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A disc-based quantitative carrier test method to assess the virucidal activity of chemical germicides
Journal of Virological Methods 112 (2003) 3 /12 www.elsevier.com/locate/jviromet
Protocol
A disc-based quantitative carrier test method to assess the virucidal activity of chemical germicides Syed A. Sattar *, V. Susan Springthorpe, Olusola Adegbunrin, A. Abu Zafer, Maria Busa Center for Research on Environmental Microbiology, University of Ottawa, Ottawa, Ont., Canada K1H8M5 Received 20 February 2003; received in revised form 10 June 2003; accepted 11 June 2003
Abstract Suspension tests for virucidal activity of chemical germicides are easier to perform, but they normally do not present the test product with a strong enough challenge. In contrast, carrier tests, where the test virus is dried on an animate or inanimate surface, offer the test formulation a higher level of challenge because it first has to penetrate successfully the inoculum to gain access to and inactivate the target organism on the carrier. Since pathogens in nature are normally found adsorbed to surfaces and/or embedded in organic or cellular debris, the results of carrier tests are more relevant to predicting the activity of chemical germicides under field situations. The method described below uses discs (1 cm in diameter) of brushed stainless steel discs as carriers. Ten ml of the test virus in a soil load is placed on each disc and the inoculum dried under ambient conditions. The dried inoculum is then exposed to 50 ml of the test formulation or a control solution for a defined contact time at the specified temperature. EBSS (0.95 ml) is added to each carrier holder to dilute/neutralize the germicide, the inoculum eluted and the eluates titrated in cell cultures to determine the degree of loss in virus viability. At least five test and three control carriers are used in each test. Controls are also included to test for toxicity of the test formulation to the host cells and any interference sub-cytotoxic levels of the formulation may have on the ability of the virus to infect the cells. The method has been used with several types of human and animal pathogenic viruses to test the activity of all major classes of chemical germicides against them. # 2003 Elsevier B.V. All rights reserved. Keywords: Virucides; Carrier test; Germicides; Metal discs; Non-enveloped viruses; Infection control
1. Background and introduction For environmental control of viruses, germicides are used mainly for the decontamination of medical devices, environmental surfaces and hands (Sattar, 2001; Sattar and Springthorpe, 1996a,b). Many chemicals used for hand antisepsis are quite different from those for the other two applications, and separate methods to assess their virucidal activity are available (Sattar and Ansari, 2002). Virucidal activity of chemical germicides is tested either in suspension (ASTM, 1996; U.S. EPA, 1981;
* Corresponding author. Tel.: /1-613-562-5800x8314; fax: /1-613562-5452. E-mail address: [email protected] (S.A. Sattar). 0166-0934/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0166-0934(03)00192-7
CEN, 2002) or carrier tests (ASTM, 1997, 2002; LloydEvans et al., 1986; Sattar and Springthorpe, 2001a,b). In suspension tests a known quantity of the test organism, with or without a soil load, is mixed with a larger volume of either the test germicide at its use dilution or a liquid (control) known to be harmless to the test virus(es). The mixtures are held for a defined contact time at a specified temperature, neutralized to stop virucidal activity, titrated for viable organisms, and the degree of loss in viability calculated (Springthorpe et al., 1986). Suspension tests are generally easier to perform but they normally do not present the test product with a strong enough challenge (Abad et al., 1997; Sattar and Springthorpe, 2001a). Since pathogens in nature are normally found adsorbed to surfaces and/or embedded in organic or cellular debris, the results of carrier tests
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are more relevant to predicting the activity of chemical germicides under field situations. With specific reference to the assessment of virucidal activity, comparative studies have found viruses more difficult to inactivate in carrier tests than in suspension tests (Sattar and Springthorpe, 2001b). Therefore, such methods are quite suitable for screening the activity of formulations under development but results based on them are of little significance in predicting the effectiveness of a given product under field conditions. Possible exceptions in this regard are products used to inactivate microbial suspensions to be made into vaccines. In the field, viruses must normally be inactivated on contaminated animate or inanimate surfaces. Therefore, in carrier tests the challenge organism is normally suspended in an appropriate soil load and a known volume of it dried on a representative surface. The dried inoculum is then exposed to the test product or a control solution for a defined contact time at the specified temperature. At the end of the contact time, the organism/germicide mixture is eluted from the surface of the carrier and the eluates titrated to determine the degree of loss in virus viability. Normally the potency of the product should be justified by meeting a defined performance criterion set by regulatory authorities. Unless a product is designed for use only in a specific apparatus, or under specified conditions, germicides are required to demonstrate potency at 20 8C. The contact time(s) are specified by the manufacturer but should be in a range that is appropriate for the practical intended use of the product. The potential of a given surface in the spread of viral infections will also depend on how often it is contacted, how readily it releases the infectious viruses it carries and what surface in the body of the host it makes contact with. This suggests that no one type of hard surface could represent the vast numbers of variations encountered under field use. Nevertheless, there are certain basic considerations in selecting a hard surface as the carrier material in testing the virucidal activity of chemical germicides; (1) it must not bind, adsorb or sequester the test virus such that virus elution from it becomes difficult, (2) its surface should not be too smooth; instead it should be reasonably uneven to simulate the topography of representative surfaces under in-use conditions, (3) if meant for reuse, it should withstand readily repeated decontamination and sterilization, (4) its surface should permit the deposition of the desired volume of the test virus as well as the test germicide and (5) the entire carrier should be submersible in a reasonably small volume of the eluent to allow efficient recovery of the virus without any wash-off as well as titration of most of the eluate. This last point is particularly problematic because many of the studies reported in the literature have carrier designs which facilitate, and handle the carriers
by such a means that, wash-off of viruses can occur during performance of the test method. In these instances, virus kill, virus wash-off and failure to recover the virus are all confounded in the data. Well-designed test methods will therefore take care to handle the carriers during performance of the tests in such a way that wash-off cannot occur. This is best done in a ‘closed’ system. Failure to kill the virus may simply result in relocating it elsewhere to a previously clean area. Also, wash-off may be more a function of the detergent content of the product than of the active ingredients, and will thus give a false evaluation, and potentially give different results between products with the same level of the same active germicidal chemicals but different detergents. To meet the above requirements, we use discs (1 cm diameter) of brushed stainless steel as carriers. The microtopography of its surface is sufficiently uneven (ridged) to provide some challenge to the virucide (Springthorpe and Sattar, 1990). The carriers are handled in a closed system so that wash-off cannot occur (ASTM, 2002; Springthorpe and Sattar, 2003). In nature, viruses are always present in an organic matrix. Even on precleaning of a surface or object enough of such ‘organic load’ or ‘soil’ remains and can interfere with the activity of a germicidal chemical by either binding to it or by preventing its access to the target virus. Any good test for virucidal activity must, therefore, simulate the presence of such soil by incorporating in the virus suspension a certain amount of organic and inorganic material, and this is now a requirement in several standard protocols (ASTM, 1996; CGSB, 1997). In practice, there are wide variations in the nature and levels of substances used and, as far as we are aware, no substance or a combination thereof can be regarded as a universal soil load. Bovine serum (5 /10%) is commonly used for this purpose, but it is relatively expensive, not readily available and may contain specific or non-specific virus inhibitors. While substances such as feces (Mbithi et al., 1990) have been used in testing the virucidal activity of chemical germicides, they are inherently variable and thus unsuitable as a soil load for standardized test protocols. In view of the above, we have developed a soil load that is a mixture of bovine mucin, peptides (tryptone) and a high molecular weight protein (bovine albumin). The concentrations of the three ingredients are designed to provide a challenge approximately equivalent to that in 5/10% serum (ASTM, 2002; Sattar and Springthorpe, 2001a). 1.1. Purpose and significance of the protocol The selection of the test viruses for this method is based on their (a) relative safety for the laboratory staff, (b) ability to grow to titers sufficiently high for testing,
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(c) property to produce cytopathic effects or plaques, or both, in cell cultures, (d) potential to spread through contaminated environmental surfaces and medical devices, and (e) relatively high resistance to a variety of chemical germicides. With these considerations in mind, all viruses included here are non-enveloped. As a general rule, enveloped viruses do not survive as well on environmental surfaces and on medical devices and are also more susceptible to chemical germicides (Sattar and Springthorpe, 2001a). Other strains or types of non-enveloped viruses may be substituted provided they meet the preceding criteria. Depending on regulatory agency and types of claims to be made, testing against two or more of the following viruses may be needed. Although polioviruses are often used for testing virucidal activity of germicides, they are not are included here because the World Health Organization is anticipated to restrict their use following the eradication of poliomyelitis. Work with vertebrate viruses requires special skills and disinfectant testing against them is also more complicated by the use of cell cultures for their cultivation and quantitation */this entails the use of additional controls (see below) to ensure that any residual effects from the disinfectant on the cells, as well as on the test virus, do not interfere with virus detection by the host cells (Table 1). Since methods for growing and titrating viruses for their infectivity vary widely, each laboratory performing virucidal testing should develop and document its own cell culture protocols and assay methods, including the controls specified below.
