BIOSAFETY

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IOSAFETY IN AND

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ICROBIOLOGICAL

B

IOMEDICAL

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ABORATORIES

 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.

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ISTORY  Published data regarding Laboratory Acquired Infections – most are aerosol Lab 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 J AN 6, 2012 S UPPLEMENT /V OL 61)    Recent MMWR reports indicate bacteria account for >40% >37 species as etiologic agents  Brucella, Shigella, Salmonella and Staph aureus are common 2005 CDC Neisseria meningitidis     General population 13/100,000 30-59 year population 0.3/100,000 30-59 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 P

REDOMINANT

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OUTES OF

I

NFECTION  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)

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ISK

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LASSIFICATION      Many agencies have classified infective microorganisms by Risk Group CDC/NIH World Health Organization Canadian Laboratory Safety Guidelines European Union  Australia/New Zealand

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ISK

G

ROUPS  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

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ISK

G

ROUPS  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

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ISK

G

ROUPS  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

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ISK

G

ROUPS  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

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ISK

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ROUP VS

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IOSAFETY

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EVEL   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)

P RINCIPLES OF C ONTAINMENT B IOSAFETY :  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

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RIMARY VS SECONDARY CONTAINMENT   Primary – protect workers in the immediate area of the lab.

Secondary – external to the lab. Usually facility design.

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IOSAFETY

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EVEL

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.”

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IOSAFETY

L

EVEL

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!!!

B

IOSAFETY

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

A

EROSOL

–

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 5 th edition)

Gross Contamination On Horizontal Surface Near Pipetting Operation

Volume of Pipetting Aid Microliter 3 5 10 25 50 200 1000 1500 6000 Total CFU On All Trial 1 4 0 3.5

27 6 49.5

205.5

138 315 Collected Plates Trail 2 0 3 6 11 21 38 1225 588 2025

Aerosol and Surface Recovery from Ten Pipetting Operations of 10

9

/ml. B. subtilis

Run Airborne CFU Settled Hands CFU Area 1 2 3 4 5 6 2,040 657 2,050 388 5,110 649 35,800 22,000 14,800 9,300 6,900 228,000 3,700 860 1700 550 2100 2900 Average 1,820 52,800 1,970

(average time 3 minutes; 1 ml. Pipette; ca. 2ml. bulb. Chatigany et. al. 1979

CFU Recovered From Operator’s Glove

Trail # 1 2 3 4 Before Pouring After Pouring Into Centrifuge Tubes 0 0 0 0 48,000 48,000 7,900 8,900 Average 0 28,000

(suspension poured contained 10 9 /ml.

Flavobacterium

.)

Aerosols From Lab Equipment

(10 10 /ml culture - 10 min. use) Blender, opened at once Sonicator with bubbling Pipetting, vigorous Dropping culture Splash on a centrifuge rotor Blender, opened after 1 minute Pipetting, carefully 10 6 10 6 10 6 3 X 10 5 10 5 2 X 10 4 10 4 Dimmick, et. al. 1973

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THER 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)

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IOSAFETY

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.

S TANDARD P RACTICES BSL 1 M ICROBIOLOGICAL            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, S

AFETY

E

QUIPMENT      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

CalvinCollege Calvin College SB 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

Ext: 6-8668 Home: 1

SECONDARY CONTACT: Lori Keen

Extension: 6-6080 Home

Elizabeth

Ext: 6-7085 Home: 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, S

AFETY

E

QUIPMENT      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 College SB 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

Ext. 6-7620

Anding

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

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ISK

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SSESSMENT    Agent Hazards Lab Procedure Hazards Staff (student) proficiency

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ISK

A

SSESSMENT            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!

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GENT

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AZARDS    

Pathogenicity

 HepB virus is 50-100 times more infectious than HIV (World Health Organization)

Route of transmission

 Inhalation, ingestion, mucous membrane?

Agent stability

  HBV- 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

R ISK OF FROM I NFECTION P ERCUTANEOUS (P ATHOGENICITY ) E XPOSURE  Virus    HBV HCV HIV Virus/mL Serum 10 2 -10 8 10 0 -10 6 10 0 -10 3 Risk 30% 2% 0.3%

T RANSMISSION EXPOSURE TO R ATES HIV INFECTED BLOOD ( ROUTE OF EXPOSURE )   Percutaneous Mucocutaneous 0.31% 0.03%

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AB

P

ROCEDURE

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AZARDS  Safety equipment available     Complexity of procedure Agent concentration and volume Aerosol generating Animals

S

TAFF

(

STUDENT

)

CONCERNS   Proficiency/competency of laboratorian Medical status

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OST

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ACTORS AFFECTING

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ISK  Deficiencies in host defenses Skin – eczema, chronic dermatitis, psoriasis Mucosa – antimicrobial therapy; bowel pathology Immune system deficiencies Asplenia Other medical conditions such as viral infections, poorly controlled Type I diabetes, pregnancy, asthma, cancer, connective tissue diseases (treatment often causes immunosuppresion)

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UMMARY

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TEPS TO

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ISK 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

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ESSONS FROM

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ALMONELLA     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

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ONCLUSIONS    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

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AKE

H

OME

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ESSONS   “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.