BIOSAFETY
BIOSAFETY IN MICROBIOLOGICAL AND BIOMEDICAL LABORATORIES
BMBL is the “Bible” for Biosafety matters Published by US Dept. of Health and Human
Services and CDC and NIH Does not have the force of law, BUT The standard for biosafety. Must comply if
receiving certain grants, etc.
HISTORY
Published data regarding Laboratory Acquired Infections – most are aerosolLab workers are infected by the agents they work
with. (The good news – “not been shown to represent a threat to the community.”)
1979 Pike concludes “the knowledge, the techniques, and the equipment to prevent most laboratory infections are available.”
The “Biosafety in Microbiological and Biomedical Labortories” (BMBL) is born
LAI’S CONTINUED, (MMWR JAN 6, 2012 SUPPLEMENT/VOL 61)
Recent MMWR reports indicate bacteria account for >40%
>37 species as etiologic agentsBrucella, Shigella, Salmonella and Staph aureus
are common 2005 CDC Neisseria meningitidis
General population 13/100,00030-59 year population 0.3/100,00030-59 year old microbiologists 20/100,000 April 2012, 25 yr old lab tech dies from infection same
sero-type as lab strain (no vaccine available for this strain)
5 PREDOMINANT ROUTES OF INFECTION
Parenteral inoculation with “sharp” Spill/splash on skin and mucous membranes Ingestion or exposure via touching mouth or eyes
with fingers or contaminated objects Inhalation of infectious aerosols Animal bites/scratches (zoonotic)
RISK CLASSIFICATION
Many agencies have classified infective microorganisms by Risk Group
CDC/NIH World Health Organization Canadian Laboratory Safety Guidelines European Union Australia/New Zealand
RISK GROUPS
RG 1 Organisms not known to cause disease in
healthy adult humans and pose minimal hazard to people and the environment. (ATCC)
Individual risk: low Community risk: low
RISK GROUPS
RG 2 Organisms that pose a moderate risk and are
associated with human disease through skin breaks, ingestion or mucous membrane exposure (ATCC)
Individual risk: moderate, potential hazard Community risk: low, limited, unlikely
RISK GROUPS
RG 3 Indigenous or exotic agents with potential for
aerosol transmission/inhalation route of exposure and have the potential for serious and even lethal effects. (ATCC)
Treatment usually available Individual risk: high, serious Community risk: Low, may be present
RISK GROUPS
RG 4 Dangerous/exotic agents which pose high risk of
life threatening disease, aerosol-transmitted lab infections; or related agents with unknown risk of transmission.
Preventive or therapeutic interventions not usually available
Individual risk: high, serious Community risk: high
RISK GROUP VS. BIOSAFETY LEVEL
Risk Group is a stable comparative descriptor of the inherent pathogenic nature of a given microorganism;
RG does not change based on how or where the agent is used.
Biosafety Level is a variable comparative descriptor of the facility, equipment and practices that serve to "contain" a microorganism while it is being handled; BSL is based on risk assessment and technical judgment and may vary with the use of the agent.
(Glenn Funk, ABSA)
PRINCIPLES OF BIOSAFETY: CONTAINMENT Laboratory practice and technique Safety Equipment Facility Design Biosafety Levels
Combination of lab practices & techniques, safety equipment and facilities specific for each of the 4 biosafety levels
PRIMARY VS SECONDARY CONTAINMENT
Primary – protect workers in the immediate area of the lab.
Secondary – external to the lab. Usually facility design.
BIOSAFETY LEVEL 1 Level 1 for agents that are “defined and
characterized strains of viable microorganisms not known to consistently cause disease in healthy adult humans.”B. subtilis, E. coli K12, S. cervasiae
Containment relies on “standard microbiological practices, no special…barriers other than a sink for handwashing.”
BIOSAFETY LEVEL 2
Level 2 for the “broad spectrum of indigenous moderate-risk agents that are present in the community and associated with disease of varying severity.” S. aureus, B. anthracis, HIV, Hep B S. aureus is a common LAI
Containment relies primarily on “good microbiological technique…”provided the potential for producing splashes and aerosols is low.”
Primary hazard is by accidental percutaneous or mucous membrane exposure. Careful with sharps!!!
BIOSAFETY 2, CONTINUED
Aerosols. If manipulations will produce aerosols, then “primary containment” such as PPE and other safety devices such as Biological Safety Cabinet (BSC) must be used.
