Guidelines for orthoebolavirus

Biosafety guidelines for the handling of specimens from patients under investigation for orthoebolaviruses, including Bundibugyo virus

This document provides information to support local risk assessments in diagnostic laboratories where specimens from patients under investigation for orthoebolaviruses are handled.

Personnel in laboratories receiving specimens from patients under investigation for orthoebolaviruses, including Bundibugyo virus (BDBV), 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 primary specimens be handled in a facility that meets the minimum requirements for Containment Level 2 (CL2) specified in the Canadian Biosafety Standard, Third Edition.^Footnote1 Due to the nature of orthoebolaviruses, additional operational practices are highly recommended when working in a CL2 facility and are outlined below.

Orthoebolaviruses are classified as Risk Group 4 human and animal pathogens. Handling and storing orthoebolaviruses (i.e., cultivating or intentionally collecting or extracting virus) is regulated under the Human Pathogens and Toxins Act (HPTA) and is only permitted in a CL4 facility operating under a Pathogen and Toxin Licence issued by the Public Health Agency of Canada (PHAC).^Footnote2

On May 17, 2026, the Director-General of the World Health Organization (WHO) declared that the Ebola disease outbreak caused by BDBV in the Democratic Republic of Congo and Uganda constitutes a public health emergency of international concern, as defined in the provisions of the International Health Regulations.^Footnote3 The rapid risk assessment conducted by PHAC indicates that the overall risk to the Canadian population of acquiring BDBV from the outbreak in the Democratic Republic of Congo and Uganda is low.^Footnote4

Given the risk group classification for orthoebolaviruses, it is important that personnel working in diagnostic laboratories coordinate sample collection, BDBV specific assays selection, and sample shipping with the National Microbiology Lab (NML) to support the safe handling of samples that may contain orthoebolaviruses.

Recommended diagnostic laboratory practices

Primary specimens (e.g., blood, serum, plasma) from patients under investigation for orthoebolaviruses are recommended to be inactivated before diagnostic testing can be conducted safely in a CL2 facility. It is recommended that inactivation of the specimens occurs in a certified biological safety cabinet (BSC).

Clinical samples needed for diagnosis through molecular tests can be inactivated by guanidine salts (50-70%) followed by ethanol, while inactivation for clinical assays can be achieved by heating blood, serum samples, or other fluids at 60°C for 60 minutes by complete submersion in a water bath.^{Footnote5,Footnote6,Footnote7} More information on the physical inactivation of orthoebolaviruses can be found in the Pathogen Safety Data Sheet (PSDS) for orthoebolaviruses.^Footnote8

It is recommended that laboratory personnel handling these types of clinical specimens don the following personal protective equipment (PPE):^Footnote9

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

Blood cultures are to be prepared in a closed system. When this is not possible, manipulations are to 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 is only to be done when essential to patient care. This is to be undertaken in a certified BSC with the use of appropriate PPE as identified above. The decision to subculture is to be predicated on the clinical status of the patient and based on an on-going risk assessment.

Sample separation (e.g., blood, serum) is to be undertaken using sealed centrifuge cups or sealed centrifuge rotors that are loaded and unloaded in a certified BSC.

Blood smears: It is important to rule out malaria from travelers returning with a fever. Only thin blood smears are to 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 orthoebolaviruses be performed only on inactivated blood. All manipulations are to be performed in a certified BSC with the use of appropriate PPE as identified above. After air drying in the BSC, thin blood smears are to be fixed with 100% methanol for 15 minutes, after which the slides are to be either dry-heat inactivated (e.g., 60°C for at least 1 hour) and/or mounted with coverslips prior to microscopy.^{Footnote6,Footnote12} It is important that all reagents are decontaminated prior to disposal. More detailed information on the physical inactivation of specific orthoebolaviruses strains can be found in the PSDS for orthoebolaviruses.^Footnote8

Polymerase chain reaction (e.g., real time multiplex PCR), if available and standardized in the facility, may be considered a safer option, as routine extraction procedures (e.g., guanidine thiocyanate-based) are often used to inactivate orthoebolaviruses. However, because inactivation is not independently verified at the facility level, it is important to conduct work in a CL2 facility using a certified BSC and appropriate PPE to manage any residual risk.

