Biosafety Directive for Human Immunodeficiency Virus (HIV) and Human T-cell Lymphotropic Virus Type 1 (HTLV-1)

January 21, 2014

TABLE OF CONTENTS

• ABBREVIATIONS
• 1.0 BACKGROUND
• 2.0 PATHOGEN DESCRIPTIONS AND RISK GROUPS
  • 2.1 HUMAN IMMUNODEFICIENCY VIRUS (HIV) DESCRIPTION AND RISK FACTORS
  • 2.2 HUMAN T-CELL LYMPHOTROPIC VIRUS TYPE 1 (HTLV-1) DESCRIPTION AND RISK FACTORS
  • 2.3 RISK GROUP (RG) DETERMINATION
• 3.0 CONTAINMENT LEVEL REQUIREMENTS
  • 3.1 CBSG CONTAINMENT ZONE OVERVIEW
  • 3.2 CONTAINMENT LEVEL (CL) DETERMINATION
• 4.0 ADDITIONAL BIOSAFETY REQUIREMENTS
  • 4.1 ADDITIONAL OPERATIONAL REQUIREMENT FOR ALL ACTIVITIES
  • 4.2 ADDITIONAL OPERATIONAL REQUIREMENTS FOR PROPAGATIVE IN VITRO ACTIVITIES
  • 4.3 ADDITIONAL OPERATIONAL REQUIREMENT FOR IN VIVO ACTIVITIES
• 5.0 BIOSAFETY CONSIDERATIONS
• 6.0 CONTACT AND ADDITIONAL INFORMATION
• 7.0 GLOSSARY
• 8.0 REFERENCES AND RESOURCES

ABBREVIATIONS

AIDS

Acquired Immunodeficiency Syndrome

ATL

Adult T-cell Leukemia

BSC

Biological Safety Cabinet

CBSG

Canadian Biosafety Standards and Guidelines

CFIA

Canadian Food Inspection Agency

CL

Containment Level (i.e., CL2, CL3, CL4)

CL2-Ag

CL2 Large Animal Facility

HAM/TSP

HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis

HEPA

High Efficiency Particulate Air

HIV

Human Immunodeficiency Virus

HTLV-1

Human T-cell Lymphotrophic Virus (type 1)

LAI

Laboratory Acquired Infection

LRA

Local Risk Assessment

PHAC

Public Health Agency of Canada

PSDS

Pathogen Safety Data Sheet

RG

Risk Group (i.e., RG1, RG2, RG3, RG4)

SIV

Simian Immunodeficiency Virus

SOP

Standard Operating Procedure

TB

Tuberculosis

1.0 BACKGROUND

Biosafety is defined as the containment principles, technologies, and practices that are implemented to prevent unintentional exposure to pathogens and toxins, or their accidental release. The application of appropriate biosafety principles and practices is fundamental to ensuring the safe and secure handling and storage of infectious materials and the protection of the public. National biosafety standards and guidelines pertaining to human and terrestrial animal pathogens and toxins are currently provided in the Government of Canada's Canadian Biosafety Standards and Guidelines (CBSG), 1st edition^{Footnote 1} referred to as 'the CBSG' for the remainder of the Directive.

The CBSG describes the physical containment and operational practices required for handling infectious material according to containment level (CL). Pathogens that have had pathogen risk and CL assessments completed have been assigned an appropriate risk group (RG) and CL. For the majority of pathogens, the CL and RG of the pathogen are the same (e.g., RG2 pathogens are handled at CL2), but there are some exceptions. As part of the pathogen risk assessments and containment level assessments conducted by the Public Health Agency of Canada (the Agency) and the Canadian Food Inspection Agency (CFIA ), or local risk assessments conducted by regulated parties, the CLs may change when the pathogen has been modified, the original conditions of use have changed, or the risks associated with a pathogen itself have changed. These CL changes reflect the risk mitigation strategies to address the specific modification of the pathogen or conditions of use. In general, many of the physical containment and operational practice requirements at CL3 are aimed at reducing the risks associated with airborne or aerosol-transmitted pathogens. As such, certain physical and/or operational requirements at CL3 for activities involving pathogens not known to be transmissible by inhalation are sometimes derogated. As stated in the CBSG, derogations are determined based on the work involved and the pathogen in question, and would be stipulated in the importation permit and/or otherwise communicated by the Agency or CFIA using another mechanism, and in this case a Biosafety Directive.