Handwashing facilities Waste decontamination
Aerosols
AEROSOL – TWO CONSIDERATIONS
Respirable-size particles that remain airborne for protracted period of time. Source of infection if inhaled.
Formation of droplets that settle rapidly on surfaces – clothing, hands, benchtops, etc. Large size droplet can contain multiple copies of the agent.
(BMBL 5th edition)
Gross Contamination On Horizontal Surface Near Pipetting Operation
Volume ofPipetting Aid
Total CFU On All
CollectedPlates
Microliter Trial 1 Trail 235
102550
200100015006000
403.5
276
49.5205.5138315
036
112138
1225588
2025
Aerosol and Surface Recovery from Ten Pipetting Operations of 109/ml. B. subtilis
Run AirborneCFU
SettledHands
CFUArea
123456
Average
2,040657
2,050388
5,110649
1,820
35,80022,00014,800
9,3006,900
228,000
52,800
3,700860
1700550
21002900
1,970(average time 3 minutes; 1 ml. Pipette; ca. 2ml. bulb. Chatigany et. al. 1979
CFU Recovered From Operator’s Glove
Trail # Before Pouring After Pouring IntoCentrifuge Tubes
1234
Average
0000
0
48,00048,0007,9008,900
28,000
(suspension poured contained 109/ml. Flavobacterium.)
Aerosols From Lab Equipment
Blender, opened at once 106
Sonicator with bubbling 106
Pipetting, vigorous 106
Dropping culture 3 X 105
Splash on a centrifuge rotor 105
Blender, opened after 1 minute 2 X 104
Pipetting, carefully 104
(1010/ml culture - 10 min. use)
Dimmick, et. al. 1973
OTHER AEROSOL GENERATING ACTIVITIES:
Flaming loops Cooling loops in culture media Subculturing and streaking culture media Expelling the last drop from a pipet Setting up cultures, inoculating media Preparing smears, performing heat fixing,
staining slides (MMWR Jan 6, 2012 Supplement/Vol 61)
BIOSAFETY 3 AND 4 Level 3 for “indigenous or exotic agents with
a potential for respiratory transmission and which may cause serious and potentially lethal infection.” M. tuberculosis.
Much higher level of secondary containment Level 4 – the really nasty stuff. Ebola,
Marburg.
STANDARD MICROBIOLOGICAL PRACTICESBSL 1
Access to laboratory limited when work with cultures is in progress.
Handwashing No eating, drinking, applying lip balm, makeup, etc. No mouth pipetting Policies for handling sharps Minimize splashes and aerosols Work surface decontaminated at least once per day Decontamination of cultures and lab wastes Biohazard sign must be posted at lab entrance Pest management program Supervisor ensures that personnel are trained
BSL 1, SAFETY EQUIPMENT
Safety Equipment – not usually required PPE such as lab coats recommended Gloves worn when skin is broken, rash Eye protection when splashes likely No special facility considerations
Calvin CollegeSB 210
BIOSAFETY LEVEL 1
ACCESS: FACULTY, STAFF AND APPROVED/BIO 321 & 336 STUDENTS
PRECAUTIONS: Standard microbiological practices. Keep doors closed when working with microbial agents.
WASH HANDS BEFORE LEAVING ROOM!!
RESPONSIBLE INVESTIGATORS:
Arlene Elizabeth
Ext: 6-8668 Ext: 6-7085Home: 1 Home: SECONDARY CONTACT: Lori KeenExtension: 6-6080Home
AGENTS USED: E.coli, E. faecalis, B. subtilis, M. luteus, M. smegmatis,
M. chloropheniclum, S. marcescens, S. cerevasie
BSL 2
Standard Microbiological practices PLUS Access limited especially to those at increased risk of
infection Biosafety manual specific to the lab including SOP’s PI’s ensure training of personnel regarding hazards,
prevention. Gloves and lab coats must be worn when working with
infectious agents. High degree of caution regarding sharps. Disinfection of work surfaces. Spills and accidents resulting in exposure are immediately
reported Biohazard sign MUST be posted stating the name of the
agent(s), the biosafety level, PI’s name and phone number, PPE required to enter, special procedures for exit.
Immunizations, medical surveillance if appropriate. Appropriate furniture/chairs – non-porous, easily cleaned.
