Draft 05 Sept

**Guidelines for implementing the global certification requirements
(Phase II) of laboratory containment of wild polioviruses**

**Introduction**

The world will be declared free of wild poliovirus transmission when the
Global Commission for the Certification of the Eradication of
Poliomyelitis is satisfied that all Regions of the World Health
Organization (WHO) have documented the absence of wild poliovirus
circulation for at least three consecutive years and all wild poliovirus
materials in laboratories are adequately contained (1).

The *WHO Global action plan for laboratory containment of wild
polioviruses, 2^nd^ Edition* (2) describes the containment requirements
for global certification of polio eradication in two phases, Laboratory
Survey and Inventory (phase I) and Global Certification (phase II). The
present phase II guidelines are provided to assist countries in
implementing and documenting the laboratory containment activities
required for Global Certification. These guidelines assume that the
following phase I requirements have been completed:

-   Qualifying biomedical institutions/laboratories have been surveyed
    and encouraged to destroy unneeded wild poliovirus infectious or
    potential infectious materials

-   Institutions/laboratories retaining such materials have been placed
    on a National Inventory and the results transmitted to the Regional
    Certification Commission through the WHO regional office

-   Laboratories listed on the National Inventory have been requested to
    implement biosafety level-2 (BSL-2/polio) measures for safe handling

-   National planning has been initiated for implementation of the
    Global Certification phase

The Global Certification Commission has requested that all countries
conduct a self assessment of the quality of the phase I activities and
submit documentation of their findings as outlined in the *Guidelines
for documenting the quality of phase I wild poliovirus laboratory
containment - laboratory survey and inventory* (3)*.*

**Phase II - Global Certification**

The Global Certification phase of containment begins when one year has
elapsed without isolation of wild poliovirus anywhere in the world (2).

During this phase nations should undertake the following:

1.  *Notify biomedical laboratories that poliovirus transmission has
    been interrupted.*

2.  *Contact laboratories on the national inventories and instruct them
    to choose one of the following options:*

*-Render materials non-infectious for poliovirus or destroy under
appropriate conditions.*

> *-Transfer wild poliovirus infectious and potential infectious
> materials to laboratories capable of meeting the required Biosafety
> standards.*

*-Implement Biosafety measures appropriate for the laboratory procedures
being performed (BSL-2/polio or BSL-3/polio).*

3.  *Document the completion of all containment requirements for global
    certification.*

1\. Notify biomedical laboratories that poliovirus transmission has been
interrupted.

The Office of the Director General, WHO, Geneva, in consultation with
its advisory bodies, will announce when one year has passed with no
evidence of wild poliovirus circulation anywhere in the world. WHO will
request nations to begin implementation of global certification
biosafety requirements for completion no later than one year from the
date of the announcement.

Nations will notify the general biomedical laboratory community that
wild poliovirus circulation has been interrupted and request agencies /
institutions and laboratories listed on national inventories to
implement the biosafety requirements for global certification. In
countries where more than one year has elapsed since the initial survey,
additional steps will be taken to search for new or other qualifying
biomedical laboratories not included in the initial survey. Special
attention will be given to identifying and communicating with
laboratories at risk of having acquired wild poliovirus after completion
of the initial survey.

[Notification of general biomedical laboratory community]{.underline}

Multiple channels and strategies will be required to notify the general
biomedical laboratory community that poliovirus transmission has been
interrupted and that global certification requirements for laboratory
containment should be implemented. Examples of mechanisms for notifying
laboratories include:

-   Publications in articles in lay and professional press

-   Announcements in professional and trade journals

-   Notification of members through professional societies

-   Direct mailings to institutions / laboratories

The national containment coordinator should choose communications
strategies that reach the highest possible number of laboratories with
the containment messages. Key messages to be communicated to the general
laboratory community are listed in Box 1.

An example of a statement suitable for press release or the core of a
general letter is shown in Annex 1.

[Notification of laboratories at risk of having acquired wild poliovirus
materials after completion of the initial survey]{.underline}

In many countries, several years may have passed from the date of the
initial laboratory survey to the date of interruption of wild poliovirus
circulation and the beginning of phase II containment. National
containment coordinators will confirm that laboratories did not acquire
wild poliovirus materials after completion of the initial survey. The
first step is to review the survey database to identify laboratories at
risk of having recently acquired poliovirus materials, including:

-   major research universities and institutions

-   reference public health laboratories

-   diagnostic laboratories performing specialized microbiological tests

-   industrial laboratories with procedures using poliovirus

-   laboratories with contacts in areas recently polio endemic

The national coordinator will communicate with responsible officials at
the institutions / laboratories so identified and provide a form for
signature confirming that all laboratory holdings have been reviewed and
that the initial negative report of wild poliovirus materials has or has
not changed (Annex 2).

[Notification of newly formed or other qualifying biomedical
laboratories not included in the initial survey]{.underline}

In some countries, new laboratories may have been established after
completion of the initial survey. The national containment coordinator
will identify new laboratories and evaluate their need to confirm that
they do not have wild poliovirus materials. The strategy for identifying
newly formed or missed laboratories will differ from country to country.
Much will depend on the results from the initial survey and the elapsed
time since it was completed. If, for example, the initial survey found
that clinical laboratories were unlikely to have wild poliovirus
materials, then searching for new clinical laboratories could be
unnecessary. On the other hand, searching for newly formed biotechnology
laboratories may be justified if the initial survey found that this
group of laboratories had worked with poliovirus in the past. Literature
searches may also assist in locating research laboratories that were not
included in the initial survey.