2. Equipment and supplies 2.1. Equipment The pieces of equipment listed below may either be already available in a standard microbiology laboratory or can be purchased readily from scientific supply houses. Air Displacement Pipettes */Eppendorf or equivalent, 100 /1000 ml with disposable tips. Analytical balance */set up to weigh small volumes of liquids-used to calibrate micropipettes and tips. Dissecting microscope */to examine cleanliness of disc carriers. Environmental Chamber/Incubator */To hold the carriers at the desired test temperature. Filter sterilization system : A membrane or cartridge filtration system (0.22-mm pore diameter; Millipore, Bedford, MA) is required for sterilizing heat-sensitive media and solutions. Freezers : A freezer at /209/2 8C is required for the storage of fetal bovine serum (FBS) and other
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additives for cell culture media. A second freezer at /70 8C or lower is required to store viruses. Hemocytometer : For counting cells. Hot Air Oven */An oven at 60 8C to dry glassware. Incubator: An incubator at 369/1 8C is needed for growing host cells and for incubating virus-infected cultures. If an open system is used for cell culture, a CO2 incubator will be required. Work with rhinoviruses will require an incubator at 339/1 8C. Laminar Flow Cabinet */A Class II (Type A) biological safety cabinet. Liquid Nitrogen Storage for Cells : Liquid nitrogen container and liquid nitrogen for cryopreservation of the stocks of cell lines. Magnetic Stir Plate and Stir Bars */Large enough for a 5-l beaker or Erlenmeyer flask for preparing culture media or other solutions. Microscope : An inverted microscope with 10 / eyepieces and 5/, 10 /, and 40/ objectives. pH meter */used to measure pH of buffers or disinfectant prior to application Positive Displacement Pipette */A pipette and pipette tips that can accurately dispense 10 ml volumes for inoculation of carriers. Refrigerator */A refrigerator at 49/2 8C for storage of prepared cell culture media and reagents. Sterile Dispenser */10 ml, for dispensing diluent/ eluent. Sterilizer */Any steam sterilizer suitable for processing culture media, reagents and labware. The steam supplied to the sterilizer must be free from additives toxic to the test organisms. Timer */Any stopwatch that can be set and read in minutes and seconds. Vortex Mixer */To vortex the eluate and rinsing fluid in the carrier to ensure efficient recovery of the test organism(s). Water bath */To hold containers with agar overlay at 45 8C. 2.2. Supplies Basic supplies such as plasticware as well as cell culture media and supplements listed below are available from a variety of commercial sources. Therefore, specific details are being given only for less commonly used items and which are considered crucial to the successful performance of the protocol described. Cell Culture Flasks: Plastic cell culture flasks of 25 or 75-cm2 capacities for culturing cells and for preparing virus pools. Cell culture Plates : 12-well cell culture plates for plaques assays. Cell Culture Media and Supplements : Culture media and the types and ratios of supplements will vary
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depending on the cell line. Eagle’s minimal essential medium (EMEM) with 5 /10% FBS is used for growing a wide variety of cells. Diluent for virus titration : EBSS with a pH of 7.2 /7.4. Eluent for virus recovery from carriers: EBSS (pH 7.2 /7.4). Flat-bottomed Teflon vials */15 ml: Catalogue # PK08936-30, Cole Parmer, Vernon Hills, Illinois; as a holder for the stainless steel discs. Miscellaneous Laboratory Ware : Automatic pipettes, pipette tips, plastic vials for storing cell and virus stocks, dilution tubes. Plastic Vials : Sterile screw-capped 2.0-ml plastic vials (cat. no.72.694.006; Sarstedt Inc., St. Laurent, Quebec, Canada) will be required for holding eluates from test and control carriers. Serological Pipettes : Sterile reusable or single-use pipettes of 10.0, 5.0, and 1.0-ml capacity. Soil Load : Either FBS, at a final concentration of 5% in the virus inoculum, or the following tripartite soil load can be used. Add 0.5 g of tryptone (Difco, Detroit, MI) to 10 ml of phosphate buffer. Add 0.5 g of bovine serum albumin (BSA; Sigma) to 10 ml of phosphate buffer. Add 0.04 g of bovine mucin (Sigma) to 10 ml of phosphate buffer. Prepare the stock solutions separately and sterilize by passage through a 0.22 mm pore diameter membrane filter, aliquot and store at either 49/2 or /209/2 8C. To obtain a 500-ml inoculum of the test inoculum, add to 340 ml of the viral suspension 25 ml BSA, 100 ml mucin and 35 ml of tryptone stock solutions. This mixture contains approximately 2 g of total protein/l, which is approximately equivalent to the protein content of a 5% solution of FBS (Sattar and Springthorpe, 2001a,b). Standard Hard Water : The quality and disinfectant (for example, chlorine) residual in tap water can vary from site to site and also at different times at the same site. The use of standard hard water, therefore, is recommended here to avoid variations in results due to differences in tap water quality. Water prepared in accordance with AOAC 960.09 E and F (AOAC International, 1990) to a standard hardness of 400 ppm as calcium carbonate (CaCO3) is used for dilution of test products. 2.3. Test viruses and cell cultures The selection of the following test viruses is based on the criteria given in Section 1.1 above. Other strains or types of viruses may be substituted provided they meet the preceding criteria. For the test viruses listed, we provide one reference which outlines the methods used successfully in the authors’ laboratory. Other published methods may be equally or more suitable in other cases. Each laboratory
performing virucidal testing must therefore develop and document its own specific standard operating procedure for each test virus used. Regardless of the methods used to prepare virus pools, or to quantitate the virus, the means of exposing virus to the germicide and the controls needed for virus assays (see below) should remain the same as specified in this paper. It should be noted however that not all methods would give the same level of sensitivity or reproducibility. 2.3.1. Human adenovirus type 4 (ATCC VR-4) Human adenoviruses cause enteric, respiratory and ocular infections. These viruses are considered to be of intermediate resistance to disinfectants. The viruses themselves are relatively sensitive to drying and somewhat sensitive to mechanical damage. This strain is considered relatively safe to work with. Recommended lines for making virus pools, and for carrying out infectivity titrations are 293 (CRL-1573) and Vero (ATCC CCL-81) cells, respectively. This does not preclude the use of other systems able to produce high titred virus pools or to quantitate the virus. For further details, see Sattar et al. (2000). 2.3.2. Hepatitis A virus strain HM-175 (ATCC VR1402) Hepatitis A virus causes infectious hepatitis and is often spread through contact with feces or through contaminated food and water. The viruses are relatively resistant to drying and mechanical damage and are among the viruses most resistant to germicides. There is now a safe and effective vaccine available against hepatitis A virus. The recommended cell line for making virus pools and for performing infectivity titrations is FRhK-4 (ATCC CRL-1688). For further details, see Mbithi et al. (1991). 2.3.3. Feline calicivirus strain F9 (ATCC VR-782) Feline calicivirus is a pathogen of cats but is harmless to humans. It belongs to a group of small round viruses, which show a higher resistance to many germicides. FCV is closely related to the Norwalk virus (NV), an important cause of acute gastroenteritis in humans. Since NV cannot be grown in the lab, FCV has been proposed as its surrogate (Doultree et al., 1999). The recommended cell line for the FCV is CrFK (ATCC CCL-94). For further details, see Bidawid et al. (2003) and Doultree et al. (1999). 2.3.4. Canine parvovirus cornell strain (ATCC VR2017) Canine parvovirus is highly infectious in dogs but is considered harmless to humans. Parvoviruses are small round viruses and among the most resistant to many germicides.
