洗手产品除病毒效果离体实验方法研究
|
摘要
目的建立一种安全、简便和费用低廉的测试洗手及洗手产品除病毒效果的离体试验方法。方法以噬菌体为替代病毒,仿真皮为载体设计试验流程,分析影响试验结果的要素,通过数据分析,研究洗手产品回收稀释液种类、噬菌体初始接种浓度及回收方式对结果稳定性的影响。使用不同洗手产品对多种噬菌体分别进行除病毒效果测试后进行统计分析,确定方法的适用性。结果以噬菌体φX174为污染病毒,0.1%蛋白胨生理盐水做为香皂和洗手液的回收稀释液使用时,其噬菌体的回收值为5.57和5.54,明显高于改良李氏肉汤吐温培养基(MLBT)、多用中和剂(PVUN)及磷酸缓冲液(PBS);对手摇瓶子2 min、摇床200 r/min振摇2 min、均质器标准强度快速拍打2 min后测得的噬菌体回收量进行单因素方差分析,P_(0.05)=0.72,表明3者无统计学差异,但是在使用均质器拍打时的变异系数(CV)为0.74%最小,数据最稳定;4个初始接种浓度中,只有在10~7 pfu/mL与10~8 pfu/mL的浓度下,平皿上的噬菌体数量符合实验设计,超过4 Log10,对这2个浓度实验结果进行t检验,其P_(0.05)=0.003,结合回收数据,认为10~8pfu/mL是最合适的加样浓度;使用上述参数,分别以噬菌体MS2、φX174、φ6为污染病毒,使用香皂、洗手液及清水进行洗手除病毒评价,所有结果 CV均<15%。结论采用10~8 pfu/mL的噬菌体悬液污染仿真皮后使用洗手产品模拟洗手,均质器拍打2 min、0.1%蛋白胨生理盐水进行回收稀释,实验结果最稳定。多次重复实验结果显示,该方法适用于多种噬菌体,也适用于多种洗手产品,是一种简单可行的洗手及洗手产品除病毒效果评价方法,具有较高的应用价值。
Objective To develop a simple, safe, and low cost in vitro method for the evaluation on virus-eliminating effectiveness of hand-washing and hand wash agents. Methods We used artificial skin as human skin surrogate and bacteriophage as virus surrogate to construct an in vitro model. We analyzed effects of various recovery diluent, initial concentration of bacteriophage inoculation, and recovery method on detection reproducibility of the method to be established.Three bacteriophage strains were used in evaluations of bar and liquid soap. Statistical analyses on experimental data were performed to assess the applicability of the method. Results The recovery(logarithmic value) of φX174 bacteriophage was 5.57 and 5.54 for washing procedure with bar soap and liquid soap when using physiological saline with 0.1% peptone as the diluent and the values were obviously higher than those when using modified Letheen broth plus Tween(MLBT), polyvalent universal neutralizer(PVUN) or phosphate buffer saline(PBS) as the diluent. The results of one-way analysis of variance demonstrated that there was no significant difference in the recovery of bacteriophage among the three patterns of simulated 2-minute hand-washing(shaking the bottle manually, shaking violently with a shaker at 200 revolutions per minute or with a homogenizer)(P_(0.05) = 0.72); however, the variation in the recovery of bacteriophage was the lowest(coefficient of variation[CV] = 0.74%) when using homogenizer in the simulated test. The bacteriophage plate count consistent with the experimental design(baseline recovery > 4 Log10, Pstudent-t test = 0.003) was achieved under two(10~8 and 10~7 plaque forming unit [pfu]/ml)of the four initial concentrations of bacteriophage inoculation; while, the simulated virus-eliminating rate was significantly higher with the initial inoculation concentration of 10~8 pfu/ml than with that of 10~7 pfu/ml, indicating that 10~8 pfu/ml was the most optimal initial inoculation concentration. All the CV of variation in bacteriophage recovery were less than 15% for the simulated virus-eliminating tests of bar soap, liquid soap and only water using φX174, MS2 and φ6 bacteriophage as virus surrogate and optimized experimental conditions. Conclusion A simple, safe, feasible, and highly applicable in vitro method was established for the evaluation on virus-eliminating effectiveness of hand wash agents using 10~8 pfu/ml bacteriophage suspension contaminated artificial skin, 2-minute shaking of a homogenizer, and physiological saline with 0.1% peptone as recovery diluent.