The national containment coordinator will contact the responsible
officials at such institutions / laboratories and request completion of
a survey form, which may be similar to that used in phase I (4).

2\. Contact laboratories on the National Inventory and instruct them to
implement one of the containment options

National authorities will notify laboratories listed on the national
inventory that wild poliovirus circulation has been interrupted and to
implement containment requirements for Global Certification. Similarly,
national vaccine regulatory authorities, in collaboration with WHO, will
notify inactivated polio vaccines (IPV) producers to implement phase II
requirements. Implementation of all biosafety requirements will be
completed in one year and documentation of containment will be submitted
the following year.

Laboratories listed on the National Inventory will be requested to
re-examine the need for long-term retention of wild poliovirus
infectious and potential infectious materials in view of the personal
and institutional responsibilities inherent in retaining a virus that
has been eradicated. All institutions/laboratories on the inventory will
be requested to submit a completed signature form (Annex 3) accompanied
by documentation that wild poliovirus materials were rendered
non-infectious, destroyed, transferred, or retained.

[Render materials non-infectious for poliovirus or destroy them under
appropriate conditions.]{.underline}

Poliovirus materials may be rendered non-infectious by heating at 56
degrees centigrade for 30 min or longer. Polioviruses may also be
inactivated by treatment with chlorine solution (bleach) or other
chemical disinfectants with proven efficacy against poliovirus. Methods
for destruction of poliovirus materials include autoclaving and
incineration, which are described in the *Global Action Plan* (2) and
listed in Annex 4.

[Transfer wild poliovirus infectious and potential infectious materials
to laboratories meeting the global certification biosafety
requirements]{.underline}

Some laboratories may have wild poliovirus materials in storage that are
no longer relevant to their current program, but may have special
scientific or historic value. The national containment coordinator will
assist laboratories in assessing the value of such materials and, if
necessary, transferring them to a laboratory that meets the Global
Certification biosafety requirements. Transfers of wild poliovirus
materials must meet national and international requirements for the safe
shipping of infectious substances (Annex 5). Details of the transfer
must be documented and the national containment coordinator notified to
amend the National Inventory.

[Implement biosafety requirements appropriate for the laboratory
procedures being carried out (BSL-2/polio or BSL-3/polio)]{.underline}

Laboratories on the National Inventory will be provided with a complete
description of biosafety requirements (2) in advance of notification to
begin phase II implementation (Annex 6 & 7). Biosafety requirements for
global certification will vary depending upon an institutional risk
assessment of types of materials and activities performed. All wild
poliovirus infectious materials must be handled under BSL-3/polio
conditions. All activities that involve inoculating
poliovirus-permissive cells or animals with potential wild poliovirus
infectious materials, i.e. any biological system in which polioviruses
replicate, must also be performed under BSL-3/polio conditions. All
other activities involving potential wild poliovirus infectious
materials may be conducted safely in a certified class I or II
biological safety cabinet in a BSL-2/polio laboratory. Standard
operating procedures (SOP) available and carefully followed in each
laboratory to ensure that the biosafety requirements are maintained
(Annex 8). The Global Certification biosafety requirements are
summarized in Box 2.

  --------------------------- ----------------------- ---------------------
  **Box 2: Global                                     
  certification biosafety                             
  requirements for wild                               
  poliovirus materials**                              

  **Category of material**    **Laboratory activity** **Biosafety level**

  Wild poliovirus infectious  All activities,         BSL-3/polio
  materials (box 2)           including storage       

  Potential wild poliovirus   Activities involving    BSL-3/polio
  infectious materials (box   poliovirus-permissive   
  3)                          cells or animals        

                              Other activities        BSL-2/polio, in
                                                      certified class I or
                                                      II biosafety cabinet
  --------------------------- ----------------------- ---------------------

> Implementation of appropriate biosafety conditions must be verified.
> Laboratories working under BSL-2 / polio conditions will provide the
> national containment coordinator with the report of a biosafety review
> by a qualified professional, who may be affiliated with the
> institution. Verification of the BSL-3 / polio laboratory should be
> done by a qualified biosafety professional(s), who is familiar with
> the operation of a BSL-3 facility (annex 9) and is not affiliated with
> the institution.
>
> Containment of wild poliovirus materials is a national as well as
> institutional responsibility. The national containment coordinator
> will need to establish a system of periodic reviews to ensure that
> laboratories on the National Inventory maintain the biosafety
> requirements and report any changes in the situation (Annex 10).
> Laboratories found not to meet the biosafety requirements must
> discontinue work with poliovirus until corrective action is
> documented. Changes in the number of institutions/laboratories listed
> on the national inventory will be reported to the WHO Regional office.
>
> **3. Document completion of all containment requirements for global
> certification.**
>
> Documentation on the state of containment will be retained by
> institutions / laboratories and national authorities. Copies of
> selected documents from phase I and II will form the basis for
> documenting national containment for global certification.
>
> [Documents to be retained by institutions/laboratories
> include:]{.underline}

