Download Biological Agents and Factors Affecting Decontamination

Biological Agents
And
Factors Affecting Decontam ination
Bruce R. Cords, Ph.D.
Ecolab Inc.
Biological Agents of Concern
B. anthracis
Anthrax
Variola major
Smallpox
Yersinia pestis
Plague
Vibrio cholera
Cholera
Filovirdia
Ebola
Aphthovirus
Foot & Mouth Disease
Botulinum Toxin
Botulism
R esistance to Biocides
PRIONS
BACTERIAL SPORES
PROTOZOA CYST/OOCYSTS
(e.g. Crytosporidium)
MYCOBACTERIUM
NON-ENVELOPED VIRUSES
FUNGI
VEGETATIVE BACTERIA
LIPID-ENVELOPED VIRUSES
R elative Biocidal Activity
Bacterial Spores
Mycobacterium
Non-enveloped Viruses
Mycobacterium
Non-enveloped Viruses
Peroxyacids, glutaraldehyde,
formaldehyde, chlorine dioxide,
ethylene oxide
Phenolics, Iodophors,
Hypochlorites
Fungi
Vegetative Bacteria
Enveloped Viruses
* There Are Exceptions!
Quaternary Ammonium
Compounds, Organic Acids
Antim icrobial Tests
(Required for EPA Registration)
Product
Test
Required Organisms
General disinfectant AOAC Use
Dilution
S. cholerasuis ATCC 10708
S. aureus ATCC 6538
Hospital disinfectant AOAC Use
Dilution
S. cholerasuis ATCC 10708
S. aureus ATCC 6538
P. aeruginosa ATCC 1542
Sporicidal
B. subtilis ATOC 19659
Cl. sporogenes ATCC 3584
AOAC
Sporicidal
Food Contact Surface Sanitizer
AOAC Germicidal Detergent Sanitizer Test
99 ml Sanitizer
Use-Solution
25ºC
Add 1 ml of E.coli
or S.aureus
(minimum of 7.5 x 107
CFU/ml)
30 second
Contact
Time
0
15
30
Required
Efficacy:
99.999% Kill in
30 seconds at
25ºC
Enumerate
Survivors
Neutralize
1 ml
Suggested Surrogates
Agent
Surrogate
Variola major (Smallpox)
Vaccinia virus
Yersinia pestis (Plague)
Yersinia pseudotuberculosis
Bacillus anthracis (Anthrax)
B. subtilis
B. globigii
Foot-and-Mouth Disease Virus
Norwalk Virus
B. cereus
?
Feline calicivirus
R egulatory 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 I ssues
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
Sm allpox / Variola M ajor
Com plex 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 Decontam ination Som ew hat
Dependent on I nfectious Dose
Smallpox
A few virons can
induce disease.
Anthrax
Infectious dose
(inhaled) may be
10,000 spores
– Smallpox decontamination to 100
virons/m2 not acceptable
– Anthrax decontamination to 100
spores/m2 probably a safe level
P lague/ 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 R NA 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 & M outh
Disease/ Apthovirus
Non Enveloped R NA 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.)
I s SAR S a P otential Bioterrorism
Agent?
1.
2.
3.
4.
5.
Available? Yes
Culturable? Yes
Infective? Highly
Deliverable? Unknown
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 anthracis
Spores vs. Vegetative Cells
Peracetic Acid 5-log
reduction
Spores
2,500 ppm for 30 min.
Vegatative
150 ppm for 30 sec.
