[Federal Register Volume 62, Number 81 (Monday, April 28, 1997)]
[Notices]
[Pages 23066-23079]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 97-10879]
[[Page 23065]]
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Part V
Department of Health and Human Services
_______________________________________________________________________
Centers for Diseases Control and Prevention
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Goals for Working Safely With Mycobacterium Tuberculosis in Clinical,
Public Health, and Research Laboratories; Notice
Federal Register / Vol. 62, No. 81 / Monday, April 28, 1997 /
Notices
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DEPARTMENT OF HEALTH AND HUMAN SERVICES
Centers for Disease Control and Prevention
Goals for Working Safely With Mycobacterium Tuberculosis in
Clinical, Public Health, and Research Laboratories
AGENCY: Centers for Disease Control and Prevention (CDC), Department of
Health and Human Services.
ACTION: Request for comments.
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SUMMARY: CDC requests comments concerning the updating of the Agent
Summary Statement for M. tuberculosis, currently in the 3rd edition of
Biosafety in Microbiological and Biomedical Laboratories published by
CDC and the National Institutes of Health. The next edition is
scheduled for the fall of 1998.
DATES: Written comments to this notice should be submitted to Vickie
Rathel, Office of Health and Safety (OHS), Centers for Disease Control
and Prevention (CDC), 1600 Clifton Road, NE., MS-F05, Atlanta, GA,
30333. Comments must be received on or before June 27, 1997. Comments
may also be faxed to Vickie Rathel at (404) 639-2294 or submitted by e-
mail to ([email protected]) as WordPerfect 5.0,5.1/.2,6.0,6.1, or ASCII
files.
FOR FURTHER INFORMATION CONTACT: Technical information may be obtained
from Jonathan Richmond, Ph.D. or Peg Tipple, MD, Office of Health and
Safety, Centers for Disease Control and Prevention (CDC), 1600 Clifton
Road, NE., MS-F05, Atlanta, GA, 30333, telephone (404) 639-2453.
SUPPLEMENTARY INFORMATION: CDC is requesting comments concerning the
update of the Agent Summary Statement for M. tuberculosis as published
in the 3rd edition of the CDC/NIH publication, Biosafety in
Microbiological and Biomedical Laboratories. The draft document ``Goals
for Working Safely with Mycobacterium tuberculosis Complex Species in
Clinical, Public Health, and Research Laboratories' presents background
information for this update and is presented below for public comment.
Comments or data may be submitted on the following topics (but not
limited to these): Existing reports of (1) laboratory-acquired skin
test conversions and infections, (2) causes of such conversions and
infections, (3) biosafety practices and procedures for manipulating
specimens containing M. tuberculosis, and (4) facility evaluations and
recommendations for improvement, including cost estimates.
Dated: April 21, 1997.
Joseph R. Carter,
Acting Associate Director for Management and Operations, Centers for
Disease Control and Prevention (CDC).
Goals for Working Safely With Mycobacterium tuberculosis Complex
Species in Clinical, Public Health, and Research Laboratories
Summary
The Mycobacterium tuberculosis complex includes four species:
Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium
africanum, and Mycobacterium microti. With the exception of M. microti,
all species are pathogenic for humans. The risk for becoming infected
with species of the M. tuberculosis complex is high for those who work
in mycobacteriological laboratories. Therefore, all cultures or
specimens suspected of containing acid-fast bacilli must be manipulated
in settings where specific engineering controls, administrative
procedures and appropriate personal work practices ensure containment
of the organism and protection of workers from exposure. When these
controls and procedures are implemented and protective measures are
followed, laboratorians can substantially reduce their risk for
becoming infected. This report updates and expands those sections of
Biosafety in Microbiological and Biomedical Laboratories (BMBL),
published by CDC and the National Institutes of Health, that address
precautions that must be taken to manipulate Mycobacterium species
safely in the laboratory.
Introduction
CDC and the National Institutes of Health (NIH) jointly issue
laboratory safety guidelines in a publication entitled Biosafety in
Microbiology and Biomedical Laboratories (BMBL) (1). The BMBL is re-
published, with updated information approximately every five years. It
provides specific guidelines for laboratories that work with infectious
organisms. The BMBL includes safety recommendations for laboratory
managers and personnel who work with M. tuberculosis complex species.
Because until recently there had been few changes in the techniques
available to laboratorians working with M. tuberculosis, these
recommendations have remained the same through the last 3 editions of
the BMBL, with no significant revisions since 1981.
Recent changes in public health recommendations for use of rapid
laboratory diagnostic procedures and the development of new
technologies led CDC and a group of consulting laboratorians to review
existing safety guidelines for working with M. tuberculosis (2,3,4).
Revisions were presented and discussed at the Second National
Conference on Laboratory Aspects of Tuberculosis, convened by the
Association of State and Territorial Public Health Laboratory Directors
(ASTPHLD) and the CDC in April 1995 (5).
This report updates and expands those sections of the BMBL that
address engineering controls, administrative practices, and specific
procedures for laboratorians who manipulate clinical specimens and
purified cultures of M. tuberculosis, M. africanum, and M. bovis (the
three species of the M. tuberculosis complex that pose an infectious
hazard to personnel in clinical and research laboratories) (6).
Intended Use of This Document
This document is intended to be used in conjunction with the BMBL
and the other references. Together these documents provide guidelines
for persons responsible for the design, maintenance and use of
laboratories doing diagnostic or research work with M. tuberculosis
complex species. It is recognized that not all current TB laboratories
have all of the facilities and equipment recommended, particularly for
activities that should be carried out under biosafety level 3 (BL-3)
conditions (1). Those laboratories should carefully review their
facilities, equipment, policies and procedures to ensure that current
activities are accomplished with the smallest risk to employees and
others, and should proceed as quickly as possible to upgrade systems as
necessary to meet the current recommendations. Those laboratories with
seriously deficient facilities should discontinue high risk procedures
until improvements are made.
Background
M. tuberculosis Complex in the Clinical Laboratory--Risks for
Laboratory Workers
The M. tuberculosis complex species are usually transmitted by the
aerosol route; percutaneous injection may lead to localized infections
before dissemination. The infectious dose of M. tuberculosis is low for
humans (i.e., 1-10 bacilli carried in 1-3 droplet nuclei (7,8)).
Specimens considered to be potential sources for laboratory
transmission are sputum, fluids collected by gastric or bronchial
lavage, cerebrospinal fluid, urine, and caseous lesions in tissues
(9,10,11).
