General Introduction to Biosafety Robert Heckert, DVM, PhD, CBSP Robert Heckert Consulting, LLC www.safevet.com
COURSE OUTLINE • Introduction • Laboratory Associated Infections • Blood-borne Pathogens • Classification of Biohazards • Infection/Biohazard Control • Spill Response • Biomedical Waste • Regulations BIOSAFETY
WHAT IS A BIOHAZARD? • A potential hazard to humans, animals or the environment caused by a biological organism, or by material produced by such an organism • Examples: • Viruses, bacteria, fungi, and parasites and their product. • Blood and body fluids, as well as tissues from humans and animals. • Transformed cell lines and certain types of nucleic acids .
WHAT IS BIOSAFETY? • Measures employed when handling biohazardous materials to avoid infecting oneself, others or the environment. • Achieved through; • Administrative Controls • Engineering Controls • Personal Protective Equipment • Practices and Procedures
WHY ARE WE CONCERNED? • Potential for acquiring a laboratory-associated infection (LAI) • Contamination of the environment • Contamination of research • Public perception
LABORATORY ASSOCIATED INFECTIONS • Percutaneous inoculation • Inhalation of aerosols • Contact of mucous membranes • Ingestion Infection Source • Cultures and stocks • Research animals • Specimens • Items contaminated with above Susceptible Host Route of Transmission • Immune system • Vaccination status • Age
LAIS • Only 20% causative or defined event • 80% of which are caused by human factors • 20% are caused by equipment failure • Top 4 accidents resulting in infection • Spillages & splashes • Needle and syringe • Sharp object, broken glass • Bite or scratch from animals or ectoparasites http://www.weizmann.ac.il/safety/bio2.html
BLOODBORNE PATHOGENS (BBP) • Sources • Blood • Semen • Vaginal Secretions • Other Bodily Fluids Cerebrospinal Amniotic Synovial • Tissue Cultures • Organ Cultures • Infected Experimental Animals
RISK OF EXPOSURE • Pathogen involved • Type of body fluid • Route of exposure • Duration of exposure • Volume of blood involved in exposure • Concentration of virus at time of exposure • PPE worn
SPECIFIC EXAMPLES OF BBPS Hepatitis B Hepatitis C HIV
ISSUES TO CONSIDER • Symptoms • Mode of transmission • Incubation period • Survival outside host • Communicability • Immunization • Prophylaxis / Treatment
IF AN EXPOSURE OCCURS • Initiate first aid • Notify your supervisor / designated person • Report to hospital emergency department or local Health Services • Report incident to regulators (if necessary)
UNIVERSAL PRECAUTIONS • Minimum standard of practice for preventing the transmission of BBP includes: • Education • Hand washing • Wearing protective barriers • Use safe work practices If samples cannot be guaranteed non-infective …… treat as infectious!
BIOHAZARD CLASSIFICATION • Conventional Agents • Recombinant DNA • Tissue Culture • Animal Work • Anatomical Specimens • Unconventional Agents
BIOHAZARD CLASSIFICATION • Organisms are categorized into a group base on the particular characteristics of each organism, such as • Pathogenicity • Infectious dose • Mode of transmission • Host Range • Availability of effective preventive measures • Availability of effective treatment
BIOHAZARD CLASSIFICATION • Organisms are categorized based on the measuresrequired for handling each organism safely in a laboratory setting, such as • Engineering Requirements • Operational Requirements • Technical Requirements • Physical Requirements
CONVENTIONAL AGENTS Unlikely to cause disease in healthy workers or animals Rarely cause serious human or animal disease May cause serious disease Likely to cause very serious disease
RECOMBINANT DNA • Genetic Engineering = in vitro incorporation of genetic material from one cell into another • The level of risk depends on source of DNA, vector and host. • The biosafety community may be able to assist the investigator in this determination.
TISSUE CULTURE • Have the potential to contain pathogenic organisms • In general; Human & non-human primate, and mycoplasma-containing cell lines Level 2 Others Level 1 A detailed risk assessment should be undertaken when using a new cell line.
ANIMAL WORK • Animals can harbour infectious organisms (naturally or introduced) • Level dependent on type of work being conducted. • Special Animal Care training is required for all personnel working with animals. • All work involving animal use must receive prior approval from the Animal Care Committee
ANATOMICAL SPECIMENS • All specimens should be considered infectious due to potential presence of infectious agents • Important to consider the type of specimen • blood, organs, tissues • Spinal sample, brain tissue • From infectious patient • In general Level 2 but it depends on the nature of the work.
UNCONVENTIONAL PATHOGENS • TSE prion diseases; lethal transmissible neurodegenerative conditions • Creutzfeld-Jakob disease, Variant C-J Disease, Mad Cow Disease, Scrapie, Chronic Wasting Disease • Resistant to destruction by procedures that normally inactivate viruses. • Contact regulatory authorities to assess requirements (containment, procedures, waste disposal, etc.)
CONTAINMENT LEVEL 2 • Clinical, diagnostic, research and teaching facilities with level 2 agents. • Requires a class I or class II biological safety cabinet if any potential for aerosol or splash exists. • An emergency plan for handling spills must be developed. • Access should be controlled.
CONTAINMENT LEVEL 3 • Specialized design and construction • primary barriers to protect the individual • secondary barriers to protect the environment • All staff must undergo special training on the agents being used, PPE, equipment, waste management as well as practices and procedures above and beyond the scope of this course.
