WHO laboratory biosafety guidelines for handling specimens suspected of containing avian influenza A virus

12 January 2005

General recommendations

The possibility that an influenza infection in humans caused by avian influenza viruses A viruses could occur following a laboratory accident is a risk to which it is crucial to be constantly alert. Efforts to minimize transmission of infection in humans will be compromised by breaches in laboratory biosafety.

Responsibility for developing a comprehensive safety policy, including a safety manual, and supporting programmes for its implementation normally rests with the director or head of an institute or laboratory. However, laboratory safety is also the responsibility of all supervisors and laboratory employees, and individual workers are responsible for their own safety and that of their colleagues.

Good microbiological technique is fundamental to laboratory safety. The use of safety equipment, combined with good procedures and practices, will help to reduce the risks involved in dealing with biosafety hazards. The most important concepts are outlined below.

  • Standard precautions should always be followed; barrier protection (gowns, gloves) should be used whenever samples are obtained from patients. In addition to these standard precautions, eyes should be protected

  • Basic containment – Biosafety Level 2 (BSL2) – practices and procedures should be the minimum requirement for handling specimens (see WHO laboratory biosafety manual, 3rd edition).

  • Examples of routine laboratory procedures that require BSL2 include:
    - routine diagnostic testing of serum and blood samples (including haematology and clinical chemistry);
    - manipulations involving neutralized or inactivated (lysed, fixed, or otherwise treated) virus particles and/or incomplete, non-infectious portions of the viral genome;
    - final packaging of specimens for transport to diagnostic laboratories for additional testing; specimens should already be in a sealed, decontaminated primary container.

  • Good laboratory practices should be followed. Eating, drinking, smoking, applying cosmetics, and handling contact lenses are prohibited in the laboratory working areas.

  • Personal protective equipment (gown, gloves, eye protection) should be worn in the laboratory when handling and processing specimens and performing diagnostic testing.

  • All technical procedures should be performed in a way that minimizes the formation of aerosols and droplets.

  • Biological safety cabinets or other physical containment devices should be used for all manipulations that may cause splashes, droplets, or aerosols of infectious materials (e.g. 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).

  • 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. Mouth pipetting must be strictly forbidden.

  • Adequate and conveniently located biohazard containers should be available for disposal of contaminated materials.

  • Work surfaces must be decontaminated after any spill of potentially dangerous material and at the end of the working day. Generally, freshly prepared bleach solutions1 are appropriate for dealing with bio-hazardous spillage. More information on disinfection and sterilization is provided in the WHO laboratory biosafety manual.

  • Personnel must wash their hands often – especially after handling infectious materials and animals, before leaving the laboratory working areas, and before eating.

  • Personal protective equipment must be removed before leaving the laboratory.

WHO biosafety guidelines for handling specimens that may contain avian influenza A virus

Laboratories must meet basic BSL2 standards and use BSL3 work practices to be able to safely:

- aliquot and/or dilute specimens
- perform diagnostic testing that does not involve propagation of viral agents in vitro or in vivo
- perform nucleic acid extractions that involve untreated specimens
- prepare smears using heat or chemical fixation.

BSL3 practices cover the following areas:

  • Any procedure that may generate aerosols or droplets should be performed in a biological safety cabinet (e.g. sonication, vortexing).

  • Laboratory workers should wear protective equipment, including disposable gloves, solid-front or wrap-around gowns, scrub suits, or coveralls with sleeves that fully cover the forearms, head coverings and, where appropriate, shoe covers or dedicated shoes, eye protection and a surgical mask, or full-face shield, because of the risk of aerosol or droplet exposure when performing specific manipulations.

  • Centrifugation of specimens should be performed using sealed centrifuge rotors or sample cups. These rotors or cups should be unloaded in a biological safety cabinet.

  • Work surfaces and equipment should be decontaminated after specimens are processed. Standard decontamination agents that are effective against non-enveloped viruses should be adequate if used according to the manufacturer’s recommendations. Generally, freshly prepared bleach solutions1 are appropriate for dealing with biohazardous spillage. More information on disinfection and sterilization is provided in the WHO laboratory biosafety manual.

  • Biological waste contaminated with suspect or confirmed influenza A/H5 specimens, should be treated as outlined in the WHO laboratory biosafety manual.

When a procedure or process cannot be conducted within a biological safety cabinet, appropriate combinations of personal protective equipment (e.g. respirators, face shields) and physical containment devices (e.g. centrifuge safety cups or sealed rotors) must be used.

WHO strongly recommends that the BSL3 precautions described above are adopted and followed for work in BSL2 laboratories with influenza A/H5 virus specimens.

Where laboratory facilities do not meet at least basic BSL2 containment conditions, specimens should be referred to suitably equipped reference laboratories for primary diagnostic tests.

For laboratories that meet BSL3 containment standards and are operated by staff trained in the use of appropriate BSL3 work practices, the following procedures can be undertaken:

- diagnostic tests that involve propagation of viral agents in vitro or in vivo
- work involving the replication of influenza A/H5 virus in cell culture and/or storage of cell culture isolates
- recovery of viral agents from cultures of influenza A/H5 specimens
- manipulations involving growth or concentration of influenza A/H5 virus.

1Work surfaces must be decontaminated after any spill of potentially dangerous material and at the end of the working day. A general all-purpose laboratory disinfectant should have a concentration of 1 g/l available chlorine (0.1%). A stronger solution, containing 5 g/l available chlorine (0.5%), is recommended for dealing with biohazardous spillage and in the presence of large amounts of organic matter. Sodium hypochlorite solutions, as domestic bleach, contain 50g/l available chlorine and should therefore be diluted 1:50 or 1:10 to obtain final concentrations of 1 g/l or 5 g/l, respectively. Bleach dilutions should be freshly prepared and allowed a contact time of at least 10 min. Chlorine is corrosive and cannot be used on all surfaces. Alternative compounds for disinfection and sterilization are provided in the WHO laboratory biosafety manual.