Consideration of the proposed research protocol procedures is one of the steps in risk assessment.

Principal Investigator's Risk Assessment

A risk assessment should always be conducted prior to initiating any work in a laboratory.

The Principal Investigator/Laboratory Director is responsible for

• Identifying potential hazards,
• Assessing risks associated with those hazards, and
• Establishing precautions and standard procedures to minimize employee exposure to those risks.  

Qualitative biological risk assessment is a subjective process that involves professional judgments. Because of uncertainties or insufficient scientific data, risk assessments sometimes are based on incomplete knowledge or information. Inherent limitations of and assumptions made in the process also exist, and the perception of acceptable risk differs for everyone. The risk is never zero, and potential for human error always exists.

A comprehensive approach for identifying hazards in the laboratory will include information from a variety of sources. No one standard approach or correct method exists for conducting a risk assessment; However, several strategies are available, such as using a risk prioritization matrix, conducting a job hazard analysis; or listing potential scenarios of problems during a procedure, task, or activity.

The process involves the following six steps:

1. Identify the hazards associated with potentially biohazardous agent or material, including human pathogens, recombinant viral vectors, and biological toxins.
2. Identify the activities that might cause exposure to the agent or material. 
3. Consider the training, competencies, and experience of laboratory personnel.
4. Evaluate and prioritize risks (evaluate the likelihood that an exposure would cause a laboratory-acquired infection [LAI] and the severity of consequences if such an infection occurs).
5. Determine your level of acceptable risk
6. Develop, implement, and evaluate controls to minimize the risk for exposure and establish plans for how to deal with an exposure, should it occur.

Step 1. Identify the hazards associated with an infectious or biohazardous agent or material. (Agent-specific hazards)--What is being worked on?

• The potential for infection, as determined by the most common routes of transmission (i.e., ingestion by contamination from surfaces/fomites to hands and mouth; percutaneous inoculation from cuts, needle sticks, non-intact skin, or bites; direct contact with mucous membranes; and inhalation of aerosols)
• The volume and concentration of organisms handled
• Intrinsic factors (if agent is known):
  • Pathogenicity, virulence, and strain infectivity/communicability “How easily can the pathogen spread and cause disease?”;
  • Mode/Route of transmission (mode of laboratory transmission may differ from natural transmission), how can the pathogen spread?  Direct or indirect contact, airborne?;
  • Infectious dose (the number of microorganisms required to initiate infection can vary greatly with the specific organism, patient, and route of exposure) or LD50 for toxic materials, or “how much of the biohazard does someone need to be exposed to for infection or illness to occur?”;
  • Genetic modifications that alter the risk, such as expression of oncogenes or siRNAs to knockdown tumor suppressors;
  • The risk of the formation of replication competent viruses when using recombinant viral vectors;
  • Form (stage) of the agent (e.g., presence or absence of cell wall, spore versus vegetation, conidia versus hyphae for mycotic agents);
  • Stability of biohazard-How long can it survive outside host?  What is its susceptibility to disinfectants and antiseptics?
  • Invasiveness of agent (ability to produce certain enzymes);
  • Host range- Zoonosis: can the pathogen infect both animals and humans? What are the natural and/or experimentally infected hosts?  If it got out, could it spread here?
  • Host factors—can biohazard cause disease in healthy adult? What populations are at greater risk (pregnant, immunocompromised, allergy-sensitized)?
  • Epidemiology—is the biohazard endemic or foreign to the geographical research area?  Is there a risk to the biohazard escaping the research facility and entering the environment?
  • Origin of the material being handled. For example human tissues or cell lines make harbor pathogens (Table 2);
  • Availability of vaccines and/or prophylactic interventions; and
  • Resistance to antibiotics.

Step 2. Identify activities that might cause exposure to the agent or material. (Procedure-based hazards):  What is being done?

The facility (e.g., BSL-2, BSL-3, open floor plan [more risk] versus separate areas or rooms for specific activities [less risk], sufficient space versus crowded space, workflow, equipment present);

The equipment (e.g., uncertified Biological Safety Cabinets [BSCs], cracked centrifuge tubes, improperly maintained autoclaves, overfilled sharps containers, Bunsen burners);

Potential for generating aerosols and droplets.

Aerosols can be generated from most routine laboratory procedures but often are undetectable. The following procedures have been associated with generation of infectious aerosols.