[[Page 23067]]
The incidence of tuberculosis among persons who work with M.
tuberculosis in the laboratory is three to five times greater than that
among laboratory personnel who do not manipulate this bacterium
(12,13,14,15). Data from one study indicate that the frequency of
infection for persons who manipulate M. tuberculosis is 100 times
greater than for the general population (12).
Kubica (16) described 13 separate incidents in which 80 of 291
(27%) exposed laboratorians developed positive tuberculin skin tests
following specific incidents. Eight of the incidents involved poor
directional airflow in the laboratory, five were associated with
failures of the biological safety cabinets, one was associated with an
autoclave failure, and the other was due to equipment failure. Two
additional incidents of poor directional airflow in clinics resulted in
64/166 (39%) conversions.
Two reports of laboratory-acquired tuberculosis tuberculin
conversions have been reported in Minnesota hospital laboratorians
(17). One case of pulmonary tuberculosis (possibly due to inadequate
compliance with safety guidelines) and a second laboratory-associated
infection (an autoinoculation incident resulting in a granuloma) have
been reported in 1995 in another hospital laboratory (18). A more
recent report (19) indicates seven laboratory-acquired skin test
conversions in nine diagnostic laboratories handling M. tuberculosis
specimens.
Under-reporting of laboratory-acquired infections appears to be the
rule, rather than the exception. Of the 15 incidents reported by
Kubica, none had been previously reported in the literature; he further
suggests from anecdotal reports that 8-30% of laboratories may
experience tuberculin conversions (16). CDC continues to periodically
receive requests to assist laboratories experiencing similar
conversions, but the facilities have been reluctant to publish their
experiences.
The risks to laboratory workers depend on how frequently specimens
positive for M. tuberculosis are processed in the laboratory, the
concentration of organisms in specimens, the number of specimens
handled by an individual worker, and safety practices in the laboratory
(19,20). Exposure to laboratory-generated aerosols created while
performing routine procedures is the most serious of the hazards
encountered by laboratory personnel (9,10,11,21,22,23). Some aerosol-
generating procedures that produce droplet nuclei in the respirable
range include: (a) Pouring liquid cultures and supernatant fluids, (b)
using fixed-volume automatic pipettors, (c) mixing liquid cultures with
a pipette, (d) preparing specimen and culture smears, (e) dropping
tubes or flasks containing cultures, (f) spilling suspensions of
bacilli, (g) breaking tubes during centrifugation, (h) preparing frozen
sections, (i) cutting or sawing through tissue specimens that have not
been fixed, and (j) homogenizing tissues for primary culture
(24,25,26,27,27A).
Needle stick and other cutaneous injuries have been uncommon causes
of laboratory acquired M. tuberculosis infection. However, with
increasing use of rapid culture techniques (e.g., BACTECTM),
recent needle stick-associated M. tuberculosis infections have been
reported (19).
Until recently, blood has not been considered a source of
laboratory transmission of M. tuberculosis (or M. bovis) partly because
mycobacteremia is transient in immunocompetent hosts. However, with the
emergence of human immunodeficiency virus/acquired immunodeficiency
syndrome (HIV/AIDS), mycobacteremia caused by M. tuberculosis has
occurred more frequently and blood is now considered a potential source
of transmission in the laboratory (29,30).
All clinical specimens suspected to be positive for M. tuberculosis
must be considered potentially infectious and must be handled according
to the recommended precautions for blood-borne pathogens (30) and in
such a way that aerosolization is minimized (9,22,23,31).
Biosafety Levels
Microbiology laboratories are special, often unique, work
environments that may pose identifiable infectious disease risks to
persons in or near them. Infections have been contracted in these
laboratories throughout the history of microbiology. A review of the
literature on such laboratory acquired infections is included in the
introductory chapter of the BMBL and in papers by Kruse and Sewell
(1,9,31). The literature, along with considerable anecdotal
information, suggests that most laboratory acquired infections occur
when the mode of transmission is unknown (as may occur with a newly
recognized pathogen), or as a result of error, accident, or
carelessness in the handling of a known pathogen.
During the 1970's, in an effort to diminish the risks of infection
in the laboratory, scientists devised a system for categorizing
etiologic agents into groups based on the mode of transmission, type
and seriousness of illness resulting from infection, availability of
treatment (e.g., antimicrobial drugs), and availability of prevention
measures (e.g., vaccination). The etiologic agent groupings were the
basis for the development of guidelines for appropriate facilities,
containment equipment, procedures and work practices to be used by
laboratorians working with the various organisms. These guidelines are
now referred to as biosafety levels (BL) 1-4.
BL-1
BL-1 defines conditions suitable for work involving well-
characterized microorganisms not known to cause disease in healthy
adult humans, and of minimal potential hazard to laboratory personnel
and the environment. The laboratory is not necessarily separated from
the general traffic patterns in the building. Work is generally
conducted on open bench tops using standard microbiological practices.
Special containment equipment or facility design is not required nor
generally used. Laboratory personnel have specific training in the
procedures conducted in the laboratory and are supervised by a
scientist with general training in microbiology or a related science.
BL-2
BL-2 is similar to BL-1 and is suitable for work involving agents
of moderate potential hazard to personnel and the environment. It
differs from BL-1 in that: (a) Laboratory personnel have specific
training in handling pathogenic agents and are directed by competent
scientists; (b) access to the laboratory is limited when work is being
conducted; (c) extreme precautions are taken with contaminated sharp
items; and (d) certain procedures in which infectious aerosols or
splashes may be created are conducted in a biological safety cabinet
(BSC) or other physical containment equipment. There is no
specification in the BMBL (1) for single-pass directional inward flow
of air for BL-2. However, most microbiology laboratories also work with
potentially hazardous chemicals. There are published recommendations
(32) for preventing build-up of chemical vapors in laboratories; this
can be accomplished by using chemical fume hood and/or having single-
pass air when recirculation would increase the ambient concentration of
hazardous materials.
BL-3
BL-3 is applicable to clinical, diagnostic, teaching, research, or
production facilities in which work is done with indigenous or exotic
agents which may cause serious or potentially
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lethal diseases as a result of exposure by the inhalation route. M.
tuberculosis is representative of microorganisms transmissible by the
aerosol route that are assigned to this level. Primary hazards to
personnel working with these agents relate to exposure to infectious
aerosols, autoinoculation, and ingestion. Laboratory personnel must
have specific training in handling pathogenic and potentially lethal
agents, and are supervised by competent scientists who are experienced
in working with these agents.
