Transcript IAFP-2007-AV-FS_Poster

Inactivation of Viruses Using a Synergistically Formulated
Alcohol-Based Hand Sanitizer
David R. Macinga1, Marcia Snyder1, Helen Rawthorne3, Alexia Taylor3, Lee-Ann Jaykus3, Karen M. Ramm2, and James W. Arbogast1
1. GOJO Industries, Inc., Akron, OH; 2. ATS Laboratories, Eagan, MN; 3. North Carolina State University, Raleigh, NC
Methods: Virucidal assays were performed by quantitative suspension test. Bacteriophage MS2 plaque titers
were determined by soft agar overlay after infection of E. coli ATCC-15597. Mammalian virus titers were
determined by infecting appropriate host cell lines and monitoring cytopathic effects.
Results: Concentrations up to 78% ethyl alcohol exhibited minimal activity against MS2. An extensive screening
program showed that numerous other ingredients provided no significant increase in efficacy (≤ 1.0 log reduction,
1 min). The synergistic combination of ethyl alcohol, quaternary polymer and organic acid produced a 4.3 log
reduction against MS2 in 1 min. A hand sanitizer formulated to contain 70% ethyl alcohol, polyquaternium and
citric acid completely inactivated feline calicivirus in 30 sec (> 4.75 log reduction); completely inactivated
rotavirus, rhinovirus, influenza A, influenza B, Avian influenza (H5N1), and respiratory syncytial virus in 30 sec;
and reduced poliovirus >3 log in 30 sec. Some degree of efficacy was attained at shorter exposure times, and
against mouse norovirus and hepatitis A virus.
Significance: Based on these results we conclude that this new synergistically formulated ethyl alcohol hand
sanitizer is a promising option for managing the transmission of viruses, including the noroviruses, in foodservice
settings. These types of control measures should be seriously considered as part of the hand hygiene and overall
restaurant infection control plans to reduce the transmission of foodborne viruses.
INTRODUCTION
The antibacterial properties of alcohol-based hand sanitizers (ABHS) containing at least 60% alcohol are well
recognized. The use of ABHS has been demonstrated to reduce Hospital Acquired Infection rates, absenteeism in
primary and secondary education settings, and absenteeism in the workplace (5-8). Various worldwide
organizations including CDC and WHO recommend ABHS as the primary means of hand hygiene when hands are
not “visibly soiled” (2,4).
The 2005 Food Code recommends handwashing as the primary means of hand hygiene (1). ABHS may be used
as an adjunct to hand washing. Two concerns about the use of ABHS in food handling environments are the heavy
soils encountered and the lack of demonstrated activity of ABHS versus viruses of concern such as Norovirus and
Hepatitis A. Current ABHS technologies have demonstrated broad spectrum bactericidal activity but have limited
virucidal activity. In general, ABHS demonstrate very good activity against enveloped viruses which contain a lipid
based membrane (4). However the activity against non-enveloped viruses varies depending on the particular virus.
The purpose of this study was to develop an ABHS with broad spectrum antiviral activity. We investigated various
chemistries, including quaternary ammonium compounds, metallic salts, and organic acids to potentiate the activity
of ethyl alcohol against the bacteriophage MS2. MS2 was previously shown to be a good model system to
evaluate hand hygiene products against non-enveloped viruses (9). Promising combinations were optimized for
activity against mammalian non-enveloped viruses.
Polymeric quaternaries are classed as quaternary ammonium compounds. Polyquaternium polymers have very
high molecular weight and a wide range of functions. Since they carry a positive charge, they are substantive to
skin and hair proteins and are widely used in personal care products for their conditioning and antistatic properties.
Many non-polymeric low molecular weight quaternary compounds have the ability to disrupt the surface
membranes of microbial species and are widely used as antibacterial agents (e.g., Chlorhexidine Gluconate or
Benzalkonium Chloride). Non-polymeric low molecular weight quaternaries may contribute to skin irritation or
sensitization.