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Table 1 Important factors and precautions in the design and performance of the quantitative carrier test for virucidal activity Factor Infectivity assay
Comments and precautions
A reliable and sensitive cell culture system for virus infectivity assays is essential and the use of animals and indirect means for testing virus infectivity are not recommended. Every effort should be made to titrate as much of the eluate as possible, especially when low levels of infective units are expected. Report limit of detection of assay system and when no infectious virus is detected, the results should be quoted as ‘less than’ the detection limit. Test virus(es) to be used As yet, there are no generally recognized surrogates for testing against viruses. The viruses listed in this protocol have been carefully selected for their safety to laboratory workers and panelists, ease of handling, relative resistance to chemical germicides as well as their potential for spread by environmental surfaces. Caution must be exercised in substituting them with other viruses, especially those without properly documented culture and passage history. Nature and level of soil loading In nature, viruses are always shed in an organic matrix and such ‘organic or soil load’ can interfere with the activity of a germicidal chemical by either interacting with it or reducing its effective concentration, or by preventing its access to the target virus through physical protection. Bovine serum (5 /10%), often used for this purpose, is relatively expensive, not readily available, and may be inhibitory to viruses such as rotaviruses. The tripartite soil load to be used in this protocol is suitable for working with a variety of viruses and other test organisms. It consists of mucin in combination with low molecular weight (peptides) and high molecular weight protein (albumin) mixture. The concentrations are designed to provide a challenge approximately equivalent to 5 /10% serum in testing for virucidal or other types of germicidal activity. Diluent, if required, for the test Unless stated otherwise on the product label, tap water is normally used to dilute germicides in the field. product However, it varies both geographically and temporally and therefore not suitable for use as a diluent in a standard test protocol. Distilled water, on the other hand, may not be reflective of field situations. In view of this, water with a standard level of hardness in it (e.g. 200 /400 ppm CaCO3) is a more desirable diluent in tests for virucidal activity. Time used for the initial drying of the Since test viruses can vary widely in their ability to survive on environmental surfaces and as the amount of inoculum infectious virus in baseline controls is crucial to determine the activity of control and test products against it, care must be taken not only to randomize the carriers in the test but also to stagger their inoculation, if necessary, to avoid false-positive or false-negative results. Contact between virus and germicide In the field, the contact time between a germicide and the surface being disinfected is 1 /3 min. Therefore, the test protocol must incorporate contact times to reflect this because longer contact times could overestimate the activity of the test product. Neutralization of virucidal activity Arresting the virucidal activity of the test formulation immediately at the end the contact time is crucial to generate meaningful results. This can be achieved by either the addition of a neutralizer or by dilution of the virus /germicide mixture, or by a combination of both. Whichever approach is adopted, its effectiveness must be properly validated before the test results can be accepted. Any toxicity of the test formulation for the cell culture system can seriously interfere with the interpretation Procedure for elimination of cytotoxicity of results. Such cytotoxicity does not always lead to readily detectable cell degeneration because apparently undamaged cell monolayers may be unable to support virus replication. Moreover, germicides with fixative properties can effectively kill host cells without detaching them or producing any apparent damage to them. Gel filtration or centrifugation of virus /germicide mixtures may be effective in the removal of cytotoxicity but such steps invariably extend the contact of the virus with the test germicide, and put into question the accuracy and relevance of claims for virucidal activity of environmental surface disinfectants. A 20 /100-fold dilution of the virus /germicide mixture at the end of the contact time is one simple and potentially widely applicable approach to reducing cytotoxicity. Product lots For greater confidence in the results it is recommended that at least two lots of the test formulation be evaluated. Product performance criterion Product performance criteria are normally set by regulatory agencies and they may differ from one jurisdiction to another. However, the true relationship between the germicidal activity of a product and its ability to prevent the spread of infections in the field is not known and remains difficult to determine. Therefore, performance criteria for potency testing of germicidal chemicals are a matter of policy and practicality rather than based on sound public health science. In tests with bacteria, it is generally feasible to measure viability reductions of 5 /6 log10. With viruses, it is more usual to aim for a 2 /4 log10 reductions in infectivity titer on hard surfaces. Some regulatory agencies also specify that, in addition to the specified level of log10 reduction in infectivity titer, no infectious virus be detectable in the highest dilution of the virus / germicide mixture titrated. Additional controls In addition to the usual cell culture controls, additional controls must be included to determine that germicide residues in the eluates do not have a negative or positive effect on the infectivity of the test virus.
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The recommended cell line for the canine parvovirus is A72 (ATCC CRL-1542). For further details, see Carmichael et al. (1981). 2.3.5. Human rhinovirus type 37 (ATCC VR-1147) or type 14 (ATCC VR-284) Rhinoviruses are major causes of the common cold. They are small viruses that are relatively sensitive to drying and acids, but somewhat more resistant to mechanical damage and many germicides. Recommended cell line MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75) or HeLa T4 cells. (Incubate infected cells at 33 8C for optimal virus replication). For further details, see Sattar et al., (2000). 2.4. Human rotavirus-Wa strain (ATCC VR-2018) Rotaviruses cause acute gastroenteritis in humans and a wide variety of animals and are often spread through contact with feces or through contaminated food and water. Almost 100% of adults have immunity to the Type A rotaviruses that include the test strain proposed. Recommended cell lines are MA-104 (CRL-2378) and CV-1 (ATCC CCL-70). For further details, see Sattar et al. (2000). Note: Prior to rotavirus inoculation, cell monolayers must be washed gently at least twice with EBSS to remove the serum from the growth medium. All diluents, maintenance media, and agar overlays also must be free from serum. Most rotaviruses also require the presence of trypsin in the medium for growth and infectivity assays. Prior determination of the appropriate trypsin concentration is needed due to wide variations in its levels in commercial preparations. The following is a general guide to preparing suitable virus pools for this test: a) Remove growth medium from cell culture vessel, wash the monolayer with EBSS as necessary and inoculate with 100 ml of thawed virus suspension. b) Allow the inoculum to remain in contact with the cells for 60/90 min. c) Add maintenance medium (EMEM with or without serum) and incubate flasks until about 75% of the monolayer shows virus-induced cytopathology. d) Freeze (/20 8C) and thaw (room temperature) the contents of the flask at least two times to release virus from infected cells. e) Centrifuge the contents of the flask at 1000 /g for 10 min and collect supernatant, which may require ultra-centrifugation or other means to concentrate the virus in it. Note: Ultracentrifugation may sometimes be needed to increase the virus titer. However use of highly purified virus pools is not recommended for testing germicides
because such purification is likely to enhance susceptibility of the virions to chemicals. Table 1. 1) Procedure (see also the Appendix A). a) Preparation of the Carriers. b) Place a sheet of filter paper on the inside bottom surface of a glass Petri dish (150 mm) and lay out up to 20 clean discs on it. 2) Sterilize the discs by autoclaving. a) Inoculation of the Carriers. b) Add soil load to virus suspension in the ratios specified above and vortex the mixture for even distribution of the contents. c) Withdraw 10 ml of the suspension with a positive displacement pipette and place it at the center of a disc carrier. For consistency, the same pipette tip can be used throughout the inoculation of a batch of carriers. d) Allow the inoculum to dry for 30/40 min under ambient conditions. e) Observe the dried inoculum on each carrier and discard any carrier in which the inoculum has run off the surface of the disc. f) Carefully pick up each disc and place it, with the inoculated side up, on the inside bottom surface of a Teflon vial. These carriers are now ready for the test procedure. 3) Exposure of the virus to the formulation under test. a) Cover the dried inoculum on each disc carrier with 50 ml of test formulation and hold the carriers at the desired temperature for the recommended contact period. b) At the end of contact time, add 0.95 ml of EBSS, with or without a neutralizer. c) Vortex the contents of the vial for 45/60 s to elute the inoculum. d) Examine each disc to ensure that the inoculum has been eluted successfully from it. Further vortexing may be needed to achieve complete elution. Sonication may be used to dislodge the inoculum from the discs as long as it produces no damage to the viability of the virus particles in the eluate. e) Proceed with the serial dilutions of the eluate. f) Inoculate serial dilutions of control and test vials into at least three monolayers each. g) Allow the viruses to adsorb to the monolayers. h) Add semisolid or liquid overlay and incubate at the required temperature. i) Record the number of infective units or plaques and calculate log10 reductions. 2.4.1. Control carriers The minimum number of control carriers in each test is three, regardless of the number of test carriers. Instead of the test formulation, add 50 ml of EBSS to each
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control carrier. The contact time and temperature for the control carriers must be the same as that for the test carriers. 2.5. Additional controls in virucidal tests The need for cell cultures when working with viruses requires the incorporation of the following additional controls in tests for virucidal activity because either the test substance or the neutralizer or a combination of both could alter the susceptibility of host cells to the virus under test. These controls must be run initially at least once and may not need to be included in subsequent tests as long as the same cell line, virus, test formulation, neutralizer and method are being used for testing. 2.5.1. Cytotoxicity control The objective of this control is to (a) determine the dilution of the test substance that causes no apparent degeneration (cytotoxicity) of the cell line to be used for measuring virus infectivity, and (b) assess if the neutralizer in any way reduces or enhances such cytoxicity. Make an initial 1:20 dilution and one further 10-folddilution of the use-dilution of the germicide in EBSS with and without the neutralizer, if any. Remove the culture medium from the monolayers of the host cell line(s) and put into each test monolayer separately the same volume of inoculum as used in virus titration; control monolayers receive an equivalent amount of EBSS (without any neutralizer) only. Hold the cultures for 30 min at the temperature normally used for the adsorption of the test virus to host cells and then examine the cells under an inverted microscope for any visible cytotoxicity. In case of cytotoxicity, a different neutralizer or alternative approaches to the removal/reduction of cytotoxicity may be needed. Sometimes it is advisable to use gel filtration to remove the disinfectant, although this procedure may lengthen the exposure time of the test organism to the disinfectant (ASTM, 1998). If no cytotoxicity is observed at either one of the dilutions, the test substance and the neutralizer should be subjected to the following interference test. 2.5.2. Control for interference with virus infectivity Levels of the test substance which show no obvious cytotoxicity could still reduce or enhance the ability of the challenge virus to infect or replicate in host cells, thus interfering with the estimation of its virucidal activity. An interference control must, therefore, be included to rule out such a possibility. Remove the culture medium from the host cell monolayers and inoculate each one of the test monolayers with a 1:20 dilution of the test substance in EBSS, or the dilution greater than the one that demonstrated
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cytotoxicity, with and without neutralizer, using the same volume as that of the inoculum used in virus titration. Controls receive EBSS alone (without the neutralizer). Hold the monolayers for 30 min at the temperature normally used for the adsorption of the test virus to host cells and then inoculate each monolayer with a countable number of infective units of the challenge virus. Incubate the monolayers for virus adsorption, place maintenance medium in the cultures, incubate them for the time required for virus replication and examine them for cytopathology or foci of virus infection. Any significant difference in virus infectivity titer is indicative of the ability of the test material or the neutralizer to affect the virus susceptibility of the host cells. In such a case, a different neutralizer or alternative approaches to the removal of the residues of the test product in the samples to be titrated for virus infectivity may be needed. In case of virus assays done using TCID50, the entire assay will need to be carried out with and without prior exposure of the monolayers to a 1:20 dilution of the test substance in EBSS, or the dilution just above the one that demonstrated cytotoxicity, with and without neutralizer, using the same volume as that of the inoculum used in virus titration.
