Laboratory Safety: Work Practices For

Laboratory Safety:Work Practices forMycobacterium tuberculosis1

Agent: Mycobacterium tuberculosis Infectious dose 1-10 organisms – No safelevel of exposure Airborne droplet nuclei can be spreadthrough normal air currents for longperiods of time and spread throughout aroom or building There is a risk to laboratorians whoprocess specimens in labs2

Overview Administrative ControlsEngineering ControlsPersonal Protective Equipment (PPE)Standard Operating Procedure (SOP)3

Laboratory Safety:Work Practices for Mycobacterium tuberculosisADMINISTRATIVE CONTROLS4

Safe Work Practices - Training How well are workers trained for the tasks? Do workers meet a level of competency beforebeing allowed to work? Training should include:– Use of safety equipment– Decontamination procedures– Spill clean-up– Use of autoclave– Waste disposal5

Safety Orientation and AnnualCompetency Includes Proper and Safe Handling PracticesUse of the biological safety cabinet (BSC)Biohazardous waste handlingUse of autoclaveDisease symptomsPost exposure managementReporting exposures and illnesses6

Link to Occupational Health andSafety Program Minimum requirements to conductwork in the laboratory:– General laboratory safety training– Familiarity with safety guidelines– Standard precautions– Training and experience7

Link to Occupational Health andSafety Program At a minimum, occupational healthprogram includes:– Offering of Interferon Gamma ReleaseAssay (IGRA) or Tuberculin Skin test(TST) who have risk of exposure to TB(two step TST on initial hire)– Ongoing evaluation of the respiratorprogram, based on expected work area8

Risk based on TB Incidence Frequency of M. tuberculosis positivespecimens encountered Concentration of organisms in specimens Number of specimens handled by anindividual worker Safety practices in the laboratory Safety equipment available in thelaboratory9

Chain of InfectionReservoir of pathogenXPortal of escapeXTransmissionXRoute of entry/infectious doseXSusceptible hostXIncubation periodXIllness10

Laboratory Biosafety PlanComponents How work is safely performedRisk AssessmentPost Exposure ManagementProtocol driven personal protective equipment(PPE) requirementsTraffic control / access restrictionUse of safety equipmentSanitation – Cleaning & DisinfectionWaste ManagementTrainingMedical Emergencies11

Risk Assessment – ExposurePrevention Employ a combination of methods:– Safe work practices– Use of containment equipment– Specially-designed laboratory facilities– What is the incidence of TB in your community?– What has been the annual incidence of TB in the last5 year?– Which tasks represent a risk to your laboratorians?12

Annual Risk Assessment Audit the program (Self audits, internal &external audits) Follow up on accidents and incidents Revise the program accordingly Monitor biosafety practices and performcompetency assessment13

Specimen Collection: All aerosol producingprocedures pose a risk of exposure14

Handling the Specimen in theLaboratory Aerosol producingprocedures pose a risk ofexposure Where can the work beperformed, biosafety level2 (BSL-2) or biosafetylevel 3 (BSL-3)? What PPE are necessary?15

Handling Clinical Specimens There are differences in literature regardingrecommended safety levels for certain mycobacteriologyprocedures. Specimen receipt and log-in can occur on the openbench. All aerosol-generating activities (any actions impartingenergy into a fluid specimen) must be conducted in abiological safety cabinet (BSC). For examples:– Surface disinfection of contaminated specimen container– Preparation of direct smear– Primary specimen digestion, decontamination,concentration– Concentrated smear preparation– Inoculation of culture media16

Risky Activities in Other LaboratorySections: Preparing frozen sections of biopsy specimens– Wear an N-95 respirator to mitigate the risk Cutting or sawing through tissue specimens thathave not been fixed– Wear an N-95 respirator or powered airpurifying respirator (PAPR) during theprocedure. Homogenizing tissues for primary culture– Use a BSC17

Laboratory Safety:Work Practices for Mycobacterium tuberculosisENGINEERING CONTROLS18

What if a BSL-3 is NOT available? BSL-2 with BSL-3 practices? A risk assessment is essential fordetermining if work with M. tuberculosiscan be conducted safely in a separate,closed BSL-2 laboratory using BSL-3practices and procedures19

Common Design of BSL-3 within aBSL-2 Laboratory BSL-2 laboratoryfacility with separateBSL-3 section Access through 2doors Arrows indicatenegative pressureair flowDoor 2Door 121

Common Design of BSL-3Laboratory BSL-3 laboratoryfacility Access through twodoor air-lock(anteroom) Pass-throughautoclaveAnteroom212

BSL-3 Laboratory Design Dedicated, single pass ventilation systemexhausts all room air to outside– 6-12 air changes per hour (ACH) removes99% of the airborne particulate matter in 2346 minutes. Time depends on ACH.– Create negative pressure, airflow should befrom “clean” to “less clean” areas– The laboratory should be kept under negativepressure at all times (alarm for failure of airhandling system)– BSL-3 facility operation should be re-verifiedat least annually22

BSL-3 Laboratory Design Interior surface of walls, floors, ceiling, andutility penetrations sealed Bench tops resistant to acids, alkalis,organic solvents, and moderate heat Foot-operated hand washing Automatic door closures Autoclave23

Safe Work Practices - Design Keep TB lab door closed andaccess limited while workingwith infectious agents Biosafety sign posted on thedoor All work must be performed ina BSC24

VentilationEquipmentFumeHoodsClass IBSCClass IIType A1BSCsBiologicalSafety CabinetsClass IIBSCClass IIType A2BSCsGloveBoxCleanBenchClass IIIBSCClass IIType B1BSCsClass TypeB2 BSCs25

