Module 02_BIOSAFETY.

Report
Module 2:
Guidance on biosafety
Global Laboratory Initiative – Xpert MTB/RIF Training Package
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Transmission of TB bacilli
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Risk assessment and precaution levels
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Safety practices in the TB microscopy laboratory and
the Xpert MTB/RIF laboratory
Safe disposal of infectious waste
Global Laboratory Initiative
Xpert MTB/RIF Training Package
At the end of this module, you will be able to:
 Describe the safety risks in laboratories using the Xpert
MTB/RIF test
 Explain what precautions should be taken to safely use
Xpert MTB/RIF
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Xpert MTB/RIF Training Package
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Following laboratory biosafety procedures prevents
laboratory staff from becoming infected with TB and
prevents microorganisms from being released into the
environment
◦ Specialized equipment may aid good laboratory practice
but does NOT replace it
◦ Always wear gloves and laboratory coats when handling
samples from patients
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Xpert MTB/RIF Training Package
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The main risks in a TB laboratory are related to the aerosols
generated during the procedures that could be inhaled by
laboratory workers.
The risk of aerosolization is associated with the:
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Type of procedure
Frequency of testing, and the laboratory’s workload
Consistency of the material and its predisposition to
aerosolize (for example, viscous liquids versus dry solids)
Bacillary load of the materials.
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Xpert MTB/RIF Training Package
Higher
risk
Doctors and nursing staff in TB ward
Healthcare workers assisting
with specimen collection
Lower
risk
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Laboratory technicians processing
specimens for smear microscopy or
the Xpert MTB/RIF assay
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Xpert MTB/RIF Training Package
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WHO has adopted an approach that assesses
the risks associated with different technical
procedures performed in different types of
TB laboratories.
◦ Risk-group classifications and
containment levels (BSLs) are no longer
used.
◦ WHO’s biosafety manual for TB laboratories
describes the MINIMUM requirements for
facilities, and the practices that can be
adopted following a risk assessment (see
http://www.who.int/tb/laboratory/resource)
Global Laboratory Initiative
Xpert MTB/RIF Training Package
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A risk assessment is simply a careful examination of what in your work
could cause harm to people.
A risk assessment must consider:
◦ The bacterial load of materials, and the viability of TB bacilli
◦ The route of transmission of TB
◦ Whether the materials handled and the manipulations required for each procedure
are likely to generate infectious aerosols
◦ The number of manoeuvres in each technique that may generate aerosols
◦ The workload of the laboratory and of individual staff members
◦ The location of the laboratory
◦ The epidemiology of the disease and the patient population
◦ The level of experience and competence of lab staff
◦ The health of lab staff (especially HIV-positive staff).
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Xpert MTB/RIF Training Package
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Identify potential hazards.
Decide who might be harmed and how.
Evaluate the risks and decide on precautions:
− Determine the suitability of the physical space
− Evaluate the staff’s proficiency in following safe practices
− Evaluate the integrity of safety equipment.
Record your findings and implement any necessary changes.
Review your assessment and update it when necessary.
A risk-assessment tool is available at
http://www.gliquality.org/
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Xpert MTB/RIF Training Package
Risk level of TB
laboratory
Low risk
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Laboratory
activities
Preparing
specimens for
smear microscopy
or Xpert MTB/RIF
testing
Assessment of risk
Procedures have a
low risk of
generating
infectious aerosols
from specimens;
the concentration
of infectious
particles is low
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Xpert MTB/RIF Training Package
Risk level of TB Laboratory
laboratory
activities
Assessment of
risk
Moderate risk
Procedures have a
moderate risk of
generating infectious
aerosols; the
concentration of
infectious particles is
low to moderate
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Processing and
concentrating
specimens for Xpert
MTB/RIF testing or
inoculation onto primary
culture media; splitting
specimens; direct DST
(for example, LPA on
processed sputum)
Global Laboratory Initiative
Xpert MTB/RIF Training Package
Risk level of TB
laboratory
Laboratory
activities
Assessment of
risk
High risk
(TB-containment
laboratory)
Culture
manipulation for
identification; DST
or LPA on cultured
isolates
Procedures have a
high risk of
generating
infectious aerosols;
the concentration
of infectious
particles is high
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Global Laboratory Initiative
Xpert MTB/RIF Training Package
The Xpert MTB/RIF test is a low-risk procedure, and
requires the same level of precautions as those used for
performing direct AFB sputum-smear microscopy.