The human immunodeficiency virus (HIV) and the human T-cell lymphotropic virus type 1 (HTLV-1) are examples of RG3 pathogens where the pathogen risk and CL assessments have been revisited by the Agency in conjunction with HIV/HTLV specialists based on current risks associated with activities involving these pathogens. It has been determined that HIV & HTLV-1 can be safely handled at CL2/CL2-Ag with specific additional operational requirements (see Section 4.0). This Biosafety Directive is intended to provide a comprehensive overview of the risk assessment outcomes, subsequent CL decisions, and considerations that have been made for those working with HIV and HTLV-1. The Biosafety Directive for HIV and HTLV-1 is to be used in conjunction with the CBSG.

2.0 PATHOGEN DESCRIPTIONS AND RISK GROUPS

2.1 Human Immunodeficiency Virus (HIV) Description and Risk Factors

HIV is an enveloped, spherical virus that is around 80–100 nm in length, and has a linear, single-stranded positive-sense RNA genome.^{Footnote 2} The retrovirus exists as two types, HIV-1 and HIV-2.^{Footnote 3} HIV-1 is the more common type, and it is divided into four groups, M, N, O, and P, based on homology of nucleotide sequences and the originating cross-species transmission.^{Footnote 4} HIV-1 groups N, O, and P are considered rare, and group M accounts for an estimated 98% of infections worldwide.^{Footnote 4}

HIV-2 is common in Portugal and West Africa, but its prevalence is increasing in other parts of the world as well. The virus is related more closely to simian immunodeficiency virus (SIV) (75% similarity) than to HIV-1 (42%).^{Footnote 5} HIV-2 infection does not appear to be as easily transmitted and immunodeficiency takes longer to develop. Unlike HIV-1 which is highly infectious in the acute stage, HIV-2 infectiousness increases in the later stages of acquired immunodeficiency syndrome (AIDS) and the highly infectious stage is shorter when compared with HIV-1.

High rates of HIV infection are observed in Africa, Asia, and Europe.^{Footnote 6} As of 2011, the Agency estimates 71,300 individuals are infected with HIV in Canada, an 11.4% increase from 2008.^{Footnote 7}

The initial, acute stage of HIV-1 infection results in flu-like symptoms that can last for 3-5 weeks and typically includes lymphadenopathy, malaise, high plasma viremia, and an initial decrease in CD4+ T cells.^8,9 As infection progresses to the clinical latency stage that can last for years, immune responses lead to a decrease in circulating virus and restoration of CD4+ T cell numbers. In the absence of treatment, viral replication continues, leading to the gradual loss of CD4+ T cells. When the number of CD4+ T cells becomes insufficient to maintain effective immunity, the likelihood of opportunistic infections and neoplasia increases. As CD4+ T cell numbers fall to very low levels, the disease progresses to acquired immunodeficiency syndrome (AIDS), with symptoms of lymphadenopathy, weight loss, chronic diarrhea, and nervous system diseases (including dementia, myopathy and pain). Progression to disease depends on viral factors such as the strain or isolate, infectious dose, and viral load. Without treatment, AIDS develops in the majority of people within 10 years after infection.^{Footnote 10} Globally, tuberculosis (TB) is the leading cause of death in patients with AIDS, as it causes mortality in one third of HIV-infected individuals worldwide, and patients with HIV are also about 30% more likely to develop TB disease than those without infection.^{Footnote 11}

The natural host of HIV is humans, and experimental infection with HIV-1 is possible in the chimpanzee, gibbon ape, and rabbit.^{Footnote 12} HIV cannot replicate outside of the host, and is extremely susceptible to drying.^{Footnote 13} The infectious dose of HIV remains unknown. Since the primary route of transmission is through sexual transmission and parenteral inoculation, it is unlikely that a major HIV epidemic would occur upon the release of the virus from the laboratory environment.