Containment Equipment - BSC Perform all work within the BSC The BSC should be certified at least annually Train staff on appropriate use:––––BSC installed away from walking traffic and doorsNo storage of items in BSCWork 4-6” from front grillClean with 10% bleach followed by 70% alcoholRemember: Don’t block the front or back grill26

BSC Maintenance Daily cleaning just the start Read the manual! Be familiar withthe performance characteristics ofthe model in use Read you annual recertificationreport tand understand what wasmeasured.AnnualPreventiveMaintenanceby whom?Are theycertified?27

TB Specimen Processing:Decontamination and Concentration Risky activities that can generate droplets &droplet nuclei Vortexing Pouring liquid cultures and supernatantfluids Using fixed-volume automatic pipettors Mixing liquid cultures with a pipette Preparing specimen and culture smears Dropping tubes or flasks containing cultures Spilling suspensions of bacilli Breaking tubes during centrifugation Heat fixing smears28

Containment Equipment Centrifugation All culture tubes sealed tightly and placed incentrifuge safety cups inside the BSC After centrifugation, open safety cups in BSC29

Laboratory Safety:Work Practices for Mycobacterium tuberculosisPERSONAL PROTECTIVE EQUIPMENT(PPE)30

Personal Protective Equipment PPE Solid front disposablegown with snug (knit)cuffs. Gloves long enough tooverlap the sleeves of thegown. N-95 or N100 respirator(must be fit tested) Remove all outerprotective clothing whenleaving the BSL-3laboratory and place intobags for autoclaving.31

Sequence for Donning PPE32http://www.cdc.gov/HAI/prevent/ppe.html

Sequence for Removing PPE33http://www.cdc.gov/HAI/prevent/ppe.html

Respiratory Protection No BSC is 100% The National Institute for Occupational Safetyand Health (NIOSH) states that respiratorsprovide greater protection– Particulate filters are more efficient– Can be fit-tested– Can be fit-checked by the user to ensure a tight sealto the face Respiratory protection program requires: SOP,training, storage, inspection, medical review,program evaluation34

Disinfect the TB LaboratoryEnvironment TB is very resistant to drying and can survive forlong periods on solid surfaces Surfaces should be disinfected daily All work in the BSC should be performed over agauze pad or paper towel soaked in disinfectant Decant fluids into a splash-proof container withdisinfectant35

Descending Order of Resistance toDisinfectantsMost Resistant Bacterial Spores - Clostridium sp. and Bacillus sp. Mycobacteria - M. tuberculosis Nonlipid or small viruses - polioviruses, coxsackievirus, rhinovirus Fungi - Trichophyton sp., Cryptococcus sp., Candida Vegetative Bacteria - Pseudomonas, S. aureus,Salmonella Lipid or medium sized viruses - Herpes simplex,Cytomegalovirus, RSV, Hepatitis B, HIVMost Sensitive36

Select the Right Level of Disinfectant forthe TB Laboratory Sterilization: complete elimination of all formsof microbial life High-level disinfection: destroys allmicroorganisms except bacterial spores Intermediate-level disinfection: inactivatesM. tuberculosis, nonspore formingbacteria, most viruses and most fungi Low-level disinfection: kills most bacteria (notTB), some viruses, and some fungi– Hospital-type germicides used primarily for housekeepingsuch as quaternary ammonium compounds (“quats”)37

Intermediate-Level Disinfection Kills M. tuberculosis and all othervegetative bacteria, all fungi, and mostviruses– Tuberculocidal chemicals PhenolicsIodophorsChlorine compoundsAlcohols38

Laboratory Safety:Work Practices for Mycobacterium tuberculosisSTANDARD OPERATING PROCEDURE(SOP)39

Spill Clean Up ProceduresMINIMAL aerosols produced Cover the spill with paper towels Saturate with disinfectant Leave the laboratory until 99% of theairborne particles have beenremoved.– The time to reenter will depend on theair changes per hour (ACH) Wear PPE to clean up Autoclave material Disinfect floors and countertops40

Spill Clean Up ProceduresMAJOR aerosols produced Evacuate immediately Do not reenter until 99.9% of droplet nucleiare removed.– The time to reenter will depend on the airchanges per hour in the laboratory May decontaminate with formaldehyde gas orother agent Reenter using appropriate respiratorprotection and PPE Do not pick up broken glass with hands41

Post Exposure ManagementInfectious Agent Exposure –Laboratory Occupational Health Protocol Laboratory staff should be trained torecognize symptoms of TB disease Report exposure events and illnesses Respond to potential exposure events Respond to respiratory illness in laboratoryworkers post exposure Initiate diagnostic testing for exposed worker42

What if There is a IGRA or TSTConversion? If a staff member converts from IGRA orTST negative to positive, an evaluation isperformed: Evaluate the BSC, repair and recertify Evaluate procedures and techniques Retrain and re-educate as indicated43

Additional References: Miller, M.J., R. Astles, T. Baszler, K. Chapin, R. Carey, L. Garcia, L.Gray, D. Larone, M. Pentella, A. Pollock, D.S. Shapiro, E. Weirich,and D. Wiedbrauk. Guidelines for safe work practices in Human andAnimal medical diagnostic laboratories. MMWR, Supplement,January 6, 2012. http://www.cdc.gov/mmwr/pdf/other/su6101.pdf Delany,J., J. Rodriguez, D. Holmes, M. Pentella, K. Baxley, and K.Shah. CDC/APHL Laboratory Biosafety Competencies for the BSL2, BSL-3, and BSL-4 Laboratories. MMWR, Supplement, April 15,2011. http://www.cdc.gov/mmwr/pdf/other/su6002.pdf44

Arrows indicate negative pressure air flow 21 or 1 Door 2 . Common Design of BSL-3 Laboratory BSL-3 laboratory facility Access through two-door air-lock . organic solvents, and moderate heat Foot-operated hand washing Automatic d