Biosafety/Bloodborne Pathogens:

Working Safely With Biological Materials in Laboratories

www.oehs.wayne.edu

This training is required annually for all Wayne State University faculty, staff, and students working in research and/or teaching laboratories with materials that are potentially infectious, including; human blood, body fluids, tissue, cell lines, animals infected with human pathogens, mammalian viruses, biological toxins, and/or recombinant DNA.

You must be registered on the Collaborative Institute Training Initiative (CITI) website and complete a quiz hosted on the CITI site in order to fulfill the requirements for this course.

When you have finished the presentation, click on the link on the last slide that will take you to the quiz on the CITI website.

What is a Biohazard?

A biohazard is an agent of biological origin that has the capacity to produce deleterious effects on humans, e.g.; microorganisms, toxins and allergens derived from those organisms; and allergens and toxins derived from higher plants and animals.

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Laboratories working with biohazards have a set of guidelines to follow from the Centers for Disease Control (CDC) and the National Institutes of Health (NIH):

The guidelines are in place to protect human health and prevent the release of pathogens into the environment.

The guidelines include: laboratory practices and procedures facility design safety equipment requirements

Click on the book to link to the CDC/NIH document

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Research using biohazards requires review and approval by the

WSU Institutional Biosafety Committee (IBC)

An IBC is mandated by the NIH at every institution.

IBC members include WSU faculty, staff and PhDs from neighboring institutions.

The IBC may increase the Biosafety Level requirements if the vector is more infectious than wild-type virus.

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The principal investigator must submit the appropriate form to the committee:

Exempt rDNA research does not need approval - researchers must sign off on exemption form.

Protocols that require IBC review & approval include: Native or recombinant mammalian viruses

Pathogenic/infectious agents (non-rDNA)

Biological toxins or rDNA encoding a toxin

Click here for additional information on Biological Agent User Application Forms.

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The appropriate biosafety level (1-4) is determined by the CDC/NIH. Selection of the biosafety level is based on

characteristics of the infectious agent:

Pathogenicity of material - disease incidence/severity

Documented route of transmission (bloodborne, airborne, ingestion)

Availability of protective immunization (HBV Vaccine) or effective therapy

Risk of exposure created by manipulation in handling the agent & caring for infected animals

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Biosafety Levels BSL1 - agents not known to cause disease.

BSL2 - agents associated with human disease.

BSL3 - indigenous/exotic agents associated with human

disease and with potential for aerosol transmission. BSL4 - dangerous/exotic agents of life threatening nature.

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This presentation will focus on the CDC/NIH guidelines for

biosafety levels 1 – 2 research laboratories.

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Biosafety Levels 1- 4 provide: Increasing levels of personnel & environmental protection,

and appropriate guidelines for:

Laboratory Practices and Techniques Standard Practices and Special Practices Knowledge of supervisor and personnel Lab specific SOPs/Biosafety manual

Safety Equipment (Primary Barriers)

Laboratory Facilities (Secondary Barriers)

Buildings (Tertiary Barriers)

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Proper handwashing is important at all biosafety levels!

Wash with warm, running water, mild, preferably liquid soap, (doesn’t have to be antibacterial).

Rub hands together vigorously for at least 15 seconds: scrub between fingers, under nails, tops & palms of hands.

Rinse with warm, running water.

Dry with disposable paper towel.

Use lotion to prevent chapping of hands. 10

LAB COAT protects clothing/skin

GLOVES: disposable latex/non-latex exam gloves change when torn or contaminated

Basic Personal Protective Equipment (PPE) is required at all Biosafety Levels

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Remove PPE before leaving the work area!

If risk of splashes or aerosols, protect the eyes & face. safety glasses/goggles face mask

If necessary, other PPE should

be worn, including: gown, face shield, booties, etc.

Basic Personal Protective Equipment (PPE) is required at all Biosafety Levels

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Biosafety Level 1 (BSL1) BSL 1 is suitable for work involving well-characterized

agents not known to cause disease in healthy adults and of minimal potential hazard to lab personnel and the environment. Some examples include:

Bacillus subtilis Naegleria gruberi Infectious canine hepatitis virus Non-entero hemorrhagic E. coli Exempt recombinant DNA experiments

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Biosafety Level 2 (BSL 2) BSL 2 is suitable for work involving agents of moderate

potential hazard to personnel and the environment. Immunization or antibiotic treatment is available Examples include: Measles virus Salmonellae Toxoplasma spp. Hepatitis B virus Adenoviruses

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All Biosafety Level 2 (or higher) labs are inspected to assure they meet the

CDC/NIH guidelines.