We present virucidal data on a new patent pending hand sanitizer based on 70% ethyl alcohol, which contains a
synergistic combination of a polyquaternium polymer and an organic acid.
METHODS
Test Substances: Two alcohol-based hand sanitizers were used in this study; one based on 62% ethyl alcohol
(PURELL® Instant Hand Sanitizer) and one based on 70% ethyl alcohol (PURELL® VF447™).
MS2 Suspension Assays: Typically, 0.1 ml phage was added to 9.9 ml of antiviral composition. After the desired
contact time at 25ºC, 0.1 ml suspension was neutralized by dilution into 9.9 ml D.E. broth. Further 10-fold serial
dilutions were prepared in D.E. broth. The remaining active phage was quantified by infecting E. coli ATCC 15597 and
using the soft agar overlay technique.
Viruses and Cell culture: Viral strains and indicator cells lines were as follows: Adenovirus type 2, ATCC VR-846 was
grown on A-549 human lung carcinoma cells; Feline calicivirus F-9, ATCC VR-782 was grown on CRFK feline kidney
cells; Hepatitis A, HM-175 was grown on Fetal Rhesus monkey kidney cells (FRhK-4) cells; Influenza A, VR-544 and
Influenza B, VR-823 were grown on Rhesus monkey kidney (RMK) cells; Avian Influenza A H5N1, NIBRG-14 was
grown on MDCK cells; Mouse Norovirus, MNV-1 was grown on RAW 264.7 cells, Rhinovirus type 37, ATCC VR-1147
was grown on MRC-5 human embryonic lung cells; Rotavirus WA, ATCC VR-2018, was grown on MA-104 rhesus
monkey kidney cells; Respiratory Syncytial Virus, VR-26 was grown on Hep-2 cells.
Virucidal Suspension Assays: Virucidal suspension tests with mammalian viruses were performed using a
modification of ASTM E1052, Standard Test Method for Efficacy of Virucidal Agents Intended for Special Applications
(3). A 4.5 ml aliquot of each test substance was dispensed into separate sterile 15 ml conical tubes and each was
mixed with a 0.5 ml aliquot of the stock virus suspension. The mixtures were vortex mixed for 10 seconds and held the
remainder of the 30 or 60 second exposure time at 33±2˚C. Immediately following the exposure period, a 0.1 ml aliquot
was removed from each tube and the mixtures were titered by 10-fold serial dilutions and assayed for the presence of
virus by infecting indicator cell lines. Cytopathic effect (CPE) was used in each case to indicate infection and TCID 50
values were calculated by the method of Spearman-Karber. Virus controls, neutralization controls, and cytotoxicity
controls were also performed.
Potentiator(s)
Exposure
Time
Log10
Reduction
100 ppm NaOCl*
None
60 sec
≥ 5.85
62% ethyl alcohol
None
60 sec
-0.10
70% ethyl alcohol
None
60 sec
-0.02
78% ethyl alcohol
None
60 sec
0.16
78% ethyl alcohol
78% ethyl alcohol
Benzethonium Chloride
Chlorhexidine Gluconate
60 sec
5.0
4.30
4.0
Log10 Reduction
Purpose: Evaluate novel hand sanitizer formulations for rapid virucidal activity against feline calicivirus (a
surrogate for human norovirus) and other relevant enveloped and non-enveloped viruses.
Active Ingredient
0.34
60 sec
3.0
2.0
0.70
1.0
0.48
0.95
78% ethyl alcohol
Copper Gluconate
60 sec
0.18
78% ethyl alcohol
Silver Zeolite
60 sec
0.58
78% ethyl alcohol
Silver Citrate
60 sec
1.25
Product
Active Ingredient(s)
78% EtOH
78% EtOH + Citric
Acid
78% EtOH +
Polyquaternium
78% EtOH +
Polyquaternium +
Citric Acid
Figure 1 depicts the efficacy results of additive versus synergistic combinations of
ingredients. A 4.3 log reduction is achieved in 60 seconds using the synergistic
combination of 78% ethyl alcohol, polyquaternium polymer and citric acid.
