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Transcript of Biological Agents And Factors Affecting Decontamination/media/Knowledge Center/Science...
Biological AgentsAnd
Factors Affecting Decontamination
Bruce R. Cords, Ph.D.Ecolab Inc.
B. anthracis Anthrax
Variola major Smallpox
Yersinia pestis Plague
Vibrio cholera Cholera
Filovirdia Ebola
Aphthovirus Foot & Mouth Disease
Botulinum Toxin Botulism
Biological Agents of Concern
MYCOBACTERIUM
PRIONS
BACTERIAL SPORES
PROTOZOA CYST/OOCYSTS(e.g. Crytosporidium)
NON-ENVELOPED VIRUSES
FUNGI
VEGETATIVE BACTERIA
LIPID-ENVELOPED VIRUSES
Resistance to Biocides
Relative Biocidal Activity
Peroxyacids, glutaraldehyde, formaldehyde, chlorine dioxide, ethylene oxide
Phenolics, Iodophors, Hypochlorites
Quaternary Ammonium Compounds, Organic Acids
Bacterial SporesMycobacteriumNon-enveloped Viruses
MycobacteriumNon-enveloped VirusesFungi
Vegetative BacteriaEnveloped Viruses
* There Are Exceptions!
Antimicrobial Tests(Required for EPA Registration)
Product Test Required Organisms
General disinfectant AOAC UseDilution
S. cholerasuis ATCC 10708S. aureus ATCC 6538
Hospital disinfectant AOAC UseDilution
S. cholerasuis ATCC 10708S. aureus ATCC 6538P. aeruginosa ATCC 1542
Sporicidal AOACSporicidal
B. subtilis ATOC 19659Cl. sporogenes ATCC 3584
Food Contact Surface SanitizerAOAC Germicidal Detergent Sanitizer Test
99 ml SanitizerUse-Solution25ºC
Add 1 ml of E.colior S.aureus
(minimum of 7.5 x 107
CFU/ml)
30 secondContactTime
Neutralize 1 mlEnumerateSurvivorsRequired
Efficacy:99.999% Kill in 30 seconds at 25ºC
0
15
30
Suggested Surrogates
Agent Surrogate Variola major (Smallpox) Vaccinia virus Yersinia pestis (Plague) Yersinia pseudotuberculosis Bacillus anthracis (Anthrax) B. subtilis B. cereus
B. globigii Foot-and-Mouth Disease Virus ? Norwalk Virus Feline calicivirus
Regulatory Hurdle
− EPA does not allow real or implied claims for any infectious agent that is not stated on the product label.
− Very few commercial products have been tested against the candidate biological agents.
Legal Issues
1) It is a violation of Federal law to use an EPA registered product in a manner inconsistent with its labeling.– Solution strength must be according to label– Applications must be on the label
2) It is a violation of Federal law for a manufacturer to make real or implied claims for efficacy against organisms which are not on the label.– Creates problems when we encounter:
• FMDV• Anthrax• Norwalk• SARS• Avian Influenza H5N1
Anthrax/ Bacillus anthracis− No products carry label claims (some have
crisis exemption)− Cutaneous, pulmonary, and
gastrointestinal transmitted by aerosols and on environmental surfaces
− Infectious dose - 5-10,000 spores (inhaled)− Effective agents (published literature)
– .25%-.50% (2500-5000 ppm) Peroxyacetic Acid– 2% (20,000 ppm) Glutaraldehyde– 4% (40,000 ppm) Formaldehyde– 1% (10,000 ppm) Sodium Hypochlorite
Smallpox/ Variola Major Complex Coat
− No products carry label claims− Transmitted through aerosols, contact
with infected person, and environmental surfaces
− Low infectious dose - a few virons− Effective agents (published literature)
– 1% (10,000 ppm) Sodium Hypochlorite– 2% (20,000 ppm) Glutaraldehyde– 2% (20,000 ppm) Formaldehyde
Levels of Decontamination Somewhat Dependent on Infectious Dose
Smallpox A few virons caninduce disease.
Anthrax Infectious dose(inhaled) may be10,000 spores
– Smallpox decontamination to 100 virons/m2 not acceptable
– Anthrax decontamination to 100 spores/m2 probably a safe level
Plague/ Yersinia pestis
− No products carry label claims− Transmitted as an aerosol through
respiratory droplets or plague-infected fleas. Organism does not survive more than a few hours outside the host.
