ASTM E2721-2010 Standard Test Method for Evaluation of Effectiveness of Decontamination Procedures for Surfaces When Challenged with Droplets Containing Human Pathogenic Viruses《评定.pdf

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1、Designation: E2721 10Standard Test Method forEvaluation of Effectiveness of Decontamination Proceduresfor Surfaces When Challenged with Droplets ContainingHuman Pathogenic Viruses1This standard is issued under the fixed designation E2721; the number immediately following the designation indicates th

2、e year oforiginal adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.INTRODUCTIONMany communicable diseases can often spread throug

3、h droplets containing infectious agents. Such“contagious droplets” may expose susceptible individuals directly or contaminate environmentalsurfaces in the immediate vicinity and render them as fomites for further spread of the disease. Thecharacteristics of the droplets (particle size and compositio

4、n) will influence the viability of themicroorganisms when exposed to environmental stresses but also shield them from physical andchemical decontaminants. The wide variations in the types and levels of such protective/shieldingingredients can impact on the effectiveness of surface decontaminants. Th

5、is test method is designedto simulate surface deposition of contagious droplets from human respiratory secretions. It is primarilyfocused on influenza viruses but other respiratory viruses or surrogates could be used. Protocols forassessing the microbicidal activity of disinfectants are also describ

6、ed.1. Scope1.1 This test method is designed to evaluate decontamina-tion methods (physical, chemical, self-decontaminating mate-rials) when used on surfaces contaminated with virus-containing droplets.1.2 This test method defines the conditions for simulatingrespiratory droplets produced by humans a

7、nd depositing thedroplets onto surfaces.1.3 The method is specific to influenza viruses but could beadapted for work with other types of respiratory viruses orsurrogates (Appendix X5).1.4 This test method is suitable for working with a widevariety of environmental surfaces.1.5 This test method does

8、not address the performance ofdecontaminants against microbes expelled via blood splatter,vomit, or fecal contamination.1.6 This test method should be performed only by thosetrained in bioaerosols, microbiology, or virology, or combina-tions thereof.1.7 The values stated in SI units are to be regard

9、ed asstandard. No other units of measurement are included in thisstandard.1.8 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine

10、 the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2E1052 Test Method for Efficacy of Antimicrobial AgentsAgainst Viruses in SuspensionE2197 Quantitative Disk Carrier Test Method for Determin-ing the Bactericidal, Virucidal, Fungicidal, Mycobacteri-c

11、idal and Sporicidal Activities of Liquid Chemical Germi-cidesE2720 Test Method for Evaluation of Effectiveness ofDecontamination Procedures for Air-Permeable Materialswhen Challenged2.2 EPA Standards:EPA 600/4-84/013 (N16) USEPA Manual of Methods forVirology31This test method is under the jurisdicti

12、on of ASTM Committee E35 onPesticides and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved Oct. 1, 2010. Published February 2011. DOI: 10.1520/E272110.2For referenced ASTM standards, visit the ASTM website, www.astm.or

13、g, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Available from United States Environmental Protection Agency (EPA), ArielRios Bldg., 1200 Pennsylvania Ave., NW, Washington, DC

14、 20460, http:/www.epa.gov.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.2.3 WHO Standards:WHO Manual on Animal Influenza Diagnosis and Surveil-lance43. Terminology3.1 Definitions:3.1.1 aerosol, na suspension of solid or liquid part

15、icles ina gas medium.3.1.2 biological aerosol, naerosol comprising particles ofbiological origin or activity which may affect living thingsthrough infectivity, allergencity, toxicity, or pharmacologicaland other processes.3.1.3 contact transmission, ninfections caused by directskin-to-skin contact o

16、r indirect contact with objects contami-nated with pathogens.3.1.4 contagious respiratory droplet, nrespiratory secre-tions containing infectious microorganisms that form largedroplets ($5 m) and settle out of the air over short distances.3.1.5 droplet transmission, ndirect transfer of pathogen-cont

17、aining droplets to conjuncitval or mucous membranes.3.1.6 influenza, nan infectious disease of birds and mam-mals caused by RNA viruses of the family Orthomyxoviridae.3.1.7 protective factor, nsoluble or insoluble materialco-deposited with microorganisms that directly protects themicroorganism from

18、environmental stresses or decontami-nants.3.1.8 self-sanitizing material, na substrate containing anantimicrobial agent that collectively acts as a germicide.4. Summary of Test Method4.1 The test method describes the steps required to depositdroplets onto surfaces and quantitatively assess decontami

19、na-tion efficiency.4.1.1 Using an aerosol device capable of meeting the dataquality objectives set for in this test method, influenza virus orsurrogates are aerosolized to form droplets that are subse-quently applied to surfaces.4.1.2 The virus-contaminated carriers are subjected to dis-infection pr

