Biosafety Guidelines for Laboratories Handling Specimens from Patients under Investigation for Ebola Virus Disease

This document is intended to support local risk assessments, specifically for a diagnostic laboratory setting.

Laboratories receiving specimens from patients under investigation for Ebola Virus Disease (EVD) must be aware that improper handling of these specimens poses serious risk to the health of laboratory personnel. At minimum, it is recommended that these types of clinical specimens be handled in a facility that meets the minimum requirements for Containment Level 2 (CL2) specified in the Canadian Biosafety Standard, 2nd edition.Footnote 1 Due to the nature of EVD, additional operational practices are highly recommended and are outlined below.

Handling and storing Ebola virus (i.e., cultivated or intentionally collected or extracted virus) is regulated under the Human Pathogens and Toxins Act and is only permitted in a Containment Level 4 facility operating under a pathogen and toxin licence issued by the Public Health Agency of Canada.Footnote 2

Recommended Laboratory Handling and Movement and Transportation of Specimens from Patients under Investigation for Ebola Virus Disease

It is recommended that laboratory personnel handling these types of clinical specimens don the following personal protective equipment (PPE):

  • two pairs of gloves (e.g., latex, nitrile, or other similar material);
  • an additional impermeable layer over the lab coat (e.g., laboratory gown, or fluid-resistant gown) ; and
  • eye and respiratory protection, such as a combination of an approved particulate respirator (e.g., N95 or N100) and eye protection (e.g. goggles, face shield, or shroud), or a powered air purifying respirator (PAPR).

Specimens from patients under investigation for EVD should only be carried out in a certified biological safety cabinet (BSC) or other primary containment device, and never on an open bench.Footnote 1Footnote 3Footnote 4 This includes activities with the potential to create infectious aerosols (e.g., pipetting, aspiration, and slide preparation). Centrifugation of infectious material should be carried out using sealed safety cups or sealed rotors that are only opened and unloaded in the BSC.

Blood cultures should be prepared in a closed system. When this is not possible, manipulations should be undertaken in a certified BSC in a CL2 laboratory with the use of appropriate PPE as identified above.

Sub-culturing of blood cultures has the potential to generate aerosols and should be done only when essential to patient care. This should be done in a certified BSC with the use of additional PPE as identified above, and the decision to subculture should be predicated on the clinical status of the patient and based on an on-going risk assessment.

Sample separation (e.g. blood, serum) should be undertaken using sealed centrifuge cups or a sealed centrifuge rotor that are unloaded in a certified BSC.

Blood smears: Malaria should be ruled out from travelers returning with a fever. Only thin blood smears should be prepared (i.e., no thick smears) and repeated as necessary (e.g., if the first thin blood smear is negative). It is recommended that dipstick tests from patients that are under investigation for EVD be performed only on inactivated blood. All manipulations should be performed in a certified BSC with the use of appropriate PPE as identified above. After air drying in the BSC, thin blood smears should be fixed with 100% methanol for 15 minutes, after which the slides should be either dry-heat inactivated (e.g., 95°C for at least 30 minutes, 60°C for at least 1 hour) and/or mounted with coverslips prior to microscopy. All reagents should be decontaminated prior to disposal.

Polymerase chain reaction (PCR), if available on-site, may be considered a safer option, as routine extraction procedures (e.g., guanidine thiocyanate-based) are often sufficient to inactivate Ebola virus, but the effectiveness of the inactivation should be confirmed. Nucleic acid extractions should be undertaken in a certified BSC in a CL2 laboratory with the use of appropriate PPE as identified above.

Automated analyzers may be used after performing a local risk assessment for the potential for aerosol generation. If ports or vents are present on the system that may generate aerosols, it is recommended that the machine be contained, either in a BSC, Plexiglas or flexible film cover, or through the use of HEPA filters. After use, analyzers should be disinfected as recommended by the manufacturer, or with a freshly prepared solution containing 0.05% sodium hypochlorite (e.g., 5 mL household bleach [5.25% sodium hypochlorite] into 495 mL water).

Additional Operational Considerations

  • Avoid activities that may generate aerosols whenever possible (e.g., mixing samples by pipetting, centrifugation);
  • Restrict the use of glass or sharps wherever possible (e.g., substitute with plastic items), and verify that staff are well trained in routine practices and biosafety;Footnote 5
  • Clearly pre-label tubes prior to the collection of patient specimens, and segregate Ebola suspect samples when handling in the laboratory;
  • Testing requisitions should be clearly labelled as Ebola Suspect, and containers should be labelled as such on the exterior of the container;
  • All samples should be stored securely and accessible only to authorized personnel;
  • Avoid unnecessary activities and personnel in the area during sample processing, whenever possible; and
  • Testing should only be performed by designated personnel, and only in designated laboratory areas and using designated equipment.

