Module 2
Safety precautions for AFB culture
and DST of Mycobacterium tuberculosis
Purpose / To provide you with an understanding of safe handling techniques and precautions while performing AFB culture and DST of Mycobacterium tuberculosis.Prerequisite modules / None
Module time / 1 hour
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 personal protective equipment;
– use appropriate disinfectants in any circumstances;
– cope with accidents in the TB laboratory;
– handle chemicals safely in the laboratory.
Content outline / • How is TB transmitted?
• What are the biohazards in a TB laboratory?
• Minimum WHO recommendations for TB culture/DST facilities
• Standard safe practices in a TB laboratory
• Personal protective equipment
• Disinfectants active on M. tuberculosis
• Safe disposal of infectious waste
• Coping with spills of infectious material
• Chemical safety
Exercises / Safety precautions for TB culture and DST
Reference / Laboratory biosafety manual, 3rd ed. Geneva, World Health Organization, 2004 (WHO/CDS/CSR/LYO/2004.11).
HOW IS TB TRANSMITTED?
The TB bacilli are almost always transmitted by patients with active pulmonary disease. A patient expels TB bacilli in small droplets of respiratory secretions, which 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 indefinitely and, following inhalation, are able to reach deep into the lungs to produce infection. Larger particles do not remain airborne for as long and do not transmit TB as efficiently.
The risk of infection depends on:
– the infectiousness of the source;
– the environment (e.g. overcrowding and inadequate ventilation promote transmission of droplet nuclei);
– the duration and intensity of exposure; and
– the susceptibility of the recipient.
Smear-positive patients who expel tubercle bacilli into the air, especially when they cough (also when they sneeze, speak or sing), are the most infectious sources.
PROPER COLLECTION OF SPUTUM
Exposure to patients collecting sputum is one of the greatest hazards to a laboratory technician – if the patient is an undiagnosed smear-positive case, he or she will produce infectious aerosols during cough.
Any coughing patient who has to come to the laboratory should be asked to cover his or her mouth with hand or tissue.
Always collect sputum specimens outdoors, where the movement of the air will rapidly dilute infectious droplets and UV rays will inactivate TB bacilli. NEVER collect sputum specimens in laboratories, toilets, waiting rooms, reception rooms, or other enclosed space.
BiohazardS in the TB laboratory
Infectious aerosols are produced in the TB laboratory whenever a liquid suspension containing tubercle bacilli is handled. Biosafety measures in the laboratory are essential to protect workers against exposure to infectious aerosols
Because of their viscosity, sputa are a minimal source of infectious aerosols. By contrast, aerosols produced during processing (especially during centrifugation steps) of homogenized sputa and during culture handling must be minimized and contained in a biological safety cabinet (BSC).
Laboratory 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 to laboratory workers. Options for reassignment of HIV-positive workers away from the high-risk areas should be considered.
MINIMUM WHO RECOMMENDATIONS FOR TB CULTURE / DST FACILITIES
WHO recommends that all specimen processing procedures are carried out in a laboratory built and equipped for biosafety level 2 (BSL2). Minimum requirements for BSL2 are that there is a BSC in the laboratory and an autoclave or other means of decontamination available in the same building.
Identification and DST should be performed in a BSL2 containment room with a directional airflow from functionally clean to dirty areas, with 6 - 12 air exchanges per hour.
The containment room may consist of the blind end of a corridor, or be formed by constructing a partition and door so that access to the laboratory is through an anteroom (e.g. double-door entry) or through the basic BSL2 laboratory. The autoclave should be in the vicinity of the laboratory so that the movement of contaminated materials is minimized.
BSCs have to be ducted to the outside or vented through a thimble. Recirculation from BSCs into the laboratory room and recirculation to other areas within the building are not permitted.
SAFETY PRACTICES IN THE TB culture AND DST LABORATORY
Good microbiological techniques (GMT) – working methods designed to eliminate or minimize exposure to pathogens via, for example, aerosols, splashes, accidental inoculation – are essential for minimizing biohazards. GMT are fundamental to laboratory safety: specialized equipment is an aid to , NOT a replacement for, GMT.