Up until 2002, 106 cases of occupationally acquired HIV infections were documented, where 57 were of healthcare workers in the United States, 35 in Europe, and 14 elsewhere (there were 3 cases from Canada).^{Footnote 14} Two hundred and thirty eight (238) cases of possible occupationally acquired infections have also been reported. Risks of acquiring infection in the laboratory environment include direct exposure of mucus membranes or breaks in the skin to the blood or bodily fluids of infected humans or animals. The most common exposure is needle stick injury, which causes about 80% of all laboratory acquired infections (LAI); however, it is reported that the infection rate associated with this type of exposure is 0.3-0.5%.^{Footnote 15}

Vaccine development has not yet been successful due to the ever-changing genetic and antigenic properties of HIV-1. There are now more than 25 effective antiviral drugs available for use; however, the emergence of drug-resistant HIV strains threatens to limit the effects of antiviral treatments.^{Footnote 16}, ^{Footnote 17} Additionally, in cases of exposure, there are effective post-exposure prophylaxis regimes and guidelines to follow.^{Footnote 18}, ^{Footnote 19}

2.2 Human T-cell Lymphotropic Virus Type 1 (HTLV-1) Description and Risk Factors

HTLV, also known as human T-cell leukemia virus is a complex human C-type retrovirus that is spherical in structure with a diameter of approximately 100 nm.^{Footnote 20} The core of HTLV is electron-dense and contains 2 positive-sense single stranded RNA genomes that upon infection are converted to DNA by reverse transcription and inserted into the host genome to cause persistent infection.^{Footnote 21} Unlike other retroviruses such as HIV, which increases its progeny by replication of the virus itself, HTLV increases its copy number by proliferation of infected cells.^{Footnote 22Footnote 23}

Four distinct HTLV types have been identified, all of which resulted from the zoonotic transmission of simian retroviruses from simian to human hosts.^{Footnote 21} HTLV-1 is the only virus among the 4 types to be linked to human disease: adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). It is estimated that 10-20 million individuals worldwide are infected with HTLV-1.^{Footnote 20Footnote 21Footnote 23Footnote 24} HTLV-1 infection is endemic in southern Japan, the Caribbean, parts of Africa (Gabon and Nigeria), the Middle East, South America (Panama, Colombia, Venezuela, Brazil and Bolivia), the Pacific Melanesian islands, and Papua New Guinea.^{Footnote 24} The number of HTLV-1 cases in Canada is unknown; however, it is reported as rare.^{Footnote 25Footnote 26} HTLV-1 infections have been described in Amerindians from coastal regions of British Colombia^{Footnote 25}, and in Nunavut.^{Footnote 26}

The main hosts of HTLV-1 are humans; however, HTLV-1 is also able to infect rabbits, rats and monkeys.^{Footnote 27} While the infectious dose of HTLV-1 remains unknown, the main mode of transmission is through direct contact of contaminated body fluids with mucous membranes or breaks in the skin (e.g., abrasions, lacerations, etc.). Even though the transmission of HTLV-1 from animals to humans would be rare, it could occur from animal bites or scratches, or contact of infected body fluids with mucous membranes. There is documentation of HTLV infection of a laboratory worker following accidental needle stick puncture.^{Footnote 28}

ATL is a serious disorder involving malignancy of T-lymphocytes. There are 4 types that are based on clinical presentation. The acute type involves increased numbers of T-cells, skin lesions, systemic lymphadenopathy and hepatosplenomegaly. The lymphoma type is characterized by prominent systemic lymphadenopathy with few abnormal cells in the blood. Both the acute and lymphoma types have poor outcomes with a median survival time of about one year. In chronic ATL, the white cell count is mildly elevated and skin lesions, lymphadenopathy and hepatosplenomegaly are sometimes present. Smoldering ATL is characterized by a few ATL cells and the absence of other clinical features commonly associated with ATL. Generally, the chronic types of ATL progress to an acute form within a few years.^{Footnote 22Footnote 25Footnote 27}

Development of ATL is strongly linked to childhood infection and has an estimated incubation period anywhere between 20-60 years, with about 2-6% of infected individuals developing the disease.^{Footnote 20Footnote 22}

HAM/TSP is a chronic demyelinating disorder characterized by muscle weakness in the extremities, sensory disturbances, urinary incontinence, impotence, and lower back pain.^{Footnote 20-22Footnote 25Footnote 27} This is a progressively disabling condition and with time, the patient may become wheelchair bound, and death may result from secondary complications.^{Footnote 22} The median incubation period of HAM/TSP is 3.3 years, with less than 1% of HTLV-1 infected patients having the condition.^{Footnote 29} Although the majority of HTLV-1 infected patients remain asymptomatic, they are still capable of transmitting the virus.^{Footnote 27} There is no cure for HTLV-1 infection, and effective and long-term therapeutic protocols are lacking for HTLV-1 associated diseases.

