Module 2

Safety precautions for AFB culture and DST of

M. tuberculosis

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Learning objectives

At the end of this module, you will be able to:

explain airborne transmission of TB; adhere to safe practice;explain the biosafety levels of laboratories for TB

activities;work safely in biological safety cabinets;use adequate protective personal equipment; use appropriate disinfectants;cope with accidents in the TB laboratory;handle chemicals safely in the laboratory . .

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Content outline

• Transmission of TB• Biohazards in a TB laboratory• Minimum WHO recommendations for TB culture / DST

facilities• Standard safe practice in a TB laboratory• Personal protective equipment• Disinfectants active against M. tuberculosis• Safe disposal of infectious waste• Coping with accidents • Chemical safety

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Transmission of tuberculosis

Mycobacterium tuberculosis is almost always transmitted by patients with active pulmonary 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.

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Coughing

Sneezing

Talking

Singing

Aerosol formation: spread of droplets

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Specimens must be collected outside the laboratory

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Biohazards in the laboratory: be aware!

• Inhalation hazards: handling of liquids containing TB bacill generates infectious aerosols: – pipetting– working with loops– centrifugation– opening tubes– vortexing suspensions

• Ingestion hazards

• Inoculation hazards

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Importance of laboratory safety

for preventing

laboratory-acquired infection

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Conclusion of of the survey::•DST is associated with the higher risk..•Smear microscopy alone does not pose a higher risk than clerical work.

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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.

10[to be adjusted to local policy][to be adjusted to local policy]

Biological safety cabinet

The biological safety cabinet, or BSC, is the single most important equipment for containing and quickly eliminating infectious aerosols generated during culture/DST procedures.

For proper functioning, the BSC must be well maintained.

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HEPA filter

• HEPA = high-efficiency particulate air.

• Traps and removes 99.97% of airborne particles equal to or larger than 0.3 µm in diameter.

• Evaluation of the performance of the HEPA filter and regular maintenance of the BSC are critical for protection

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Work in a BSC

BSC work zone should be divided into three areas to minimize contamination over items: – a clean area: supplies;– working area: specimen;– contaminated area:

waste container.

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Work 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.

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Is flame allowed in the BSC?

• Avoid continuous flame = permanent source of heat

• Use intermittent burner

• Can be used in BSC I or II

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Biosafety levels

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WHO recommendations for BSL2 (specimen processing for TB cultures)

• BSCs should be ducted or vented to the outside.

• Air from BSCs must not be allowed to recirculate into the room in TB laboratories.

• An adequate budget for regular maintenance and servicing is essential.

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WHO recommendations for handling TB cultures

In addition to BSL2 recommendations:

• Isolation of the laboratory (containment room).

• Anteroom with a double-door entry; may consist of a BSL2 room

• Ventilation: directional airflow, controlled ventilating system.

• Autoclave on site (but not mandatory in the containment room).

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Laboratory for handling TB cultures

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Which biosafety level for TB laboratory activities?

Microscopy in a well-ventilated room

Culture = specimen processing in a BSC in a BSL2

Culture identification and DST = BSL2 containment room with ventilation:

directional airflow from functionally clean to dirty areas, controlled ventilating system ensuring 6 to 12 air exchanges per hour.

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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.

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Standard practices

• Hand-washing (dry with disposable paper).

• Work surfaces to be decontaminated at least once a day.

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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.

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Personnel qualifications: proper trainingFor culture procedure:

• TB disease and transmission;• wearing and use of protective equipment and clothing;• handling of infectious materials;• laboratory design, including air flow conditions;• use of BSC, centrifuge (operation, identification of malfunctions,

maintenance);• preventing and coping with incidents;• good laboratory practice and good microbiological techniques;• organization of work flow procedures; • waste management;• importance of laboratory results for patient management;

• importance of laboratory results for the national TB programme.24

Personal protective equipment

• Masks/respirators

• Gloves

• Gowns

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Personal protective equipment – masks

• Surgical masks prevent the spread of microorganisms from the wearer (protection from exhalation).

• Masks do not provide protection to the wearer against inhaling small infectious aerosols.

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Personal protective equipment – respirators

N95/FFP2 FFP3

N95/FFP2 respirators effectively filter out more than 95% (98% for FFP3) of particles 0.3 µm or more in diameter.

Respirators have to be fitted to the face. Facial hair causes the respirator to be ineffective.

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Personal protective equipment – respirators

Respirators may be worn in settings of high MDR-TB and/or HIV prevalence, where staff may be HIV-infected and highly susceptible to infectious aerosols.