Factors Contributing to Failure
of Decontam ination
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 Decontam ination
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 Equal
Reduced Temperature Sanitizer Efficacy
70oF. Well Water (250 ppm)
30 Seconds
L
o
g
6
5
R
e
d
u
c
t
i
o
n
4
3
2
1
0
0.20%
0.35%
0.13%
0.26%
Peracetic Acid
Organic Acid
Acid
Anionic
S. aureus
Quat
E. coli
Iodophor Chlorine
All Sanitizers Are NOT Created Equal
Reduced Temperature Sanitizer Efficacy
55oF. Well Water (250 ppm)
30 Seconds
L
o
g
6
5
R
e
d
u
c
t
i
o
n
4
3
2
1
0
0.20%
0.35%
0.13%
0.26%
Peracetic Acid
Organic Acid
Acid
Anionic
S. aureus
Quat
E. coli
Iodophor Chlorine
All Sanitizers Are NOT Created Equal
Reduced Temperature Sanitizer Efficacy
40 oF. Well Water (250 ppm)
30 Seconds
L
o
g
6
R
e
d
u
c
t
i
o
n
5
4
3
2
1
0
0.20%
0.35%
Peracetic Acid
0.13%
0.26%
Organic Acid
Acid
Anionic
S. aureus
Quat
E. coli
Iodophor Chlorine
All Sanitizers Are NOT Created Equal
Reduced Temperature Sanitizer Efficacy
40 oF. Well Water (250 ppm)
2 Minutes
L
o
g
6
R
e
d
u
c
t
i
o
n
5
4
3
2
1
0
0.20%
0.35%
Peracetic Acid
0.13%
0.26%
Organic Acid
Acid
Anionic
S. aureus
Quat
E. coli
Iodophor Chlorine
Effect of Tem perature Organic Load
and Concentration on Disinfectant
Activity
Quaternary Disinfectant:
(Log Reduction for P. aeruginosa)
.03%
BSA
x0.5
F
20°C
x1.0
P
x2.0
P
F
P
P
QUAT
1000 ppm
5 minutes .3%
BSA
Taylor et al 1999
x0.5
F
10°C
x1.0
F
x2.0
P
F
F
F
P (Pass) = > 5 log reduction
F (Fail) = < 5 log reduction
Effect of Tem perature Organic Load
and Concentration on Disinfectant
Activity
Peracetic Acid Disinfectant:
(Log Reduction for P. aeruginosa)
Peracetic
Acid
300 ppm
5 minutes
.03%
BSA
.3%
BSA
Taylor et al 1999
x0.5
P
20°C
x1.0
P
x2.0
P
x0.5%
P
F
P
P
F
10°C
x1.0 x2.0
P
P
P
P
P (Pass) = > 5 log reduction
F (Fail) = < 5 log reduction
Treatm ent of a 48 Hour
Biofilm of P seudom onas aeruginosa*
70
60
1 Minute Contact Time
50
40
30
20
10
0
80 ppm
160 ppm
Peracetic/Organic Acid
320 ppm
Peracetic Acid
Chlorine
*Fatemi and Frank. 1999. Journal of Food Protection. Vol. 62 (761-765)
Effect of R elative Hum idity on
Sporicidal Activity (B. globigii) of
Form aldehyde
Log
Reduction
Concentration
RH
Temp
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
Time
Effect of R elative Hum idity on
Sporicidal Activity (B. subtilis v
niger) of Peracetic Acid Vapor
Concentration
1mg/L
1mg/L
1mg/L
RH
Temp
80%
60%
40%
RT
RT
RT
Ref: Emerging Infectious Disease Vol 9 No 6 p. 626
Log
Reduction
>5 log
>4 log
>3 log
Time
10’
10’
10’
I nactivation Of Bacillus spp. By Boiling I n
Tap W ater
Initial log
CFU/ml
C
UNC
After 5’
Boiling
C
UNC
B. anthracis
(sterne)
4.95
4.92
0
2.01
B. cereus
(commercial)
4.62
4.59
0
1.46
B. cereus
ATCC 9592
4.54
4.76
0
0.48
B. thuringensis
ATCC 35646
4.63
4.46
0
1.47
C = Covered
Temp above surface:
C = 98.9°C
Ref: Emerging Infectious Disease Vol 10 No 10 2004
UNC = Uncovered
UNC = 77.3°C
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 – Ecolab
Diez – University of Minnesota
General M ethod
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
I m pact of Food Soil on I nactivation
of B. cereus spores by Perox yacetic
Acid (Prelim inary R esults)
Conditions = 10 minute exposure
20°C
B. cereus ATCC 10987
No Soil
Whole Milk
Egg Yolk
Flour
Ref: Ecolab, NCFPD Project
Log Reduction
5,000 ppm
10,000 ppm
>7.0
>7.0
1.5
4.0
2.0
4.0
2.0
7.0
I m pact of Food Soil and Tem perature on
I nactivation of B. cereus Spores by
Perox yacetic Acid (Prelim inary Results)
Conditions =
No Soil
Whole Milk
Egg Yolk
Flour
Ref: Ecolab, NCFPD Project
10 minute exposure
10,000 ppm Peroxyacetic acid
B. cereus ATCC #10987
10°C
6.0
1.5
2.0
1.5
Log Reduction
20°C
30°C
>7.0
>7.0
4.0
>7.0
4.0
5.5
7.0
7.0
I m pact of Food Soil on I nactivation
of B. cereus spores by Sodium
Hypochlorite (Prelim inary R esults)
Conditions = 10 minute exposure
20°C
B. cereus ATCC 10987
No Soil
Whole Milk
Egg Yolk
Flour
Log Reduction @ 50,000 ppm
>7.0
1.2
1.9
>7.0
Note: Flour @ 1,000 ppm - >7.0 log reduction
Ref: Ecolab, NCFPD Project
Other R esults 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 I nclude:
− B. anthracis (nonvirulent strain)
− Yersinia Pestis