− Infectious dose - 50-1500− Effective agents (published literature)
– 1% (10,000 ppm) Sodium Hypochlorite– 70% Ethanol– 2% (20,000 ppm) Glutaraldehyde– Many general disinfectants
Cholera/ Vibrio cholera
− No products carry label claims− Transmitted by contact and in water and
food contaminated with excreta from infection individuals
− Infectious dose 106
− Effective agents– General disinfectants
Ebola/ Filovirida Enveloped RNA Virus
− No products carry label claims− Transmitted by contact with infected
individuals or their blood/secretions. Environmental?
− Low infectious dose− Effective agents (published literature)
– 2% (20,000 ppm) Sodium Hypochlorite– 2% (20,000 ppm) Glutaraldehyde– .5% (5,000 ppm) Peracetic Acid– 1% (10,000 ppm) Formaldehyde
Foot & Mouth Disease/ Apthovirus
Non Enveloped RNA Virus− Some products carry Label Claims− Transmitted by aerosols, environmental
surfaces and direct contact between animals− Low infectious dose− Effective agents (published literature and
product approvals)– .03% (300 ppm) Peracetic Acid– .1% (10,000 ppm) Peroxymonosulphate– .05% (500 ppm) Sodium Hypochlorite– Other products (Ministry of Agriculture Fisheries
and Food; U.K.)
Is SARS a Potential Bioterrorism Agent?
1. Available? Yes2. Culturable? Yes3. Infective? Highly4. Deliverable? Unknown5. Decontamination? Likely to be
susceptible to common disinfectants
Lessons Learned from the Anthrax Case
1. We were not prepared for decontamination (e.g. Hart Building).
2) It took three treatments with ClO2 to achieve adequate results.
3) Many items were destroyed as opposed to decontaminated.
4) Ambulance chasers with “cure-alls” were abundant (e.g. spores vs. vegetative).
Bacillus anthracisSpores vs. Vegetative Cells
Peracetic Acid 5-logreduction
Spores 2,500 ppm for 30 min.
Vegatative 150 ppm for 30 sec.
Factors Contributing to Failure of Decontamination
Disinfectant/Biocide:
− Selection of biocide not effective against infectious agent
− Biocide too dilute− Insufficient contact time− Temperature too low*− Relative humidity too low
− (gaseous disinfectants)
Factors Contributing To Failure of Decontamination
Environmental Factors:
− Presence of organic matter*− Inactivation of QAC’s by residual soaps
and detergents− Incorrect application/coverage− Inadequate treatment of water supply− “Wettability” of surface
All Sanitizers Are NOT Created EqualReduced Temperature Sanitizer Efficacy
70oF. Well Water (250 ppm)30 Seconds
0123456
0.20% 0.35% 0.13% 0.26% AcidAnionic
Quat Iodophor Chlorine
S. aureus E. coli
Log
Reduction Peracetic Acid Organic Acid
All Sanitizers Are NOT Created EqualReduced Temperature Sanitizer Efficacy
55oF. Well Water (250 ppm)30 Seconds
0123456
0.20% 0.35% 0.13% 0.26% AcidAnionic
Quat Iodophor Chlorine
S. aureus E. coli
Log
Reduction Peracetic Acid Organic Acid
All Sanitizers Are NOT Created EqualReduced Temperature Sanitizer Efficacy
40 oF. Well Water (250 ppm)30 Seconds
0123456
0.20% 0.35% 0.13% 0.26% AcidAnionic
Quat Iodophor Chlorine
S. aureus E. coli
Log
Reduction
Peracetic Acid Organic Acid
0123456
0.20% 0.35% 0.13% 0.26% AcidAnionic
Quat Iodophor Chlorine
S. aureus E. coli
All Sanitizers Are NOT Created EqualReduced Temperature Sanitizer Efficacy
40 oF. Well Water (250 ppm)2 Minutes
Log
Reduction
Peracetic Acid Organic Acid
Effect of Temperature Organic Load and Concentration on Disinfectant
ActivityQuaternary Disinfectant:
(Log Reduction for P. aeruginosa)
20°C 10°Cx0.5 x1.0 x2.0 x0.5 x1.0 x2.0
.03% F P P F F PBSA
.3% F P P F F FBSA