Aerosol Generating procedures:

• Manipulating needles, syringes and sharps
  • Subculturing positive blood culture bottles, making smears
  • Expelling air from tubes or bottles
  • Withdrawing needles from stoppers
  • Separating needles from syringes
  • Aspirating and transferring body fluids
  • Harvesting tissues
• Manipulating inoculation needles, loops, and pipettes
  • Flaming loops
  • Cooling loops in culture media
  • Subculturing and streaking culture media
  • Expelling last drop from a pipette (including Eppendorff pipettes)
• Manipulating specimens and cultures
  • Centrifugation
  • Setting up cultures, inoculating media
  • Mixing, blending, grinding, shaking, sonicating, and vortexing specimens or cultures
  • Pouring, splitting, or decanting liquid specimens
  • Removing caps or swabs from culture containers, opening lyophilized cultures, opening cryotubes
  • Spilling infectious material
  • Filtering specimens under vacuum
  • Preparing smears, performing heat fixing, staining slides
  • Performing serology, rapid antigen tests, wet preps, and slide agglutinations
  • Throwing contaminated items into biohazardous waste
  • Cleaning up spills

Additional Hazards

• Use of animals;
  • The route of inoculation or challenge--How is the biohazard being introduced to an animal? E.g., injection, aerosol challenge, absorption
  • Handling of the animal--How will the animal be restrained? E.g., physical or chemical restraints
  • Shedding of the biohazard--Is the biohazard shed by the animal being treated? What are the possible routes of shedding? E.g., urine, feces, dander, saliva Also consider how long the animal is infectious for and how long shedding may occur. Can the biohazardous agent replicate in the animal host? If applicable, what is the metabolic half-life of the biohazard (e.g., a biotoxin)?
  • Type of caging and housing used--For example, are the animals in open-caging systems or HEPA-filtered cage racks? How often are the cages changed?
  • Education and competency of personnel--Are the laboratory personnel who handle the animals well trained? How experienced are the staff?
• Use of sharps;
• Production of large volumes or concentrations of potential pathogens or agents;
• Improperly used or maintained equipment;
  • Examples of possible hazards are decreased dexterity or reaction time for workers wearing gloves, reduced ability to breathe when wearing N95 respirators, or improperly fitting personal protective equipment (PPE).
• Working alone in the laboratory.
  • No inherent biologic danger exists to a person working alone in the laboratory; however, the supervisor is responsible for knowing if and when a person is assigned to work alone. Because assigning a person to work alone is a facility-specific decision, a risk assessment should be conducted that accounts for all safety considerations, including type of work, physical safety, laboratory security, emergency response, potential exposure or injury, and other laboratory-specific issues.

Step 3. Consider the competencies and experience of laboratory personnel.

• Age (younger or inexperienced employees might be at higher risk);
• Genetic predisposition and nutritional deficiencies, immune/medical status (e.g., underlying illness, receipt of immunosuppressive drugs, chronic respiratory conditions, pregnancy, non-intact skin, allergies, receipt of medication known to reduce dexterity or reaction time);
• Education, training, experience, competence;
• Stress, fatigue, mental status, excessive workload;
• Perception, attitude, adherence to safety precautions; and
• The most common routes of exposure or entry into the body (i.e., skin, mucous membranes, lungs, and mouth)

Step 4. Evaluate and prioritize risks.

Risks are evaluated according to the likelihood of occurrence and severity of consequences.

• Severity of consequences:

Consequences may depend on duration and frequency of exposure and on availability of vaccine and appropriate treatment. Following are examples of consequences for individual workers:

• Colonization leading to a carrier state
• Asymptomatic infection
• Toxicity, oncogenicity, allergenicity
• Infection, acute or chronic
• Illness, medical treatment
• Disease and sequelae
• Death

• Likelihood/Probability of occurrence:

• Almost certain: expected to occur
• Likely: could happen sometime
• Moderate: could happen but not likely
• Unlikely: could happen but rare
• Rare: could happen, but probably never will

Step 5.  Determine your level of risk and acceptable level of risk.

Use the risk matrix to determine your level of risk, and decide what your acceptable level of risk is.  Not all risk can be eliminated, the goal is to minimize risk to acceptable levels.

Step 6. Develop, implement, and evaluate controls to minimize the risk for exposure.

Engineering controls:

If possible, first isolate and contain the hazard at its source.

• Primary containment: BSC, sharps containers, centrifuge safety cups, splash guards, safer sharps (e.g., auto-retracting needle/syringe combinations, disposable scalpels), and pipette aids
• Secondary containment: building design features (e.g., directional airflow or negative air pressure, hand washing sinks, closed doors, double door entry)

Administrative and work practice controls

• Strict adherence to standard and special microbiological practices
• Adherence to signs and standard operating procedures
• Frequently washing hands
• Wearing PPE only in the work area
• Minimizing aerosols
• Prohibiting eating, drinking, smoking, chewing gum
• Limiting use of needles and sharps, and banning recapping of needles
• Minimizing splatter (e.g., by using lab "diapers" on bench surfaces, covering tubes with gauze when opening)
• Monitoring appropriate use of housekeeping, decontamination, and disposal procedures
• Implementing "clean" to "dirty" work flow
• Following recommendations for medical surveillance and occupational health, immunizations, incident reporting, first aid, post-exposure prophylaxis
• Training
• Implementing emergency response procedures