CONTAINMENT LEVEL 4 • Only few level 4 facilities in the world • Design specifications are extremely stringent, worker is completely isolated from infectious material.
BIOLOGICAL SAFETY CABINETS • Effective means of primary physical containment for biological agents, especially when aerosols are generated. • HEPA filters remove particles (min 0.3 microns) with 99.97% efficiency. • There are 3 main classes of cabinets (I, II, III) which provide various levels of protection.
BIOLOGICAL SAFETY CABINETS • Biological Safety Cabinet • HEPA filtered laminar air flow • Exhaust • Personnel, environment & product protection • Laminar flow hoods • Vertical or horizontal laminar flow • HEPA filtered air (intake only) • Product protection only VS
WORKING SAFELY IN A BSC Before using the cabinet: • Ensure BSC is certified • Turn off UV lamp; turn on fluorescent lamp • Disinfect work surfaces with appropriate disinfectant • Place essential items inside cabinet • Allow the blower to run for 5-10 min before work
WORKING SAFELY IN A BSC While using the cabinet: • Ensure material and equipment is placed near the back of the hood, especially aerosol-generating equipment. Do not block any vents • Use techniques that reduce splatter and aerosols. • General work flow should be from clean to contaminated areas • Minimize movement so as not to impede air flow • Open flame in BSC’s is controversial
WORKING SAFELY IN A BSC After using the cabinet: • Leave blower on at least 5 minutes to purge cabinet • Remove and decontaminate equipment and materials • Disinfect cabinet surfaces • Turn off blower and fluorescent lamp, turn on UV lamp
WORKING SAFELY IN A BSC Maintenance: • Before and after each use - Work surfaces wiped down • Weekly - UV lamp should be wiped clean • Monthly - All vertical surfaces wiped down • Annually - UV lamp intensity verified - Decontamination with formaldehyde gas - Certification
PERSONAL PROTECTIVE EQUIPMENT (PPE) • PPE can become an important line of defence (last line of defense) • Responsibility of both the user and the supervisor to ensure that PPE is worn
PPE • Criteria for consideration • Routes of exposure that need to be blocked • Degree of protection offered • Ease of use • Only effective if correctly selected, fitted, used and cared for, and the individual is trained • Ensure PPE is removed before leaving the lab
PPE Footwear • Closed toed shoes should always be worn. Booties are worn in some higher containment labs and animal facilities. Lab Coats/Gowns • Long-sleeved, knee length with snaps • Elastic cuffs • Back-closing gowns • Periodic cleaning required
PPE Gloves • Latex, nitrile & vinyl for work with biological agents. • Exam gloves should not be reused, change frequently. Utility gloves can be disinfected and reused if they show no sign of degradation. • Consider tensile characteristics, length of cuff. • Double gloving. • BSO can provide assistance finding an alternative for people with allergies.
PPE Eye & Face Protection • Goggles, safety glasses to protect the eyes • Full face shield to protect facial skin. Respirators • Only personnel who have been fit-tested and trained should wear respirators.
PRACTICES AND PROCEDURES • General Safety Guidelines • Good Microbiological Practice • Handwashing • Suspicious Packages • Specific Procedures • Centrifuges • Needles & Syringes and other sharps • Pipettes • Blenders, Grinders, Sonicators & Lyophilizers • Inoculation Loops • Cryostats
GENERAL LABORATORY SAFETY GUIDELINES • Mostly common sense, but you must understand the hazards you face in the laboratory and be adequately trained to deal with them. • Basic must be known for all labs. b i o s a f e t y
GOOD MICROBIOLOGICAL PRACTICE (GMP) • Basic code of practice that should be applied to all types of work involving microorganisms. • Objectives: • prevent contamination of laboratory workers and the environment • prevent contamination of the experiment/samples • Application of aseptic technique, minimization of aerosols, contamination control, personal protection, emergency response
HANDWASHING • One of the single effective means of preventing infections if done properly and frequently • When to wash? • Before starting any manipulations • Before leaving the lab • When hands are obviously soiled • Before and after completing any task in a BSC • Every time gloves are removed • Before contact with one’s face or mouth • At the end of the day
SAFE USE OF CENTRIFUGES • Before use • Stress lines? Overfilled? Balanced? • Caps or stoppers properly in place? • Run conditions achieved? • Use sealable buckets (safety cups) or sealed rotors • After run • Centrifuge completely stopped? • Spills or leaks? • Allow aerosols to settle (30 min) or open in a BSC.
Avoid use whenever possible Use a BSC for all operations with infectious material Fill syringes carefully Shield needles when withdrawing from stoppers Do not bend, shear or recap needles. Dispose of all used needles/syringes in yellow sharps containers NEEDLES AND SYRINGES
PIPETTES • Mouth pipetting is prohibited. • Never force fluids out. • To avoid splashes, allow discharge to run down dispense the receiving container wall. • Never mix material by suction and expulsion. • Reusable pipettes should be placed horizontally in a disinfectant filled pan.
BLENDERS, GRINDERS, SONICATORS, AND LYOPHILIZERS • Operate in a BSC whenever possible. Allow aerosols to settle for 5 minutes before opening. • Safety Blender • Do not use glass blender jars • Decontaminate immediately after use • Lyophilizers • Use glassware designed for vacuum work, ensure there is no damage before using • All surfaces should be disinfected after use • Use vapour traps whenever possible