P (Pass) = > 5 log reductionF (Fail) = < 5 log reduction
QUAT1000 ppm5 minutes
Taylor et al 1999
Effect of Temperature Organic Load and Concentration on Disinfectant
ActivityPeracetic Acid Disinfectant:
(Log Reduction for P. aeruginosa)
20°C 10°Cx0.5 x1.0 x2.0 x0.5% x1.0 x2.0
.03% P P P P P PBSA
.3% F P P F P PBSA
P (Pass) = > 5 log reductionF (Fail) = < 5 log reduction
Peracetic Acid300 ppm5 minutes
Taylor et al 1999
Treatment of a 48 HourBiofilm of Pseudomonas aeruginosa*
0
10
20
30
40
50
60
70
80 ppm 160 ppm 320 ppm
Peracetic/Organic Acid Peracetic Acid Chlorine
1 Minute Contact Time
*Fatemi and Frank. 1999. Journal of Food Protection. Vol. 62 (761-765)
Effect of Relative Humidity on Sporicidal Activity (B. globigii) of
Formaldehyde
LogConcentration RH Temp Reduction Time
400 mg/m3 30% 25°C 1 22’
400 mg/m3 98% 25°C 1 9’
Ref: Emerging Infectious Disease Vol 9 No 6 p. 625
Effect of Relative Humidity on Sporicidal Activity (B. subtilis v niger) of Peracetic Acid Vapor
LogConcentration RH Temp Reduction Time
1mg/L 80% RT >5 log 10’1mg/L 60% RT >4 log 10’1mg/L 40% RT >3 log 10’
Ref: Emerging Infectious Disease Vol 9 No 6 p. 626
Inactivation Of Bacillus spp. By Boiling In Tap Water
Initial log After 5’CFU/ml Boiling
C UNC C UNC
B. anthracis 4.95 4.92 0 2.01(sterne)
B. cereus 4.62 4.59 0 1.46(commercial)
B. cereus 4.54 4.76 0 0.48ATCC 9592
B. thuringensis 4.63 4.46 0 1.47ATCC 35646
C = Covered UNC = Uncovered
Temp above surface: C = 98.9°C UNC = 77.3°C
Ref: Emerging Infectious Disease Vol 10 No 10 2004
National Center for Food Protection and Defense
Study No: 910F2376745 – Ecolab
Development of Time/Temperature Concentration Matrix for Inactivation of Infectious Bioterrorism Agents by Chemical Biocides
Student: Hilgren – Ecolab
Co-Advisors: Swanson – EcolabDiez – University of Minnesota
General Method
Bacillus Spores
Food Soil• Egg Yolk Emulsion 8.8% protein
• Whole Milk 3.5% fat
• Flour Slurry 7.6% starch
Spore/soil mixture dried on stainless steel carrier
Carrier immersed in candidate biocide for 10 minutes
Impact of Food Soil on Inactivation of B. cereus spores by Peroxyacetic
Acid (Preliminary Results)
Conditions = 10 minute exposure20°CB. cereus ATCC 10987
Log Reduction5,000 ppm 10,000 ppm
No Soil >7.0 >7.0Whole Milk 1.5 4.0Egg Yolk 2.0 4.0Flour 2.0 7.0
Ref: Ecolab, NCFPD Project
Impact of Food Soil and Temperature on Inactivation of B. cereus Spores by
Peroxyacetic Acid (Preliminary Results)
Conditions = 10 minute exposure10,000 ppm Peroxyacetic acidB. cereus ATCC #10987
Log Reduction10°C 20°C 30°C
No Soil 6.0 >7.0 >7.0Whole Milk 1.5 4.0 >7.0Egg Yolk 2.0 4.0 5.5Flour 1.5 7.0 7.0
Ref: Ecolab, NCFPD Project
Impact of Food Soil on Inactivation of B. cereus spores by Sodium
Hypochlorite (Preliminary Results)
Conditions = 10 minute exposure20°CB. cereus ATCC 10987
Log Reduction @ 50,000 ppmNo Soil >7.0Whole Milk 1.2Egg Yolk 1.9Flour >7.0
Ref: Ecolab, NCFPD Project
Note: Flour @ 1,000 ppm - >7.0 log reduction
Other Results to Date:
− Quarternary Ammonium Chloride− Not effective at 75,000 ppm (7.5%), 20°C− Normal disinfection level = 200-800 ppm
− Iodophor− Not effective at 19,000 ppm (1.9%), 20°C− Normal disinfection level = 25 ppm
− Acidified Sodium Chlorite− Somewhat effective− Egg yolk and milk results not as good as flour
− Hydrogen Peroxide− Effective at 25-30%; 20°C− Not effective at 10°C
− Mixed Peroxyacid System− Results similar to peroxyacetic acid
Future Testing to Include:
− B. anthracis (nonvirulent strain)− Yersinia Pestis