2.3 Risk Group (RG) Determination

RG determination is based on the outcome of a pathogen risk assessment which assesses the inherent risk of a pathogen utilizing risk factors such as; pathogenicity, infectious dose, and communicability. The categories range from RG1 (low individual and low community risk) to RG4 (high individual and high community risk). A full list of pathogen risk factors and the definitions of the RG levels can be found in the CBSG.

In collaboration with a group of experts, the Agency performed pathogen risk assessments for both HIV and HTLV-1 and based on the risk factors associated with the pathogens outlined in the previous sections, it was determined that both viruses will remain in the RG3 category. The CBSG defines a RG3 pathogen as one that poses a high risk to the health of individuals and/or animals and a low risk to public health. RG3 pathogens are likely to cause serious disease in a human or animal. Effective treatment and preventive measures are usually available and the risk of spread of disease caused by these pathogens is low for the public.^{Footnote 1} The RG determination for HIV and HTLV-1 is in line with assessments that have been completed by international counterparts as shown in Table 1.

 From: Anthology of Biosafety XIII - Risk Group Classification for Infectious Agents

3.0 CONTAINMENT LEVEL REQUIREMENTS

3.1 CBSG Containment Zone Overview

The CBSG reflects the harmonization of the operational and physical containment requirements for human and terrestrial animal pathogens and toxins. This section will reiterate the types of work that are included for each containment column (i.e., CL2) and the distinction between the animal containment zones. More detailed information can be found in the CBSG.

An animal containment zone refers to a series of co-located animal rooms/cubicles, as well as associated corridors and support rooms (e.g., storage and preparation areas) of equal CL, serviced by a single entry/exit. In a "small animal containment zone" (SA zone), animals are housed in primary containment caging. The room encompassing these cages is referred to as an "animal room". Generally, SA zones follow the same requirements as those for laboratory work areas and are represented under the CL2 or CL3 columns (Table 2).

In a "large animal containment zone" (LA zone), the room itself provides the primary containment. The room or space housing the animals is referred to as an "animal cubicle". When small-sized animals are not housed in primary containment caging (e.g., chickens in pens), they are considered to be housed in an LA zone and the room and containment requirements provide the appropriate risk mitigation. The CBSG provides distinct columns for LA zones at CL2 and CL3, labelled CL2-Agriculture (CL2-Ag) and CL3-Ag, respectively.

3.2 Containment Level (CL) Determination

CL determination provides the end-user with a description of the minimum physical containment and operational practices required for handling pathogens safely in a laboratory setting. CLs range from a basic laboratory to CL4 (maximum containment facility). Please see the CBSG for full descriptions of the CLs.

For the majority of pathogens, the CL and RG of the pathogen are the same (e.g., RG2 pathogens are handled at CL2), but there are some exceptions. For example, certain physical and/or operational requirements at CL3 for activities involving RG3 pathogens not known to be transmissible by inhalation can sometimes be derogated.

The risk assessments for HIV and HTLV-1 clearly indicate that both pathogens have the ability to produce serious disease in humans. However, the assessments also report that transmission is largely dependent on parenteral inoculation and contact with mucous membranes; survival of the virus outside the host is limited and occurs only under ideal conditions; airborne transmission is not possible; and the risk for laboratory users as well as the environment is limited. It has also been determined that the culturing (propagation) or in vivo work involving either pathogen does not increase the risk for the laboratory user and that proper handling and operational practices can mitigate the risk.

Based on the risk assessments completed by the Agency in conjunction with HIV and HTLV-1 specialists, it has been determined that HIV and HTLV-1 can be safely handled at CL2/CL2-Ag with specific additional operational requirements. See Table 3 for the containment categories, Section 4 for the additional requirements that are to be followed in order to work at CL2/CL2-Ag, and Section 5 for additional biosafety considerations for working with HIV and HTLV-1. For a complete listing of the operational and physical containment requirements, please refer to Part I Chapters 3 and 4 of the CBSG.