3. Experimental and results The method described has been used successfully in working with all the viruses listed above (Sattar and Springthorpe, 2001b) and with the Sabin vaccine strain of poliovirus type 1 (Sattar et al., 1998). The results presented here are more recent examples of tests with a variety of chemical germicides against a human rotavirus. In all these tests, the virus was suspended in the soil load described, the test germicides were diluted in sterile water with a hardness of 400 ppm Ca2CO3 and the contact temperature was 20 8C. Domestic bleach, which contains about 5.25% sodium hypochlorite (52 500 ppm available chlorine), was tested at chlorine concentrations ranging from 500 to 5250 ppm. Rotavirus-contaminated stainless steel discs were exposed to 50 ml of the test solution and the discs were held for 10 min at 20 8C. Control discs received an equivalent volume of EBSS. At the end of the contact time, each disc was eluted in 950 ml of EBSS containing 0.1% sodium thiosulfate. The eluates were diluted and plaque assayed in MA-104 cell monolayers. As shown in Fig. 1, it required nearly 2000 ppm of chlorine to show a 3 log10 reduction in the infectivity titre of the virus, while about 3000 ppm of chlorine was needed to reduce the virus titre by 6 log10. When a high-level disinfectant based on ortho -phthalaldehyde was tested (Fig. 2) with a contact time of 5
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Fig. 1. Activity of sodium hypochlorite (bleach) against a human rotavirus as determined in the disc-based quantitative carrier test.
min, it showed a 6 log10 reduction in rotavirus infectivity even at 1000 ppm, which is /5-fold lower than its recommended in-use concentration of 5500 ppm. Since the product is sold for reuse in the decontamination of semi-critical medical devices, it would be expected to retain strong virucidal activity even after several days of reuse. The results obtained with the phenolic used in this study (Fig. 3) showed very little kill at the use-dilution (775 ppm). However, because we wanted to see if the classic kill response curve would hold, and it was
possible to do so, we increased the phenolic concentration until a response was obtained against the rotavirus. This was only obtained at many times the recommended use-dilution.
4. Discussion The results presented here are just an illustration of how the method described can be applied to test a variety of chemical germicides for their virucidal activ-
Fig. 2. The effect of an ortho -phthalaldehyde-based germicide against a human rotavirus as determined in the disc-based quantitative carrier test.
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Fig. 3. The effect of a phenolic-based germicide against a human rotavirus as determined in the disc-based quantitative carrier test.
ity. It can not only give good dose /response curves but can also distinguish between formulations with weak and strong activity against a given test virus. In the results reported here the virus titre was deliberately kept higher than 6 log10 to show that the test method is capable of testing germicides if the product performance criterion calls for a ]/million-fold reduction in the viability of the test virus. However, the test method is equally capable of working with lower starting titres of the test virus to allow testing for product performance criteria of 3 /4 log10. Standardization of virucide tests, nationally and internationally, will promote confidence among disinfectant users and the general public. Anyone reading the literature will realize that there are almost as many published virucide tests as there are authors of papers on virucides. In this paper, we have made no attempt to detail the similarities and differences in these publications, or to critique them. What we have tried to do is provide the basis for a general understanding of the potential pitfalls in testing virucides, and suggest the basic protocols and controls that should be present in generic methods. We hope this will allow the reader to understand better this field and to be able to critique the published literature independently. Perhaps what is needed most of all are decisions by policy makers */the acceptance of surrogate virus(es) to permit a general virucidal claim, and a defined performance standard. Some jurisdictions already have one or both of these in place (CGSB, 1997). We have made some suggestions regarding the former, but much debate is still required in this area. The major advance that is perhaps required here is to accept that any product with
a virucide claim must be able to kill a wide range of nonenveloped viruses. Many products currently on the market list only enveloped viruses among the organisms on the label. This can easily mislead persons unfamiliar with virus classification, especially if the enveloped viruses listed are among those most feared, such as HIV and HBV. Under the current stage of knowledge and approaches to risk assessment it is not possible to determine with any degree of certainly the desired level of reduction in the virus load in a given setting to significantly reduce disease transmission. There are also obvious practical limitations to how high a level of challenge virus(es) one can present to the product under evaluation. By the same token, what would one regard as too low a level of challenge? Experience accumulated over the past two decades clearly indicates that, if test viruses are chosen carefully, it is feasible to determine a 3/4 log10 reduction in virus infectivity titer after its exposure to a test germicide in a proper carrier test.
Appendix A: Main steps in the quantitative carrier test for virucidal activity Step
Time (min) Inoculate each carrier with 10 ml of test virus, 50 allow inoculum to dry and then place one carrier each in a Teflon vial with the inoculated side up ¡/
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Place 50 ml of germicide on 3/5 test carriers; place an equivalent volume of EBSS on each of at least 3 control carriers; hold carriers for required contact time ¡/ Add 950 ml of EBSS, with/without a neutralizer, to each disc carrier ¡/ Elute the inoculum from the carriers ¡/ Subject eluates to 10-fold dilutions in EBSS as necessary ¡/ Inoculate samples onto cell monolayers and incubate for virus adsorption ¡/ Examine the cultures and determine the log10 reduction in the viability titer of the test organism in relation to control carriers ¡/ Determine if the test formulation meets the specified performance criterion
5/15
2
5/10 10
70/100
30/60
10
References Abad, F.X., Pinto, R.M., Bosch, A., 1997. Disinfection of human enteric viruses on fomites. FEMS Microbiol. Lett. 156, 107 /111. AOAC International (1990). Official methods of analysis. AOAC, Washington, D.C., USA. ASTM International, 1996. Standard Test Method for Efficacy of Antimicrobial Agents against Viruses in Suspension, Designation E1052-96, West Conshohocken, PA. ASTM International, 1997. Standard Test Method for Efficacy of Virucidal Agents Intended for Inanimate Environmental Surfaces, Designation E1053-97, West Conshohocken, PA. ASTM International, 1998. Standard Test Method for Neutralization of Virucidal Agents in Virucidal Efficacy Evaluations, Designation E1482-92 (1998), West Conshohocken, PA. ASTM International, 2002. Standard Quantitative Disk Carrier Test Method for Determining the Bactericidal, Virucidal, Fungicidal, Mycobactericidal and Sporicidal Activities of Liquid Chemical Germicides, Designation E2197-02, West Conshohocken, PA. Bidawid, S., N. Malik, N., Adegbunrin, O, Sattar, S.A., Farber, J.M., 2003. Feline kidney cell line-based plaque assay for feline calicivirus, a surrogate for Norwalk virus. J. Virol. Methods. 107, 163 / 167. Canadian General Standards Board (CGSB), 1997. Assessment of the efficacy of antimicrobial agents for use on environmental surfaces and medical devices, National Standard of Canada #CAN/CGSB2.161.97, Canadian General Standards Board, Ottawa, Ont., Canada. Carmichael, L.E., Joubert, J.C., Pollock, R.V.H., 1981. A modified canine parvovirus strain with novel plaque characteristics. 1. Viral attenuation and dog response. Cornell Vet. 71, 408 /427. Comite´ Europe´en de Normalisation, 2002. Chemical disinfectants and antiseptics */Virucidal quantitative suspension test for chemical
disinfectants and antiseptics used in human medicine */Test method requirements (phase 2/step 1), Designation CEN TC 216/ WG 1 N 239, Paris, France. Doultree, J.C., Druce, J.D., Birch, C.J., Bowden, D.S., Marshall, J.A., 1999. Inactivation of feline calicivirus, a Norwalk virus surrogate. J. Hosp. Infect. 41, 51 /57. Lloyd-Evans, N., Springthorpe, V.S., Sattar, S.A., 1986. Chemical disinfection of human rotavirus-contaminated inanimate surfaces. J. Hyg. 97, 163 /173. Mbithi, J.N., Springthorpe, V.S., Sattar, S.A., 1990. Chemical disinfection of hepatitis A virus on environmental surfaces. Appl. Environ. Microbiol. 56, 3601 /3604. Mbithi, J.N., Springthorpe, V.S., Sattar, S.A., 1991. The effect of relative humidity and air temperature on the survival of hepatitis A virus on environmental surfaces. Appl. Environ. Microbiol. 57, 1394 /1399. Sattar, S.A., 2001. Survival of microorganisms on animate and inanimate surfaces and their disinfection. In: Rutala, W.A. (Ed.), Disinfection, Sterilization, and Antisepsis: Principles and Practices in Healthcare Facilities, Association of Practitioners in Infectitous Control (APIC), Washington, DC, pp. 195 /205. Sattar, S.A., Ansari, S.A., 2002. The fingerpad protocol to assess hygienic hand antiseptics against viruses. J. Virol. Methods 103, 171 /181. Sattar, S.A., Springthorpe, V.S., 1996a. Transmission of viral infections through animate and inanimate surfaces and infection control through chemical disinfection. In: Hurst, C.J. (Ed.), Modeling Disease Transmission and its Prevention by Disinfection. Cambridge University Press, New York, NY, pp. 224 /257. Sattar, S.A., Springthorpe, V.S., 1996b. Environmental spread and germicide control of viruses in hospitals. Infect. Control Steriliz. Technol. 2, 30 /36. Sattar, S.A., Springthorpe, V.S., 2001a. Methods for testing the virucidal activity of chemicals. In: Block, S.S. (Ed.), Disinfection, Sterilization and Preservation, fifth ed.. Lippincott Williams & Wilkins, Philadelphia, PA, pp. 1391 /1412. Sattar, S.A., Springthorpe, V.S., 2001b. New methods for efficacy testing of disinfectants and antiseptics, In: Rutala, W.A. (Ed.), Disinfection, Sterilization, and Antisepsis: Principles and Practices in Healthcare Facilities, Association of Practitioners in Infectitous Control (APIC), Washington, DC, pp. 173 /186. Sattar, S.A., Springthorpe, V.S., Rochon, M., 1998. A product based on accelerated and stabilized hydrogen peroxide: evidence for broad-spectrum germicidal activity. Can J. Infect. Control 13 (4), 123 /130. Sattar, S.A., Abebe, M., Bueti, A.J., Jampani, H., Newman, J., Hua, S., 2000. Activity of an alcohol-based hand gel against human adeno-, rhino-, and rotaviruses using the fingerpad method. Infect. Control Hosp. Epidemiol. 21, 516 /519. Springthorpe, V.S., Sattar, S.A., 1990. Chemical disinfection of viruscontaminated surfaces. CRC Crit. Rev. Environ. Control 20, 169 / 229. Springthorpe, V.S., Sattar, S.A., 2003. Quantitative Carrier Test (QCT) to Assess the Germicidal Activities of Chemicals: Rationales and Procedures. ISBN 0-88927-298-0, Center for Research and Environment Microbiology, 451 Smyth Road, Ottawa, Ont., Canada. Springthorpe, V.S., Grenier, J.L., Lloyd-Evans, N., Sattar, S.A., 1986. Chemical disinfection of human rotaviruses: efficacy of commercially available products in suspension test. J. Hyg. 97, 139 /161. US Environmental Protection Agency, 1981. Efficacy Test Requirements: Virucides, Designation DIS/TSS-7/Nov, 12, 1981, Washington, DC.
