Biosafety for TB

laboratories

Korean Institute of Tuberculosis

Chang-Ki Kim, MD, PhD

1

WHO TB laboratory biosafety manual

• WHO & CDC

• The Expert Group meeting

• WHO biosafety manual, 3rd ed.

• Basic requirements

• Website:

– http://apps.who.int/iris/bitstrea

m/10665/77949/1/9789241504

638_eng.pdf

Transmission of tuberculosis

MTB is almost always transmitted by patients with active pul disease:

– TB patient expels bacilli in small droplets of respiratory secretions.

– Secretions quickly evaporate leaving “droplet nuclei” less than 5 μm in

diameter.

– Droplet nuclei of this size, containing 1–3 bacilli, can remain suspended

in the air.

– Following inhalation, droplet nuclei are able to reach deep into the

lungs to produce infection.

– Low infective dose for humans: Infectious dose = 10 bacilli

3

Coughing

Sneezing

Talking

Singing

Aerosol formation: spread of droplets

4

Laboratory safety

Diameter of particles (microns)

Procedures Generating Infective Aerosols

1. Flaming of transfer loop+MTB

2. Homogenizing/blending/spattering/

breaking of tube/flask/conainer+MTB

3. Pipetting/opening centrifuge tube

4. Sputum collection

Prevention for Aerosol Production,

Release or Inhalation

1. Controlled airflow in the laboratory

2. Safety equipment/supplies/arrangement

3. Training in safe/correct procedures

Upper

respiratory

tract

Pulmonary

space

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5

Occupational infectious diseases among

HCWs in Korea, 1998-2004

• Compensated by the Industrial Accident Compensation Insurance

• 1998. 1 - 2004. 12

Ahn YS et al, Industrial Health 2008;46:448

Kinds of infection No. of workers (%)

Tuberculosis 219 (71.3)

Hepatitis 42 (13.7)

Chickenpox 11 (3.6)

AIDS 8 (2.6)

Scabies 7 (2.3)

Measles 5 (1.6)

Pneumonia 4 (1.3)

Other 11 (3.6)

Total 307 (100)

Lab technician (11)

6

Laboratory Biosafety

To enable laboratory staff to work safely

• Administrative controls

• Containment principles

• Practice and procedures

• Safety equipment

• Emergency preparedness

• Facilities

7

Procedures of Risk assessment

1. Identify the inherent hazards

– e.g: drug resistant TB (MDR-TB, XDR-TB)

2. Decide who might be harmed and how

– Generation of aerosols

• Procedure, frequency, workload, consistency, bacillary load, viability

– Susceptibility to TB of lab workers

• Immunity level: HIV infection or pregnancy

8

Factors to be considered for risk

assessment-1

9

Factors relevant to all TB laboratories Considerations

Pathogenicity Untreated TB has a mortality rate of 30–50%; about 30% of persons with prolonged exposure to an infectious TB case become infected; 5–10% of infected persons develop TB

Primary route of transmission Inhalation of infectious droplet nuclei

Secondary routes of transmission (uncommon in laboratory)

Ingestion, direct inoculation

Stability Tubercle bacilli can remain viable for extended periods in the environment

Infectious dose Estimated to be 10 bacilli by inhalation

Factors to be considered for risk

assessment-2

10

Factors relevant to all TB laboratories Considerations

Susceptibility of immunocompetent persons to developing TB

5–10% develop TB during their lifetime

Susceptibility of immunocompromised persons to developing TB

5–10% develop TB per year

Risk of community-acquired TB in high-burden settings

High

Effective vaccine No, none available

Effective treatment for strains susceptible to different medicines

Yes

Effective treatment for MDR strains Yes, but more difficult to treat than susceptible strains

Effective treatment for XDR strains Few treatment options

Relative risk for TB of lab

procedures

11

Considerations for Risk assessment

12

Procedures of Risk assessment

3. Evaluate the risks and decide on precautions

– Determine the suitability of the physical structure

– Evaluate the staff’s proficiency in following safe practices

– Evaluate the integrity of safety equipment

4. Record your finding and implement them

5. Review your assessment and update it if necessary

13

Biosafety measures for TB lab

1. Code of practice

2. Equipment

3. Laboratory design and facilities

4. Health surveillance

5. Training

6. Waste handling

14

Codes of practice

Lab practices and procedures essential for implementing GMT

• Laboratory access

– Biohazard sign, only authorized persons

• Responsibilities of the Laboratory manager

– Development of biosafety system and SOP, training, evaluation, maintenance planning

