Use of Vaporized Hydrogen Peroxide for Decontamination American Biological Safety Association October 9, 2007 Barbara Johnson, PhD, RBP Claire Fritz

Agenda • Introduction to Vaporized Hydrogen Peroxide • Review of Microbiological Decontamination Data – Animal and Public Health Organisms – Bioterrorism Agents

• Conducting Safe and Effective Decontamination • Conclusions

Hydrogen Peroxide Vapor

Hydrogen Peroxide 35%

Vaporization

Room Temperature Sterilization Process 2H

2H2O2

2O

+ O2

Sporicidal at Low Concentrations (Typically 70 – 1000 ppm)

Non-Toxic Residues

Why H2O2 Vapor? Hydrogen Peroxide

Chlorine Dioxide

Formaldehyde

Skin/eye irritant

Severe irritant

Human Carcinogen

PEL 1.0 ppm

PEL 0.1 ppm STEL 0.3 ppm IDLH 5.0 ppm

PEL 0.75 ppm STEL 2 ppm IDLH 10 ppm

IDLH 75 ppm

__________________________________________________________ PEL - Personal Exposure Limit (8 hours) STEL - Short-term Exposure Limit (15 min.) IDLH – Immediately Dangerous to Life or Health

Typical H2O2 Vapor Cycle

__________________________________________________________

Titer of virus in Liquid Suspension 9 8

mean Log10/ml

7 6 5

Out of Box In Box VHP + Box

4 3 2 1 0 AIV

ASFV

BTV

__________________________________________________________ AIV – Avian Influenza Virus BTV- Blue Tongue Virus ASFV – African Swine Fever Virus

Titer of virus in Liquid Suspension 9 8

mean Log10/ml

7 6 5

Out of Box In Box VHP + Box

4 3 2 1 0 HCV-CC

HCV-WB

NDV

PRV

__________________________________________________________ HCV-CC Hog Cholera Virus – Cell Culture HCV-WB Hog Cholera Virus – Whole Blood

NDV- Newcastle Disease Virus PRV- Pseudorabies Virus

Titer of virus in Liquid Suspension 9 8 7

mean Log10/ml

6 5 4 3

Out of Box In Box VHP + Box

2 1 0

SVDV VEV VSV-CC VSV-AF __________________________________________________________

SVDV – Swine Vesicular Disease Virus VEV – Vesicular Exanthema Virus VSV-CC Vesicular Stomatitis Virus in Cell Culture VSV-AF Vesicular Stomatitis Virus in Allantoic Fluid

Titer of virus on Slides 9 8

mean Log10/ml

7 6 5

control 2 min

4 3 2 1 0 Herpes I

Rhinovirus

Adenovirus

Titer of virus on Slides 9 8

mean Log10/ml

7 6 control 2 min 3 min 5 min

5 4 3 2 1 0 Vaccinia

Influenza A2

Polio I

Average M. tuberculosis CFU/10ul After 60 Minutes VHP Exposure Genotype Strains Control No-VHP Front BSC (corner) Mid Room near floor

H37Rv H37Rv MaxPlanck NY 6.0x106 2.27x106

Beijing/W

0.0

0.0

0.0

0.0

0.0

0.0

8.0x104

Top of 0.0 0.0 0.0 BSC __________________________________________________________ BSC – Biosafety Cabinet

Inactivation of B. anthracis Spores 9

9 x 107

8

Log CFU/Slide

7

3.4 x 106

6

4 x 104

5

Control VHP Treated

4 3 2

1.1 x 101

1

0.0

0.0

0 PBS Rep 1

PBS Rep 2

H20

__________________________________________________________ PBS – Spores Dried in Phosphate Buffer Saline H20 – Spores dried in Water

Log10 CFU/Slide

Inactivation of Vegetative Y. Pestis 9 8 7 6 5 4 3 2 1 0

6 X 107

1.7 X 103

Control VHP Treated 0.0

Non-Air Dried Control

Air Dried Control 10% FBS

Air Dry VHP Treated 10% FBS

__________________________________________________________ FBS – Fetal Bovine Serum

Inactivation of C. burnetii (Renografin Purified) Optical Density (405 mm)