More emphasis is placed on primary and secondary barriers at BL-3
to protect personnel in contiguous areas and in the community from
exposure to potentially infectious aerosols, and to prevent
contamination of the environment. The laboratory has special
engineering and design features to provide a total environment aimed at
the control of infectious aerosols.
The BL-3 laboratory is separated from other parts of the building
by an anteroom with two sets of doors, or by access through a BL-2
area. Because of the potential for aerosol transmission, air movement
is unidirectional into the laboratory (i.e., from clean areas into the
BL-3 area) and all exhaust air from the BL-3 area is directed outside
the building without any recirculation. All procedures at BL-3
involving the manipulation of infectious materials are conducted within
BSCs or other physical containment devices. Personnel wear appropriate
personal protective clothing and equipment while in the BL-3
laboratory.
BL-3 facilities have solid floors and ceilings and sealed
penetrations. They are designed and maintained to allow appropriate
decontamination in the event of a significant spill.
BL-3 laboratories have single pass air, i.e., non-recirculating air
ventilation systems, to protect personnel. Filtration of exhaust air
through high efficiency particulate air (HEPA) filters is neither
required nor recommended in most situations. Single pass air that mixes
with outside air allows for the rapid dilution of the small numbers of
microorganisms that may be released in the laboratory.
All waste from the BL-3 laboratory must be autoclaved before being
discarded into routine disposal containers.
BL-4
BL-4 is required for work with dangerous and exotic agents which
pose a high individual risk of aerosol-transmitted laboratory
infections and life-threatening diseases. Within work areas of the
facility, all activities are confined to Class III biological safety
cabinets, or Class II biological safety cabinets used by workers
wearing one-piece positive-pressure body suits ventilated by a life
support system. Members of the laboratory staff have specific and
thorough training in handling extremely hazardous infectious agents;
and they understand the primary and secondary containment functions of
the standard and special practices, the containment equipment, and the
laboratory design characteristics. They are supervised by competent
scientists who are trained and experienced in working with these
agents.
All wastes are decontaminated before leaving the BL-4 laboratory,
and air is exhausted from the BL-4 area through HEPA filters.
Relationship of the BMBL BL to the American Thoracic Society Levels of
Service
The current agent summary statement published in BMBL recognizes
the ``levels of service'' concept for clinical mycobacteriology
laboratories that was first proposed in 1967 (33) and accepted in 1983
by the American Thoracic Society (ATS)(21,34). The ``levels of
service'' approach to laboratory services remains standard today,
although increased workloads, new techniques, need for faster results
for management of complicated cases, and economic considerations are
forcing reconsideration of the concept (2,4, 5, 35, 36, 37). However,
BSL recommendations are based on risks related to laboratory
procedures, so if/when a laboratory changes the services it provides,
laboratory activities can be re-assessed and facilities, equipment and
work practices modified, if necessary, using the BMBL as a guideline.
Determining the Type of Tuberculosis Laboratory Needed for a Facility
Decisions on the type of laboratory for a given facility must be
based on an assessment of the extent of tuberculosis activities that
will be carried out in that laboratory. The assessment must include
issues such as expected workload, personnel training and experience,
risks of the various laboratory procedures, and availability of
appropriate space and required equipment.
Assessment of Proficiency in the Mycobacteriology Laboratory
Although this document emphasizes appropriate facilities,
equipment, and safe work practices, the laboratory workload must also
be considered in deciding what to include in a new or renovated
mycobacteriology laboratory.
Laboratories that receive fewer than 20 specimens per week to
process for isolating, identifying, and testing for M. tuberculosis
drug susceptibility are unlikely to maintain proficiency in the
required procedures and would be unlikely to maintain proficiency at
Mycobacteriology Level II. Usually 20 processed specimens per week will
only produce an average of one M. tuberculosis isolation per week. If
requests fall below this level, specimens should be sent to a
laboratory that processes a larger number of specimens (5,36,37).
Assessment of Risk in the Mycobacteriology Laboratory
Specific risks associated with many laboratory activities that
involve specimens and cultures of M. tuberculosis have been assessed in
recent publications (22,38). These publications recommend that
laboratory workers evaluate all procedures for risks related to aerosol
generation and injury from contaminated sharp objects (e.g., needle
sticks) and develop a strategy for safe, step-by-step manipulation of
both specimens and cultures.
Recommendations for safe practices associated with specific
procedures are detailed in other publications (1,22,39).
The Limited Service Laboratory
A small facility that only occasionally is asked to support the
evaluation and management of possible M. tuberculosis cases may opt to
package specimens for shipment to a reference laboratory. The
originating laboratory will require personnel who can collect an
adequate specimen and know how to handle the specimen properly. The
required laboratory facility will be equivalent to the BL-2 space found
in a general microbiology laboratory (1,36). Supplies for correctly
packaging the specimen for shipment to the full-service laboratory must
be available. See Shipment of Clinical Specimens and Cultures for more
information on packaging and shipping specimens.
Some small hospital laboratories may opt to do smears for acid-fast
bacilli (AFB) on inactivated specimens, then send additional specimens
to a larger laboratory for culture. ``Stat'' laboratories in emergency
rooms or other locations, where AFB status of a patient is urgently
needed, but only the simplest equipment is available, can also be
equipped to do direct AFB smears on inactivated samples. This allows
prompt service and some diagnostic assistance to clinicians, without
requiring a BL-3 laboratory.
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The laboratory that intends to do only AFB smears on inactivated
specimens will require only a BL-2 laboratory with a BSC, but will
require knowledgeable personnel working under close supervision.
The Full-service Laboratory
The laboratory that provides all diagnostic services will require
both BL-2 and BL-3 areas of sufficient size to accommodate all required
equipment and personnel.
Facilities and Equipment
Relating Laboratory Activities to BL
Laboratory activities required for the evaluation of a patient with
possible tuberculosis include: specimen collection; transport of
specimens to the laboratory; verifying labels and logging in specimens;
initial processing that may include transferring specimens to tubes for
centrifugation; preparation, staining and reading of smears;
preparation of specimens for culture; and preparation of isolates for
further study, including antimicrobial susceptibility testing.