-   A full description of all wild poliovirus infectious and potential
    infectious materials including origin, number of vials and volumes,
    storage location, including building, room, and freezer

-   The name(s) of principal investigator and persons authorized to
    access the materials

-   A description of current work involving polio materials

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-   Copies of all correspondence, signed statements, and biosafety
    reports submitted to the national containment coordinator

> [Documents to be retained by national authorities
> include]{.underline}:
>
> Documentation of national inventory process

1)  Report describing implementation of Phase I activities **(**Result
    from Guidelines for documenting the quality of Phase I wild
    poliovirus laboratory containment activities: Laboratory survey and
    National inventory)

2)  Report describing implementation of Phase II activities including
    descriptions of the following:

-   process for notifying the laboratory community that wild polio
    circulation has been interrupted

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-   efforts to identify newly formed laboratories

-   repeat surveys of certain laboratories and criteria for selection

-   notification of laboratories on the national inventory to implement
    containment

3)  National laboratory database (list of laboratories surveyed)

4)  Results of national laboratory survey (paper records and electronic
    database)

5)  Records of follow-up with laboratories (letters and other
    correspondence)

6)  Signed statements from laboratories that reported destruction of
    wild poliovirus materials after completion of the initial survey
    process (annex 3)

7)  Signed statements from laboratories reporting transfer of wild
    poliovirus materials and signed receipt from recipient laboratories
    (annex 4)

8)  National Inventory of laboratories holding wild poliovirus
    infectious and potential infectious materials (annex 11)

-   Name of institution, address, and contact information for
    responsible official

-   Names and locations of laboratories retaining materials

-   Types (wild poliovirus infectious or potential infectious) and
    respective amounts of materials being held (e.g. number of vials)

9)  Description from each institution of the process for periodic
    biosafety review to ensure the laboratory continues to meet
    biosafety requirements (annex 10)

> Documentation of biosafety requirements

10) Signed statements from institutions that such materials are
    maintained under BSL-2/polio or BSL-3/polio biosafety conditions as
    appropriate

11) BSL-2/polio: Report of qualified professional biosafety officer that
    laboratory meets specified conditions, only potential infectious
    materials are being handled, and only low-risk procedures are being
    performed (annex 5,9)

12) BSL - 3/polio: Report of external professional biosafety review that
    laboratory meets all high containment requirements including storage
    of materials listed on inventory (Annex 6,9)

13) A description of the national process for post global certification
    periodic review for maintaining the national inventory and ensuring
    that the listed laboratories meet the biosafety requirements (annex
    10)

14) Summary of collaboration with WHO and other international bodies to
    ensure that eligible IPV producers within country comply with the
    WHO *Guidelines for containment in IPV production facilities*

> Documentation of certification process

15) Records on follow-up from Regional Certification Commission reviews
    and recommendations

16) Signed final endorsement from the national certification committee

> [Documentation to be submitted to the Regional Certification
> Commission]{.underline}
>
> At the end of the first year after notification, all countries should
> submit documentation of containment to its National Certification
> Committee for review. When endorsed by the National Committee,
> documentation will be forwarded to the highest political authority
> overseeing laboratory containment activities (e.g. Minister of
> Health). This authority will submit the documentation to the WHO
> Regional Office for review by the Regional Commission. When satisfied
> that the containment requirements for all countries have been
> fulfilled, the Regional Commission will submit Regional documentation
> through WHO Headquarters to the Global Commission.
>
> *Reports to the Regional Commission from countries with no wild
> poliovirus materials will include:*

-   Documentation of the national inventory process (numbers 1, 2, 6,
    and 7 in the list of documents retained by the national authorities)

-   Documentation of the certification process (number 16)

> *Reports to the Regional Commission from countries with laboratories
> retaining wild poliovirus materials will include:*

-   Statement from the national authority having responsibility for
    laboratories with wild poliovirus materials

-   Documentation of the national inventory process (numbers 1, 2, 6, 7,
    and 8 in the list of documents retained by the national authorities)

-   Documentation of biosafety requirements (numbers 11, 12, 13, and 14)

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-   Documentation of the certification process (number 16)

> **References:**
>
> **Annexes**

1.  Sample statement for notification of the general biomedical
    laboratory community that poliovirus transmission has been
    interrupted and that the global requirements for laboratory
    containment are now in effect (Walt)

2.  Sample signature form that all laboratory holdings have been
    reviewed and that the initial negative report of wild poliovirus
    materials has or has not changed (Walt)

3.  Sample signature form for institutions/laboratories on phase I
    inventory to indicate that wild poliovirus materials were rendered
    non-infectious, destroyed by autoclaving or incineration,
    transferred to a laboratory that meets the appropriate containment
    level, or retained (Walt)

4.  **Methods for disposal of wild poliovirus infectious or potential
    infectious materials**

5.  **Requirements for the safe transport of wild poliovirus infectious
    or potential infectious materials**

6.  **BSL-2/polio biosafety requirements**

7.  **BSL-3/polio biosafety requirements**

8.  **Model of standard operating procedure (Maureen)**

9.  **Protocol for verification and reporting of BSL-2/polio and
    BSL-3/polio conditions (Maureen)**

10. Sample of the national process for post global certification
    periodic review that laboratories on the national inventory maintain
    biosafety requirements (Maureen)

11. Sample national inventory of laboratories holding wild poliovirus
    infectious or potential infectious materials (Chris)

**ANNEX 1**

**ANNEX 2**

**ANNEX 3**

**ANNEX 4**

Methods for disposal of wild poliovirus infectious materials or
potential wild poliovirus infectious materials

#### 

#### Sterilization (use of autoclaves)

The use of moist steam under pressure is the most effective method for
sterilizing laboratory materials.

