animal room from access corridors or other
contiguous areas. Physical separation of the animal
room from access corridors or from other activities
may also be provided by a double-doored clothes
change room (showers may be included), airlock, or
other access facility that requires passage through
two sets of doors before entering the animal room.
2. The interior surfaces of walls, floors, and
ceilings are water resistant so that they can be cleaned
easily. Penetrations in these surfaces are sealed or
capable of being sealed to facilitate fumigation or
space decontamination.
3. A foot-, elbow-, or automatically operated
sink for handwashing is provided near each animal-
room exit door.
sealed.
4. Windows in the animal room are closed and
5. Animal room doors are self-closing and are
kept closed when infected animals are present.
6. An autoclave for decontaminating wastes is
available, preferably within the animal room. Mate-
rials to be autoclaved outside the animal room are
transported in a covered, leakproof container.
7. An exhaust-air ventilation system is provided
This system creates directional airflow that draws air
into the animal room through the entry area. The
building exhaust can be used for this purpose if the
exhaust air is not recirculated to any other area of the
building, is discharged to the outside, and is dis-
persed away from occupied areas and air intakes.
Personnel must verify that the direction of the air-
flow is proper (i.e., into the animal room). The ex-
haust air from the animal room that does not pass
through biological safety cabinets or other primary
containment equipment can be discharged to the out-
side without being filtered or otherwise treated.
8. The HEPA-filtered exhaust air from Class I
or Class II biological safety cabinets or other primary
OCR for page 152
152
containment devices is discharged directly to the
outside or through the building's exhaust system.
Exhaust air from these primary containment devices
may be recirculated within the animal room if the
cabinet is tested and certified at least every 12 months.
If the HEPA-f~tered exhaust air from Class I or
Class II biological safety cabinets is discharged to
the outside through the building exhaust system, it is
connected to this system in a manner (e.g., thimble-
unit connection) that avoids any interference with the
air balance of the cabinets or building exhaust sys
tem.
ADDENDUM 2:
CDC CAUTIONARY NOTICE
CDC cautionary notice for all human-serum-derived
reagents used as controls:
WARNING: Because no test method can
offer complete assurance that laboratory
specimens do not contain HIV, hepatitis B
virus, or other infectious agents, this speci-
men should be handled at the BSL 2 as rec-
ommended for any potentially infectious
human serum or blood specimen in the CDC-
NIH manual, Biosafety in Microbiological
and Biomedical Laboratories, 1984, pages
11-13.
If additional statements describing the results of
any heat treatment or serologic procedures already
performed on the human-serum reagent or control
are used in conjunction with the above cautionary
notice, these statements should be worded so as not
to diminish the impact of the warning that empha-
sizes the need for universal precautions.
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APPENDIX B
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APPENDIX B
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APPENDIX B
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Representative terms from entire chapter:
biological safety