Log10 Reduction
MS2
FCV
PURELL® VF447™
70% Ethyl Alcohol
2.08
≥2.80
PURELL® Instant Hand Sanitizer
62% Ethyl Alcohol
0.00
1.25
Clorox Anywhere Hand Sanitizing Spray
71% Ethyl Alcohol
0.00
n.t.
Dial Hand Sanitizer
62% Ethyl Alcohol
0.00
n.t.
Kay Actigel
60% Ethyl Alcohol
0.00
n.t.
0.00
1.60
1.79
≥3.2
70% Ethyl alcohol,
1,3 - Butanediol
54.1% Ethyl alcohol
10.0% 1-Propanol
21.6% Glycol Blend
Manorapid Synergy
0.0
Table 1 illustrates in vitro results from 60 second efficacy screenings against MS2
Bacteriophage. Ethyl alcohol concentrations from 62% to 78% exhibit minimal activity.
No significant increase in efficacy is seen with the addition of many different potentiators.
Table 4: In Vitro Virucidal Activity of Various
Hand Sanitizers
Manorapid Hand Antiseptic
0.16
Soaptronic Germstar
70% Isopropyl Alcohol
0.05
0.90
Sterillium Virugard
95% Ethyl Alcohol
0.90
n.t.
Sterillium Rub
45% Isopropyl Alcohol,
30% 1-Propanol,
0.2% Mecetronium etilsulfate
0.00
1.40
Viraguard Antiseptic Hand Spray
75% Isopropyl Alcohol
0.18
0.70
Zero+ Hand Sanitizer
Amosilk
0.12
0.20
*standard commercial bleach, diluted
Table 4 shows the virucidal activity of a spectrum of hand sanitizer products against MS2
bacteriophage and Feline Calicivirus, a surrogate for Norovirus. The data shown is a 60 second in
vitro assay. n.t. = not tested
In Vitro VIRUCIDAL ACTIVITY AGAINST MAMMALIAN VIRUSES
Table 2: Activity Against Mouse Norovirus
and Feline Calicivirus
Mouse Norovirus
Log10 Reduction
Product
30 Sec
60 Sec
Feline Calicivirus
Log10 Reduction
30 Sec
®
PURELL Instant Hand
Sanitizer
1.16
1.44
1.00
®
PURELL Food Code
Compliant
PURELL® VF447™
1.37
≥ 3.68
1.56
≥4.25
n.t.
≥ 4.7
Table 2 shows the increased virucidal activity of Purell® VF447™ against Mouse
Norovirus and Feline Calicivirus as compared to original Purell Instant Hand
Sanitizer and Purell Food Code Compliant Instant Hand Sanitizer. Values listed
as “ ≥ ” denote that the virus was inactivated to below the detection limit of the
assay. The log reduction was calculated based on the starting viral titer.
n.t. = not tested.
SUMMARY
Table 3: Activity Against Additional Key
Enveloped and Non-Enveloped Viruses
Type
Non-Enveloped
Introduction: The CDC estimates that Noroviruses are the leading cause of foodborne illness in the U.S. and
reports indicate that hands are the most important vector for transmission. The 2005 Food Code does not
recommend hand sanitizers for prevention of Norovirus transmission, likely in large part because current products
are relatively ineffective against Norovirus surrogates.