-   All cultures and contaminated materials should normally be
    autoclaved in leak-proof containers, e.g. autoclavable colour-coded
    plastic bags, before disposal.

-   Packaging should allow the penetration of steam.

-   After being autoclaved the materials may be placed in transfer
    containers for transportation to the disposal point.

-   Autoclaves should be validated in order to ensure that sterilizing
    conditions are fulfilled under all loading patterns.

#### Incineration

Incineration is the method of choice for the final disposal of
contaminated waste, including carcasses of laboratory animals,
preferably after autoclaving. The incineration of infectious materials
is an alternative to autoclaving only if the incinerator:

-   is under laboratory control;

-   has an efficient means of temperature control and a secondary
    burning chamber.

#### Final disposal

The disposal of laboratory and medical waste is subject to various
national regulations. In general, ash from incinerators may be treated
in the same way as normal domestic waste and removed by local
authorities. Autoclaved waste may be disposed of by off-site
incineration or in licensed landfill sites.

> **ANNEX 5**
>
> Requirements for the safe transport of wild poliovirus infectious or
> potential infectious materials
>
> The transportation of wild poliovirus materials must comply with
> national and international regulations for the transport of infectious
> materials by any mode of transport. These regulations are based upon
> the Recommendations of the United Nations Committee of Experts on the
> Transport of Dangerous Goods (UN). The International Civil Aviation
> Organization (ICAO) and the International Air Transport Association
> (IATA) have incorporated the UN Recommendations in their respective
> regulations, as have other international and national transport
> organizations and regulatory bodies. Detailed information on the
> requirements necessary for air transportation are found in the ICAO
> *Technical instructions for the safe transport of dangerous goods by
> air* and the *Dangerous goods regulations* of IATA. The principles of
> safe transport by local surface means is the same as for air or
> international transport and may be regulated by national authorities.
> Sending materials by mail may also be restricted
> nationally/internationally. Prior contact should be made with the
> local post office to ascertain whether the packaged material will be
> accepted by the postal service.
>
> It is the responsibility of the sender to ensure the correct
> packaging, labelling and documentation for all shipments of wild
> poliovirus infectious or potential infectious materials. The sender
> must make advance arrangements with the receiver of the shipment
> including investigating the need for an import permit and obtaining
> confirmation that the receiving laboratory meets the containment
> requirements for BSL-2/polio of BSL-3/polio as appropriate. An export
> licence may also be required by national authorities. Good
> coordination between the sender, the carrier and the receiving
> laboratory will ensure the safe and expeditious transport of the
> material. Practical guidance to facilitate compliance with
> international regulations can be found in *Guidelines for the Safe
> Transport of Infectious Substances and Diagnostic Specimens.*
>
> New requirements to improve the current regulations are under
> consideration for adoption by regulatory bodies from January 1, 2005.
> Background and technical information to explain the comprehensive
> amendments can be found in *New International Regulations on the
> Transport of Infectious Substances.*
>
> References:
>
> International Civil Aviation Organization. *Technical Instructions for
> the Safe Transport of Dangerous Goods by Air. 2003-2004.* Doc 9284,
> 2003.
>
> International Air Transport Association. *Dangerous Goods
> Regulations.* 44^th^ edition. January 2003.
>
> World Health Organization. *Guidelines for the Safe Transport of
> Infectious Substances and Diagnostic Specimens.* WHO/EMC/97.3. Geneva,
> 1997
>
> World Health Organization. *New International Regulations on the
> Transport of Infectious Substances.* WHO/CDS/CSR/LYO/LAB/2003. Geneva,
> 2003.

**ANNEX 6**

BSL-2/polio biosafety requirements

##### 

##### BSL-2/polio consists of the standard BSL-2 requirements as described in the *WHO Laboratory biosafety manual* with additional specific requirements for wild poliovirus:

###### Specific requirements for wild poliovirus

Access to the laboratory is restricted.

All persons entering the laboratory are fully immunized against polio.

All open manipulations with wild poliovirus infectious or potential
infectious materials are performed using a certified class I or II
biological safety cabinet.

Wild poliovirus infectious and potential infectious materials are stored
in secure areas with limited access.

Freezers and refrigerators are locked with limited access to the key
mechanism and clearly marked as containing wild poliovirus materials.

Freezer inventories are current and complete, including nature of
material, volume or amount, location in freezer.

Documentation is current on all materials, including geographical source
and date of collection.

All materials are transferred to and from the freezer in leak-proof,
unbreakable secondary containers.

Standard operating procedures (SOP) are established and regular training
provided on responses to all spills, breakage of virus-containing
vessels, and accidents where virus may have been released.

###### Standard biosafety level 2 requirements *(WHO Laboratory biosafety manual*, 2^nd^ edition revised):

Access

The international biohazard warning symbol and sign must be displayed.

Laboratory doors should be kept closed.