4.0 ADDITIONAL BIOSAFETY REQUIREMENTS

HIV and HTLV-1 can be safely handled in a CL2 or CL2-Ag zone that meets the physical and operational requirements described in Part I Chapters 3 and 4 of the CBSG, as well as the additional requirements noted below.

4.1 Additional Operational Requirement for All Activities

• Centrifugation of infectious material to be carried out in sealed safety cups (or rotors) that are unloaded in a BSC (CBSG R4.6.27). Where this is not possible, additional precautions (e.g., PPE and/or safety equipment, splash guards) are to be in place to prevent exposure to infectious materials due to spills, splashes, or similar events.

4.2 Additional Operational Requirements for Propagative in vitro Activities

• All activities involving open vessels of infectious material to be conducted in a certified BSC or other appropriate primary containment device (CBSG R4.6.24); and
• An additional layer of protective clothing to be donned prior to work with infectious material in accordance with entry procedures (CBSG R4.4.6). An additional layer of protective clothing (e.g., solid-front gowns with tight-fitting wrists, second pair of gloves, waterproof aprons or head covers) provides additional protection and guards against exposure following a tear that compromised, or a spill that contaminated, the outer protective layer.

4.3 Additional Operational Requirement for in vivo Activities

• An additional layer of protective clothing to be donned prior to work with infected animals, in accordance with entry procedures (CBSG R4.4.6).

5.0 BIOSAFETY CONSIDERATIONS

The importance of requirements surrounding the completion of LRAs is provided in the CBSG. Many of the requirements in the CBSG are risk and performance-based and as such, are dependent on the LRA performed. Based on the risks associated with work involving HIV and/or HTLV-1, the following list of CBSG requirements are being highlighted to assist with the development of LRAs and standard operating procedures (SOPs). Although these requirements or parts thereof are listed as CL2/CL2-Ag operational requirements in Part I Chapter 4 of the CBSG, they are critical for the safe handling of HIV and HTLV-1 and are highlighted and expanded upon here to facilitate the development of LRAs and associated procedures.

• Good microbiological practices to be employed (CBSG R4.6.18);
• Use of needles, syringes, and other sharp objects to be strictly limited (CBSG R4.6.9). Where possible, plasticware should be used instead of glassware; Applicable provincial/territorial legislation should be consulted to determine local requirements for the use of safety-engineered needles;
• A certified BSC to be used for procedures that may produce infectious aerosols or aerosolized toxins when aerosol generation cannot be contained through other methods; or that involve high concentrations of infectious material or toxins (CBSG R4.6.23);
• Face protection to be used where there is a risk of exposure to splashes or flying objects (CBSG R4.4.2);
• Open wounds, cuts, scratches, and grazes to be covered with waterproof dressings (CBSG R4.6.6);
• Procedures to be in place to prevent a leak, drop, spill, or similar event, during the movement of infectious material within the containment zone, or between containment zones within a building (CBSG R4.6.29);
• A medical surveillance program, based on an overarching risk assessment and LRAs, to be developed, implemented, and kept up to date (CBSG R4.2.1). The surveillance plan should include a protocol for the evaluation of potentially exposed personnel to receive post exposure prophylactic treatment; and
• Disinfectants effective against the infectious material in use to be available and used in the containment zone (CBSG R4.8.2).

6.0 CONTACT AND ADDITIONAL INFORMATION

Please note that this Directive is based on currently available scientific evidence and is subject to review and change as new information becomes available. If the Directive is amended, the Agency will communicate the updated information to the impacted regulated parties and distribute the amended Directive. For more information on this Directive please contact:

Public Health Agency of Canada
Centre for Biosecurity
Standards and Guidelines Program
Email: standards.normes@phac-aspc.gc.ca
CBSG Website
PHAC PSDS Website

7.0 GLOSSARY

The following list is an excerpt from the CBSG pertaining to terms referenced in this Directive. A comprehensive list of terms and their definitions can be found in the Glossary in Part II Chapter 21 of the CBSG.

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