20、otocols and incubated for the specified time andconditions. Control samples are incubated under identicalconditions but are not exposed to the disinfection protocols.NOTE 1Carriers with incorporated microbicides do not receive anyadditional disinfection treatment. An untreated control is needed to a

21、ssessantimicrobial efficacy.4.1.3 Virus particles are eluted from the test and controlcarriers and viability is assessed by 50 % tissue cultureinfectious dose assay (log10TCID50).NOTE 2Nonviable techniques for viral enumeration such as poly-merase chain reaction (PCR) or hemagglutination cannot be u

22、sed.4.1.4 The virucidal activity of the decontamination proce-dure is determined from the log difference in viability betweentreated and test carriers.5. Significance and Use5.1 The efficacy of disinfection technologies can be evalu-ated on finished products, as well as on developmental items.5.2 Th

23、is test method defines procedures for validation of thedroplet generator, preparation of the test specimen, applicationof the challenge virus, enumeration of viable viruses, assessingdata quality, and calculation of decontamination efficiency.5.3 This test method provides defined procedures for crea

24、t-ing droplets that approximate those produced by humanrespiratory secretions, with particular emphasis on droplet sizedistribution and aerosolization media.5.4 Safety concerns associated with aerosolizing microbialagents are not addressed as part of this test method. Individualusers should consult

25、with their local safety authority, and adetailed biological aerosol safety plan and risk assessmentshould be conducted prior to using this method. Users areencouraged to consult the manual Biosafety in Microbiologicaland Biomedical Laboratories5published by the U.S. Centersfor Disease Control and Pr

26、evention (CDC).5.5 This test method differs from Test Methods E1052 andE2197 in the presentation of virus to the surface. The afore-mentioned test methods use liquid inoculum to contaminatecarrier surfaces, whereas this method presents the virus indroplets that are representative of human respirator

27、y secretions5.6 This method differs from Test Method E2720, because(1) larger droplets are being formed, (2) the droplets will not becompletely dried prior to application to surfaces, (3) thedroplets can be applied to any surfaces, not just those that areair permeable, and (4) unique equipment is re

28、quired to createdroplets.6. Apparatus6.1 Droplet ApparatusThe apparatus used to load micro-organisms onto a substrate is composed of several commer-cially available components and can be readily constructed.6,7,8The overall design of the apparatus can take various forms andcan be fashioned in differ

29、ent dimensions while meeting thevalidation requirements and data quality objectives listedbelow. Appendix X1 contains the description of a prototypicaldevice that can be used to load droplets onto surfaces.However, it is the responsibility of the user of this standard tovalidate the performance of t

30、he device prior to use.6.1.1 Validation requirements and baseline testing.6.1.1.1 Environmental ConditionsGenerator must be ca-pable of delivering air with a relative humidity of 50 6 10 %.6.1.1.2 Loading uniformity across the diameter of the testspecimen is required to ensure the even distribution

31、of the4Webster, R., Cox, N., Stohr, K. WHO Manual on Animal Influenza Diagnosisand Surveillance. World Health Organization, Department of Communicable Dis-ease Surveillance and Response. WHO/CDS/CDR/2002.5 Rev. 1.5CDC-NIH, Biosafety in Microbiological and Biomedical Laboratories , 5thEdition, U.S. D

32、epartment of Health and Human Services, Washington, D.C., 2009.6Vo, E., Rengasamy, S., Shaffer, R., “Development of a Test System to EvaluateDecontamination Procedures for Viral Droplets on Respirators.” Applied andEnvironmental Microbiology, Vol 75, No. 23, 2009, pp. 73037309.7Woo, M. H., Hsu, Y. M

33、., Wu, C. Y., Heimbuch, B. K., Wander, J. D., “ADevicefor a Consistent and Controlled Delivery of Aerosolized Droplets Containing ViralAgents Onto Surfaces.” Journal of Aerosol Science, Vol 41, 2010, pp. 941-952.8Heimbuch B. K., Wallace, W. H., Kinney, K., Lumley, A. E., Wu, C-Y, Woo,M-H, Wander, J.

34、 D., “A Pandemic Influenza Preparedness Study: Use of EnergeticMethods to Decontaminate Filtering Facepiece Respirators Contaminated withH1N1 Aerosols and Droplets,” American Journal of Infection Control, 2010, DOI10.1016/j.ajic.2010.07.004.E2721 102droplets over the surface of the carrier. A standa

35、rd deviation of60.5 log10TCID50is desired.6.1.1.3 Sample-to-Sample Variation ObjectiveThe vari-ability of virus loading for multiple samples loaded for a singletest must have a standard deviation of 60.5 log10TCID50.6.1.1.4 Droplet CharacteristicsThe droplets generated forthis method will have a num

36、ber median diameter (CMD) of15 6 5 m. The virus will be aerosolized in a saliva substitute(Table 1) that will add the appropriate “protective factors.”This method would be suitable for simulating other fluids ofinterest; however, if a different fluid is used, the formulationand recipe listing the pr