Children under the age of 16 years should not be allowed to enter
laboratory working areas.

"No smoking" "No eating" and "No drinking" signs should be displayed
clearly inside and outside the laboratory.

Personal protection

Laboratory coveralls, gowns or uniforms must be worn at all times for
work in the laboratory.

Appropriate gloves must be worn for all procedures that may involve
direct or accidental contact with blood, infectious materials or
infected animals. After use, gloves should be removed aseptically and
hands must then be washed.

Personnel must wash their hands after handling infectious materials and
animals, and before they leave the laboratory working areas.

Safety glasses, face shields (visors) or other protective devices must
be worn when it is necessary to protect the eyes and face from splashes,
impacting objects and sources of artificial ultraviolet radiation.

It is prohibited to wear protective laboratory clothing outside of the
laboratory, e.g. in canteens, coffee rooms, offices, libraries, staff
rooms and toilets.

Open-toed footwear should not be worn in laboratories.

Eating, drinking, applying cosmetics and handling contact lenses is
prohibited in the laboratory working areas.

Storing human foods or drinks anywhere in the laboratory working areas
is prohibited.

Protective laboratory clothing is not stored in the same lockers or
cupboards as street clothing.

Procedures

Pipetting by mouth must be strictly forbidden.

Materials must not be placed in the mouth. Labels must not be licked.

All technical procedures should be performed in a way that minimizes the
formation of aerosols and droplets. Whenever there is an increased risk
of aerosolization, work should be conducted in a biological safety
cabinet.

The use of hypodermic needles and syringes should be limited. They must
not be used as substitutes for pipetting devices or for any purpose
other than parenteral injection or aspiration of fluids from laboratory
animals.

All spills, accidents and overt or potential exposures to infectious
materials must be reported to the laboratory supervisor. A written
record of such accidents and incidents should be maintained.

A written procedure for the clean up of all spills must be developed and
followed.

Laboratory working areas

The laboratory should be kept neat, clean and free of materials not
pertinent to the work.

Work surfaces must be decontaminated after any spill of potentially
dangerous material and at the end of the working day.

All contaminated materials, specimens and cultures must be
decontaminated before disposal or cleaning for reuse.

Packing and transportation must follow applicable national and/or
international regulations.

When windows can be opened, they should be fitted with arthropod-proof
screens.

Biosafety management

It is the responsibility of the laboratory director (the person who has
immediate responsibility for the laboratory) to ensure the development
and adoption of a biosafety management plan and a safety or operations
manual.

The laboratory supervisor (reporting to the laboratory director) should
ensure that regular training in laboratory safety is provided.

Personnel should be advised of special hazards and required to read the
safety or operations manual and follow standard practices and
procedures. The laboratory supervisor should make sure that all
personnel understand these. A copy of the safety or operations manual
should be available in the laboratory.

When appropriate, there should be an arthropod and rodent control
programme.

Appropriate medical evaluation, surveillance and treatment should be
provided for all personnel in case of need, and adequate medical records
should be maintained.

Baseline serum samples may be collected from laboratory staff and other
persons at risk. These should be stored appropriately according to
national or local guidelines. Additional specimens may be collected
periodically depending on the organisms handled and the function of the
laboratory.

Design features

Ample space must be provided for the safe conduct of laboratory work and
for cleaning and maintenance.

Walls, ceilings and floors should be smooth, easy to clean, impermeable
to liquids and resistant to the chemicals and disinfectants normally
used in the laboratory. Floors should be slip-resistant. Exposed pipes
and ducting should be avoided where possible.

Bench tops should be sealed to the walls, impervious to water and
resistant to disinfectants, acids, alkalis, organic solvents and
moderate heat.

Illumination should be adequate for all activities. Undesirable
reflections and glare should be avoided.

Laboratory furniture should be sturdy. Open spaces between and under
benches, cabinets and equipment should be accessible for cleaning.

Storage space must be adequate to hold supplies for immediate use and
thus prevent clutter on bench tops and in aisles. Additional long-term
storage space, conveniently located outside the laboratory working
areas, should also be provided.

Space and facilities should be provided for the safe handling and
storage of solvents, radioactive materials, and compressed and liquefied
gases.

Facilities for storing outer garments and personal items should be
provided outside the laboratory working areas.

Facilities for eating and drinking and for rest should be provided
outside the laboratory working areas.

Hand-wash basins, with running water if possible, should be provided in
each laboratory room, preferably near the exit door.

Doors should have vision panels, be self-closing and have appropriate
fire ratings.

An autoclave should be available in the same building as the laboratory.

Safety systems should cover fire, electrical emergencies, emergency
shower and eyewash facilities.

First-aid areas or rooms suitably equipped and readily accessible should
be available.

There are no specific ventilation requirements for BSL-2 laboratories.
However, consideration should be given to the provision of mechanical
ventilation systems that provide an inward flow of air without
recirculation. If there is no mechanical ventilation, windows should be
able to be opened and should be fitted with arthropod-proof screens.

A dependable supply of good quality water is essential. There should be
no cross-connections between sources of laboratory and drinking-water
supplies. An anti-backflow device should protect the public water
system.

There should be a reliable and adequate electricity supply and emergency
lighting to permit safe exit. A stand-by generator is desirable for the
support of essential equipment, such as incubators, biological safety
cabinets, freezers, etc., and for the ventilation of animal cages.

