Goals for Working Safely With Mycobacterium Tuberculosis in Clinical, Public Health, and Research Laboratories

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).

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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

[[Page 23068]]

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.

[[Page 23069]]

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 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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