Objective To develop a simple, safe, and low cost in vitro method for the evaluation on virus-eliminating effectiveness of hand-washing and hand wash agents. Methods We used artificial skin as human skin surrogate and bacteriophage as virus surrogate to construct an in vitro model. We analyzed effects of various recovery diluent, initial concentration of bacteriophage inoculation, and recovery method on detection reproducibility of the method to be established.Three bacteriophage strains were used in evaluations of bar and liquid soap. Statistical analyses on experimental data were performed to assess the applicability of the method. Results The recovery(logarithmic value) of φX174 bacteriophage was 5.57 and 5.54 for washing procedure with bar soap and liquid soap when using physiological saline with 0.1% peptone as the diluent and the values were obviously higher than those when using modified Letheen broth plus Tween(MLBT), polyvalent universal neutralizer(PVUN) or phosphate buffer saline(PBS) as the diluent. The results of one-way analysis of variance demonstrated that there was no significant difference in the recovery of bacteriophage among the three patterns of simulated 2-minute hand-washing(shaking the bottle manually, shaking violently with a shaker at 200 revolutions per minute or with a homogenizer)(P_(0.05) = 0.72); however, the variation in the recovery of bacteriophage was the lowest(coefficient of variation[CV] = 0.74%) when using homogenizer in the simulated test. The bacteriophage plate count consistent with the experimental design(baseline recovery > 4 Log10, Pstudent-t test = 0.003) was achieved under two(10~8 and 10~7 plaque forming unit [pfu]/ml)of the four initial concentrations of bacteriophage inoculation; while, the simulated virus-eliminating rate was significantly higher with the initial inoculation concentration of 10~8 pfu/ml than with that of 10~7 pfu/ml, indicating that 10~8 pfu/ml was the most optimal initial inoculation concentration. All the CV of variation in bacteriophage recovery were less than 15% for the simulated virus-eliminating tests of bar soap, liquid soap and only water using φX174, MS2 and φ6 bacteriophage as virus surrogate and optimized experimental conditions. Conclusion A simple, safe, feasible, and highly applicable in vitro method was established for the evaluation on virus-eliminating effectiveness of hand wash agents using 10~8 pfu/ml bacteriophage suspension contaminated artificial skin, 2-minute shaking of a homogenizer, and physiological saline with 0.1% peptone as recovery diluent.
引文
[1]Tuladhar E,Hazeleger WC,Koopmans M,et al.Reducing viral contamination from finger pads:hand washing is more effective than alcohol-based hand disinfectants[J].Journal of Hospital Infection,2015,90(3):226-234.
[2]American Society for Testing and Materials.ASTM E2870-13,standard test method for evaluating relative effectiveness of antimicrobial handwashing formulations using the palmar surface and mechanical hand sampling[S].United States:American Society for Testing and Materials International,2013.
[3]American Society for Testing and Materials.ASTM E1838-10,standard test method for determining the virus-eliminating effectiveness of hygienic handwash and handrub agents using the fingerpads of adults[S].United States:American Society for Testing and Materials International,2010.
[4]American Society for Testing and Materials.ASTM E1174-13,standard test method for evaluation of the effectiveness of health care personnel handwash formulations[S].United States:American Society for Testing and Materials International,2013.
[5]American Society for Testing and Materials.ASTM E2011-13,standard test method for evaluation of hygienic handwash and handrub formulations for virus-eliminating activity using the entire hand[S].United States:American Society for Testing and Materials International,2013.
[6]Rotter M,Sattar S,Dharan S,et al.Methods to evaluate the microbicidal activities of hand-rub and hand-wash agents[J].Journal of Hospital Infection,2009,73(3):191-199.
[7]World Health Organization.Guidelines on hand hygiene in health care[S].World Health Organization,2009,Part 1:3.
[8]Adams MH.Bacteriophages[N].Interscience Publishers INC,New York,1959.
[9]Tamimi AH,Carlino S,Edmonds S,et al.Impact of an alcoholbased hand sanitizer intervention on the spread of viruses in homes[J].Food and Environmental Virology,2014,6(2):140-144.
[10]Casteel MJ,Schmidt CE,Sobsey MD.Chlorine disinfection of produce to inactivate hepatitis A virus and coliphage MS2[J].International Journal of Food Microbiology,2008,125(3):267-273.
[11]Shirasaki N,Matsushita T,Matsui Y,et al.Investigation of enteric adenovirus and poliovirus removal by coagulation processes and suitability of bacteriophages MS2 andφX174 as surrogates for those viruses[J].Science of the Total Environment,2016,563-564:29-39.
[12]Kosel J,Gutiérrez-Aguirre I,Ra?ki N,et al.Efficient inactivation of MS-2 virus in water by hydrodynamic cavitation[J].Water Research,2017,124:465-471.
[13]Elving J,Emmoth E,Albihn A,et al.Composting for avian influenza virus elimination[J].Applied and Environmental Microbiology,2012,78(9):3280-3285.
[14]International Standards Organization.International Standards Organization 107050.Water quality-detection and enumeration of bacteriophages.Part 1[S].Switzerland,1995.