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Biosafety Levels 1 – 2 Facility Design Guidelines

Lab doors are lockable. Sinks available for hand washing. Work surfaces easily cleaned,

impervious to water.

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BSL2 labs should be under negative pressure. Air flows into lab and doesn’t re-circulate to non-lab areas.

Biosafety Level 2 Additional Requirements

Biosafety Cabinet Autoclave Eye Wash 17

Biosafety Levels 1 – 2 Standard Microbiological Practices

Restrict/limit access when working

No eating, drinking, storing food, etc.

No mouth pipetting

Minimize splashes and aerosols

Decontaminate wastes

Decontaminate work surfaces daily

Maintain insect & rodent control program 18

Extreme precaution with SHARPS

Gloves and additional PPE

Use of mechanical pipetting devices

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Biosafety Levels 1 – 2 Standard Microbiological Practices +

Extra care should be taken when using any contaminated sharp item, including needles and syringes, slides, pipettes, capillary tubes, razor blades, lancets, and scalpels. Plastic should be substituted for glass whenever possible.

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Biosafety Level 2 Precautions with SHARPS

Used disposable needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal. Always dispose of whole unit in SHARPS containers!

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Biosafety Level 2 Precautions with SHARPS

Biohazard warning signs posted on lab entrance.

WSU Biosafety Manual available.

Biosafety SOPs are read & signed by all staff.

Principal Investigator must train staff annually.

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Biosafety Level 2 Additional precautions

Cultures, tissue, specimens should be placed in a container with a cover that prevents leakage during collection, handling, processing, transport, storage or shipping.

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Biosafety Level 2 Additional precautions

OSHA Occupational Exposure to Bloodborne Diseases Standard All research involving the use of human blood, body fluids,

tissue, cell lines, etc. is carried out at Biosafety Level 2 because it is unknown if these materials contain bloodborne diseases such as Hepatitis B, Hepatitis C, HIV.

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Based on the concept of UNIVERSAL PRECAUTIONS:

treat all human blood, tissue, cells, etc. as if they

ARE infectious

Bloodborne Diseases

HIV: Human Immunodeficiency Virus causes AIDS - no cure or vaccination

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HBV: Hepatitis B virus causes liver disease vaccination available

HCV: Hepatitis C virus causes liver disease

no vaccination available

Bloodborne diseases are only spread when blood and certain other body fluids from an infected source get into the bloodstream of an uninfected person.

An occupational exposure is contact with infected material through:

needlestick or cut with contaminated sharp object

cuts/breaks in skin

mucous membranes (eyes, nose, mouth)

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Bloodborne Diseases are NOT transmitted through:

Kissing or hugging

Sneezing or coughing

Food or water

Sharing eating utensils, cups, etc.

Casual contact

Saliva, tears, perspiration, urine, feces (when there is no blood present)

How is HIV Transmitted? Blood and body fluids

serum semen vaginal secretions fluids around internal organs/systems

IV drug use vaginal or anal intercourse mother to child in utero * There are no documented cases of HIV being transmitted through saliva, tears or perspiration, saliva.

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HIV Transmission in Healthcare Currently there are 57 documented cases

and 140 possible cases of occupationally acquired HIV among healthcare workers in the U.S. since reporting began in 1985.

(There have been no confirmed cases reported since 1999)

http://www.cdc.gov/hiv/resources/factsheets/PDF/hcw.pdf

Healthcare workers with documented & possible occupationally acquired AIDS/HIV infection, by occupation, 1981 - 2006. No new documented cases of occupationally acquired HIV/AIDS have been reported since December 2001. One new case of possible occupational transmission has been reported.

Occupation Documented Possible

Nurse 24 35

Laboratory worker, clinical 16 17

Physician, nonsurgical 6 12

Laboratory technician, nonclinical 3 -

Housekeeper/maintenance worker 2 13

Technician, surgical 2 2

Embalmer/morgue technician 1 2

Health aide/attendant 1 15

Respiratory therapist 1 2

Technician, dialysis 1 3

Dental worker, including dentist - 6

Emergency medical technician/paramedic - 12

Physician, surgical - 6

Other technician/therapist - 9

Other healthcare occupation - 6

Total 57 140

HIV Exposure Risk Rate of seroconversion after needlestick exposure

to infective material from HIV+ person is 0.3% or about 1 in 300.

