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Inactivation of Viruses Using a Synergistically Formulated Alcohol-Based Hand Sanitizer David R. Macinga 1, Marcia Snyder 1, Helen Rawthorne 3, Alexia.

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Presentation on theme: "Inactivation of Viruses Using a Synergistically Formulated Alcohol-Based Hand Sanitizer David R. Macinga 1, Marcia Snyder 1, Helen Rawthorne 3, Alexia."— Presentation transcript:

1 Inactivation of Viruses Using a Synergistically Formulated Alcohol-Based Hand Sanitizer David R. Macinga 1, Marcia Snyder 1, Helen Rawthorne 3, Alexia Taylor 3, Lee-Ann Jaykus 3, Karen M. Ramm 2, and James W. Arbogast 1 1. GOJO Industries, Inc., Akron, OH; 2. ATS Laboratories, Eagan, MN; 3. North Carolina State University, Raleigh, NC ABSTRACT 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. 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. 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 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 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. 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. 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 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. SUMMARY 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 non- enveloped 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. 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 non- enveloped 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. REFERENCES 1.Anonymous Food Code. Food and Drug Administration and U.S. Public Health Service. U.S. Government Printing Office, Washington, D.C. 2.Anonymous WHO guidelines on hand hygiene in health care (advanced draft). The World Health Organization. Geneva, Switzerland. 3.Anonymous ASTM E (2002). Standard Test Method for Efficacy of Virucidal Agents In Suspension. Annual Book of ASTM Standards. 4.Boyce, J. M. and D. Pittet 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:S White, C., R. Kolble, R. Carlson, N. Lipson, M. Dolan, Y. Ali, and M. Cline The effect of hand hygiene on illness rate among students in university residence halls. Am. J. Infect. Control 31: Hammond, B., Y. Ali, E. Fendler, M. Dolan, and S. Donovan Effect of hand sanitizer use on elementary school absenteeism. Am. J. Infect. Control 28: Fendler, E. J., Y. Ali, B. S. Hammond, M. K. Lyons, M. B. Kelley, and N. A. Vowell The impact of alcohol hand sanitizer use on infection rates in an extended care facility. Am. J. Infect. Control 30: Hilburn, J., B. S. Hammond, E. J. Fendler, and P. A. Groziak Use of alcohol hand sanitizer as an infection control strategy in an acute care facility. Am. J. Infect. Control 31: Jones, M. V., K. Bellamy, R. Alcock, and R. Hudson The use of bacteriophage MS2 as a model system to evaluate virucidal hand disinfectants. J. Hosp. Infect. 17: Anonymous Food Code. Food and Drug Administration and U.S. Public Health Service. U.S. Government Printing Office, Washington, D.C. 2.Anonymous WHO guidelines on hand hygiene in health care (advanced draft). The World Health Organization. Geneva, Switzerland. 3.Anonymous ASTM E (2002). Standard Test Method for Efficacy of Virucidal Agents In Suspension. Annual Book of ASTM Standards. 4.Boyce, J. M. and D. Pittet 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:S White, C., R. Kolble, R. Carlson, N. Lipson, M. Dolan, Y. Ali, and M. Cline The effect of hand hygiene on illness rate among students in university residence halls. Am. J. Infect. Control 31: Hammond, B., Y. Ali, E. Fendler, M. Dolan, and S. Donovan Effect of hand sanitizer use on elementary school absenteeism. Am. J. Infect. Control 28: Fendler, E. J., Y. Ali, B. S. Hammond, M. K. Lyons, M. B. Kelley, and N. A. Vowell The impact of alcohol hand sanitizer use on infection rates in an extended care facility. Am. J. Infect. Control 30: Hilburn, J., B. S. Hammond, E. J. Fendler, and P. A. Groziak Use of alcohol hand sanitizer as an infection control strategy in an acute care facility. Am. J. Infect. Control 31: Jones, M. V., K. Bellamy, R. Alcock, and R. Hudson The use of bacteriophage MS2 as a model system to evaluate virucidal hand disinfectants. J. Hosp. Infect. 17: 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. 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. Figure 1: In Vitro Efficacy Results versus MS2 CONCLUSION 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. Table 1: Hydroalcoholic Mixtures Versus MS2 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. 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. 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 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 25 º C, 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 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 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. 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 25 º C, 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 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 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. Table 2: Activity Against Mouse Norovirus and Feline Calicivirus In Vitro VIRUCIDAL ACTIVITY AGAINST MAMMALIAN VIRUSES 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. Table 4: In Vitro Virucidal Activity of Various Hand Sanitizers Table 3: Activity Against Additional Key Enveloped and Non-Enveloped Viruses Table 3 shows the virucidal activity of Purell ® VF447 against additional key enveloped and non-enveloped viruses compared to original Purell Instant Hand Sanitizer. *Tested at Retroscreen Virology Ltd., London, UK. Non-mammalian 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. *Tested at Retroscreen Virology Ltd., London, UK. Non-mammalian virus. *standard commercial bleach, diluted For additional information contact David Macinga, Ph. D., at ©2007 GOJO Industries, Inc., all rights reserved


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