The Mycobacteriology Laboratory Facility and Equipment
The tuberculosis laboratory should be isolated from other
laboratory areas (Figure 1). Access to the area should require passage
through two doors equipped with self-closing devices. This may be
achieved with an anteroom, by having the BL-3 isolation room accessible
only from the BL-2 laboratory, or by other design arrangements (9).
The BL-2 laboratory area is where work with specimens that has a
low potential for creating aerosols can be performed. A BSC is provided
for working with the specimens (see Handling Specimens).
Work that may create infectious aerosols is performed in the BL-3
area. The BL-3 laboratory is also where M. tuberculosis complex species
are cultured for identification, drug susceptibility testing, and other
tests that require concentrated cell suspensions. Specific facility
design recommendations are contained in Table 1 (1).
Air Handling in the Mycobacteriology Laboratory
The entire mycobacteriology laboratory suite should have a
unidirectional negative air flow in relation to the corridor so that in
case of an accident, no aerosols of infectious materials can escape
into non-laboratory areas. Exhaust air must be discharged directly to
the outside. Discharge from the outside exhaust must be directed away
from occupied areas and air supply intakes of any building.
HEPA filtration of exhaust air is not routinely required for BL-3
laboratories. However, laboratory facility designers and managers
should determine whether unusual or high risk situations are present
(e.g., proximity of laboratory exhaust system outlet to air intake for
patient care areas, with no way to correct problem), and make a site-
specific determination on the need for HEPA filtration.
Similarly, different air pressure gradients within the laboratory
are needed depending on the relative risk of the activities to be
performed. For example, a ``clean room'' used for the preparation of
media or other materials, is maintained at a slightly higher pressure
than the BL-2 laboratory area. The ``isolation room'', or BL-3
laboratory area, is maintained negative to the BL-2 area. Thus, airflow
is from the least contaminated to the most, and air is then exhausted
to the outside without recirculation. Air movement can be tested with a
simple indicator (e.g., a strip of tissue paper placed in a 1.5-inch by
12-inch slot in the door) or with more complex devices (e.g.,
magnehelic gauges) (2,9,39A,39B).
Ten to twelve exchanges per hour are recommended for laboratory
facilities (39A,39B,39C).
Under ideal conditions of maximal air mixing (2), 12 changes of
room air per hour will remove approximately 99% of airborne
particulates in 23 minutes; in laboratories that have only six air
changes per hour, 46 minutes are required to achieve 99% removal,
assuming uniform mixing of air in the room. However, removal can be
slowed even further by convectional mixing and by air turbulence
resulting from furniture placement.
Air flow should be measured to determine the characteristic of
aerosol clearance in the specific BL-2 or BL-3 laboratory. Ideal
conditions for air mixing in laboratories rarely exist, and clearance
may take 3-10 times longer than calculated, a factor that should be
considered in determining when it is safe to reenter a laboratory after
a spill.
Engineering personnel should document at least annually that the
specified number of air changes occur.
Floors, Ceilings and Utilities--Building for Ease of Decontamination in
Case of Spills
Interior surfaces of walls, monolithic floors and ceiling of the
BL-3 laboratory should be sealed to allow for formaldehyde gas
decontamination in the event of a major spill or aerosol release. All
air spaces surrounding a pipe, electrical conduit, or other device that
passes through a wall, floor, or ceiling should be sealed to prevent
air from leaking out of the laboratory.
Biological Safety Cabinets in the Mycobacteriology Laboratory
The most crucial piece of equipment in all diagnostic
mycobacteriology laboratories is the biological safety cabinet (BSC).
BSCs are used at both BL-2 and BL-3.
BSC's are of several types. Class II BSCs, recommended for use in
tuberculosis laboratories, provide a clean work environment, protect
workers against potentially infectious aerosols, and keep infectious
agents from entering the environment. A recent publication, Primary
containment for biohazards: selection, installation and use of
biological safety cabinets (40) details operating procedures for safely
working in BSCs.
The installation of the BSC must conform to accepted specifications
(41). It should be located away from doors, air-supply fans, drafts,
and areas frequented by personnel (40). Improperly positioned BSCs have
contributed to laboratory-associated skin-test conversions (16). A
Class ll, Type A BSC that exhausts HEPA filtered air into the room is
acceptable at BL-2 and BL-3 when a 12-inch or greater clearance exists
above the cabinet and when the use of toxic chemicals (e.g., generation
of cyanogen bromide in the niacin test) is strictly prohibited in the
BSC. Thimble adaptors that loosely connect the BSC to the building
exhaust system may be used.
Ensuring That Air Handling Systems and BSCs Work Properly
BSCs must be certified at least annually by personnel trained in
the certification process (1,16,23,40).
More frequent BSC certification is recommended for laboratories in
which operations create substantial aerosols or when dust accumulates
on the HEPA filter, thereby rapidly decreasing the cabinet's
efficiency. The uninterrupted operation of the BSC should be assured
with a back-up source of power and, where applicable, redundant power
supply to room air exhaust fans. Preventive maintenance operations that
should be routine in every laboratory include daily monitoring of room
and BSC air flow direction and, when present, the magnehelic gauge that
measures the pressure differential across the exhaust HEPA filter (40).
Laboratory operations involving aerosolization or culture-amplified
suspensions of bacilli must incorporate
[[Page 23070]]
additional preventive maintenance and safety checks, which can include
smoke testing or other means for detecting direction of air flow and
velocity. Anemometer readings should be taken before working with new
configurations of instruments and devices in the BSC. Laboratorians
working in BSCs must keep air intake and exhaust grilles free, avoid
overcrowding of the cabinet, and understand the operational parameters
of the cabinet (38,40). Where aerosolization of large volumes of
culture-amplified fluids can occur, a Class III BSC may be used to
ensure total containment of droplet nuclei (1,40).
Centrifugation and Other Aerosol-producing Procedures
As a rule, all procedures that can lead to aerosol production must
be conducted inside a BSC in a BL-3 laboratory as specified in the BMBL
(1). Centrifuges present unique problems for aerosol containment.
Table-top centrifuges placed inside BSCs disrupt the cabinet's
containment airflow. Were a tube to break or leak, aerosolized material
would be expelled into the room with considerable force. Whenever
(potentially) infectious materials are centrifuged, bioaerosol-
containing equipment should be used.