HIV in high concentration during period prior to antibody development.

Much less infective than HBV, HCV, Herpes

Source: Centers for Disease Control

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HIV Transmission in Healthcare Workers

Factors associated with HIV transmission in healthcare: Deep injury Device visibly contaminated with source patient’s blood Procedures involving a needle placed directly in a vein or artery Terminal illness in source patient No zidovudine (AZT) prophylaxis

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Hepatitis B Virus Inflammation of the liver – most

common bloodborne disease

Symptoms range from flu-like to none at all

No symptoms – person can still be infectious and can spread the disease

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Hepatitis B Facts Incubation period from 28-160 days Symptoms may include:

loss of appetite fatigue fever possible jaundice and dark urine

HBV is a much greater risk on the job than HIV, especially if you are not vaccinated.

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Fluids that pose risk of infection:

blood and blood products

body fluids containing visible blood

semen and vaginal secretions

breast milk

saliva (through a human bite)

Hepatitis B Facts

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How can Hepatitis B be transmitted in the workplace? Needlsticks or cuts with sharp, contaminated

objects

splashes to eyes/nose/mouth

contact with broken skin

human bites that break the skin

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Hepatitis B in Healthcare Workers (HCWs) in the U.S. Prior to vaccination, it was estimated that more than

12,000 HCWs were occupationally infected with HBV annually, resulting in 250 deaths. 1983 – Incidence of HBV among HCWs was 3 times higher

than in the general population. By 1995 it was 5 times lower.

Advent of HBV vaccine was a major advance in preserving

health and lives of HCWs. Source: Arch Intern Med 1997; 157:2601-2605

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Administered in 3 shots over 6 months. Engerix-B Vaccine is yeast derived - no chance of infection

from vaccination Provides long term protection against HBV for 96% of

healthy adults: no booster recommendation by CDC at this time. Post exposure vaccination is 70-88% effective when started

within one week.

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Hepatitis B Vaccination

Hepatitis B Vaccination Vaccination is given at 4K University Health Center (Detroit

Receiving Hospital), Monday – Friday 9:00 – 4:30.

Must present certificate of training to be vaccinated.

Must sign declination form if you choose to not be vaccinated.

Call 577-1200 for more information.

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Hepatitis C Virus Spread primarily through

blood/blood products. Less likely to be spread sexually.

If left untreated, 80-85% of cases become chronic

There is no vaccine for HCV.

Based on limited studies, risk for infection after needlestick is approximately 1.8%. 40

Hepatitis C Virus Identified in 1988, formerly called non-A

non-B hepatitis - called “silent epidemic” Blood supply not tested until early 90s.

Incubation period from 2-26 weeks. Most

people never have symptoms

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Incidence of HCV infection in U.S.

Est. number of chronic HCV cases in the U.S. is 2.7 – 3.9 million.

Persons newly infected with HCV are usually asymptomatic, so acute hepatitis C is rarely identified or reported.

Most prevalent among those born during 1945–1965, who were likely infected during the 1970s and 80s when rates were highest.

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Reported Acute (New) Cases of Hepatitis C Virus (HCV)

Estimated Actual New Cases of HCV (range) in 2011*

2005 2006 2007 2008 2009 2010 2011 2011 (estimated)* 694 802 849 878 781 853 1,229 16,500 (7,200- 43,400)

* Actual acute cases estimated to be 13.4 times the number of reported cases in any year

What is the incidence of HCV infection in the United States? Although only 802 cases of confirmed acute hepatitis

C were reported in the U.S. in 2006, CDC estimates that approx. 19,000 new HCV infections occurred that year, after adjusting for asymptomatic infection and underreporting.

Persons newly infected with HCV are usually asymptomatic, so acute hepatitis C is rarely identified or reported.

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Presenter

Presentation Notes

Prior to 2002, incidence rates for acute hepatitis C decreased for all age groups (excluding the 0–19 year age group); rates remained fairly constant from 2002 through 2010. In 2011, the overall rate of acute hepatitis C increased from 2010, with the largest increases among persons aged 0-19 years (from 0.05 to 0.10 cases per 100,000 population) and 20–29 years (from 0.75 to 1.18 cases per 100,000 population). When compared to all age groups, persons aged 20–29 years had the highest rate and persons ≥60 years of age (0.07 cases per 100,000 population) had the lowest rate.

Sources of Infection for Persons with Hepatitis C Virus in the U.S.