The following precautions are essential to protect both you and all other laboratory personnel.
Standard practices
• Access to the laboratory must be limited.
• Assume that all specimens are potentially infectious.
• Never smoke, eat, or drink in the laboratory.
• Wash hands with soap and water before and after performing any procedures.
• No mouth pipetting, no sucking pencils or pens.
• Do not perform work for which you are not trained.
• Always follow safety procedures.
• Perform all procedures carefully, following GMT, to minimize the creation of splashes or aerosols.
• Decontaminate work surfaces and equipment at least once a day and after any spill of viable material.
• Decontaminate all cultures, stocks, and other regulated wastes before disposal by autoclaving. Materials to be decontaminated outside the immediate laboratory must be placed in durable, leakproof containers, closed for transport from the laboratory. Containers must be packaged in accordance with applicable local, state, and federal regulations before removal from the facility.
Work in a BSC
The BSC is the most important safety equipment in a TB laboratory performing culture and DST.
A Class I BSC is an open-fronted, ventilated cabinet with a unidirectional inward airflow away from the operator. Exhaust air is hard-ducted through HEPA filters which remove particles of diameter 0.3 µm and above with an efficiency of at least 99.95%. The Class I BSC provides protection for the worker and the environment but does not protect products (specimens, strain cultures) against contamination.
A Class II BSC additionally provides protection against contamination of the product (specimens, strain cultures) by recirculating part of the HEPA-filtered air in a laminar vertical flow inside the cabinet so that a “curtain” of clean air descends across the whole working surface. Class II BSCs should be vented to the outside through a thimble or with hard ducting.
Switch on the BSC 15 minutes before use to implement the air flow. Assemble all items needed for work in the BSC to avoid frequent displacements, and organize the work to minimize arm movements.
Do not overcrowd the working area as this disturbs the air flow. Conduct all manipulations within the BSC as far as possible towards the back of the unit. In Class II BSCs, work in the BSC away from the grille; never allow the grille to be covered by anything.
Do not use large open flames in the BSC: they create turbulence which disrupts the pattern of air supplied to the work surface. Special Bunsen burners for use in BSCs are recommended.
The BSC should be switched off 15 minutes after the end of work to eliminate all infectious aerosols.
PERSONAL PROTECTIVE EQUIPMENT
The decision of use of additional PPE should be based on risk assessment (e.g.. workload, prevalence of tuberculosis, prevalence of multi-drug resistant TB, rate of failure/relapse). Risk assessment should be reviewed routinely and revised when necessary.
Masks and respirators
One of the commonest misconceptions is that a standard surgical mask will protect the wearer from becoming infected with TB. Surgical masks are made from porous material that will not trap droplets nuclei, and they fit poorly, leaving large gaps between the face and mask. They are designed to prevent the spread of microorganisms from the wearer to others by capturing the large wet particles in the exhaled air. They are not designed to protect the wearer from inhaling small infectious aerosols.
Although BSC and air flow in the laboratory are the main means of protection against exposure to contaminated aerosols generated during culture and DST activities, additional personal protection must be risk-assesses and e.g. considered in settings where MDR-TB and/or HIV are prevalent. Staff may be HIV-infected and highly susceptible to contaminated aerosols.
Protection against inhalation of infectious aerosols can be provided by respirators, which are devices with the capacity to filter particles of 0.3–0.4 µm diameter and which fit closely to the face to prevent leakage around the edges. The N95 (or FFP2) respirator is a lightweight, disposable nose and mouth respirator; it effectively filters out more than 95% of the particles of diameter 0.3 µm and above. The FFP3 respirator removes more than 98% of such particles.
Each user should be instructed in the proper use of the respirator and informed about its limitations. Respirators should be correctly fitted to the face to prevent leakage around the face seal. This is done by placing the mask over the nose and mouth with the top elastic band over the crown of the head and the bottom elastic band over the back of the neck. The metal strip covering the nose should be firmly moulded over the bridge of the nose. Facial hair between the wearer's skin and the sealing surfaces of the respirator will prevent a good seal.
Respirators should also be worn during emergency cleaning of spillages that involve the release of viable organisms in the work area.