There should be a reliable and adequate supply of gas. Good maintenance
of the installation is mandatory.

Three aspects of waste disposal need special attention to meet
performance and pollution-control requirements:

-   autoclaves for the treatment of solid waste need specially designed
    accommodation and services;

-   incinerators should be of special design, equipped with afterburners
    and smoke-consuming devices;

-   contaminated wastewater must be decontaminated.

Laboratories are occasionally the targets of vandals. Physical and fire
security must be considered. Strong doors, screened windows, and
restricted issue of keys are compulsory. Other measures should be
considered and applied, as appropriate, to augment security.

Essential biosafety equipment

Pipetting aids -- to avoid mouth pipetting. Many different designs are
available.

Biological safety cabinets, to be used whenever:

-   infectious materials are handled; such materials may be centrifuged
    in the open laboratory if sealed centrifuge safety cups are used and
    if they are loaded and unloaded in a biological safety cabinet;

-   there is an increased risk of airborne infection;

-   procedures with a high potential for producing aerosols are used;
    these may include centrifugation, grinding, blending, vigorous
    shaking or mixing, sonic disruption, opening of containers of
    infectious materials whose internal pressure may be different from
    the ambient pressure, intranasal inoculation of animals, and
    harvesting of infectious tissues from animals and eggs.

Plastic disposable transfer loops. Alternatively, electric transfer loop
incinerators may be used inside the biological safety cabinet (BSC) to
reduce aerosol production.

Screw-capped tubes and bottles.

Autoclaves to decontaminate infectious materials.

Plastic disposable Pasteur pipettes, whenever available, to avoid glass.

Equipment such as autoclaves and biological safety cabinets must be
validated with appropriate methods (usually by a certified examiner)
before being taken into use. Recertification should take place at
regular intervals, according to the manufacturer's instructions.

**ANNEX 7**

BSL-3/polio containment requirements

##### BSL-3/polio consists of the standard BSL-3 requirements as described in the *WHO Laboratory biosafety manual* with additional specific requirements for wild poliovirus:

###### Specific requirements for wild poliovirus

Access to the laboratory is restricted.

All persons entering the laboratory are fully immunized against polio.

All open manipulations are performed using a certified class I or II
biological safety cabinet.

All wild poliovirus materials are stored in a secure area, preferably
inside the BSL-3/polio laboratory.

Current documentation is maintained on all wild poliovirus materials,
including:

-   geographical source and date of collection/isolation;

-   nature of collection source;

-   cell passage history;

-   genomic sequence of isolate;

-   complete composition, history, and properties of virus if research
    product.

Laboratory maintains a full and updated inventory of all wild poliovirus
materials including:

-   nature of material;

-   volume or amount;

-   position in freezer.

All wild poliovirus materials are stored in locked freezers with limited
access to the key mechanism

All wild poliovirus materials are stored in leak-proof, screw-cap
containers bearing a unique identification number and the name of the
responsible person

The laboratory has written procedures for response to spillage

###### Biosafety level 3 requirements (*WHO Laboratory biosafety manual*, 2^nd^ edition revised):

Biosafety level 3 includes all biosafety level 2 requirements with the
following modifications:

Access

The two-person rule should apply, whereby no individual ever works alone
in the laboratory.

The international biohazard warning symbol and sign displayed on
laboratory access doors must identify the microorganism(s) handled and
the name of the laboratory supervisor who controls access, and indicate
any special conditions for entry into the area, e.g. immunization.

Personal protection

Laboratory protective clothing must be of the type with solid-front or
wrap-around gowns, scrub suits, coveralls, head covering and, where
appropriate, shoe covers or dedicated shoes. Front-buttoned standard
laboratory coats are unsuitable. Laboratory protective clothing must not
be worn outside the laboratory, and it must be decontaminated before it
is laundered.

Laboratory design and facilities

The laboratory should be separated from the areas that are open to
unrestricted traffic flow within the building. Additional separation may
be achieved by placing the laboratory at the blind end of a corridor, or
constructing a partition and door or access through an anteroom or basic
laboratory -- biosafety level 2.

Entry for personnel must be through a vestibule (i.e. double-door
entry).

Access to the laboratory area must be designed to prevent entrance of
arthropods and other vermin.

Access doors must be self-closing and interlockable. A break-through
panel may be provided for emergency exit use.

The surfaces of walls, floors and ceilings should be water-resistant and
easy to clean. Openings in these surfaces (e.g. for service pipes)
should be sealed to facilitate decontamination of the room(s).

The laboratory room must be sealable for decontamination. Air-ducting
systems must be constructed to permit gaseous decontamination.

Windows must be closed, sealed and break-resistant.

A foot- or elbow-operated or automatically controlled water source at
the hand-wash basin should be provided near to each exit door.

There must be a ventilation system that establishes a directional air
flow from access spaces into the laboratory room. Staff must at all
times ensure that proper directional air flow into the laboratory room
is maintained.

The building ventilation system must be so constructed that air from the
laboratory is not recirculated to other areas within the building. Air
may be HEPA filtered, reconditioned and recirculated within that
laboratory. Exhaust air from the laboratory (other than from biological
safety cabinets) must be discharged to the outside of the building, so
that it is dispersed away from occupied buildings and air intakes. It is
recommended that this air is discharged through high-efficiency
particulate air (HEPA) filters.