1.5

1

0.5

0 -2 VHP -4 VHP -5 -7 -9 -10 -11 Treated Treated Control Control Control Control Control Pre-test Serum (1:80)

30 Days post-test serum (1:80)

Inactivation of Botulism Toxin 500 > LD50 400

BOT (µg/kg)

400 300 Controls VHP Treated

200 100 LD50 .04

LD50 8

0 Non-Air Dried Control

Air Dried Control

VHP Treated

Inactivation of SEB 10

9.3

SEB (µg/slide)

8.0

8 6 4

3.4

Standard Solution Control VHP Treated

2 0 Standard Solution

Control

VHP Treated

__________________________________________________________ SEB – Staphlococcal Enterotoxin B

Room Decontamination A step-by-step process should be followed:

• • • • • •

Room Preparation and Sealing Room Preconditioning (Humidity Control) Fumigation Dwell or “Hold Time” Aeration Biological Indicator Removal and Incubation

Room Preparation • Remove and/or reduce clutter – Spores are Harder to Kill on Paper and Absorbent Materials • Clean Surfaces, especially where spills occur • Shut off Air Supply; Shut off or decrease exhaust • Recirculate Biological Safety Cabinets • Place Biological Indicators and Chemical Indicators • Place Fans for Turbulence • Seal Doors with Tape • Place Warning Signs

Room Preparation Chemical and Biological Indicators: • Geobacillus stearothermophilus • 106 spores per sample are typical for Bio-Safety Applications • A minimum of 10-12 samples are used for most small applications. Large applications use 1 sample per 100 ft2 • Chemical Indicators are also available

Room Preparation Ordinary fans are used for gas distribution !!

Room Preconditioning • Humidity makes it easier to kill spores for all gas fumigation methods • Formaldehyde and Chlorine Dioxide protocols usually require humidification to between 60 and 75% RH before the decontamination process • High humidity limits the quantity of Hydrogen Peroxide that can be vaporized and maintained in the gaseous state – Vaporized Hydrogen Peroxide decontamination is usually started with a initial humidity of less than 60%

Fumigation Ports installed through the wall

Temporary door frame adapter

Aeration • Aeration can be achieved using Hydrogen Peroxide Generator • Most use their Exhaust System to Aerate • Rooms should be ≤ 1.0 ppm before occupying • Dräger pump or handheld sensor

Dräger PAC III Meter

Dräger pump

Conclusions • Hydrogen Peroxide Vapor has been shown to be Highly Effective Against Many Organisms and can Denature Toxins and Other Chemicals • Successful Decontamination Depends on Proper Room Preparation and Control • Remember Safety Comes First

References AMSCO Internal Document, 1989, Determination of the Virucidal Activity of Hydrogen Peroxide Gas. Heckert, R.A., M. Best, L. T. Jordan, G. C. Dulac, D. L. Eddington And W. G. Sterritt, 1997, Efficacy of Vaporized Hydrogen Peroxide against Exotic Animal Viruses, Applied and Environmental Microbiology, Vol. 63, No. 10 , p. 3916–3918

References Johnson, B., B. Harper, A.J. Mohr, D. Winters and I.G. Resnick; 1993, Potential Use of Vaporized Hydrogen Peroxide Treatment in the Inactivation of B. anthracis, Y. pestis, C. burnetii, Botulinum Toxin and Staphylococcal Enterotoxin B. American Biological Safety Association Conference, 11 Oct 1993. Kahnert A., P Seiler, M. Stein, B Aze, G. McDonnell, S.H.E. Kaufmann, 2005, Decontamination with vaporized hydrogen peroxide is effective against Mycobacterium tuberculosis, Letters in Applied Microbiology, 2005, doi:10.1111/j. 1472-765X.2005