[15]Turgeon N,Toulouse MJ,Martel B,et al.Comparison of five bacteriophages as models for viral aerosol studies[J].Applied and Environmental Microbiology,2014,80(14):4242-4250.
[16]Rheinbaben FV,Schünemann S,Gross T,et al.Transmission of viruses via contact in a household setting:experiments using bacteriophage fX174 as a model virus[J].Journal of Hospital Infection,2000,46(1):61-66.
[17]Verreault D,Rousseau GM,Gendron L,et al.Comparison of polycarbonate and poly tetrafluoroethylene filters for sampling of airborne bacteriophages[J].Aerosol Science and Technology,2010,44(3):197-201.
[18]American Society for Testing and Materials.F1671/F1671M,standard test method for resistance of materials used in protective clothing to penetration by blood-borne pathogens using Phi-X174bacteriophage penetration as a test system[S].United States:American Society for Testing and Materials International,2013.
[19]Wu B,Wang R,Fane AG.The roles of bacteriophages in membrane-based water and wastewater treatment processes:a review[J].Water Research,2017,110:120-132.
[20]Vidaver AK,Koski RK,Van Etten JL.Bacteriophage Phi 6:a lipid-containing virus of Pseudomonas phaseolicola[J].Journal of Virology,1973,11(5):799-805.
[21]Aranha-Creado H,Peterson J,Huang PY.Clearance of murine leukaemia virus from monoclonal antibody solution by a hydrophilic PVDF microporous membrane filter[J].Biologicals,1998,26(2):167-172.
[22]Lytle CD,Tondreau SC,Truscott W,et al.Filtration sizes of human immunodeficiency virus type 1 and surrogate viruses used to test barrier materials[J].Applied and Environmental Microbiology,1992,58(2):747-749.
[23]Phillpotts RJ,Thomas RJ,Beedham RJ,et al.The Cystovirus,phi6as a simulant for Venezuelan equine encephalitis virus[J].Aerobiologia,2010,26(4):301-309.
[24]Sassi HP,Ikner LA,Abd-Elmaksoud S,et al.Comparative survival of viruses during thermophilic and mesophilic anaerobic digestion[J].Science of the Total Environment,2017,615:15-19.
[25]Otter JA,Donskey C,Yezli S,et al.Transmission of SARS and MERS coronaviruses and influenza virus in healthcare settings:the possible role of dry surface contamination[J].Journal of Hospital Infection,2016,92(3):235-250.
[26]Sassi HP,Sifuentes LY,Koenig DW,et al.Control of the spread of viruses in a long-term care facility using hygiene protocols[J].American Journal of Infection Control,2015,43(7):702-706.
[27]Oxford J,Berezin EN,Courvalin P,et al.The survival of influenza A(H1N1)pdm09 virus on 4 household surfaces[J].American Journal of Infection Control,2014,42(4):423-425.
[28]L'Huillier AG,Tapparel C,Turin L,et al.Survival of rhinoviruses on human fingers[J].Clinical Microbiology and Infection,2015,21(4):381-385.
[29]Jr GJ,Hendley JO.Transmission of experimental rhinovirus infection by contaminated surfaces[J].American Journal of Epidemiology,1982,16(5):828-833.
[30]Thomas Y,Boquetesuter P,Koch D,et al.Survival of influenza virus on human fingers[J].Clinical Microbiology and Infection,2013,20(1):O58-O64.
[31]谢小保,李彩玲,李素娟,等.离体和在体消毒方法相关性的初步研究[J].中国卫生检验杂志,2011,21(8):1942-1946.
[2]American Society for Testing and Materials.ASTM E2870-13,standard test method for evaluating relative effectiveness of antimicrobial handwashing formulations using the palmar surface and mechanical hand sampling[S].United States:American Society for Testing and Materials International,2013.
[3]American Society for Testing and Materials.ASTM E1838-10,standard test method for determining the virus-eliminating effectiveness of hygienic handwash and handrub agents using the fingerpads of adults[S].United States:American Society for Testing and Materials International,2010.
[4]American Society for Testing and Materials.ASTM E1174-13,standard test method for evaluation of the effectiveness of health care personnel handwash formulations[S].United States:American Society for Testing and Materials International,2013.
[5]American Society for Testing and Materials.ASTM E2011-13,standard test method for evaluation of hygienic handwash and handrub formulations for virus-eliminating activity using the entire hand[S].United States:American Society for Testing and Materials International,2013.
[6]Rotter M,Sattar S,Dharan S,et al.Methods to evaluate the microbicidal activities of hand-rub and hand-wash agents[J].Journal of Hospital Infection,2009,73(3):191-199.
[7]World Health Organization.Guidelines on hand hygiene in health care[S].World Health Organization,2009,Part 1:3.
[8]Adams MH.Bacteriophages[N].Interscience Publishers INC,New York,1959.