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Work in a well ventilated area.
Wear gloves and a laboratory coat at all times when
handling samples from patients.
In settings with a high burden of MDR-TB, a risk assessment
may determine that additional biosafety precautions are
required in Xpert MTB/RIF laboratories; these precautions
may include the use of N95 respirators or biosafety cabinets
(BSCs).
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Xpert MTB/RIF Training Package
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Add sample reagent to the specimen in a ratio of 2:1. This
step inactivates M. tuberculosis bacilli in the sample,
significantly reducing the biosafety risk to the laboratory
technician.
The sample reagent is provided in 8 ml vials. Processing
sputum specimens that have a volume greater than 4 ml
requires using more than one vial of reagent, splitting the
specimen into smaller volumes or concentrating the
specimen.
Because of the risk of generating aerosols, splitting
specimens and handling concentrated specimens must be
performed within a certified BSC.
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Xpert MTB/RIF Training Package
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Smear preparation and staining: [Dirty activity]
Processing samples for Xpert MTB/RIF testing, and
inoculating cartridges:
[Dirty activity]
Microscopic examination of stained smears: [Clean activity]
Loading cartridges into the GeneXpert instrument:
[Clean activity]
Record keeping and storage: [Clean activity]
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Xpert MTB/RIF Training Package
Adapt according to the NTP’s guidelines in your country
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Use of bench spaces: The bench used to process
specimens for direct sputum-smear microscopy or the
Xpert MTB/RIF assay should be separate from areas where
specimens are received and from areas where paperwork
is completed and telephones are used.
Ventilation: When appropriate microbiological techniques
are used, direct smear testing and direct processing of
specimens for the Xpert MTB/RIF assay may both be
carried out on an open bench in an adequately ventilated
area.
Global Laboratory Initiative
Xpert MTB/RIF Training Package
Adapt according to the NTP’s guidelines in your country
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For low-risk procedures natural ventilation should be sufficient, providing that
air flows away from the technician and across the work area to the outside.
When the climate prevents windows from being opened, consideration should be
given to using mechanical ventilation systems, such as an exhaust fan.
• Where natural or mechanical ventilation is
not practical, ventilated work stations are an
optional solution for managing aerosol
containment during direct sputum-smear
microscopy or Xpert MTB/RIF testing
(http://www.aphl.org/aphlprograms/global/D
ocuments/GH_2011July_VentilatedWorkstation
Guidance.pdf).
• A BSC is not essential for Xpert MTB/RIF
testing on direct specimens (i.e., specimens
that do not require splitting or concentration).
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Xpert MTB/RIF Training Package
Adapt according to the NTP’s guidelines in your country
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Adequate ventilation for TB laboratories is typically
described as directional airflow with 6–12 air exchanges per
hour (ACH).
Directional airflow refers to air moving from clean areas
across areas where aerosols may be generated (dirty areas)
and then to the outside.
Global Laboratory Initiative
Xpert MTB/RIF Training Package
Adapt according to the NTP’s guidelines in your country
Ways to determine whether ventilation is adequate
MEASURING AIR VELOCITY
DETERMINING AIR
DIRECTION
-Anemometer
Precision instrument
Assessments and verifications
- Smoke tubes
-Vaneometer
Simple and affordable
Measures air velocity and direction
Can be used routinely
Courtesy of P. Jensen - CDC
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Global Laboratory Initiative
Xpert MTB/RIF Training Package
Adapt according to the NTP’s guidelines in your country
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Gloves: essential (use gloves that are disposable and
powder-free).
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Lab coats: essential.
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Respirators: normally not required but
◦ May be needed based on a risk assessment
◦ Must always be included in a spill kit
◦ Recommended respirators are N95 (NIOSH N95)
and FFP2 (EN 149:2001).
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Global Laboratory Initiative
Xpert MTB/RIF Training Package
Adapt according to the NTP’s guidelines in your country
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Gloves are required for processing specimens for
Xpert MTB/RIF testing.
Use disposable powder-free gloves.
Wearing gloves may give technicians a false
sense of safety:
◦ Regular and thorough handwashing remains
essential.
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To avoid contamination remove gloves before
using computer terminals or telephones.
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DO NOT reuse gloves.
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DO NOT wear gloves outside the laboratory.