Protocol
A disc-based quantitative carrier test method to assess the virucidal activity of chemical germicides Syed A. Sattar *, V. Susan Springthorpe, Olusola Adegbunrin, A. Abu Zafer, Maria Busa Center for Research on Environmental Microbiology, University of Ottawa, Ottawa, Ont., Canada K1H8M5 Received 20 February 2003; received in revised form 10 June 2003; accepted 11 June 2003
Abstract Suspension tests for virucidal activity of chemical germicides are easier to perform, but they normally do not present the test product with a strong enough challenge. In contrast, carrier tests, where the test virus is dried on an animate or inanimate surface, offer the test formulation a higher level of challenge because it first has to penetrate successfully the inoculum to gain access to and inactivate the target organism on the carrier. Since pathogens in nature are normally found adsorbed to surfaces and/or embedded in organic or cellular debris, the results of carrier tests are more relevant to predicting the activity of chemical germicides under field situations. The method described below uses discs (1 cm in diameter) of brushed stainless steel discs as carriers. Ten ml of the test virus in a soil load is placed on each disc and the inoculum dried under ambient conditions. The dried inoculum is then exposed to 50 ml of the test formulation or a control solution for a defined contact time at the specified temperature. EBSS (0.95 ml) is added to each carrier holder to dilute/neutralize the germicide, the inoculum eluted and the eluates titrated in cell cultures to determine the degree of loss in virus viability. At least five test and three control carriers are used in each test. Controls are also included to test for toxicity of the test formulation to the host cells and any interference sub-cytotoxic levels of the formulation may have on the ability of the virus to infect the cells. The method has been used with several types of human and animal pathogenic viruses to test the activity of all major classes of chemical germicides against them. # 2003 Elsevier B.V. All rights reserved. Keywords: Virucides; Carrier test; Germicides; Metal discs; Non-enveloped viruses; Infection control
1. Background and introduction For environmental control of viruses, germicides are used mainly for the decontamination of medical devices, environmental surfaces and hands (Sattar, 2001; Sattar and Springthorpe, 1996a,b). Many chemicals used for hand antisepsis are quite different from those for the other two applications, and separate methods to assess their virucidal activity are available (Sattar and Ansari, 2002). Virucidal activity of chemical germicides is tested either in suspension (ASTM, 1996; U.S. EPA, 1981;
* Corresponding author. Tel.: /1-613-562-5800x8314; fax: /1-613562-5452. E-mail address: [email protected] (S.A. Sattar). 0166-0934/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0166-0934(03)00192-7
CEN, 2002) or carrier tests (ASTM, 1997, 2002; LloydEvans et al., 1986; Sattar and Springthorpe, 2001a,b). In suspension tests a known quantity of the test organism, with or without a soil load, is mixed with a larger volume of either the test germicide at its use dilution or a liquid (control) known to be harmless to the test virus(es). The mixtures are held for a defined contact time at a specified temperature, neutralized to stop virucidal activity, titrated for viable organisms, and the degree of loss in viability calculated (Springthorpe et al., 1986). Suspension tests are generally easier to perform but they normally do not present the test product with a strong enough challenge (Abad et al., 1997; Sattar and Springthorpe, 2001a). Since pathogens in nature are normally found adsorbed to surfaces and/or embedded in organic or cellular debris, the results of carrier tests
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are more relevant to predicting the activity of chemical germicides under field situations. With specific reference to the assessment of virucidal activity, comparative studies have found viruses more difficult to inactivate in carrier tests than in suspension tests (Sattar and Springthorpe, 2001b). Therefore, such methods are quite suitable for screening the activity of formulations under development but results based on them are of little significance in predicting the effectiveness of a given product under field conditions. Possible exceptions in this regard are products used to inactivate microbial suspensions to be made into vaccines. In the field, viruses must normally be inactivated on contaminated animate or inanimate surfaces. Therefore, in carrier tests the challenge organism is normally suspended in an appropriate soil load and a known volume of it dried on a representative surface. The dried inoculum is then exposed to the test product or a control solution for a defined contact time at the specified temperature. At the end of the contact time, the organism/germicide mixture is eluted from the surface of the carrier and the eluates titrated to determine the degree of loss in virus viability. Normally the potency of the product should be justified by meeting a defined performance criterion set by regulatory authorities. Unless a product is designed for use only in a specific apparatus, or under specified conditions, germicides are required to demonstrate potency at 20 8C. The contact time(s) are specified by the manufacturer but should be in a range that is appropriate for the practical intended use of the product. The potential of a given surface in the spread of viral infections will also depend on how often it is contacted, how readily it releases the infectious viruses it carries and what surface in the body of the host it makes contact with. This suggests that no one type of hard surface could represent the vast numbers of variations encountered under field use. Nevertheless, there are certain basic considerations in selecting a hard surface as the carrier material in testing the virucidal activity of chemical germicides; (1) it must not bind, adsorb or sequester the test virus such that virus elution from it becomes difficult, (2) its surface should not be too smooth; instead it should be reasonably uneven to simulate the topography of representative surfaces under in-use conditions, (3) if meant for reuse, it should withstand readily repeated decontamination and sterilization, (4) its surface should permit the deposition of the desired volume of the test virus as well as the test germicide and (5) the entire carrier should be submersible in a reasonably small volume of the eluent to allow efficient recovery of the virus without any wash-off as well as titration of most of the eluate. This last point is particularly problematic because many of the studies reported in the literature have carrier designs which facilitate, and handle the carriers
by such a means that, wash-off of viruses can occur during performance of the test method. In these instances, virus kill, virus wash-off and failure to recover the virus are all confounded in the data. Well-designed test methods will therefore take care to handle the carriers during performance of the tests in such a way that wash-off cannot occur. This is best done in a ‘closed’ system. Failure to kill the virus may simply result in relocating it elsewhere to a previously clean area. Also, wash-off may be more a function of the detergent content of the product than of the active ingredients, and will thus give a false evaluation, and potentially give different results between products with the same level of the same active germicidal chemicals but different detergents. To meet the above requirements, we use discs (1 cm diameter) of brushed stainless steel as carriers. The microtopography of its surface is sufficiently uneven (ridged) to provide some challenge to the virucide (Springthorpe and Sattar, 1990). The carriers are handled in a closed system so that wash-off cannot occur (ASTM, 2002; Springthorpe and Sattar, 2003). In nature, viruses are always present in an organic matrix. Even on precleaning of a surface or object enough of such ‘organic load’ or ‘soil’ remains and can interfere with the activity of a germicidal chemical by either binding to it or by preventing its access to the target virus. Any good test for virucidal activity must, therefore, simulate the presence of such soil by incorporating in the virus suspension a certain amount of organic and inorganic material, and this is now a requirement in several standard protocols (ASTM, 1996; CGSB, 1997). In practice, there are wide variations in the nature and levels of substances used and, as far as we are aware, no substance or a combination thereof can be regarded as a universal soil load. Bovine serum (5 /10%) is commonly used for this purpose, but it is relatively expensive, not readily available and may contain specific or non-specific virus inhibitors. While substances such as feces (Mbithi et al., 1990) have been used in testing the virucidal activity of chemical germicides, they are inherently variable and thus unsuitable as a soil load for standardized test protocols. In view of the above, we have developed a soil load that is a mixture of bovine mucin, peptides (tryptone) and a high molecular weight protein (bovine albumin). The concentrations of the three ingredients are designed to provide a challenge approximately equivalent to that in 5/10% serum (ASTM, 2002; Sattar and Springthorpe, 2001a). 1.1. Purpose and significance of the protocol The selection of the test viruses for this method is based on their (a) relative safety for the laboratory staff, (b) ability to grow to titers sufficiently high for testing,