• Personal protective equipment

• Procedures

– To minimize or prevent the formation of aerosols

• Work areas

– “Functionally clean” and “potentially contaminated”

– Neat, clean and free of stuffs no used routinely, decontamination

15

Good microbiological techniques

• Good microbiological techniques (GMT):

– working methods applied to minimize exposure to pathogens via, for

example, aerosols, splashes, accidental inoculation.

• GMT are fundamental to laboratory safety.

• Specialized equipment may support good laboratory practice but

does not replace it.

16

Standard practices

• Limited access to the laboratory.

• No eating, drinking, smoking, etc.

• No mouth-pipetting, no chewing pencils, etc.

• Assume that ALL specimens are potentially

infectious.

17

Standard practices

• Hand-washing (dry with

disposable paper).

• Work surfaces to be

decontaminated at least once

a day.

18

Equipment

• Designed to prevent or limit contact between the operator and the

infectious material;

• Constructed of materials that are impermeable to liquids and

resistant to corrosion;

• be smooth and without sharp edges and unguarded moving parts;

• Designed, constructed and installed to facilitate simple operation,

and provide for easy maintenance, cleaning, decontamination and

certification testing;

19

Design and facilities

Secondary containment measures protecting lab workers and providing

barrier for the community from TB aerosols

[Basic recommended design features]

• Adequate ventilation and directional airflow

• Ample space for the safe work, and for cleaning and maintenance.

• Walls, ceilings and floors should be smooth and easy to clean.

• Adequate Illumination

• Furniture made impervious materials and easy decontamination

• Open spaces between and under equipments for cleaning

20

Design and facilities

• Adequate storage space to hold supplies for immediate use

• Additional Space for long-term storage outside work areas.

• An area for the safe preparation, handling and storage of acids, stains and

solvents

• Facilities for storing outer garments and personal items outside work areas.

• Facilities for easting and drinking, and personal item outside work areas.

• A sink for handwashing and soap in each room in the lab

• Laboratory doors with a glass window panel and appropriate fire ratings:

self-closing.

21

Waste handling-Incineration

• Useful for disposing of laboratory waste

• In low risk laboratories, plastic sputum container, Xpert cartridge,

and wooden sticks should be removed in sealed bags and

incinerated.

• Efficient means of temperature control and a secondary burning

chamber

– Primary chamber: at least 800°C, secondary chamber: at least 1,000°C

• Incineration vs autoclave

22

Waste handling-autoclave

[Materials suitable for autoclaving]

1. Instruments, glassware, media or solutions for sterile use in the

general diagnostic TB laboratory;

2. Mycobacterial cultures for waste disposal;

3. All infected materials, including closed specimen containers, should

be placed in the BSC in autoclavable bags.

• All positive TB cultures must be autoclaved before disposal.

• Separate autoclaves for clean and for dirty

• Record the time, temperature and pressure each time.

• Use biological indicators

23

Waste handling-Disinfectants

Because of their specific cell wall structure, TB bacilli are resistant to most

standard disinfectants.

• Phenol (5%): Very irritant to skin, use derivatives.

• Chlorine:

– widely used, corrosive to metals

– Bleach: 50 g/L -> diluted to 1:50 or 1:10 (Stock ;3 mon, working solution; daily)

• Alcohol (70%): No residue, use on skin and work surfaces.