At a minimum, tubes should be equipped with O-rings. Floor-standing
centrifuges that have bioaerosol containment heads are currently
available. Centrifuges can also be placed in secondary containment
devices (especially constructed cabinets/enclosed areas) equipped with
HEPA-filtered exhaust air systems (23,38).
New Growth Detection and Molecular Biological Techniques
After two decades with relatively few changes, new techniques and
new equipment are being added to tuberculosis laboratories. Biosafety
issues related to newer equipment have been reviewed recently (22). As
additional equipment and procedures become available, and they are
considered for inclusion in clinical and research laboratories, a risk
assessment should be done, reviewing manufacturer's specifications and
warnings, adequacy of existing facility for new equipment, need for
revision of existing procedures, and personnel training. As with older
equipment, potential for aerosol generation and risk of needle stick or
other injury should be specifically addressed.
Policies and Procedures in the Mycobacteriology Laboratory
Handling Specimens--Tasks and Risks
Risks associated with many laboratory activities that involve
specimens and cultures of M. tuberculosis have been assessed in recent
publications (22). These publications recommend that laboratory workers
evaluate procedures for relative risk of aerosolization and develop a
strategy for safe, step-by-step manipulation of both specimens and
cultures. The guidelines published in the BMBL (1) and here are
considered to be adequate, based on current knowledge and standard
practice. However, laboratory directors should routinely evaluate the
risks and adjust the level of safety upwards as indicated.
Specimen Collection
Collection of appropriate and adequate specimens and prompt
transport of those specimens to the laboratory are critical first steps
in the laboratory evaluation of the tuberculosis patient. These
procedures involve very significant bio-containment and personnel
protection issues. Guidelines for these activities are included in
(2,9,10,11).
Specimen Receipt and Initial Processing
Sputum specimens collected from patients who have clinical signs of
tuberculosis (2,36) are sent to the laboratory in closed containers
that are opened in a BSC. Transfer of patient information, labeling
containers, and other paperwork can be done safely by trained
laboratory personnel at BL-2.
AFB Smears
The first step in the diagnostic process is to determine if the
specimen contains AFB. In most U.S. laboratories, smears are prepared--
either directly from specimens (e.g., sputum judged likely to have
large numbers of AFB), or after digestion, decontamination of other
microorganisms in the specimen, and centrifugation to concentrate the
mycobacteria in the specimen. Use of rapid-detection systems may
eventually reduce the need to make smears, but may pose a new set of
potential hazards.
Direct Smears
Direct smears are useful only for the examination of specimens
likely to contain large numbers of AFB (e.g., sputum). Because of the
potential for aerosol generation, specimen containers must be opened
and direct smears prepared and air dried in a Class I or II BSC. Smears
may be dried and heat-fixed by placing the slide on a warmer in the BSC
and heating it at 65-75 deg. C (149-167 deg. F) for at least 2 hours.
Heat-fixed smears may contain viable tubercle bacilli (Allen), but they
are not easily aerosolized if dried on a slide. Personnel may remove
fixed slides from the BSC and stain them without wearing respiratory
protective devices or following special engineering controls (i.e., in
the BL-2 laboratory). Stain reagents for both light and fluorescence
microscopy contain phenol, which kills tubercle bacilli during the
staining process (42).
Smears From Concentrated Specimens
Specimens concentrated by centrifugation may contain very large
numbers of AFB. These specimens may be handled in one of two ways.
Use of Tuberculocidal Agents To Allow Processing of Concentrated
AFB Smears in the BL-2 Laboratory
A working group of the 1995 ASTPHLD/CDC Conference (5) affirmed
that if AFB smears are made at BL-2, specimens must have been treated
with a tuberculocidal disinfectant. Specimen containers must be opened
and disinfectant added in the BSC. Specimens treated with an equal
volume of 5% sodium hypochlorite solution (i.e., undiluted household
bleach) for 15 minutes (43,44) may be centrifuged and subsequently
handled outside the BSC at BL-2. Other tuberculocidal agents may affect
staining characteristics; if such agents are used, the laboratory must
confirm that the stain result is accurate. The major disadvantage to
this method is that the treated specimen cannot subsequently be used
for cultures.
Preparation of Concentrated AFB for Smear and Culture in the BL-3
Laboratory
Sputum specimen containers must be opened, chemicals for digestion
added, and the processed specimen placed in appropriate centrifuge
tubes in a BSC.
Centrifugation of diagnostic specimens suspected of containing live
tubercle bacilli must be done in a BL-3 laboratory. Centrifuge tubes
must be placed into rotors or biocontainment cups designed to contain
aerosols that will be generated if a tube leaks or breaks; tubes must
be removed from the cups only in the BSC. O-rings on the centrifuge
caps must be examined daily to assure that the seal is intact and that
the integrity of the unit is maintained; cracked or otherwise faulty O-
rings must be replaced before equipment is reused. (23,38) Concentrated
specimens should be returned to a properly maintained and certified BSC
(40) (see Biological Safety Cabinets) in the BL-3 laboratory. In the
BSC the centrifuge
[[Page 23071]]
tubes can be removed from the safety cups, and smears can be made or
primary cultures can be inoculated. As with direct smears (above),
smears made from concentrated material may be dried and heat-fixed by
placing the slide on a warmer in the BSC and heating it at 65-75 deg. C
(149-167 deg. F) for at least 2 hours.
AFB Cultures--Conventional Techniques
BL-3 practices, containment equipment, and facilities are required
for manipulating cultures known or suspected to be positive for AFB.
In addition to centrifugation, other aerosol-generating procedures
such as blending, mixing, pipetting, inoculation of media, and
sonication must be performed in a BSC at BL-3. A working group of the
ASTPHLD/CDC Conference (5) recognized that activities such as
inoculation of both liquid and solid medium for primary isolation,
identification of all Mycobacterium species using rapid methods, and
susceptibility testing of M. tuberculosis must be done at BL-3.
When tubercle bacilli are inoculated onto a solid medium contained
in a test tube, the screw cap is left loose for up to one week to allow
water vapor, oxygen, and carbon dioxide to diffuse. Droplet nuclei do
not form in the undisturbed tube.
Examining closed culture vessels (e.g., slant tubes, sealed agar
plates) may be done at BL-2. All cultures of specimens must be assumed
to contain M. tuberculosis until tests prove otherwise, and specimens
from patients having mixed infections with two Mycobacterium species
can occur.