Biological safety cabinets should be sited away from walking areas and
out of cross-currents from doors and ventilation systems

The exhaust air from Class I or Class II biological safety cabinets,
which will have been passed through HEPA filters, must be discharged in
such a way as to avoid interference with the air balance of the cabinet
or the building exhaust system. All HEPA filters must be installed in a
manner that permits gaseous decontamination and testing.

An autoclave for the decontamination of contaminated waste material
should be available in the containment laboratory. If infectious wastes
have to be transported out of the containment laboratory for disposal,
they must be transported in sealed, unbreakable and leak-proof
containers according to national or international regulations, as
appropriate.

Anti-backflow devices must be fitted to the water supply.

Effluents should be decontaminated before being discharged to the
sanitary sewer.

Biosafety management

Medical examination of all laboratory personnel who work in biosafety
level 3 containment laboratories is mandatory. This should include
recording of a detailed medical history and a physical examination.

A baseline serum sample should be obtained and stored for future
reference.

Individuals who are immunocompromised should not be employed in
facilities with biosafety level 3 containment laboratories.

Special consideration should be given to the employment of pregnant
women

After a satisfactory clinical assessment, the examinee should be
provided with a medical contact card stating that he or she is employed
in a facility with a containment laboratory -- biosafety level 3. It is
suggested that this card should include a picture of the card holder,
should be wallet-sized and should always be carried by the holder.

> **ANNEX 8**
>
> Model of BSL-3/polio Standard Operating Procedures
>
> Standard operating procedures (SOP) should be prepared for each
> BSL-3/polio laboratory and carefully followed to ensure that the
> required biosafety and containment requirements are maintained.
>
> Sample table of contents for BSL-3/polio SOP:
>
> 1\. Description of Program

-   include a description of the program for work with wild poliovirus
    materials (list of wild poliovirus infectious materials, nature of
    activities)

-   list all staff authorized to work with wild polivirus materials

-   outline the roles and responsibilities for the management of
    biosafety and laboratory containment

> 2\. Description of Physical Facility

-   include a detailed floor plan

-   describe the facility in detail including laboratory location,
    security and access devices, signage on doors/freezers, room layout,
    surface finishes, sealing of containment perimeter, laboratory
    services, ventilation system, biological safety cabinets and other
    primary containment devices, decontamination systems

> 3\. Operational Practices

-   outline restricted access policy and procedures including
    immunization requirements

-   outline procedures to verify correct operation of the
    BSL-polio/facility prior to entry (e.g. confirm directional airflow
    on visual monitoring device)

-   describe in detail the entry/exit procedures for staff including the
    removal of jewelry and street clothing, and the donning and doffing
    of protective clothing

-   describe when showering on exit is required (e.g. after spill and
    potential contamination with wild poliovirus materials )

-   describe in detail the entry/exit procedures for samples, supplies
    and equipment

-   include description of good microbiological practices to be followed
    (e.g. no eating/drinking in laboratory, routine handwashing,
    minimizing creation of droplets/aerosols and subsequent surface
    contamination of laboratory, safe use of sharps)

-   describe procedures for routine decontamination of work surfaces

-   outline the specific procedures for manipulating all wild poliovirus
    materials in the biological safety cabinet and other primary
    containment devices (e.g. use of sealed centrifuge cups)

-   outline procedures for transferring materials to and from freezers,
    biological safety cabinets, incubators and other areas (e.g. in
    leak-proof unbreakable secondary containers to avoid breakages and
    spillages)

-   detail the procedures for controlling insects and rodents in the
    facility

> 4\. Storage of Wild Poliovirus Materials

-   describe in detail the storage policies and procedures for wild
    poliovirus materials

-   list all personnel authorized to access wild poliovirus materials

-   include a list of all freezers and other locations where materials
    are stored

-   include sample record-keeping forms for maintaining inventory
    current

> 5\. Decontamination and Disposal of Waste

-   describe in detail the procedures for the decontamination of
    potentially contaminated materials before disposal or reuse (e.g.
    autoclaving, incineration)

-   outline procedures for decontamination of protective clothing before
    laundering and reuse, or disposal

-   outline procedures for decontamination and removal of heat-sensitive
    materials that cannot be autoclaved out of the containment
    laboratory (e.g. use of gaseous or liquid disinfectants)

-   include a description of the procedures for the routine efficacy
    monitoring of autoclaves using biological indicators (include a
    sample of the record log of results)

-   provide efficacy information on the disinfectants used against wild
    poliovirus materials

> 6\. Emergency Procedures

-   detail the procedures to be followed in the event of spillages,
    breakages and other releases of wild poliovirus materials that occur
    within the biological safety cabinet, within the laboratory
    perimeter but outside the biological safety cabinet, and outside of
    the laboratory perimeter

-   describe procedures to respond to losses of containment (e.g.
    failure of biological safety cabinet, failure of ventilation system,
    autoclave failure) and the potential release of wild poliovirus
    materials from the facility

-   develop and describe procedures for accidents and life-threatening
    emergencies (e.g. use of emergency exit procedures and bypassing of
    routine exit procedures to give priority to health and safety where
    necessary)