[9]Tamimi AH,Carlino S,Edmonds S,et al.Impact of an alcoholbased hand sanitizer intervention on the spread of viruses in homes[J].Food and Environmental Virology,2014,6(2):140-144.
[10]Casteel MJ,Schmidt CE,Sobsey MD.Chlorine disinfection of produce to inactivate hepatitis A virus and coliphage MS2[J].International Journal of Food Microbiology,2008,125(3):267-273.
[11]Shirasaki N,Matsushita T,Matsui Y,et al.Investigation of enteric adenovirus and poliovirus removal by coagulation processes and suitability of bacteriophages MS2 andφX174 as surrogates for those viruses[J].Science of the Total Environment,2016,563-564:29-39.
[12]Kosel J,Gutiérrez-Aguirre I,Ra?ki N,et al.Efficient inactivation of MS-2 virus in water by hydrodynamic cavitation[J].Water Research,2017,124:465-471.
[13]Elving J,Emmoth E,Albihn A,et al.Composting for avian influenza virus elimination[J].Applied and Environmental Microbiology,2012,78(9):3280-3285.
[14]International Standards Organization.International Standards Organization 107050.Water quality-detection and enumeration of bacteriophages.Part 1[S].Switzerland,1995.
[15]Turgeon N,Toulouse MJ,Martel B,et al.Comparison of five bacteriophages as models for viral aerosol studies[J].Applied and Environmental Microbiology,2014,80(14):4242-4250.
[16]Rheinbaben FV,Schünemann S,Gross T,et al.Transmission of viruses via contact in a household setting:experiments using bacteriophage fX174 as a model virus[J].Journal of Hospital Infection,2000,46(1):61-66.
[17]Verreault D,Rousseau GM,Gendron L,et al.Comparison of polycarbonate and poly tetrafluoroethylene filters for sampling of airborne bacteriophages[J].Aerosol Science and Technology,2010,44(3):197-201.
[18]American Society for Testing and Materials.F1671/F1671M,standard test method for resistance of materials used in protective clothing to penetration by blood-borne pathogens using Phi-X174bacteriophage penetration as a test system[S].United States:American Society for Testing and Materials International,2013.
[19]Wu B,Wang R,Fane AG.The roles of bacteriophages in membrane-based water and wastewater treatment processes:a review[J].Water Research,2017,110:120-132.
[20]Vidaver AK,Koski RK,Van Etten JL.Bacteriophage Phi 6:a lipid-containing virus of Pseudomonas phaseolicola[J].Journal of Virology,1973,11(5):799-805.
[21]Aranha-Creado H,Peterson J,Huang PY.Clearance of murine leukaemia virus from monoclonal antibody solution by a hydrophilic PVDF microporous membrane filter[J].Biologicals,1998,26(2):167-172.
[22]Lytle CD,Tondreau SC,Truscott W,et al.Filtration sizes of human immunodeficiency virus type 1 and surrogate viruses used to test barrier materials[J].Applied and Environmental Microbiology,1992,58(2):747-749.
[23]Phillpotts RJ,Thomas RJ,Beedham RJ,et al.The Cystovirus,phi6as a simulant for Venezuelan equine encephalitis virus[J].Aerobiologia,2010,26(4):301-309.
[24]Sassi HP,Ikner LA,Abd-Elmaksoud S,et al.Comparative survival of viruses during thermophilic and mesophilic anaerobic digestion[J].Science of the Total Environment,2017,615:15-19.
[25]Otter JA,Donskey C,Yezli S,et al.Transmission of SARS and MERS coronaviruses and influenza virus in healthcare settings:the possible role of dry surface contamination[J].Journal of Hospital Infection,2016,92(3):235-250.
[26]Sassi HP,Sifuentes LY,Koenig DW,et al.Control of the spread of viruses in a long-term care facility using hygiene protocols[J].American Journal of Infection Control,2015,43(7):702-706.
[27]Oxford J,Berezin EN,Courvalin P,et al.The survival of influenza A(H1N1)pdm09 virus on 4 household surfaces[J].American Journal of Infection Control,2014,42(4):423-425.
[28]L'Huillier AG,Tapparel C,Turin L,et al.Survival of rhinoviruses on human fingers[J].Clinical Microbiology and Infection,2015,21(4):381-385.
[29]Jr GJ,Hendley JO.Transmission of experimental rhinovirus infection by contaminated surfaces[J].American Journal of Epidemiology,1982,16(5):828-833.
[30]Thomas Y,Boquetesuter P,Koch D,et al.Survival of influenza virus on human fingers[J].Clinical Microbiology and Infection,2013,20(1):O58-O64.
[31]谢小保,李彩玲,李素娟,等.离体和在体消毒方法相关性的初步研究[J].中国卫生检验杂志,2011,21(8):1942-1946.