AFB Culture and Identification--Newer Techniques
Droplet nuclei may be formed while centrifuging or vortexing liquid
culture materials (as might be done in preparing suspensions before
examination with a probe or high-performance liquid chromatography
[HPLC]) and disrupting cells by sonication or shearing procedures (as
required for some procedures of molecular biology), and such activities
must be done in a BL-3 laboratory using BL-3 procedures.
Waste Disposal
All cultures, glass and plasticware, used protective clothing and
other potentially contaminated materials from the tuberculosis
laboratory must be decontaminated before disposal or reprocessing.
Waste should be decontaminated as close to the point of use as
possible, ideally before materials are removed from the laboratory
area. Materials to be decontaminated outside of the laboratory must be
placed in a durable leakproof container and closed for transport from
the laboratory. Materials to be decontaminated off site must be
packaged in accordance with applicable local, state, and federal
regulations before removal from the facility.
Autoclaves
The BMBL (1) recommends that an autoclave be located in the
facility containing the BL-3 laboratory. If this is not possible, all
wastes that contain mycobacteria should be placed in a leak-proof
discard pan (the pan can be lined with an autoclavable plastic bag)
that contains disinfectant solution to a depth of approximately 2-3 cm;
the pan should be covered with a solid lid before being removed from
the BSC. The lid should be adjusted to allow steam penetration during
autoclaving.
The autoclave must be of sufficient size to handle infectious waste
generated by the laboratory without undue delay, and located so it can
be loaded and unloaded safely and conveniently. Laboratories that are
adding or renovating BL-3 space may wish to consider equipping the
laboratory with through-the-wall autoclaves to minimize movement of
infectious materials throughout the facility.
An improperly operated autoclave contributed to at least one
laboratory-acquired tuberculin skin-test conversion (16). Proper
training in the use of autoclaves and routine proficiency testing are
necessary components of the laboratory safety program.
Safety Strategies
Prevention of Aerosols
In most cases, the ``laboratory accident'' that results in an
exposure and thus a tuberculin skin-test conversion is not as overt as
the breakage of a bottle; more often, lapses in technique allow droplet
nuclei to be released from culture-amplified materials. Therefore, all
laboratory equipment and procedures should be evaluated when put into
use and periodically thereafter to ensure that opportunities for
generation of aerosols are minimized.
Spill Avoidance
A spill can occur at any time during the processing of specimens.
If a culture containing M. tuberculosis complex, whether in liquid or
on solid medium, is dropped and broken, an aerosol is generated.
Laboratory personnel should avoid practices that can result in
spills (e.g., hand-carrying tubes, vials, and bottles, or improperly
stacking racks or baskets). All tubes, plates, and other containers
should be transported on carts in protected racks or baskets.
Spill Response Plan
A written exposure-control plan should be prepared by the director
of the mycobacteriology laboratory. Specified clean-up materials and
personal protective equipment (PPE) should be stored and a copy of the
plan posted outside of the appropriate rooms in both BL-2 and BL-3
laboratories. Although plans will vary according to individual
facilities and practices, all plans should contain the following
information (9,13,22,31):
Instructions on evacuation of the laboratory;
Instructions for notifying the biosafety office, building
engineers, security personnel and others needed to manage the spill;
Instructions on how to manage air-handling equipment,
particularly in the event that a space-decontamination is needed (e.g.,
the cubic volume of the room would be required);
Spill clean-up procedures that will be employed in various
spaces in the laboratory, the sequencing of each procedure, and the
relevant administrative controls, engineering controls, and personal
protective equipment required (1);
Other decontamination procedures, including steps to
control associated problems (e.g., formaldehyde fumes that may not be
contained in the sealed rooms during gas decontamination);
Provisions for follow-up tuberculin skin testing and other
medical intervention procedures;
Provision for spill-response drills to ensure appropriate
action in response to an emergency.
Recommended Management of a Spill
When a spill occurs, all persons should leave the room immediately
so that an assessment of the spill and exposure can be made without
further personnel exposure. Two hours or more later, depending on the
number of air changes in the laboratory, the degree of convectional
mixing in the room air and the turbulence resulting from furniture and
equipment placement, a person wearing a HEPA or N100 respirator
(National Institute for Occupational Safety and Health (45),
Occupational Safety and Health Administration (46)) and protective
clothing should reenter the room to cover the spill with towels soaked
with a tuberculocidal disinfectant. After soaking for at least 2 hours,
the spill should be cleaned up by
[[Page 23072]]
a person wearing a respirator and protective clothing. When more
intensive aerosolization of culture-amplified fluids occurs, the room
should be sealed and decontaminated with formaldehyde gas.
Personnel Protection
Principles
The fundamental principle of personal protection is the consistent
use of appropriate personal protective equipment while manipulating
materials that might contain infectious tubercle bacilli. Training,
monitoring, and medical surveillance are integral to personal
protection. Laboratory supervisors are responsible for educating all
laboratory personnel in the concepts of biosafety and for ensuring that
safety procedures are followed; when a new procedure is introduced,
each step of the operation should be evaluated for potential
biohazards.
Training and Monitoring of Equipment
Laboratorians who manipulate M. tuberculosis complex species must
be taught appropriate procedures and be trained to monitor all
equipment (especially the BSC) for proper operation. Personnel must
confirm that air flow is unidirectional through the facility and that
negative air-pressure gradients are maintained (9,23,40).
Medical Surveillance
Tuberculin Skin Testing
Personnel should be monitored for delayed-type hypersensitivity to
tuberculin. All new personnel should receive a two-step tuberculin skin
test by the Mantoux procedure (2,47); if the tuberculin skin-test
results are positive, a reference chest roentgenogram should be made.
Tuberculin-positive personnel should be advised of the symptoms of
active tuberculosis so that they will know to seek medical attention if
such symptoms occur.
Tuberculin skin test by the Mantoux procedure (but not
roentgenogram) should be performed at least annually and should be used
for surveillance of laboratory personnel whose tuberculin skin test
results were negative. This frequency of skin testing is adequate for
persons who manipulate specimens from tuberculosis patients or who
perform simple procedures on cultures that are unlikely to generate
aerosols.
When the risk for aerosolizing bacterial cultures and suspensions
is high, performing a skin test at shorter intervals is necessary
(i.e., every 3-6 months depending on the degree of exposure).
Records of tuberculin skin-test application, the results of the
reaction (measurement of the zone of induration in millimeters) and the
reference chest roentgenogram should be maintained in the employee
health clinic or in the laboratory's safety records.