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(c) property to produce cytopathic effects or plaques, or both, in cell cultures, (d) potential to spread through contaminated environmental surfaces and medical devices, and (e) relatively high resistance to a variety of chemical germicides. With these considerations in mind, all viruses included here are non-enveloped. As a general rule, enveloped viruses do not survive as well on environmental surfaces and on medical devices and are also more susceptible to chemical germicides (Sattar and Springthorpe, 2001a). Other strains or types of non-enveloped viruses may be substituted provided they meet the preceding criteria. Depending on regulatory agency and types of claims to be made, testing against two or more of the following viruses may be needed. Although polioviruses are often used for testing virucidal activity of germicides, they are not are included here because the World Health Organization is anticipated to restrict their use following the eradication of poliomyelitis. Work with vertebrate viruses requires special skills and disinfectant testing against them is also more complicated by the use of cell cultures for their cultivation and quantitation */this entails the use of additional controls (see below) to ensure that any residual effects from the disinfectant on the cells, as well as on the test virus, do not interfere with virus detection by the host cells (Table 1). Since methods for growing and titrating viruses for their infectivity vary widely, each laboratory performing virucidal testing should develop and document its own cell culture protocols and assay methods, including the controls specified below.
2. Equipment and supplies 2.1. Equipment The pieces of equipment listed below may either be already available in a standard microbiology laboratory or can be purchased readily from scientific supply houses. Air Displacement Pipettes */Eppendorf or equivalent, 100 /1000 ml with disposable tips. Analytical balance */set up to weigh small volumes of liquids-used to calibrate micropipettes and tips. Dissecting microscope */to examine cleanliness of disc carriers. Environmental Chamber/Incubator */To hold the carriers at the desired test temperature. Filter sterilization system : A membrane or cartridge filtration system (0.22-mm pore diameter; Millipore, Bedford, MA) is required for sterilizing heat-sensitive media and solutions. Freezers : A freezer at /209/2 8C is required for the storage of fetal bovine serum (FBS) and other
5
additives for cell culture media. A second freezer at /70 8C or lower is required to store viruses. Hemocytometer : For counting cells. Hot Air Oven */An oven at 60 8C to dry glassware. Incubator: An incubator at 369/1 8C is needed for growing host cells and for incubating virus-infected cultures. If an open system is used for cell culture, a CO2 incubator will be required. Work with rhinoviruses will require an incubator at 339/1 8C. Laminar Flow Cabinet */A Class II (Type A) biological safety cabinet. Liquid Nitrogen Storage for Cells : Liquid nitrogen container and liquid nitrogen for cryopreservation of the stocks of cell lines. Magnetic Stir Plate and Stir Bars */Large enough for a 5-l beaker or Erlenmeyer flask for preparing culture media or other solutions. Microscope : An inverted microscope with 10 / eyepieces and 5/, 10 /, and 40/ objectives. pH meter */used to measure pH of buffers or disinfectant prior to application Positive Displacement Pipette */A pipette and pipette tips that can accurately dispense 10 ml volumes for inoculation of carriers. Refrigerator */A refrigerator at 49/2 8C for storage of prepared cell culture media and reagents. Sterile Dispenser */10 ml, for dispensing diluent/ eluent. Sterilizer */Any steam sterilizer suitable for processing culture media, reagents and labware. The steam supplied to the sterilizer must be free from additives toxic to the test organisms. Timer */Any stopwatch that can be set and read in minutes and seconds. Vortex Mixer */To vortex the eluate and rinsing fluid in the carrier to ensure efficient recovery of the test organism(s). Water bath */To hold containers with agar overlay at 45 8C. 2.2. Supplies Basic supplies such as plasticware as well as cell culture media and supplements listed below are available from a variety of commercial sources. Therefore, specific details are being given only for less commonly used items and which are considered crucial to the successful performance of the protocol described. Cell Culture Flasks: Plastic cell culture flasks of 25 or 75-cm2 capacities for culturing cells and for preparing virus pools. Cell culture Plates : 12-well cell culture plates for plaques assays. Cell Culture Media and Supplements : Culture media and the types and ratios of supplements will vary
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depending on the cell line. Eagle’s minimal essential medium (EMEM) with 5 /10% FBS is used for growing a wide variety of cells. Diluent for virus titration : EBSS with a pH of 7.2 /7.4. Eluent for virus recovery from carriers: EBSS (pH 7.2 /7.4). Flat-bottomed Teflon vials */15 ml: Catalogue # PK08936-30, Cole Parmer, Vernon Hills, Illinois; as a holder for the stainless steel discs. Miscellaneous Laboratory Ware : Automatic pipettes, pipette tips, plastic vials for storing cell and virus stocks, dilution tubes. Plastic Vials : Sterile screw-capped 2.0-ml plastic vials (cat. no.72.694.006; Sarstedt Inc., St. Laurent, Quebec, Canada) will be required for holding eluates from test and control carriers. Serological Pipettes : Sterile reusable or single-use pipettes of 10.0, 5.0, and 1.0-ml capacity. Soil Load : Either FBS, at a final concentration of 5% in the virus inoculum, or the following tripartite soil load can be used. Add 0.5 g of tryptone (Difco, Detroit, MI) to 10 ml of phosphate buffer. Add 0.5 g of bovine serum albumin (BSA; Sigma) to 10 ml of phosphate buffer. Add 0.04 g of bovine mucin (Sigma) to 10 ml of phosphate buffer. Prepare the stock solutions separately and sterilize by passage through a 0.22 mm pore diameter membrane filter, aliquot and store at either 49/2 or /209/2 8C. To obtain a 500-ml inoculum of the test inoculum, add to 340 ml of the viral suspension 25 ml BSA, 100 ml mucin and 35 ml of tryptone stock solutions. This mixture contains approximately 2 g of total protein/l, which is approximately equivalent to the protein content of a 5% solution of FBS (Sattar and Springthorpe, 2001a,b). Standard Hard Water : The quality and disinfectant (for example, chlorine) residual in tap water can vary from site to site and also at different times at the same site. The use of standard hard water, therefore, is recommended here to avoid variations in results due to differences in tap water quality. Water prepared in accordance with AOAC 960.09 E and F (AOAC International, 1990) to a standard hardness of 400 ppm as calcium carbonate (CaCO3) is used for dilution of test products. 2.3. Test viruses and cell cultures The selection of the following test viruses is based on the criteria given in Section 1.1 above. Other strains or types of viruses may be substituted provided they meet the preceding criteria. For the test viruses listed, we provide one reference which outlines the methods used successfully in the authors’ laboratory. Other published methods may be equally or more suitable in other cases. Each laboratory
performing virucidal testing must therefore develop and document its own specific standard operating procedure for each test virus used. Regardless of the methods used to prepare virus pools, or to quantitate the virus, the means of exposing virus to the germicide and the controls needed for virus assays (see below) should remain the same as specified in this paper. It should be noted however that not all methods would give the same level of sensitivity or reproducibility. 2.3.1. Human adenovirus type 4 (ATCC VR-4) Human adenoviruses cause enteric, respiratory and ocular infections. These viruses are considered to be of intermediate resistance to disinfectants. The viruses themselves are relatively sensitive to drying and somewhat sensitive to mechanical damage. This strain is considered relatively safe to work with. Recommended lines for making virus pools, and for carrying out infectivity titrations are 293 (CRL-1573) and Vero (ATCC CCL-81) cells, respectively. This does not preclude the use of other systems able to produce high titred virus pools or to quantitate the virus. For further details, see Sattar et al. (2000). 2.3.2. Hepatitis A virus strain HM-175 (ATCC VR1402) Hepatitis A virus causes infectious hepatitis and is often spread through contact with feces or through contaminated food and water. The viruses are relatively resistant to drying and mechanical damage and are among the viruses most resistant to germicides. There is now a safe and effective vaccine available against hepatitis A virus. The recommended cell line for making virus pools and for performing infectivity titrations is FRhK-4 (ATCC CRL-1688). For further details, see Mbithi et al. (1991). 2.3.3. Feline calicivirus strain F9 (ATCC VR-782) Feline calicivirus is a pathogen of cats but is harmless to humans. It belongs to a group of small round viruses, which show a higher resistance to many germicides. FCV is closely related to the Norwalk virus (NV), an important cause of acute gastroenteritis in humans. Since NV cannot be grown in the lab, FCV has been proposed as its surrogate (Doultree et al., 1999). The recommended cell line for the FCV is CrFK (ATCC CCL-94). For further details, see Bidawid et al. (2003) and Doultree et al. (1999). 2.3.4. Canine parvovirus cornell strain (ATCC VR2017) Canine parvovirus is highly infectious in dogs but is considered harmless to humans. Parvoviruses are small round viruses and among the most resistant to many germicides.