37、otective factors and droplet size mustbe reported.6.2 Other EquipmentThe list is specific for influenzavirus. Other equipment may be needed if a different virus isused.6.2.1 Autoclave, capable of maintaining 121 to 123C and15 to 17 lbs per in.2-gauge (psig).6.2.2 CO2Incubator, capable of maintaining

38、 35 to 37C and5 6 0.5 % CO2.6.2.3 Vortex Mixer.6.2.4 Analytical Balance, capable of weighing 0.001 g.6.2.5 Refrigerator, capable of maintaining 2 to 8C.6.2.6 Stopwatch or Electronic Timer.6.2.7 Pipettor, with a precision of 0.001 mL.7. Reagents and Materials7.1 ReagentsThe list is specific for influ

39、enza use. Otherreagents may be needed if a different virus is used.7.1.1 Influenzavirus (H1N1; A/PR/8/34)cell cultureadapted, ATCC VR-1469.7.1.1.1 The WHO Manual on Animal Influenza Diagnosisand Surveillance contains specific procedures for preparinginfluenza virus and titering samples. Appendix X2

40、also hasspecific information on titrating viable influenza viruses. Otherviruses may be used, but conditions for propagation andenumeration are not provided in this method.7.1.2 MadinDarby Canine Kidney (MDCK) Cell Line,ATCC CRL-34.7.1.3 Artificial Saliva, see Table 1 in section 6.1.1.4.7.1.4 Minima

41、l Essential Medium With Earles BalancedSalts (EMEM).7.1.5 Heat-Inactivated Fetal Bovine Serum (45 min at56C).7.1.6 Penicillin/Streptomycin, 10 000 units penicillin and 10mg streptomycin per mL.7.1.7 L-Glutamine, 200 mM in 0.85 % NaCl.7.1.8 Crystal Violet.7.1.9 Glutaraldehyde.7.1.10 TPCKTrypsin.7.1.1

42、1 Phosphate Buffered Saline (PBS).7.1.12 Bovine Serum Albumin.7.1.13 TrypsinEDTA Solution0.05 % trypsin, 0.53 mMEDTA in Hanks balanced salts solution without sodium bicar-bonate, calcium, and magnesium.7.1.14 Distilled Water and Purified Water.7.1.15 Ethanol, laboratory grade.7.1.16 Bleach.7.2 Mater

43、ialsThe list is specific for influenza use. Otherreagents may be needed if a different virus is used.7.2.1 Tissue Culture Treated FlasksT-75, T-175, 12-well,and 96-well plates.7.2.2 Pipettes, 1, 5, 10, and 25 mL.7.2.3 Test Tube Rack.7.2.4 Micropipettes, capable of delivering 0.001 mL accu-rately and

44、 consistently.7.2.5 1.7-mL Sterile Microcentrifuge Tubes.7.2.6 15-mL Sterile Centrifuge Tubes.7.2.7 50-mL Sterile Centrifuge Tubes.7.2.8 Test Materials.8. Sampling, Test Specimens, and Test Units8.1 Cut test specimens from finished products or fromspecimens that can be documented as representative o

45、f finishedproducts. The configuration of the particular aerosol devicedictates the size and type of each specimen. Place specimensinto the droplet loader in the proper orientation. In some casesthe complete finished product may be used, which obviates theneed for cutting “coupons.”9. Experimental De

46、sign9.1 A minimum of three independent test and controlsamples must be evaluated so that fundamental statisticalanalysis of the data can be performed.10. Test Procedure10.1 Apparatus OperationAppendix X1 describes a drop-let loading device and details the standard protocols foroperation of the devic

47、e. General information that is indepen-dent of the droplet devices is listed below.10.2 Perform Neutralizer Effectiveness TestThe objectiveof this test is to determine whether toxic effects from thechemical or physical decontamination method have beenneutralized by the extraction buffer prior to vir

48、us enumeration.Treat a test specimen not exposed to virus with the decontami-nation procedure following the experimental protocol. Follow-ing the completion of the decontamination procedure, place thetest specimen in 10 mL of the extraction buffer and perform theextraction procedure following the ex

49、perimental protocol.Remove and discard the test specimen, then split the sampleinto two equal volumes. Set aside sample A as it will be usedto determine toxicity to the MDCK host cells. Add 10 L of aTABLE 1 Artificial SalivaReagent AmountMgCl27 H2O0.04gCaCl2H213NaHCO30.42 g0.2MKH2PO47.70 mL0.2MK2HPO412.3 mLNH4Cl 0.11 gKSCN 0.19 g(NH2)2CO 0.12 gNaCl 0.88 gKCl 1.04 gMucin 3.00 gDistilled water 1000 mLpH7E2721 103virus suspension of known titer (for example, 105TCID50permL) to sample B and incubate at room temperature (18 to24C) for a minimum o