Biological Agents And Factors Affecting Decontamination

1
Biological Agents And Factors Affecting
Decontamination

• Bruce R. Cords, Ph.D.
• Ecolab Inc.

2
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

3
Resistance to Biocides
PRIONS
BACTERIAL SPORES
PROTOZOA CYST/OOCYSTS (e.g. Crytosporidium)
MYCOBACTERIUM
NON-ENVELOPED VIRUSES
FUNGI
VEGETATIVE BACTERIA
LIPID-ENVELOPED VIRUSES
4
Relative Biocidal Activity
Peroxyacids, glutaraldehyde, formaldehyde,
chlorine dioxide, ethylene oxide
Phenolics, Iodophors, Hypochlorites
Quaternary Ammonium Compounds, Organic Acids
There Are Exceptions!
5
Antimicrobial Tests(Required for EPA
Registration)
6
Food Contact Surface SanitizerAOAC Germicidal
Detergent Sanitizer Test
99 ml Sanitizer Use-Solution 25ºC
Add 1 ml of E.coli or S.aureus
30 second Contact Time
(minimum of 7.5 x 107 CFU/ml)
Enumerate Survivors
Neutralize 1 ml
Required Efficacy 99.999 Kill in 30 seconds at
25ºC
7
Suggested Surrogates
8
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.

9
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

10
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

11
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

12
Levels of Decontamination Somewhat Dependent on
Infectious Dose

• Smallpox decontamination to 100 virons/m2 not
  acceptable
• Anthrax decontamination to 100 spores/m2 probably
  a safe level

13
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

14
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

15
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

16
Foot Mouth Disease/ApthovirusNon 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.)

17
Is SARS a Potential Bioterrorism Agent?

• 1. Available? Yes
• 2. Culturable? Yes
• 3. Infective? Highly
• 4. Deliverable? Unknown
• 5. Decontamination? Likely to be susceptible
  to common disinfectants

18
Lessons Learned from the Anthrax Case

• 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).

19
Bacillus anthracisSpores vs. Vegetative Cells
20
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)

21
Factors Contributing To Failure of Decontamination

• Environmental Factors
• Presence of organic matter
• Inactivation of QACs by residual soaps and
  detergents
• Incorrect application/coverage
• Inadequate treatment of water supply
• Wettability of surface

22
All Sanitizers Are NOT Created EqualReduced
Temperature Sanitizer Efficacy70oF. Well Water
(250 ppm)30 Seconds
Log Reduction
Peracetic Acid
Organic Acid
23
All Sanitizers Are NOT Created EqualReduced
Temperature Sanitizer Efficacy55oF. Well Water
(250 ppm)30 Seconds
Log Reduction
Peracetic Acid
Organic Acid
24
All Sanitizers Are NOT Created EqualReduced
Temperature Sanitizer Efficacy40 oF. Well Water
(250 ppm)30 Seconds
Log Reduction
Peracetic Acid
Organic Acid
25
All Sanitizers Are NOT Created EqualReduced
Temperature Sanitizer Efficacy40 oF. Well Water
(250 ppm)2 Minutes
Log Reduction
Peracetic Acid
Organic Acid
26
Effect of Temperature Organic Load and
Concentration on Disinfectant Activity

• Quaternary Disinfectant
• (Log Reduction for P. aeruginosa)
• 20C 10C
• x0.5 x1.0 x2.0 x0.5 x1.0 x2.0
• .03 F P P F F P
• BSA
• .3 F P P F F F
• BSA
• P (Pass) gt 5 log reduction
• F (Fail) lt 5 log reduction

QUAT 1000 ppm 5 minutes
Taylor et al 1999
27
Effect of Temperature Organic Load and
Concentration on Disinfectant Activity

• Peracetic Acid Disinfectant
• (Log Reduction for P. aeruginosa)
• 20C 10C
• x0.5 x1.0 x2.0 x0.5 x1.0 x2.0
• .03 P P P P P P
• BSA
• .3 F P P F P P
• BSA
• P (Pass) gt 5 log reduction
• F (Fail) lt 5 log reduction

Peracetic Acid 300 ppm 5 minutes
Taylor et al 1999
28
Treatment of a 48 HourBiofilm of Pseudomonas
aeruginosa
Average (CFU/cm2)
Fatemi and Frank. 1999. Journal of Food
Protection. Vol. 62 (761-765)
29
Effect of Relative Humidity on Sporicidal
Activity (B. globigii) of Formaldehyde

• Log Concentration RH
  Temp Reduction Time
• 400 mg/m3 30 25C 1 22
• 400 mg/m3 98 25C 1 9Ref
  Emerging Infectious Disease Vol 9 No 6 p. 625

30
Effect of Relative Humidity on Sporicidal
Activity (B. subtilis v niger) of Peracetic Acid
Vapor

• Log Concentration RH
  Temp Reduction Time
• 1mg/L 80 RT gt5 log 10
• 1mg/L 60 RT gt4 log 10
• 1mg/L 40 RT gt3 log 10
• Ref Emerging Infectious Disease Vol 9 No 6 p.
  626

31
Inactivation Of Bacillus spp. By Boiling In Tap
Water

• Initial log After 5 CFU/ml
  Boiling
• C UNC C UNCB. 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 0.48 ATCC 9592
• B. thuringensis 4.63 4.46 0 1.47 ATCC 35646

C Covered UNC Uncovered
Temp above surface C 98.9C
UNC 77.3C
Ref Emerging Infectious Disease Vol 10 No 10
2004
32
National Center for Food Protection and Defense

• Study No 910F2376745 EcolabDevelopment 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

33
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
34
Impact of Food Soil on Inactivation of B. cereus
spores by Peroxyacetic Acid (Preliminary Results)

• Conditions 10 minute exposure 20C B. cereus
  ATCC 10987
• Log Reduction
• 5,000 ppm 10,000 ppm
• No Soil gt7.0 gt7.0
• Whole Milk 1.5 4.0
• Egg Yolk 2.0 4.0
• Flour 2.0 7.0

Ref Ecolab, NCFPD Project
35
Impact of Food Soil and Temperature on
Inactivation of B. cereus Spores by Peroxyacetic
Acid (Preliminary Results)

• Conditions 10 minute exposure 10,000 ppm
  Peroxyacetic acid B. cereus ATCC 10987
• Log Reduction
• 10C 20C 30C
• No Soil 6.0 gt7.0 gt7.0
• Whole Milk 1.5 4.0 gt7.0
• Egg Yolk 2.0 4.0 5.5
• Flour 1.5 7.0 7.0

Ref Ecolab, NCFPD Project
36
Impact of Food Soil on Inactivation of B. cereus
spores by Sodium Hypochlorite (Preliminary
Results)

• Conditions 10 minute exposure 20C B. cereus
  ATCC 10987
• Log Reduction _at_ 50,000 ppm
• No Soil gt7.0
• Whole Milk 1.2
• Egg Yolk 1.9
• Flour gt7.0

Note Flour _at_ 1,000 ppm - gt7.0 log reduction
Ref Ecolab, NCFPD Project
37
Other Results to Date

• Quarternary Ammonium Chloride
• Not effective at 75,000 ppm (7.5), 20C
• Normal disinfection level 200-800 ppm
• Iodophor
• Not effective at 19,000 ppm (1.9), 20C
• 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 20C
• Not effective at 10C
• Mixed Peroxyacid System
• Results similar to peroxyacetic acid

38
Future Testing to Include

• B. anthracis (nonvirulent strain)
• Yersinia Pestis

?