Figure 1: In Vitro Efficacy Results versus MS2
Enveloped
ABSTRACT
Table 1: Hydroalcoholic Mixtures Versus MS2
ATCC / Strain
Number
Exposure
Time
Adenovirus type 2
VR-846
Hepatitis A
Virus
Log10 Reduction
PURELL® Instant
Hand Sanitizer
PURELL®
VF447™
30 sec
0.5
2.75
HM-175
30 sec
0
1.75
Poliovirus type 1
VR-1000
30 sec
0
3.5
Rhinovirus type 37
VR-1147
30 sec
2.75
≥ 3.25
Rotavirus WA
VR-2018
30 sec
≥ 5.75
≥ 4.75
Influenza A
VR-544
30 sec
≥ 6.25
≥ 5.25
Influenza B
VR-823
30 sec
≥ 5.75
≥ 4.25
NIBRG-14
15 sec
≥ 3.50
≥ 3.75
VR-26
30 sec
≥ 2.50
≥ 1.50
Avian Influenza (H5N1)*
Respiratory Syncytial
Virus
Table 3 shows the virucidal activity of Purell® VF447™ against additional key enveloped and
non-enveloped viruses compared to original Purell Instant Hand Sanitizer.
 Very few compounds potentiate the efficacy of ethyl alcohol against the
non-enveloped bacteriophage MS2. This is consistent with other data
which demonstrates ethyl alcohol hand sanitizers are typically
ineffective versus many non-enveloped viruses.
 The combination of ethyl alcohol, citric acid and polyquaternium
exhibits synergistic virucidal activity.
 A hand sanitizer (PURELL® VF447™ ) based on 70% ethanol,
polyquaternium polymer & citric acid exhibited:
► increased virucidal activity versus critical food service industry nonenveloped viruses, including Mouse Norovirus and Feline Calicivirus
(surrogates for human norovirus) and Hepatitis A,
► uniquely high levels and broad spectrum viral efficacy relative to
current hand sanitizer benchmarks.
 This new technology should be considered for risk modeling and as a
means to better control infections and manage risks in food service.
*Tested at Retroscreen Virology Ltd., London, UK. Non-mammalian virus.
REFERENCES
CONCLUSION
1.
2.
3.
4.
5.
PURELL® VF447™, a synergistically formulated ethyl alcohol-based hand sanitizer, is a promising option for
managing the transmission of viruses, including noroviruses, in foodservice settings.
6.
7.
8.
9.
Anonymous. 2005 Food Code. Food and Drug Administration and U.S. Public Health Service. U.S. Government Printing Office, Washington, D.C.
Anonymous. 2006. WHO guidelines on hand hygiene in health care (advanced draft). The World Health Organization. Geneva, Switzerland.
Anonymous. 2002. ASTM E1052-96 (2002). Standard Test Method for Efficacy of Virucidal Agents In Suspension. Annual Book of ASTM Standards.
Boyce, J. M. and D. Pittet. 2002. Guideline for Hand Hygiene in Health-Care Settings. Recommendations of the Healthcare Infection Control Practices
Advisory Committee and the HIPAC/SHEA/APIC/IDSA Hand Hygiene Task Force. Am. J. Infect. Control 30:S1-46.
White, C., R. Kolble, R. Carlson, N. Lipson, M. Dolan, Y. Ali, and M. Cline. 2003. The effect of hand hygiene on illness rate among students in university
residence halls. Am. J. Infect. Control 31:364-370.
Hammond, B., Y. Ali, E. Fendler, M. Dolan, and S. Donovan. 2000. Effect of hand sanitizer use on elementary school absenteeism. Am. J. Infect. Control
28:340-346.
Fendler, E. J., Y. Ali, B. S. Hammond, M. K. Lyons, M. B. Kelley, and N. A. Vowell. 2002. The impact of alcohol hand sanitizer use on infection rates in an
extended care facility. Am. J. Infect. Control 30:226-233.
Hilburn, J., B. S. Hammond, E. J. Fendler, and P. A. Groziak. 2003. Use of alcohol hand sanitizer as an infection control strategy in an acute care facility.
Am. J. Infect. Control 31:109-116.
Jones, M. V., K. Bellamy, R. Alcock, and R. Hudson. 1991. The use of bacteriophage MS2 as a model system to evaluate virucidal hand disinfectants. J.
Hosp. Infect. 17:279-285.
For additional information contact David Macinga, Ph. D., at [email protected]
©2007 GOJO Industries, Inc., all rights reserved