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Global Laboratory Initiative
Xpert MTB/RIF Training Package
Adapt according to the NTP’s guidelines in your country
Laboratory coats are essential:
◦ Leave coats at the worksite
(do not take coats home)
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Fasten the laboratory coat when worn
Use appropriate size and type
DO NOT wear outside of the laboratory
Launder at least weekly, and after any overt
contamination (do not take the coat home); disinfect
before laundering.
Global Laboratory Initiative
Xpert MTB/RIF Training Package
Adapt according to the NTP’s guidelines in your country
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N95 and FFP2 respirators effectively filter out >94-95% of
particles ≥0.3-0.4 µm in diameter.
Respirators must be fitted to the face! (Use the FIT-TEST)
Respirators protect the wearer
from inhaling droplet nuclei
(they protect against inhalation).
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Surgical masks do not protect the wearer against inhaling
infectious aerosols.
Surgical masks prevent
microorganisms spreading from the
wearer (they protect others from
exhalation).
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Global Laboratory Initiative
Xpert MTB/RIF Training Package
 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 before autoclaving for predecontamination treatment.
NOTE: Because of the specific cell-wall structure of TB
bacilli, they are resistant to most standard disinfectants –
that is, quaternary ammonium compounds are ineffective.
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Global Laboratory Initiative
Xpert MTB/RIF Training Package
Adapt according to the NTP’s guidelines in your country
Select disinfectants that are effective against mycobacteria based on the material to be
disinfected.
 PHENOL 2-5% in deionized water is highly irritating, and caution must be used in
preparation. It is preferable to use phenolic derivatives:
◦ Decontaminate equipment, surfaces and items or liquids before disposal (wear
gloves)
◦ Prepare the solution daily and leave in contact with the surface for at least 15
minutes to ensure decontamination.
 CHLORINE (sodium hypochlorite, or bleach with 0.72% active chlorine) is an irritant,
and is corrosive to metals and plastics:
◦ It is a general purpose disinfectant; also can be used to soak contaminated items
◦ Allow at least 15 minutes to ensure decontamination
◦ Prepare daily and store in a well-ventilated area (toxic gas). Do not autoclave.
 ALCOHOL 70% leaves no residue but is volatile and flammable (keep far from open
flames):
◦ Use as a disinfectant on skin (follow by washing with soap) and work surfaces
(including metals).
 PERACETIC ACID leaves no residue, but is stable for only 48 hours after preparation:
◦ Rapid action against all microorganisms.
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Xpert MTB/RIF Training Package
 Diluted solutions should be
prepared daily.
 Store stock solutions according to
the manufacturer's
recommendations.
 Use commercial solutions for dirty
situations or the worst possible
situations (check to be certain
that the product is
tuberculocidal).
NEVER ADD WATER TO ACID
ALWAYS ADD ACID TO WATER
WATER
ACID
WATER
Results in excessive heat,
foaming, splashing!
Pour slowly to avoid
excessive heat build-up.
Fire in BSC:
Bunsen burner
ignited 70% alcohol
used for cleaning
 Follow national chemical safety
guidelines.
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Xpert MTB/RIF Training Package
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All laboratories handling samples for TB diagnostic testing should
have a spill kit containing:
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Instructions (SOPs) for cleaning up spills
A large biohazard bag (autoclavable)
Suitable tuberculocidal disinfectant, such as hypochlorite
(freshly prepared) or phenol-derivatives, stored in
opaque bottles
Laboratory gowns (disposable) and goggles
Box of gloves (different sizes)
Respirators (N95 or FFP2)
Paper towels, cotton wool or absorbent cloths
Soap and chloramine tablets
Dustpan
Sharps container
“DO NOT ENTER” sign.
Check the contents of the spill kit regularly and replace items
after use or when they have expired.
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Xpert MTB/RIF Training Package
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All personnel in the laboratory must be trained in the
procedures for handling spills.
The actions required depend on where the spill occurs:
◦ Outside a BSC
◦ Within a BSC
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Xpert MTB/RIF Training Package
Spill outside the BSC (major event) (1 of 2)
◦ Immediately vacate and secure the laboratory, and inform the
laboratory manager.
◦ Leave the laboratory’s ventilation or exhaust systems on, including in
the BSC.
◦ Do not re-enter the laboratory for at least 1 hour (post DO NOT
ENTER signs).