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Transfusions * 3%

Occupational 3%

No Identified Risks

10%

Household 3% Sexual 21%

Illegal Drug Use

60%

*None since 1994

Source: http://www.cdc.gov/hepatitis/statistics.htm

Generation of Aerosols

If aerosols may be generated, work must be performed in Class II biosafety cabinet. Procedures that may generate aerosols include: Pipetting Centrifugation Tissue culture Sonication Blending of tissues Animal innoculations

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Biological Safety Cabinets these cabinets provide:

Product protection Personal protection Environmental protection

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Laminar Flow “Clean Benches” This is not a Biosafety Cabinet –

it does NOT protect the worker!

Filtered air flows from back of

cabinet, across work surface, and towards user. Protects samples and work but

NOT the user

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Biological Safety Cabinets

Contain HEPA Filters (high efficiency particulate air). These trap particulates as small as 0.3u.

Does not protect from chemicals: fumes and vapors pass through

Chemicals and heat may damage filter.

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Biological Safety Cabinets May use for non-volatile toxic

chemicals or low-level radioactive materials.

May use for “minute” amounts of volatile chemicals.

Ensure annual certification from OEH&S.

Place BSC away from high traffic areas, airflow ducts and lab entrance doors.

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Biological Safety Cabinets Working Safely

Disinfect work surfaces with iodine based disinfectant (e.g. Wescodyne).

Load cabinet with all needed supplies.

Allow cabinet to run 10-15 minutes before beginning work.

Check inward airflow with a piece of tissue.

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Biological Safety Cabinets Working Safely

Always enter straight into cabinet – no sweeping motions.

Perform work in a slow, methodical manner.

Place materials well within the cabinet – don’t block grills.

Place discard pan within cabinet – discard pipettes inside in disinfectant tray

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When finished, decontaminate all items to be removed from cabinet.

Decon work surfaces again.

Allow cabinet to run 10-15 minutes before turning off.

Only use UV light as secondary disinfection.

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Biological Safety Cabinets Working Safely

Safe Centrifuge Use Check tubes for cracks/chips.

Use matched sets of tubes, buckets, etc. Tightly seal all tubes and safety cups. Ensure that rotor is locked to spindle and bucket seated. Close lid during operation.

Allow to come to complete stop before opening.

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Emergency Response Surface Decontamination

Alert co-workers

Define/isolate contaminated area

Put on appropriate PPE

Remove glass with forceps or scoop

Apply absorbent towel(s) to spill; remove bulk & reapply if needed

Apply disinfectant on top of towels.

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Emergency Response Surface Decontamination

If using bleach, mix FRESH 1:9 solution.

Allow adequate contact time (20 minutes).

Remove towel & mop up.

Clean again with soap/water or alcohol.

Properly dispose of materials in biohazard bins.

Wash your hands last.

For larger spills or questions, contact OEHS. 56

Minimum strength of cleaners must be tuberculocidal (kills M. tuberculosis var. bovis and all vegetative

bacteria, fungi, and most viruses) Require minimum 20 minute exposure

Examples: quaternary ammonium detergents,

phenolics, iodophores, chlorine compounds, alcohols

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Emergency Response Surface Decontamination – other acceptable disinfectants

Emergency Response responding to exposures

Occupational exposure is contact with blood or other potentially infectious materials:

needlestick or cut with sharp, contaminated object

contact with broken skin through cuts, rashes, other breaks in skin

splashes to eyes, nose, mouth

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Thoroughly wash affected area. Do not wash with bleach or other strong cleaners. If eyes/face exposed, flush in eyewash immediately.

Report incident to supervisor: Get medical attention!

4K University Health Center: includes assessment, blood tests, titer, counseling, follow-up visits…all confidential.

Complete a WSU Report of Injury form: Risk Management

577-3110. 59

Emergency Response responding to exposures

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Biological Waste Disposal

Request Waste Pick-Ups & Replacement Containers on-line at:

www.oehs.wayne.edu

SHARPS containers Red Bins

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Must be used for all SHARPS (contaminated or not)

Don’t overfill containers!

Locate containers conveniently.

Never recap needles: #1 cause of needlesticks!

Correct SHARPS Disposal Get free containers from OEH&S

Request SHARPS pick-up and replacement containers on OEH&S website: http://www.oehs.wayne.edu/forms/biowaste-pickup.php

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Hypodermic needles, with syringe

IV tubing with needles attached

Contaminated Pasteur pipettes

Razors and microtome blades

Scalpels

Lancets

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Correct SHARPS Disposal What goes into SHARPS container?