-   include sample form for documenting and reporting all spills,
    breaches of containment, accidents and other emergencies

> 7\. Laboratory Housekeeping, Cleaning and Maintenance

-   outline procedures to maintain tidiness of laboratory (e.g. no
    storage of books, paperwork and materials not pertinent to work
    within the laboratory, no personal items brought into the
    laboratory)

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-   describe routine procedures for maintaining containment (e.g.
    ensuring doors are kept closed, filling drainage traps with
    disinfectant)

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-   describe policies and procedures for routine cleaning of laboratory
    (e.g. by laboratory staff or specific personnel dedicated and
    trained for this task, use of dedicated cleaning supplies)

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-   outline requirements for maintenance of laboratory equipment and
    systems (e.g. entry of maintenance personnel, decontamination of
    equipment prior to servicing, decontamination of laboratory prior to
    maintenance work being performed on laboratory systems)

8\. Staff Training

-   provide details of training provided to all staff on practices and
    procedures intended to interrupt potential for transmission of wild
    poliovirus to persons outside the laboratory (e.g. directly through
    contaminated workers' skin or clothing, through contaminated
    effluents and wastes, indirectly through silent infection of
    laboratory workers)

-   outline training requirements to ensure staff have a good
    understanding of the laboratory design and physical operation, the
    standard operational procedures, and emergency procedures

-   outline requirements for regular retraining programs

-   include sample training forms and other documentation showing
    evidence that staff participated and understood training

**ANNEX 9**

Protocol for verification and reporting of BSL-2/polio and BSL-3/polio
conditions

To ensure that the requirements of the intended containment level have
been met, each laboratory should undergo a detailed verification
process. This process involves reviewing the physical laboratory layout,
facility construction and the performance of critical containment
components. Operational protocols intended to interrupt transmission to
the community should also be carefully reviewed. Verification that
laboratory staff working in these areas are trained on the practices to
prevent the release of infectious agents from the facility, the physical
containment systems, and emergency procedures should also be a part of
the verification process. Finally, the on-site review should confirm the
accuracy of the inventory of wild poliovirus materials.

ACTIVITIES TO BE UNDERTAKEN DURING THE VERIFICATION PROCESS

1\. Establish a review team. The team should number at least 2 people
and in the case of BSL-3/polio should include a qualified
biosafety/containment specialist who is familiar with the design and
operation of a BSL-3/polio facility and is external to the institution.

2\. Collect and review all available documentation, including laboratory
plans and specifications, reports of testing critical containment
equipment and systems (see below), biosafety and operational protocols,
training records, polio immunization records, and inventories of wild
poliovirus materials.

3\. Conduct an on-site visit and review the operational protocols with
laboratory staff. Particular attention should be given to practices
intended to interrupt potential for transmission to persons outside the
laboratory either directly (e.g. though contaminated laboratory
effluents, wastes transported to landfills, contaminated workers' skin
or clothing) or indirectly (e.g. silent infection of laboratory workers
and subsequent secondary transmission).

4\. Visually examine the physical laboratory layout and facility
construction, and verify results of selected containment system tests
(e.g. inward directional airflow, control sequences of operation during
exhaust fan failure, proper alarm functioning).

5\. Review training records of laboratory staff to ensure a good
understanding of the laboratory design and operation, and biosafety
practices to contain wild poliovirus materials.

5\. Examine all freezers and storage areas claimed to contain wild
poliovirus materials to ensure materials listed on the inventory can be
located and access to these materials is restricted.

6\. Prepare a written report outlining all personnel on the review team,
details of the activities carried out, details of the findings and
review team recommendations including any corrective action that may be
required.

Checklist of BSL-2/polio Critical Containment Equipment and Systems
Testing

-   verification of signage and correct operation of access control
    devices

-   visual inspection of laboratory to ensure surfaces and coatings
    (floors, walls, ceilings, benchtops) facilitate cleaning and
    disinfection

-   verification of correct operation of water supply backflow
    preventers

-   verification of operation of autoclave using biological indicators
    to ensure complete decontamination of wild poliovirus materials in
    wastes

-   certification of biological safety cabinets

Checklist of BSL-3/polio Critical Containment Equipment and Systems
Testing

-   verification of signage and correct operation of access control
    devices

-   verification of correct operation of interlocking doors and/or
    operational protocols to ensure doors located on either side of
    entry area cannot be opened at the same time

-   visual inspection of laboratory to ensure surfaces and coatings
    (floors, walls, ceilings, benchtops) facilitate cleaning and
    disinfection

-   visual inspection (e.g. using a smoke pencil) of the integrity of
    the containment barrier (floors, walls, ceiling, floor/wall joints,
    wall/ceiling joints, seals, service penetrations)

-   verification of correct operation of water supply backflow
    preventers

-   verification of operation of autoclave using biological indicators
    to ensure complete decontamination of wild poliovirus materials in
    wastes

-   demonstration of inward directional airflow (e.g. using a smoke
    pencil)

-   testing of ventilation control system and alarms for fail-safe
    operation by failure of system components (e.g. fan failure)

-   certification of biological safety cabinets

-   integrity testing of exhaust air HEPA filters

-   verification that contaminated ductwork is sufficiently sealed to
    allow for the decontamination

**ANNEX 10**