◦ Before re-entering the laboratory, put on clean gloves, a clean
laboratory coat and a respirator.
◦ Cover the spill (or spills) with cloths or absorbent paper, and soak the
paper with a suitable disinfectant; apply the disinfectant
concentrically, from the outer margin towards the centre of the spill.
◦ Allow sufficient time for the disinfectant to act (at least 30-60
minutes) before disposing of any material.
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Spill outside the BSC (major event) (2 of 2)
◦ Collect all containers and clean-up material and place them in
the disposal bag provided in the spill kit; tie the disposal bag
and place it in a container to be autoclaved (use an appropriate
container for sharp objects).
◦ Change gloves if they have been contaminated; dispose of
these along with other infectious waste.
◦ Clean and disinfect the area of the spill.
◦ Staff exposed to the spill should be referred for medical advice,
and details recorded in the incident logbook.
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Xpert MTB/RIF Training Package
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Spill within the BSC (1 of 2)
◦ Cover the spill area with cloths or absorbent paper, and apply a suitable
disinfectant concentrically – that is, from the outer margin towards the
centre of the spill:
 Any equipment or material that has been splashed must be cleaned
(including the interior surfaces and walls of the BSC, or safety buckets)
 Do not use bleach to disinfect metal parts (it is corrosive).
◦ Allow sufficient time for the disinfectant to act (30-60 minutes) before
disposing of any material.
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Spill within the BSC (2 of 2)
◦ Within the BSC, place all containers and clean-up material
into the disposal bag provided in the spill kit; tie the bag,
and place it in a container to be autoclaved.
◦ Change gloves if they have been contaminated; dispose of
these along with other infectious waste.
◦ Staff exposed to the spill should be referred for medical
advice, and details recorded in the incident logbook.
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Breakage of tubes inside sealed buckets
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Always use sealed centrifuge buckets.
Load and unload them in a BSC.
Global Laboratory Initiative
Xpert MTB/RIF Training Package
Adapt according to the NTP’s guidelines in your country
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At the end of each day, seal
contaminated material (such as used All used materials should be
sputum containers, transfer pipettes considered to be contaminated!
and used cartridges) in a biohazard
bag, and autoclave or incinerate or bury
it as soon as possible.
◦ Warning: incinerating plastics can
release toxins that are harmful to
breathe.
Decontaminate transfer pipettes using
an appropriate disinfectant prior to
disposal.
If the NTP’s guidelines do not adequately describe the safe disposal of infectious waste, general
guidance that can be modified for this slide can be found on page 31 of:
http://www.finddiagnostics.org/export/sites/default/resourcecentre/reports_brochures/docs/malaria_rdt_transport_healthclinics_may09.pdf
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The Xpert MTB/RIF test is a low-risk procedure that requires the same
level of precautions as those used for performing direct AFB sputumsmear microscopy.
The main risks in a TB laboratory are related to the aerosols generated
during the procedures that could be inhaled by laboratory workers.
The risk of aerosolization is associated with the type of procedure, the
frequency of testing, the workload, the consistency of the material and
its predisposition to aerosolize (for example, viscous liquids versus dry
solids), and the bacillary load of the materials.
A BSC is required only when splitting and handling concentrated
specimens.
Disinfectants should be selected based on their tuberculocidal activity
and the material to be disinfected.
Ensure that the laboratory is prepared for emergencies and has standard
operating procedures for handling accidents such as spills.
Global Laboratory Initiative
Xpert MTB/RIF Training Package
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What are the sources of infectious aerosols in a TB laboratory?
What precautions must be taken when handling specimens for
performing Xpert MTB/RIF testing (direct and indirect)?
What additional safety precautions are needed to perform Xpert MTB/RIF
testing compared with smear microscopy?
What are the critical items and equipment needed to safely process
specimens?
What are the most effective disinfectants in TB laboratories?
What procedures should be followed when coping with accidents?
What are the contents of a spill kit?
What are the steps for safely disposing of infectious waste?
Global Laboratory Initiative
Xpert MTB/RIF Training Package
Acknowledgements
The Xpert MTB/RIF Training Package has been developed by a consortium of
GLI partners, including FIND, KNCV, US CDC, USAID, TB CARE I and WHO, with
funding from USAID.
The modules are based on materials originally developed by FIND, KNCV and
Cepheid.

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