BSL 2, SAFETY EQUIPMENT
PPE and equipment (BSC) used when procedures with a potential for significant aerosols and/or splashes are conducted or high concentrations or large volumes of the agent are in use.
Lab coats routinely worn in the laboratory Gloves worn when hands may come in
contact with agent, contaminated equipment or surfaces.
PPE remains in lab and is removed before leaving the lab!!!
Facility requirements
Calvin CollegeSB 210
Biosafety Level 2
ALL UNAUTHORIZED PERSONS KEEP OUT!
ACCESS: Allowed for faculty, staff and Bio 207 students.
PRECAUTIONS: Standard microbiological practices; lab coats must be worn during all procedures; additional precautions may apply. WASH HANDS AND REMOVE LAB COATS AND GLOVES BEFORE LEAVING ROOM!!
RESPONSIBLE INVESTIGATORS: Amy Anding Ext. 6-7620 Ext. 6-6025
AGENTS IN USE: may include S.aureus, P.aeruginosa, P. vulgaris, S. choleraesius
Also RG1 agents - E.coli, E. faecalis, B. subtilis, M. luteus, M. smegmatis,
S. marcescens, S. cerevasie
RISK ASSESSMENT
Agent Hazards Lab Procedure Hazards Staff (student) proficiency
RISK ASSESSMENT BSL assessments need to be done on case by
case basis. Consider the following: Procedures to be performed Pathogenicity Route of transmission Agent stability Infectious dose Concentration Experience and skill level Effective prophylaxis Health status of the worker Always err on the side of caution!
AGENT HAZARDS Pathogenicity
HepB virus is 50-100 times more infectious than HIV (World Health Organization)
Route of transmission Inhalation, ingestion, mucous membrane?
Agent stabilityHBV- at least 7 days and still be capable of
causing infection.HIV- evidence suggests only hours of
viability http://www.cdc.gov/hiv/resources/qa/transmission.htm
Infectious dose
RISK OF INFECTIONFROM PERCUTANEOUS EXPOSURE(PATHOGENICITY )
Virus Virus/mL Serum Risk
HBV 102-108 30% HCV 100-106 2% HIV 100-103 0.3%
TRANSMISSION RATESEXPOSURE TO HIV INFECTED BLOOD (ROUTE OF EXPOSURE)
Percutaneous 0.31% Mucocutaneous 0.03%
LAB PROCEDURE HAZARDS
Safety equipment available Complexity of procedure Agent concentration and volume Aerosol generating Animals
STAFF (STUDENT) CONCERNS
Proficiency/competency of laboratorian Medical status
HOST FACTORS AFFECTING RISK
Deficiencies in host defensesSkin – eczema, chronic dermatitis, psoriasisMucosa – antimicrobial therapy; bowel pathologyImmune system deficienciesAspleniaOther medical conditions such as viral infections, poorly controlled Type I diabetes, pregnancy, asthma, cancer, connective tissue diseases
(treatment often causes immunosuppresion)
SUMMARY STEPS TO RISK ASSESSMENT…START TODAY
Identify the hazards associated with the agent Identify the activities that might cause exposure Consider competency/experience of personnel Evaluate and prioritize risks and severity of
consequences Develop and IMPLEMENT controls to minimize the
risk for exposure
LESSONS FROM SALMONELLA Aug 2010-June 2011 109 individuals in 38
states infected with strain X of Salmonella Typhimurium. Ages 1-91, median 21.
Exposure to clinical and teaching microbiology labs was a possible source. 60% of ill persons had exposure to a microbiology lab in the week prior to illness. Some specifically worked with Salmonella in micro labs.
Several children living in households with a person who worked or studied in micro labs became ill with the outbreak strain.
http://www.cdc.gov/salmonella/typhimurium-laboratory/011712/index.html
CONCLUSIONS
Investigated two groups of laboratories – labs associated with illness and labs w/out illness
Lab practices and safety policies were similar BUT Labs associated with illness had less knowledge of
biosafety training materials Those free of illness were more likely to train
employees regarding signs and symptoms of illness
Enforcement of policies may be difficult to enforce or monitor
TAKE HOME LESSONS
“If you work in a laboratory it is possible to bring bacteria home through contaminated lab coats, pens, notebooks, and other items…”
“Leave food, drinks or personal items like car keys, cell phones and mp3 players outside of the laboratory.”
Wear a lab coat, leave the lab coat in the lab. Train for signs and symptoms (illness) of agents in
use.