If a tuberculin skin-test conversion occurs, the laboratory
supervisor must schedule retesting of all laboratory personnel at 3-
month intervals until no further conversions are found. The standard
interval of testing may then be resumed. Engineering controls,
laboratory procedures, and safety practices must be carefully reviewed
when a tuberculin skin-test conversion occurs in laboratory personnel.
New procedures, additional training, or other appropriate
administrative controls may be indicated as a result of this review.
Certain immunocompromised persons (including HIV-positive persons
with or without AIDS-defining illness) are at increased risk for
developing active tuberculosis when infected with M. tuberculosis.
Supervisors of personnel who work in laboratories that process
specimens for isolation of M. tuberculosis should educate their workers
about the risk of occupationally-acquired tuberculosis to
immunocompromised persons.
BCG Vaccine
An attenuated live vaccine strain derived from M. bovis (Bacille de
Calmette et Guerin {BCG}) is used in many countries as a live vaccine
against tuberculosis. BCG is not routinely used to vaccinate laboratory
personnel or other health care workers in the United States (48).
However, when health care workers are employed in workplaces where the
risk of infection with multiple drug resistant strains of M.
tuberculosis is high and where other infection control measures have
been unsuccessful, ACET/ACIP recommends consideration be given to BCG
immunization for persons who have a reaction of <5 mm induration after
skin testing with 5 TU of PPD tuberculin.
Work With BCG in the Laboratory or Clinical Setting
BCG is administered for cancer immunotherapy, as well as to protect
against tuberculosis. The infectious vaccine is often prepared in a
hospital pharmacy or clinic rather than in a laboratory. Personnel can
develop delayed-type hypersensitivity to tuberculin as a result of
inhalation of aerosols containing the bacilli; therefore reconstitution
of the vaccine in open containers must be done aseptically by persons
wearing gloves and working in a Class I or II BSC. The package insert
provides instructions for safe vaccine administration.
The BCG strain of M. bovis may be done safely in a BL-2 facility
using BL-2 practices and procedures. However, should laboratories be
asked to attempt culture of BCG from clinical materials, these should
be handled as though they contained M. tuberculosis organisms.
Personal Protective Equipment
Certain protective clothing and equipment must be worn by personnel
entering BL-2 and BL-3 laboratories.
Supervisors must emphasize the availability and use of personal
protective equipment through training and control procedures.
Clothing
BL-2 Laboratory
Laboratorians working at BL-2 should wear a laboratory coat or gown
over their street clothes; the coat or gown must be removed when
leaving the laboratory. Gloves must be worn when handling specimens or
any other vessel that may contain tubercle bacilli.
BL-3 Laboratory
Laboratorians working at BL-3 must wear protective laboratory
clothing such as a solid-front or wrap-around gown. Scrub suits may be
worn under the protective gowns, particularly in research or other
situations where there is potential exposure to large volumes of liquid
culture material. The scrub suits should be changed daily. The
protective gown worn in BL-3 laboratories must have long sleeves with
snug (knit) cuffs. Gloves must be worn and must be long enough to
overlap the sleeves of the gown. Caps and booties are recommended.
Laboratorians should remove all outer protective clothing when leaving
the BL-3 laboratory and place the clothing into bags for autoclaving.
Respirators
Recommendations for respirator use are based on recently published
guidelines for particulate respirators (NIOSH) and evaluations of the
risk for infection by aerosol inhalation associated with work
performed. Engineering controls, safe work practices, including use of
personal protective equipment (Table 2), and common sense are combined
to minimize risk.
OSHA Standard
The respiratory protection standard of the Occupational Safety and
Health Administration (46) requires that all respiratory protective
devices used in the workplace be certified by the
[[Page 23073]]
National Institute for Occupational Safety and Health (45). CDC
published recommendations for selection of respirators for protection
against tuberculosis in 1994 (2). Four criteria govern the use of these
respirators:
The ability of an unloaded respirator to filter particles
0.3 in size with a filter efficiency of 95% (i.e., filter
leakage of 5%), given flow rates of up to 50 L per minute.
The ability to be qualitatively or quantitatively fit-
tested to obtain a face-seal leakage rate of no more than 10%.
The ability to fit different facial sizes and
characteristics, which can usually be attained by making the
respirators available in at least three sizes.
The ability to check for face piece fit by the person
wearing the respirator each time it is worn in accordance with OSHA
standards.
NIOSH Procedures for Certification of Respirators
Since publication of the CDC recommendations for selection of
respirators for M. tuberculosis in 1994, the NIOSH procedures for
certification of respirators have been revised (45). The revised
guidelines for certification of air-purifying respirators enable users
to select from a broader range of certified models that meet the
performance criteria. NIOSH certifies three classes of filters,
designated as the N-, R-, and P-series, using newly available
particulate filter tests. Each series contains three levels of filter
efficiency, 95%, 99%, and 99.97%, respectively. All tests for
classification of the filter employ the most penetrating aerosol size
(i.e., 0.3 aerodynamic mass median diameter). Respirators in
the N-series are tested against an aerosol of sodium chloride (NaCl),
and the R- and P-series filters are tested against an aerosol of
dioctylphalate (DOP). Currently available HEPA respirators or any of
the respirators that are certified by NIOSH for use in laboratory
settings under the Code of Federal Regulations 42, Part 84 are
recommended (45).
Respirator Program in the Mycobacteriology Laboratory
The respirator program, in accordance with the OSHA standard (46),
should be implemented by the laboratory's safety officer or person
designated to perform this task and should include written procedures
concerning how to: (a) select the appropriate respirator, (b) conduct
fit-testing, and (c) train personnel on the use, fit checking, and
storage of the respirator. Surgical masks are not NIOSH certified
respirators and must not be worn to provide respiratory protection.
Use of Respirators in the Mycobacteriology Laboratory
When sputum specimens are collected in a laboratory setting, either
the patient must be in a negative air-pressure booth equipped with a
HEPA filter on the exhaust, or the laboratorian must wear a HEPA
respirator (which may be a powered air purifying respirator equipped
with N100 respirator cartridges (2)).