PPE (as a last resort in providing a barrier to the hazard)

• Gloves for handling all potentially contaminated materials, containers, equipment, or surfaces
• Face protection (face shields, splash goggles worn with masks, masks with built-in eye shield) if BSCs or splash guards are not available. Face protection, however, does not adequately replace a BSC. At BSL-2 and above, a BSC or similar containment device is required for procedures with splash or aerosol potential.
• Laboratory coats and gowns to prevent exposure of street clothing, and gloves or bandages to protect nonintact skin
• Additional respiratory protection if warranted by risk assessment

Job safety analysis

One way to initiate a risk assessment is to conduct a job safety analysis for procedures, tasks, or activities performed at each workstation or specific laboratory by listing the steps involved in a specific protocol and the hazards associated with them and then determining the necessary controls, on the basis of the agent/organism. Precautions beyond the standard and special practices for BSL-2 may be indicated in the following circumstances:

• Organisms transmitted by inhalation
• Work with vectors expressing oncogenes or toxins
• Work with large volumes or highly concentrated cultures
• Compromised immune status of staff
• Training of new or inexperienced staff
• Technologist preference

Monitoring effectiveness of controls

Risk assessment is an ongoing process that requires at least an annual review because of changes in new and emerging pathogens and in technologies and personnel.

• Review reports of incidents, exposures, illnesses, and near-misses.
• Identify causes and problems; make changes, provide follow-up training.
• Conduct routine laboratory inspections.
• Repeat risk assessment routinely.

The Principal Investigator must conduct a comprehensive risk assessment to:

• Determine risk group of the agent - Appendix B of the NIH Guidelines or ABSA website
• Evaluate agent factors
  • Virulence, pathogenicity, infectious dose, environmental stability, route of spread, communicability, quantity, availability of vaccine or treatment
    • Strain that is known to be more hazardous than wild type should be considered for handling at a higher containment level.
    • Attenuated strains may be able to be handled at lower containment.
• Evaluate gene product effects:
  • Toxicity, physiological activity and allergenicity
• Evaluate how the agent will be used:
  • Animal experiments may require higher containment, or large quantity production (>10 L) may require higher containment.
• Determine appropriate laboratory containment and practices required based on the above information.
  • Containment levels are described in Appendix G of the NIH Guidelines

Risk Groups

There are four risk groups:

Risk Group 1: Agents are not associated with disease in healthy adult humans. Risk Group 2: Agents are associated with human disease which is rarely serious and for which preventative or therapeutic interventions are often available. Risk Group 3: Agents are associated with serious or lethal human disease for which preventative or therapeutic interventions may be available. Risk Group 4: Agents are likely to cause serious or lethal human disease for which preventative or therapeutic interventions are usually not available.

Risk Group 1

Agents are not associated with disease in healthy adult humans.

Examples

1. E. coli K-12
   1. That does not possess a complete lipopolysaccharide
   2. That does not carry any active virulence or colonization factors or genes encoding these factors
2. Bacillus subtilis host vector systems
3. Adeno-associated virus (AAV) types 1-4
4. Recombinant AAV constructs produced in the absence of helper virus transgene cannot encode for tumorigenic gene product or toxin molecule

Animal Viral Etiologic Agents in Common Use (Risk Group 1) Animal Viral Etiologic Agents in Common Use Not associated with disease in healthy human adults

Examples

1. Murine Cytomegalovirus
2. Bovine Papilloma virus
3. Feline leukemia virus
4. Murine leukemia virus (exception: amphotropic and xenotropic strains are infectious to humans and are considered Risk Group 2).

Risk Group 2

Agents are associated with human disease which is rarely serious and for which preventative or therapeutic interventions are often available.

Examples

1. Staphylococcus aureus
2. Streptococcus
3. Cryptococcus neoformans
4. Giardia sp.
5. Human adenoviruses
6. Herpesviruses (expect Herpes B)
7. Influenza viruses

Risk Group 3

Agents are associated with serious or lethal human disease for which preventative or therapeutic interventions may be available.

Examples

1. Mycobacterium tuberculosis
2. Human immunodeficiency virus (HIV) types 1 and 2
3. Venezuelan equine encephalomyelitis (VEE) virus
4. West Nile virus (WNV)

Risk Group 4

Agents are likely to cause serious or lethal human disease for which preventative or therapeutic interventions are usually not available.

Examples

1. Ebola virus
2. Herpes B virus