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 are to 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 practice considerations

• Avoid activities that may generate aerosols whenever possible (e.g., mixing samples by pipetting, centrifugation).
• Strictly limit 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.
• Clearly pre-label tubes prior to the collection of patient specimens, and segregate orthoebolavirus suspect samples when handling in the laboratory.
• Clearly label testing requisitions as orthoebolavirus suspect and label containers as such on the exterior.
• Store all samples securely and limit access only to authorized personnel. Patient specimens that have been confirmed positive for orthoebolavirus are to be safely disposed of.
• Avoid unnecessary activities and the presence of unnecessary personnel in the area during sample processing, whenever possible.
• Perform testing using designated equipment in designated laboratory areas. Testing is to be performed by designated personnel.

Occupational health

Potential exposures to these specimens must be reported immediately according to your institution's policy and procedures. Reporting exposure to primary specimens not confirmed to contain orthoebolaviruses may be done on a voluntary basis to PHAC. Exposure reports may be submitted online or by email to pathogens.pathogenes@phac-aspc.gc.ca.

Decontamination

It is important to surface decontaminate specimen containers that will be transported to another location using an effective disinfectant prior to packaging. Effective disinfectants are described in the PSDS for orthoebolaviruses.^Footnote8

Orthoebolaviruses are susceptible to 3% acetic acid, 1% glutaraldehyde, 70% alcohol-based products, and 0.5% calcium hypochlorite (bleach powder).^{Footnote9,Footnote10,Footnote13,Footnote14} WHO states 10 minutes of contact time with 0.5% hypochlorite is effective on nonporous, contaminated surfaces without visible spills. For surfaces with visible spills, complete disinfection can be achieved by covering the spill with a paper towel or cloth and pouring 0.5% hypochlorite solution on it and allowing 15 minutes of contact time.

Laboratory tests have demonstrated that the use of 70% ethanol for 1 minute is effective at inactivating Mayinga and Kikwit variants of Orthoebolavirus zairense, whereas 2.5 minutes is required to inactivate the Makona variant.^Footnote15 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, which are the most important outbreak variants of Orthoebolavirus zairense.^Footnote15 0.5% chlorine solution is recommended by the WHO as a suitable disinfectant for surfaces contaminated with orthoebolaviruses.^Footnote10

PPE to be removed in a manner that minimizes contamination of the skin and hair, and avoids any contact between soiled items (e.g., gloves, gowns, respirators) and any area of the face. Contaminated and potentially contaminated clothing and PPE to be decontaminated using an effective method.

Hands to 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.^Footnote16

Spill procedures

Following a spill, the area is to 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 an effective disinfectant such as 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).^{Footnote9,Footnote17} Paper towel and other waste material are to be removed to a waste container using tongs or other appropriate tools. Following the removal of the initial material, the disinfection process is to be repeated.

It is important that individuals attending to this task wear protective attire to prevent exposure to pathogens and toxins. As per standard laboratory spill response procedures, appropriate PPE (e.g., PAPR, approved N95 or N100 respirator, eye protection) to 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 and decontaminated immediately after completion of the process. This can be achieved by using decontamination technologies and processes that have been demonstrated to be effective, such as chemical disinfectants, autoclaving, or incineration.^Footnote8

Transportation considerations

• Ship clinical samples from patients under investigation for orthoebolaviruses separately from other samples.
• Maintain a log of all individuals who have handled, decontaminated, and transported these types of clinical specimens, including waste associated with the specimens.
• Place samples in a leak-proof secondary container for movement within hospitals.^Footnote11

Shipping samples to the National Microbiology Laboratory

Shipments of specimens containing infectious substances must comply with the requirements of the Transportation of Dangerous Goods Regulations and Transport Canada requirements including classification, documentation (i.e., shipping documentation), training, markings (i.e., labels and placards), and packaging.^Footnote18

Orthoebolaviruses are infectious substances of Category A. Therefore, any material that contains orthoebolaviruses (e.g., cultures) or may contain orthoebolaviruses (e.g., primary specimens) is classified as a Category A infectious substance and is assigned to UN2814, INFECTIOUS SUBSTANCE, AFFECTING HUMANS, Class 6.2, Category A. Shipments containing Category A infectious substances must comply with the requirement for an approved emergency response assistance plan. For guidance, refer to the Transport Canada guidance on shipping infectious materials.^Footnote19

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

The NML OCD will work with the requesting provincial jurisdiction to initiate the emergency response assistance plan. 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 Ebola disease 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 biosafety and biosecurity inquiries may be addressed to pathogens.pathogenes@phac-aspc.gc.ca.

References