• Peracetic acid:

– Less harmful decomposition products, removal of organic material, leave no

residue (Working solution (2%) stable for 48 hours)

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Classification of TB laboratories

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Risk level of TB

laboratory Laboratory activities Assessment of risk

Low risk Direct sputum-smear microscopy;

preparation of specimens for use

in an automated nucleic acid

amplification test cartridge

Low risk of generating infectious

aerosols from specimens; low

concentration of infectious particles

Moderate risk Processing and concentration of

specimens for inoculation on

primary culture media; direct DST

(for example, LPA on sputum)

Moderate risk of generating

infectious aerosols from specimens;

low concentration of infectious

Particles

High risk

(TB-containment

laboratory)

Culture manipulation for

identification; DST or LPA on

cultured isolates

High risk of generating infectious

aerosols from specimens; high

concentration of infectious particles

Low-risk TB laboratories

• Manipulate sputum specimens for direct sputum-smear microscopy

or Xpert MTB/RIF

• Factors that increase risk of infection

– Leak, careless procedure, mixed use of bench

• Use separate bench for smear or Xpert

• Adequate ventilation

– Directional airflow with 6-12 air exchanges per hour (ACH)

– Natural ventilation??, air flow at least 0.5 m/s

• Respirators are not required.

26

Determining adequate ventilation in TB

laboratory with mechanical ventilation

1. Identify the air exhaust vent or vents.

2. Cover the vent with a piece of cardboard with opening of 10 cm x 10 cm;

3. Measure the outflowing air velocity with a vaneometer or anemometer;

4. Calculate the volumetric airflow rate for each air exhaust port

– Q = V x A x 3,600

– Q = Volumetric airflow rate, V = Velocity of air, A = Area of opening in m2

5. Sum up all the exhausts for the room;

6. Measure the volume of the room

– Vol = Length x Width x Height = m3 (measure in metres);

7. Calculate the ACH: ACH = Q/Vol.

27

Moderate-risk TB laboratories

• Process specimens for inoculation on primary solid-culture media or

direct DST

• Factors that increase risk of infection

– Poor ventilation, poor illumination, poorly working BSC, blockage of

HEPA filter, careless manipulation, not following SOP, opening

centrifuge buckets outside the BSC, inadequate warning or lack of

information on an emergency.

• All procedures with sputum samples must be conducted in a BSC.

(Class I or II BSC are recommended.)

28

Moderate-risk TB laboratories

• Class II type A2 vs. Class II type B

– Class II type B requires hard-ducting

-> balance and maintenance are difficult.

• Unidirectional airflow with 6-12 ACHs

• Expelled air from BSC

– Release into lab or exhausted outside lab

• Glove should be changed regularly.

• Respirator are not required with properly maintained BSC and GMT

• Separate from other areas

29

High-risk TB laboratories

• TB-containment laboratory

– Manipulate cultures to identify M. tuberculosis

– Manipulate cultures or suspensions of MTB for indirect DST & molecular assays

– Biosafety level 3 laboratory??? Maybe or maybe not

• Factors that increase risk of infection

– Opening positive culture vial

– Preparing smear from positive cultures

– DNA extraction from positive culture

– Manipulation of cultures for ID and DST

– Accident or spill

30

High-risk TB laboratories

• Lab design:

– Two sets of entry doors (anteroom)

– Unidirectional flow of air into lab

– Self closing and interlocking

– A glass panel should be installed to give a view

• PPE

– Protective lab gown, glove should be worn.

– Respirator : additional protection, not substitute for BSC

31

Safety equipments

• Biological Safety Cabinets (BSC)

• Centrifuges with safety buckets

• Autoclaves

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Biological Safety Cabinet

• The BSC is the most important safety equipment.

• Class I BSC

– Open-fronted, a unidirectional inward airflow

– HEPA filters which remove particles of diameter 0.3 μm

– Protection for the worker but not products against contamination.

• Class II BSC

– Protection against contamination of the product

– HEPA-filtered air in a laminar vertical flow

– Class II type A2 is preferred

– Class II type B are not recommended for new TB lab

• hard-ducted to the outside (Certification, operation and maintenance are more difficult)

33

Class I Class II type A2

70% recirculated

30% exhausted

F: plenum under

negative pressure

Exhausted air can

be recirculated to

the room or

discharged to

the outside of the

building through a

thimble connected

to a dedicated duct

34

Thimble connection

• Used with Class II type A2 BSC

• Small opening between the

thimble and cabinet’s exhaust

housing

• Performance of a thimble-

connected BSC is not affected

much by fluctuation in the

building’s airflow

• Damper control for the exhaust

• No need for adjustment of BSC

35

Biological Safety Cabinet

• Operation

– Switch on the BSC 15 min before use

– In Class II BSCs, work in the BSC away from the grille

– The BSC should be switched off 15 min after the end of work

• Location

– clearance of 30 cm should be provided behind and on each side of the

cabinet to allow easy access for maintenance.