Respirators should be stored in a convenient, clean, and sanitary location and should be discarded after 8 hours (cumulative) of use and not be kept for more than one week.
Gloves
In accordance to universal precautions, appropriate gloves should be worn for all procedures that involve handling of body fluids. Gloves must be worn in case of hand injury/skin disease or when the risk of exposure to blood-borne pathogens is high; specimens resulting from invasive clinical investigation must be handled with gloves.
Gloves must be worn while handling cultures.
Gloves may give a false sense of protection. Contaminated gloves may in fact be the source of hazards for other staff members if used to handle or operate equipment in the laboratory. Change gloves after every session that requires their use and after every interruption of the activity. Never wear gloves outside the laboratory.
Change gloves after every activity session that requires their use and after every interruption of the activity. Every time hands are removed from the BSC, gloves must be pulled out (as indicated below) and discarded in a waste container in the BSC.
Disposable latex, vinyl or nitrile gloves can be used, and the correct size (small, medium or large) should be available for all individuals. Hypoallergenic gloves should be provided in case of allergy to latex proteins and/or to the cornstarch used for powder.
Reusing single-use gloves is not advised, discard used gloves as contaminated material.
Following the safe removal of gloves, wash hands immediately with water and liquid soap. Proper hand-washing with soap and adequate care in the handling of contaminated materials are critical elements of safe laboratory practice.
Gowns
Always use a gown inside the laboratory (never outside) and change at least weekly.
Long-sleeved with narrow cuffs, back-opening gowns or overalls give better protection than laboratory coats and are preferred in microbiology laboratories. When worn, laboratory coats should be fully buttoned.
An area of the laboratory must be designated for storage of used and new clothing. Laboratory gowns must be stored apart from personnel clothing. Laundering services should be provided at/near the facility.
Consideration should be given to having extra clothing suitable for visitors, maintenance, and emergency response personnel.
Removing personal protective equipment
Always remove in the following order:
1. Respirator/mask
2. Gown/coat/suit/overalls
3. Disposable gloves
Personal protective equipment – summary
• Surgical masks do NOT protect against TB infection. TB bacilli pass through them, and they may give a false sense of security.
• Effective respiratory protection is provided by an N95 respirator.
• Hand-washing and careful technique are acceptable practices for most countries.
• The additional protection conferred by respirators must be assessed for each setting.
DISINFECTANTS
Phenol should be used at a concentration of 2–5%, with a contact time of 15–30 minutes, depending on the type and volume of material to be disinfected. It is useful in soaked paper towels to cover working surfaces: this minimizes spatter and aerosol formation in the event of spillage.
Phenol is highly irritating to the skin, eyes and mucous membranes. It is also considered to be very toxic through oral exposure. Because of this toxicity and the odour, phenol derivatives are now generally used in its place. Many phenolic compounds are used for the decontamination of surfaces and some (e.g. triclosan, chloroxylenol, orthophenylphenol) are among the more commonly used antiseptics. Commercially available solutions should be used according to the manufacturer's instructions for "dirty or worst-possible situations".
Hypochlorite is a widely available disinfectant. It is normally sold as bleach, an aqueous solution of sodium hypochlorite (NaOCl), which can be diluted with water to provide various concentrations of available chlorine. It should be used at concentrations of 0.1% and 0.5% (1 and 5 g/l); the lower concentration is recommended for “clean conditions” and the higher for “dirty conditions”, with a contact time of 15–30 minutes, depending on the type and volume of material to be disinfected.
Bleach can be used as a general-purpose disinfectant and for soaking contaminated metal-free materials (it is highly alkaline and can be corrosive to metal).
Sodium hypochlorite solutions, as domestic bleach, contain 50 g/l available chlorine and should therefore be diluted 1:50 or 1:10 to obtain final concentrations of 1 g/l and 5 g/l, respectively. Bleach, whether stock or in working solutions, must be stored in a well-ventilated, cool, dark area. In good storage conditions, the 50 g/l solution may last as long as 3 months; diluted solutions should be prepared daily. Storage of stock or working solutions in open containers releases chlorine gas, thus weakening the germicidal potential. Moreover, chlorine gas is highly toxic.