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.