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Table 1 Important factors and precautions in the design and performance of the quantitative carrier test for virucidal activity Factor Infectivity assay
Comments and precautions
A reliable and sensitive cell culture system for virus infectivity assays is essential and the use of animals and indirect means for testing virus infectivity are not recommended. Every effort should be made to titrate as much of the eluate as possible, especially when low levels of infective units are expected. Report limit of detection of assay system and when no infectious virus is detected, the results should be quoted as ‘less than’ the detection limit. Test virus(es) to be used As yet, there are no generally recognized surrogates for testing against viruses. The viruses listed in this protocol have been carefully selected for their safety to laboratory workers and panelists, ease of handling, relative resistance to chemical germicides as well as their potential for spread by environmental surfaces. Caution must be exercised in substituting them with other viruses, especially those without properly documented culture and passage history. Nature and level of soil loading In nature, viruses are always shed in an organic matrix and such ‘organic or soil load’ can interfere with the activity of a germicidal chemical by either interacting with it or reducing its effective concentration, or by preventing its access to the target virus through physical protection. Bovine serum (5 /10%), often used for this purpose, is relatively expensive, not readily available, and may be inhibitory to viruses such as rotaviruses. The tripartite soil load to be used in this protocol is suitable for working with a variety of viruses and other test organisms. It consists of mucin in combination with low molecular weight (peptides) and high molecular weight protein (albumin) mixture. The concentrations are designed to provide a challenge approximately equivalent to 5 /10% serum in testing for virucidal or other types of germicidal activity. Diluent, if required, for the test Unless stated otherwise on the product label, tap water is normally used to dilute germicides in the field. product However, it varies both geographically and temporally and therefore not suitable for use as a diluent in a standard test protocol. Distilled water, on the other hand, may not be reflective of field situations. In view of this, water with a standard level of hardness in it (e.g. 200 /400 ppm CaCO3) is a more desirable diluent in tests for virucidal activity. Time used for the initial drying of the Since test viruses can vary widely in their ability to survive on environmental surfaces and as the amount of inoculum infectious virus in baseline controls is crucial to determine the activity of control and test products against it, care must be taken not only to randomize the carriers in the test but also to stagger their inoculation, if necessary, to avoid false-positive or false-negative results. Contact between virus and germicide In the field, the contact time between a germicide and the surface being disinfected is 1 /3 min. Therefore, the test protocol must incorporate contact times to reflect this because longer contact times could overestimate the activity of the test product. Neutralization of virucidal activity Arresting the virucidal activity of the test formulation immediately at the end the contact time is crucial to generate meaningful results. This can be achieved by either the addition of a neutralizer or by dilution of the virus /germicide mixture, or by a combination of both. Whichever approach is adopted, its effectiveness must be properly validated before the test results can be accepted. Any toxicity of the test formulation for the cell culture system can seriously interfere with the interpretation Procedure for elimination of cytotoxicity of results. Such cytotoxicity does not always lead to readily detectable cell degeneration because apparently undamaged cell monolayers may be unable to support virus replication. Moreover, germicides with fixative properties can effectively kill host cells without detaching them or producing any apparent damage to them. Gel filtration or centrifugation of virus /germicide mixtures may be effective in the removal of cytotoxicity but such steps invariably extend the contact of the virus with the test germicide, and put into question the accuracy and relevance of claims for virucidal activity of environmental surface disinfectants. A 20 /100-fold dilution of the virus /germicide mixture at the end of the contact time is one simple and potentially widely applicable approach to reducing cytotoxicity. Product lots For greater confidence in the results it is recommended that at least two lots of the test formulation be evaluated. Product performance criterion Product performance criteria are normally set by regulatory agencies and they may differ from one jurisdiction to another. However, the true relationship between the germicidal activity of a product and its ability to prevent the spread of infections in the field is not known and remains difficult to determine. Therefore, performance criteria for potency testing of germicidal chemicals are a matter of policy and practicality rather than based on sound public health science. In tests with bacteria, it is generally feasible to measure viability reductions of 5 /6 log10. With viruses, it is more usual to aim for a 2 /4 log10 reductions in infectivity titer on hard surfaces. Some regulatory agencies also specify that, in addition to the specified level of log10 reduction in infectivity titer, no infectious virus be detectable in the highest dilution of the virus / germicide mixture titrated. Additional controls In addition to the usual cell culture controls, additional controls must be included to determine that germicide residues in the eluates do not have a negative or positive effect on the infectivity of the test virus.
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The recommended cell line for the canine parvovirus is A72 (ATCC CRL-1542). For further details, see Carmichael et al. (1981). 2.3.5. Human rhinovirus type 37 (ATCC VR-1147) or type 14 (ATCC VR-284) Rhinoviruses are major causes of the common cold. They are small viruses that are relatively sensitive to drying and acids, but somewhat more resistant to mechanical damage and many germicides. Recommended cell line MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75) or HeLa T4 cells. (Incubate infected cells at 33 8C for optimal virus replication). For further details, see Sattar et al., (2000). 2.4. Human rotavirus-Wa strain (ATCC VR-2018) Rotaviruses cause acute gastroenteritis in humans and a wide variety of animals and are often spread through contact with feces or through contaminated food and water. Almost 100% of adults have immunity to the Type A rotaviruses that include the test strain proposed. Recommended cell lines are MA-104 (CRL-2378) and CV-1 (ATCC CCL-70). For further details, see Sattar et al. (2000). Note: Prior to rotavirus inoculation, cell monolayers must be washed gently at least twice with EBSS to remove the serum from the growth medium. All diluents, maintenance media, and agar overlays also must be free from serum. Most rotaviruses also require the presence of trypsin in the medium for growth and infectivity assays. Prior determination of the appropriate trypsin concentration is needed due to wide variations in its levels in commercial preparations. The following is a general guide to preparing suitable virus pools for this test: a) Remove growth medium from cell culture vessel, wash the monolayer with EBSS as necessary and inoculate with 100 ml of thawed virus suspension. b) Allow the inoculum to remain in contact with the cells for 60/90 min. c) Add maintenance medium (EMEM with or without serum) and incubate flasks until about 75% of the monolayer shows virus-induced cytopathology. d) Freeze (/20 8C) and thaw (room temperature) the contents of the flask at least two times to release virus from infected cells. e) Centrifuge the contents of the flask at 1000 /g for 10 min and collect supernatant, which may require ultra-centrifugation or other means to concentrate the virus in it. Note: Ultracentrifugation may sometimes be needed to increase the virus titer. However use of highly purified virus pools is not recommended for testing germicides
because such purification is likely to enhance susceptibility of the virions to chemicals. Table 1. 1) Procedure (see also the Appendix A). a) Preparation of the Carriers. b) Place a sheet of filter paper on the inside bottom surface of a glass Petri dish (150 mm) and lay out up to 20 clean discs on it. 2) Sterilize the discs by autoclaving. a) Inoculation of the Carriers. b) Add soil load to virus suspension in the ratios specified above and vortex the mixture for even distribution of the contents. c) Withdraw 10 ml of the suspension with a positive displacement pipette and place it at the center of a disc carrier. For consistency, the same pipette tip can be used throughout the inoculation of a batch of carriers. d) Allow the inoculum to dry for 30/40 min under ambient conditions. e) Observe the dried inoculum on each carrier and discard any carrier in which the inoculum has run off the surface of the disc. f) Carefully pick up each disc and place it, with the inoculated side up, on the inside bottom surface of a Teflon vial. These carriers are now ready for the test procedure. 3) Exposure of the virus to the formulation under test. a) Cover the dried inoculum on each disc carrier with 50 ml of test formulation and hold the carriers at the desired temperature for the recommended contact period. b) At the end of contact time, add 0.95 ml of EBSS, with or without a neutralizer. c) Vortex the contents of the vial for 45/60 s to elute the inoculum. d) Examine each disc to ensure that the inoculum has been eluted successfully from it. Further vortexing may be needed to achieve complete elution. Sonication may be used to dislodge the inoculum from the discs as long as it produces no damage to the viability of the virus particles in the eluate. e) Proceed with the serial dilutions of the eluate. f) Inoculate serial dilutions of control and test vials into at least three monolayers each. g) Allow the viruses to adsorb to the monolayers. h) Add semisolid or liquid overlay and incubate at the required temperature. i) Record the number of infective units or plaques and calculate log10 reductions. 2.4.1. Control carriers The minimum number of control carriers in each test is three, regardless of the number of test carriers. Instead of the test formulation, add 50 ml of EBSS to each