– A clearance of 30-35 cm above the cabinet may be required to accurately

measure air velocity across the exhaust filter, and to change exhaust filters.

• Ultraviolet lights

– Not recommended

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Working in a BSC

• BSC work zone should be divided into three areas to

minimize contamination over items:

1. Clean area: supplies

2. working area: specimen

3. Contaminated area: waste container

37

Working in a BSC

• Organize a realistic workload in the BSC.

• Do not overload, e.g. no more than 6–8 specimens for processing at

a time, according to centrifuge capacity.

• All needed material should be present in the BSC so that work is not

interrupted and moves in and out of the BSC are minimized.

• Avoid continuous flame = permanent source of heat

– Electric incinerator, disposable loops

38

Personal protective equipments

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Personal Protective equipment

• Surgical mask

– Fit poorly, leaving large gaps between the face and mask.

– Prevent the spread of microorganisms from the wearer to others

– Do not protect the wearer from inhaling infectious aerosols.

• Respirator

– Protection against inhalation of infectious aerosols

– Fit closely to the face to prevent leakage around the edges.

– The N95 (or FFP2) respirator is a lightweight, disposable nose and mouth respirator;

– effectively filters out ≥95% of the particles of diameter ≥0.3 μm.

• Respirator are not normally required for work in a TB lab.

– May be recommended after risk assessments

– Included in spill kit

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N95 Respirators

41

Respirator Fit Test

42

Infectious spills (outside BSC)

The laboratory manager should be informed of the incident immediately, and staff

must be prevented from re-entering the laboratory for at least 1 hour.

1. Put on gloves, a protective laboratory gown and respirator, and re-enter.

2. Cover the spill with cloth or paper towels to contain it.

3. Pour an appropriate disinfectant over the paper towels and the immediate

surrounding area

4. Apply disinfectant concentrically beginning at the outer margin of the spill.

5. Allow sufficient time for the disinfectant to act before clearing away any material.

6. Place other contaminated material in a sealed bag for appropriate disposal.

7. Clean and disinfect the area of the spill.

• Anyone who was exposed to the spill should be referred for medical advice.

• Record should be kept of the incident. 43

Infectious spills (inside BSC)

When a spill of infectious material occurs within a BSC, a clean-up procedure should

begin immediately, and the cabinet should continue to operate.

1. Place absorbent tissue over the spill area, and apply disinfectant solution liberally.

2. If the walls of the BSC have been splashed, clean with a layer of absorbent paper

towel liberally soaked in disinfectant solution.

3. Leave affected areas covered with disinfectant for 30 minutes to 1 hour.

4. Carefully collect contaminated sharps material, and place in appropriate container.

5. Any equipment or reusable material that has been splashed should be cleaned.

6. Electrical equipment should be checked carefully before it is used;

7. Collect other contaminated material in a sealed bag.

44

Spill clean-up kit

• Hypochlorite solution stored in an opaque bottle

• Respirators (1 box), Gloves (1 box), Goggles ( 2 pairs)

• Laboratory gowns (4-6 disposable gowns)

• Dustpan and brush (for disposal if necessary)

• Paper towels, Soap, Sharps container, Biohazard bags

Hypochlorite in solution has a limited shelf life.

For a large spill, it may be better to prepare the disinfectant solution at the

time of clean up.

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Medical fitness of laboratory staff

• In accordance with national laws and practices, health surveillance of TB

laboratory workers should be performed:

– before enrolment in the TB laboratory;

– at regular intervals thereafter;

– after any biohazard incident.

• Workers should be educated about the symptoms of TB and provided

with ready access to free medical care if symptoms arise.

• Confidential HIV counselling and testing should be offered.

Reassignment of HIV-positive workers away from high-risk environments

should be considered.

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Health Supervision

• Tuberculin test

• Annual medical check-up (chest radiography)

• Training in safe laboratory procedures.

• Regular monitoring of equipment.

• Record all laboratory accidents.

• Staff should be educated about TB signs and symptoms to report

promptly for evaluation

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Thank you for your attention

48