All manipulations of M. tuberculosis cultures create splatter or
aerosol and must be performed in a BSC located in a BL-3 facility. All
workers in BL-3 laboratories should wear an N95 respirator and other
protective clothing (see Clothing) to minimize potential exposure when
infectious materials are being manipulated. Laboratory infections are
nearly always caused by either poorly monitored BSCs or a BSC in which
normal aerosol containment capability is compromised, thereby
permitting escape of droplet nuclei (38,40). The respirator then acts
as an additional barrier to reduce the likelihood that tubercle bacilli
will enter the lung.
Research
Research procedures involving the M. tuberculosis complex species
should be carefully evaluated. Large volumes of fluids and suspensions
of concentrated mycobacteria must be manipulated at BL-3 using
procedures approved by the institution's biosafety representative
knowledgeable in containment of M. tuberculosis. Filtering exhaust
laboratory air is not required; however, overriding local conditions
may make it prudent to install HEPA filters.
Research Involving Animals
Experiments involving induced M. tuberculosis or M. bovis
infections in animals pose hazards during certain stages of the study.
The animals are challenged (i.e., intentionally infected with tubercle
bacilli) by either intravenous injection (mice) or by inhalation of an
aerosol (mice and other animals). During this process, laboratory
personnel are at risk for being self-inoculated or exposed to aerosols.
Primates are likely to produce an infectious aerosol by coughing.
Therefore, all infected primates must be housed in an animal biosafety
level 3 (ABL-3) facility (1).
Rodents are unlikely to produce aerosols by coughing, but they
should be housed in bonnet-top or similar containment cages because of
the risk for aerosolizing AFB from contaminated bedding. Rodent cages
can be held in an Animal Biosafety Level 2 (ABL-2) facility (1) that
has single-pass, unidirectional inward air flow and that exhausts all
air to the outside. Litter must be handled as if infectious. Laboratory
and animal-care personnel should always follow ABL-3 practices and
procedures. An ABL-3 facility also may be used for work with other
rodent species.
Shipment of Clinical Specimens and Cultures
Specimens that may contain species of the M. tuberculosis complex,
including clinical specimens and cultures, must be packaged, labeled,
and shipped in accordance with Public Health Service (PHS), Department
of Transportation (DOT), and International Air Traffic Association
(IATA) regulations (50,51,52,53). PHS shipping regulations are being
revised to reflect varying risks of disease transmission during
shipment of infectious agents, and to conform more closely to DOT and
IATA regulations. An NPRM will be published for comments in mid-1997.
Under the proposed PHS shipping regulation, clinical specimens sent
for initial diagnosis should be placed in a water-tight primary
container (e.g., screw-capped container). The primary container should
be placed in a watertight secondary container (e.g., sealable plastic
bag). The primary container should be surrounded by sufficient
absorbent material to completely soak up the liquid in the clinical
specimen. The secondary container should be placed into a sturdy outer
container that bears the address label and a label indicating
``clinical specimen''.
Mycobacterial cultures, and other materials known to contain M.
tuberculosis complex species should be enclosed in a watertight primary
container (e.g., a screw-capped tube or plastic vial). The primary
container should be placed in a watertight, durable secondary container
(e.g., rigid aluminum can with a sealable top). The space between the
primary container and secondary container should contain sufficient
absorbent material to completely soak up the liquid in the culture or
specimen in the event of leakage or breakage. The secondary container
should be placed into a sturdy outer container that bears the address
label and PHS infectious substance label. Packages containing cultures
of M. tuberculosis species must also bear DOT's infectious substance
label on the outer package. All packages containing infectious
substances must meet DOT performance standards.
[[Page 23074]]
The importation of materials containing species of the M.
tuberculosis complex into the United States requires an import permit
(50). An application to import etiologic agents or vectors, federal
regulations regarding importation, and other information may be
obtained by calling CDC/OHS voice/FAX information system at (404) 639-
3883.
Packages containing M. tuberculosis complex species should be
opened in a BSC in the receiving laboratory. Damaged packages should be
reported to CDC/OHS at (800) 232-0124.
The Mycobacteriology Laboratory in Need of Improvement
It is recognized that some laboratories may not currently meet
these guidelines because of certain facility limitations, (e.g., not
having a complete BL-3 laboratory). In those laboratories, the
laboratory director and biosafety officer should evaluate the facility,
available equipment and work practices to determine what services can
be provided without compromising employee health and safety. Activities
must be modified or discontinued if necessary. For example, personnel
working in a BL-2 laboratory can inactivate the tubercle bacilli before
centrifugation and other activities that could generate aerosols. Some
laboratory directors may choose to temporarily refer some work to other
laboratories until improvements to their own facility have been made.
In some situations, it may not be possible to suspend or
significantly alter current laboratory activities. In that case, the
laboratory director and biosafety officer should develop policies and
procedures to allow those activities to continue following full BL-3
practices and procedures while working in a BL-2 laboratory (1).
However, the pursuit of achieving optimum good laboratory practices
must include the timely development of a plan to achieve appropriate
facility upgrades. When a temporary program is implemented to continue
routine work in a BL-2 facility with BL-3 procedures, all work
practices should be closely monitored, and all employees should receive
tuberculin skin tests at recommended intervals.
Conclusions
Although the incidence of tuberculosis is higher in laboratory
workers than for the general population, the risk of becoming infected
with M. tuberculosis in the laboratory can be minimized through the use
of the engineering controls, administrative procedures, and specific
work-place practices that are presented in these guidelines.
Full biosafety level 3 is recommended for laboratories performing
work with live tubercle bacilli that may generate infectious aerosols.
Currently available procedures for preparing AFB smears, preparing
samples for culture, identification and antimicrobial susceptibility
testing of AFB all have the potential for generation of aerosols and
must be done using BL-3 practices and procedures.
Biosafety level 2 facilities and procedures are sufficient for
laboratories performing direct AFB smears on samples that have been
treated to inactivate the tubercle bacilli.
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Advisory Council for the Elimination of Tuberculosis and the
Advisory Committee on Immunization Practices. MMWR 1996;45,RR-4.
50 U.S. Department of Health and Human Services, Public Health
Service; Foreign Quarantine; Etiologic Agents and Vectors. 42 CFR
Part 71, 54, 1996.
51 U.S. Department of Health and Human Services, Public Health
Service; Interstate Shipment of Etiologic Agents. 42 CFR Part 72,
1980.
52 International Air Transport Association (IATA). Dangerous
Goods Regulations, 1996
53 U.S. Department of Transportation; Hazardous Materials
Regulations ( HMR). 49 CFR Part 171-180, 1996.
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