NEVER dispose of SHARPS in glass waste boxes or in the trash!

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Biological Waste (Red) Bins Free 28 gallon red plastic bins and a

liner bag are provided by OEH&S. Labs must purchase their own

autoclavable biohazard bags Autoclave materials before placing

into bins. Always place bags in bins or other

leak-proof secondary containers. DO NOT autoclave the red bin or the

liner bag! 65

Items contaminated with human or animal blood, body fluids or tissue. Cultures/stocks of infectious agents: including waste from

production of biologicals, discarded vaccines, and culture dishes. Materials/microorganisms used in recombinant DNA research.

NO SHARPS (needles, razor blades, etc!)

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Biological Waste (Red) Bins what goes into bins?

When bin is full:

close & tie liner bag shut

place lid on securely

do not overfill bins. They shouldn’t weigh more than 35 lbs.

request pick-up and replacement containers from OEH&S website: http://www.oehs.wayne.edu/forms/biowaste-pickup.php

Only properly prepared bins will be accepted!

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Waste glass & plastic disposal Use a sturdy, durable box.

Label the box “broken glass” or “glass waste”

Line the box with plastic bag.

Decontaminate items with bleach or autoclave before putting into box.

Keep weight reasonable – 25 lbs. or less!

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When box is full, tape shut securely.

Place box in an area for the custodial staff to dispose.

Custodians reserve the right to refuse pick up of boxes that aren’t prepared properly.

Waste glass & plastic disposal

Autoclave Decontamination Place items in secondary containers:

stainless steel or autoclavable plastic bins.

Temps of 121 degrees C for 20 minutes for most recombinant organisms and pathogens.

Larger loads require more time.

Autoclaves are usually in shared areas – clean up after yourself!

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Do not cap or plug vessels.

Do not add excessive amounts of liquid to load.

Usually steam remains in the chamber at end of a liquid cycle, so be careful!

Wear eye and face protection.

Stand behind door when opening it – keep face away from door.

Slowly open door only a crack to allow residual steam to escape.

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Autoclave Decontamination

Liquid Biological Waste Disposal Aqueous biological solutions such as blood, urine, cells, microbial

cultures must be autoclaved or chemically disinfected (treated with bleach)

1 part bleach to 9 parts contaminated liquid – let stand for 20 to 30 minutes.

After treating, dispose down drain with lots of H2O.

Do not autoclave bleach treated waste.

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Working with research animals Animals release airborne allergens which can cause

allergy symptoms in some people.

Mild symptoms include: itchy/watery eyes, runny nose, skin rash/itchy skin, nasal congestion, and cough.

Reduce your exposure by using PPE, including: Gloves Lab Coat N-95 Mask for your nose/mouth (requires a fit-test)

For more information on WSU’s Animal Contact program, call the OEH&S Occupational Health Nurse at 577-5917.

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Dangerous Goods Shipping Overview By law, anyone who packs, ships, transports or receives dangerous

goods must be properly trained. These slides do NOT count as your initial training!

Who’s the law? US Department of Transportation (DOT)

Training must be renewed every 2 years.

The shipper bares ultimate legal responsibility and liability for properly performing these tasks.

Penalties for non-compliance with shipping regulations can result in the following fines: • Up to $250,000 and up to a year jail sentence for individuals • Up to $500,000 per incident for organizations

There are nine classes of Dangerous Goods or Hazardous Materials

Class 1: Explosives Class 2: Gases Class 3: Flammable Liquids Class 4: Flammable Solids Class 5: Oxidizers and

Organic Peroxides

Class 6: Toxic (poisonous) and Infectious Substances 6.1 Toxic Substances 6.2 Infectious Substances

Class 7: Radioactive Class 8: Corrosive Class 9: Miscellaneous Dangerous

Goods (includes dry ice)

Class 6 and Class 9 are most commonly shipped by labs.

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Shipping Dangerous Goods • Common materials shipped include: human

and animal samples, bacteria, DNA, proteins, chemicals (solids or liquids), dry ice, etc.

• Shipping ANY material on dry ice by air is considered a Dangerous Good/Hazardous Material

• OEH&S is the designated authority for shipping Dangerous Goods from WSU.

• Contact OEH&S at 577-1200 to ship any Dangerous Goods.

Take the Quiz! To verify your completion of this course through WSU, you must return to the CITI site and take the quiz.

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If you are unable to register on CITI, you may download the quiz here. Questions? Call 577-1200 or e-mail: fracassa@wayne.edu