Respirators must be available in all culture/DST labs for coping with spillages outside the BSC.

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Personal protective equipment – gloves

• Gloves should always be worn while specimen processing and handling TB cultures.

• Wearing gloves can give technicians a false sense of safety.

• Frequent hand-washing and care in the handling of contaminated materials are good laboratory practices.

• Gloves should be removed at every interruption of work and should not be reused. Hands should be washed after removal of gloves.

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Personal protective equipment – gowns

• Gowns must always be worn inside the laboratory (but never outside) and should be changed regularly, at least weekly.

• Gowns should preferably be fully buttoned, long-sleeved with narrow cuffs, and back-opening.

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Remove PPE in the following order:

1.Disposable gloves

1.Respirator/mask

1.Gown/coat/suit/overalls

Removing PPE

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Personal protective equipment – conclusion

• Hand-washing and GMT are acceptable practices for most countries.

• Each country must evaluate the risks and decide on the level of personal protection equipment that is appropriate, given the available resources.

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Disinfectants

• A disinfectant is a chemical or mixture of chemicals used to kill microorganisms.

• Disinfectants are usually applied to surfaces or inanimate objects.

• Disinfectants may be used for pre-decontamination treatment, before autoclaving.

Because of their specific cell wall structure,TB bacilli are resistant to most standard disinfectants, e.g. quaternary ammonium compounds are ineffective.

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Disinfectants

The only adequate disinfectants against TB bacilli are:• Phenol, 2–5%: very irritant to skin, use derivatives.• Hypochlorite (bleach): corrosive to metals.• Alcohol 70%, no residue, use on skin and work surfaces.• Iodophores, 3–5%, iodine plus inert polymer.

• Glutaraldehyde, requires an activator, highly irritant to skin.

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Disinfectants

• Diluted solutions should be prepared daily.• Stock solutions should be stored according to

manufacturer's recommendations. • Use commercial solutions for “dirty or worst possible

situations”.• Follow national chemical safety guidelines.

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[to be adjusted to local policy][to be adjusted to local policy]

Waste disposal

• All infected materials, including closed specimen containers, should be placed in the BSC in autoclavable bags.

• All cultures and related materials should be autoclaved.

• All material handled in the BSC should be considered as infected.

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Incident: spill

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Spill in the BSC – what to do

• Cover the spill with absorbent paper towel.

• Pour over disinfectant and leave for at least 2 hours.

• Discard absorbent tissue and all clean-up material in an autoclavable bag and autoclave.

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Spill outside the BSC

• EVACUATE the room and stay outside with the door closed for at least 2 hours.

• Using appropriate respiratory protection devices, return to the accident area to clean the spill.

• Cover the spill with paper towel and pour over disinfectant. Leave for at least 2 hours.

• Once disinfection is complete, discard all waste into suitable waste containers and autoclave.

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Spill outside the BSC

Emergency spill kit should be available in all culture/DST laboratories, with:

•all personal protective equipment (overalls, over-shoes, gloves, respirators);

•Paper towels;

•large quantities of disinfectant.

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Chemical safety

• Alcohols are flammable: avoid flame

• Phenol is corrosive:– avoid direct contact with the skin or mucous

membranes;– limit exposure to phenolic fumes.

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NEVER ADD WATER TO ACIDALWAYS ADD ACID TO WATER

WATER

ACID

ACID

WATER

Chemical safety: handling acids

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SAFETY FIRST!

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True and false exercise

1. Surgical masks protect you from TB infection.

2. Remove personal protective equipment in the following order:

respirator/mask

disposable gloves

gowns/coats/suits/overalls.

3. In case of spills outside the BSC, you should evacuate the room and stay outside with the door closed for at least 30 minutes.

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Module review: take-home messages

Never smoke, eat, or drink in the laboratory. Wash your hands frequently with soap and water

before and after performing any procedures. Avoid hazards in a TB laboratory by paying

careful attention to safety procedures. Always work carefully and in a safe manner

following good microbiological technique. The additional protection conferred by

respirators must be assessed for each setting.

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Self-assessment• How is TB transmitted from person to person?

• What are the sources of infectious aerosols in a laboratory?

• What is the critical equipment for safe culture and DST?

• What personal protective equipment is recommended for TB laboratory activities?

• What are the most efficient disinfectants in TB laboratories?

• What procedures should be followed when coping with accidents?

• What precautions should be taken when handling acids?

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