Occupational Health

Potential exposures to these specimens must be reported immediately according to your institution's policy and procedures. Licensed facilities are to report exposures to the PHAC without delay. Facilities that are not regulated by the PHAC may do so on a voluntary basis. Exposure reports may be submitted online or by email to PHAC.pathogens-pathogenes.ASPC@canada.ca

Decontamination

  • Specimen containers that will be transported to another location should be surface decontaminated using an effective disinfectant prior to packaging. A list of effective disinfectants can be found within the Pathogen Safety Data Sheet for Ebola virusFootnote 6 :
    • Ebola virus is susceptible to 3% acetic acid, 1% glutaraldehyde, alcohol-based products, calcium hypochlorite (bleach powder), and dilutions of 5.25% household bleach (i.e., 0.525% to 0.0525% sodium hypochlorite for ≥ 10 min).Footnote 3Footnote 4Footnote 7Footnote 8Footnote 9 The World Health Organization (WHO) recommendations for cleaning up spills of blood or body fluids suggest flooding the area with a 1:10 dilution of 5.25% household bleach (i.e., 1 part household bleach diluted in 9 parts water, or 0.525% sodium hypochlorite) for 10 minutes for surfaces that can tolerate stronger bleach solutions (e.g., cement, metal).Footnote 9 For surfaces that may corrode or discolour, they recommend careful cleaning to remove visible stains followed by contact with a 1:100 dilution of 5.25% household bleach (i.e., 1 part household bleach diluted in 99 parts water, or 0.0525% sodium hypochlorite) for more than 10 minutes.Footnote 9 The cleaning material can be soaked in 0.5% sodium hypochlorite or decontaminated using other effective means (e.g., autoclaving, incineration). Bleach breaks down quickly when diluted, so solutions should be freshly prepared.
    • Laboratory tests have demonstrated that the use of 70% ethanol for 1 minute is effective at inactivating Mayinga and Kikwit variants of the virus, whereas 2.5 minutes is required to inactivate the Makona variant. Ethanol concentrations in the 60 to 95% range are suggested by the United States Food and Drug Administration.Footnote 9 Use of 0.5% and 1% sodium hypochlorite solutions (i.e., 50 mL household bleach into 450 mL or 200mL water, respectively) for 5 minutes is effective at inactivating all three variants.Footnote 9Footnote 10 A 0.5% chlorine solution is also recommended by the WHO to disinfect surfaces contaminated with Ebolavirus.Footnote 9
  • PPE should be removed in a manner that minimizes contamination of the skin and hair, and avoiding any contact between soiled items (e.g., gloves, gowns, respirators) and any area of the face. Contaminated and potentially contaminated clothing and PPE are to be decontaminated using an effective method.
  • Hands should be washed thoroughly immediately after the removal of PPE.

Disposal

All potentially contaminated liquid and solid materials must be appropriately decontaminated before disposal, reuse or removal from the laboratory.Footnote 11

Spill Consideration

The area should be evacuated and secured, and aerosols allowed to settle for a minimum of 30 minutes. The recommendations for cleaning up spills of blood or body fluids is to cover the spill with absorbent material (e.g., paper towel) and, starting from the outer edges, flooding the area with a 0.5% sodium hypochlorite solution and allowing to sit for 10 minutes for surfaces that can tolerate stronger bleach solutions (e.g., flooring, concrete, steel).Footnote 9 Paper towel and other waste material should be removed to a waste container using tongs. Following the removal of the initial material, the disinfection process should be repeated.

Individuals attending to this task should wear protective attire. As per standard laboratory spill response procedures, appropriate PPE (e.g., PAPR, approved N95 or N100 respirator, and eye protection) should be worn by those involved in the clean-up activity. All waste and PPE, including disposable gloves, impermeable gowns, and protective eye wear, is to be removed immediately after completion of the process, placed in an autoclave bag, and sterilized prior to disposal.Footnote 6

Transportation

Clinical samples from patients under investigation for Ebola virus disease should be shipped separately from other samples.

Laboratories should maintain a log of all individuals who have handled, decontaminated and transported these types of clinical specimens, including waste associated with the specimens.

If transportation delays are expected, samples should be refrigerated or frozen at -70°C.

Within Hospital

Specimens should be placed in a durable, leak-proof secondary container for transport within a facility. To reduce the risk of breakage or leaks, do not use any pneumatic tube system for transporting specimens from patients under investigation for EVD.Footnote 4

Shipping Samples to the National Microbiology Laboratory

Packaging, shipping and transport of specimens must comply with the requirements of the Transportation of Dangerous Goods Regulations, Transport Canada and the Dangerous Goods Regulations, International Air Transport Association.Footnote 12Footnote 13

  • For shipments, patient/primary sample specimens should be shipped as (UN2814, Category 6.2), and Emergency Response Assistance Plan (ERAP) must be initiated.

Liaise with the provincial public health laboratory of your jurisdiction to coordinate with the National Microbiology Laboratory (NML) Operations Center Director (OCD) at 1-866-262-8433. The OCD is staffed 24/7.

The NML OCD will work with the requesting provincial jurisdiction to initiate the ERAP. If you require assistance with the shipping process, sample requirements, sample shipping conditions, the NML OCD will connect you with the appropriate subject matter experts.

Concurrent with a request for laboratory services for EVD or other viral hemorrhagic fevers, provinces and territories are requested to notify and provide a clinical history of the patient's illness to the Public Health Agency of Canada Health Portfolio Operations Centre (HPOC) at 1-800-545-7661. Clarification or further information may be requested from the patient's clinician in order to optimize the delivery of the requested laboratory service(s).

Contact Information

Please note that this information is based on currently available scientific evidence and is subject to review and change as new information becomes available. Further general biosafety information may be obtained from the Public Health Agency of Canada by calling 1-800-545-7661.

References and Resources

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