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Validating Autoclave Cycles for Carcass Disposal in ABSL-2/3 Containment Laboratories

Rebecca McGirr1, James Burch2, Leslee Arwood2, Christopher Sample2, Scott Alderman2

1. Sanofi Pasteur Limited. 2. Duke Human Vaccine Institute/Duke Regional Biocontainment Laboratory

Disclaimer• This worked was sponsored by and conductedThis worked was sponsored by and conducted

at the DHVI/Duke-RBL

• Sanofi Pasteur Limited was not involved in this work

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Background• Infectious waste, including carcasses, needs to beInfectious waste, including carcasses, needs to be

sterilized before leaving the ABSL-3 facility for downstream disposal

• With start of ferret work, autoclave cycles need to be validated to ensure sterilization of potentially p yinfectious carcasses

Background: Initial Results• Standard cycle parameters

Cycle Parameters Used:Standard cycle parameters

used for solid biohazardous waste not sufficient for sterilizing intact ferret carcasses

• Biological indicators (BIs)

Used:Time: 90 minutesTemperature: 123.1oCPrevac: 15 PSI# of Prevacs: 3

placed in and around carcasses were not inactivated

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Objectives of Study• To develop and validate autoclave cycle to

f /ensure sterilization of small/medium-sized carcasses

• To develop a method for implanting BIs into intact carcasses

• To determine maximum carcass load per• To determine maximum carcass load per biohazard bag and number of bags per pan

• To determine total maximum load size/cycle

Experimental Design: Water Test• Based on previous results:p

• Chose a 4-hour cycle• 123oC• 3 prevacs at 20 PSI

• Stainless steel pans used:p• Durable, inexpensive, easy to clean• Filled with water• Placed biological indicators (BIs)

inside pans

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Experimental Design: Water Test

id

Fill to 2”

FrontFront Top

Lid

Lid

Back Top

Lid

Lid

Cycle Parameters:Time: 239.30 minutesTemperature: 123.1oC

Fill to 2” from top D

oorFront Bottom

Lid

Back Bottom

Lidp

Prevac: 20 PSI# of Prevacs: 3

Results of Water Test

• All BIs passed (no growth)

• Next step: test cycle on p yferret carcasses

Control vial

Exp. vial

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Experimental Design: Ferret Test

G stearothermophilusAmpoule placed in carcass

G. stearothermophilusampoule tied to label w/wire

via small incisions; wire affixed to spine

Ferret ID

Each pan contains 4

Experimental Design: Ferret Test

Each pan contains 4 ferret carcasses total (each weighing ~ 2kg)

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Experimental Design: Ferret Test

FLid Lid F

ront

D

Back Top

2/bag 2/bag

Front Top

4/bag

Front BottomBack Bottom

Door

1/bag 1/bag 1/bag 1/bag4/bag

Experimental Design: Ferret Test

Cycle Parameters:Time: 239.30 minutesTemperature: 123.1oCPrevac: 20 PSIPrevac: 20 PSI# of Prevacs: 3

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Results• All but one validation passed

• Vial placed in between carcasses failed (top, front pan)

Control vial

Failed

Results

FFront

Door

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Experimental Design: Modification• Increase temperatureIncrease temperature

• Increase number of prevacs and lower PSI

• Used fresh or frozen carcasses

Cycle Parameters:Time: 239.30 minutesTemperature: 125oC (123.1oC)

Fr

Lid

Temperature: 125 C (123.1 C)Prevac: 15 PSI (20 PSI)# of Prevacs: 5 (3)

ont

Do

4/b

Top

4/bag

Bottom

or

4/bag

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Results• All validations passed with either fresh or frozen

carcasses

Control vial‘In-between’

vials

Conclusion• Autoclave cycle time, temperature and numberAutoclave cycle time, temperature and number

of pre-vacuum pulses is critical to ensuring sterilization of carcasses

• Sterilization was validated with up to 4 ferret carcasses/pan, 4 pans per cycle (16 total)p , p p y ( )

• Multiple carcasses per load can be sterilized under validated autoclave conditions

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Future Directions• Repeat experiment to confirm resultsRepeat experiment to confirm results

• Increase number of pans per autoclave to 6 (24 carcasses total)

• Test with other types of carcasses (e.g., rabbits)

Acknowledgements

DHVI Safety Team Duke RBL-Virology TeamDHVI Safety Team

Scott AldermanJim Burch

Duke RBL Virology Team

Chris SampleLeslee ArwoodAriel MasonRebecca Sahm

Duke RBL Team:Rebecca SahmCharles McGeeElizabeth Fallon

Doug ElliottThank You!