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control carrier. The contact time and temperature for the control carriers must be the same as that for the test carriers. 2.5. Additional controls in virucidal tests The need for cell cultures when working with viruses requires the incorporation of the following additional controls in tests for virucidal activity because either the test substance or the neutralizer or a combination of both could alter the susceptibility of host cells to the virus under test. These controls must be run initially at least once and may not need to be included in subsequent tests as long as the same cell line, virus, test formulation, neutralizer and method are being used for testing. 2.5.1. Cytotoxicity control The objective of this control is to (a) determine the dilution of the test substance that causes no apparent degeneration (cytotoxicity) of the cell line to be used for measuring virus infectivity, and (b) assess if the neutralizer in any way reduces or enhances such cytoxicity. Make an initial 1:20 dilution and one further 10-folddilution of the use-dilution of the germicide in EBSS with and without the neutralizer, if any. Remove the culture medium from the monolayers of the host cell line(s) and put into each test monolayer separately the same volume of inoculum as used in virus titration; control monolayers receive an equivalent amount of EBSS (without any neutralizer) only. Hold the cultures for 30 min at the temperature normally used for the adsorption of the test virus to host cells and then examine the cells under an inverted microscope for any visible cytotoxicity. In case of cytotoxicity, a different neutralizer or alternative approaches to the removal/reduction of cytotoxicity may be needed. Sometimes it is advisable to use gel filtration to remove the disinfectant, although this procedure may lengthen the exposure time of the test organism to the disinfectant (ASTM, 1998). If no cytotoxicity is observed at either one of the dilutions, the test substance and the neutralizer should be subjected to the following interference test. 2.5.2. Control for interference with virus infectivity Levels of the test substance which show no obvious cytotoxicity could still reduce or enhance the ability of the challenge virus to infect or replicate in host cells, thus interfering with the estimation of its virucidal activity. An interference control must, therefore, be included to rule out such a possibility. Remove the culture medium from the host cell monolayers and inoculate each one of the test monolayers with a 1:20 dilution of the test substance in EBSS, or the dilution greater than the one that demonstrated
9
cytotoxicity, with and without neutralizer, using the same volume as that of the inoculum used in virus titration. Controls receive EBSS alone (without the neutralizer). Hold the monolayers for 30 min at the temperature normally used for the adsorption of the test virus to host cells and then inoculate each monolayer with a countable number of infective units of the challenge virus. Incubate the monolayers for virus adsorption, place maintenance medium in the cultures, incubate them for the time required for virus replication and examine them for cytopathology or foci of virus infection. Any significant difference in virus infectivity titer is indicative of the ability of the test material or the neutralizer to affect the virus susceptibility of the host cells. In such a case, a different neutralizer or alternative approaches to the removal of the residues of the test product in the samples to be titrated for virus infectivity may be needed. In case of virus assays done using TCID50, the entire assay will need to be carried out with and without prior exposure of the monolayers to a 1:20 dilution of the test substance in EBSS, or the dilution just above the one that demonstrated cytotoxicity, with and without neutralizer, using the same volume as that of the inoculum used in virus titration.
3. Experimental and results The method described has been used successfully in working with all the viruses listed above (Sattar and Springthorpe, 2001b) and with the Sabin vaccine strain of poliovirus type 1 (Sattar et al., 1998). The results presented here are more recent examples of tests with a variety of chemical germicides against a human rotavirus. In all these tests, the virus was suspended in the soil load described, the test germicides were diluted in sterile water with a hardness of 400 ppm Ca2CO3 and the contact temperature was 20 8C. Domestic bleach, which contains about 5.25% sodium hypochlorite (52 500 ppm available chlorine), was tested at chlorine concentrations ranging from 500 to 5250 ppm. Rotavirus-contaminated stainless steel discs were exposed to 50 ml of the test solution and the discs were held for 10 min at 20 8C. Control discs received an equivalent volume of EBSS. At the end of the contact time, each disc was eluted in 950 ml of EBSS containing 0.1% sodium thiosulfate. The eluates were diluted and plaque assayed in MA-104 cell monolayers. As shown in Fig. 1, it required nearly 2000 ppm of chlorine to show a 3 log10 reduction in the infectivity titre of the virus, while about 3000 ppm of chlorine was needed to reduce the virus titre by 6 log10. When a high-level disinfectant based on ortho -phthalaldehyde was tested (Fig. 2) with a contact time of 5
10
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Fig. 1. Activity of sodium hypochlorite (bleach) against a human rotavirus as determined in the disc-based quantitative carrier test.
min, it showed a 6 log10 reduction in rotavirus infectivity even at 1000 ppm, which is /5-fold lower than its recommended in-use concentration of 5500 ppm. Since the product is sold for reuse in the decontamination of semi-critical medical devices, it would be expected to retain strong virucidal activity even after several days of reuse. The results obtained with the phenolic used in this study (Fig. 3) showed very little kill at the use-dilution (775 ppm). However, because we wanted to see if the classic kill response curve would hold, and it was
possible to do so, we increased the phenolic concentration until a response was obtained against the rotavirus. This was only obtained at many times the recommended use-dilution.
4. Discussion The results presented here are just an illustration of how the method described can be applied to test a variety of chemical germicides for their virucidal activ-
Fig. 2. The effect of an ortho -phthalaldehyde-based germicide against a human rotavirus as determined in the disc-based quantitative carrier test.
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11
Fig. 3. The effect of a phenolic-based germicide against a human rotavirus as determined in the disc-based quantitative carrier test.
ity. It can not only give good dose /response curves but can also distinguish between formulations with weak and strong activity against a given test virus. In the results reported here the virus titre was deliberately kept higher than 6 log10 to show that the test method is capable of testing germicides if the product performance criterion calls for a ]/million-fold reduction in the viability of the test virus. However, the test method is equally capable of working with lower starting titres of the test virus to allow testing for product performance criteria of 3 /4 log10. Standardization of virucide tests, nationally and internationally, will promote confidence among disinfectant users and the general public. Anyone reading the literature will realize that there are almost as many published virucide tests as there are authors of papers on virucides. In this paper, we have made no attempt to detail the similarities and differences in these publications, or to critique them. What we have tried to do is provide the basis for a general understanding of the potential pitfalls in testing virucides, and suggest the basic protocols and controls that should be present in generic methods. We hope this will allow the reader to understand better this field and to be able to critique the published literature independently. Perhaps what is needed most of all are decisions by policy makers */the acceptance of surrogate virus(es) to permit a general virucidal claim, and a defined performance standard. Some jurisdictions already have one or both of these in place (CGSB, 1997). We have made some suggestions regarding the former, but much debate is still required in this area. The major advance that is perhaps required here is to accept that any product with
a virucide claim must be able to kill a wide range of nonenveloped viruses. Many products currently on the market list only enveloped viruses among the organisms on the label. This can easily mislead persons unfamiliar with virus classification, especially if the enveloped viruses listed are among those most feared, such as HIV and HBV. Under the current stage of knowledge and approaches to risk assessment it is not possible to determine with any degree of certainly the desired level of reduction in the virus load in a given setting to significantly reduce disease transmission. There are also obvious practical limitations to how high a level of challenge virus(es) one can present to the product under evaluation. By the same token, what would one regard as too low a level of challenge? Experience accumulated over the past two decades clearly indicates that, if test viruses are chosen carefully, it is feasible to determine a 3/4 log10 reduction in virus infectivity titer after its exposure to a test germicide in a proper carrier test.
Appendix A: Main steps in the quantitative carrier test for virucidal activity Step
Time (min) Inoculate each carrier with 10 ml of test virus, 50 allow inoculum to dry and then place one carrier each in a Teflon vial with the inoculated side up ¡/
12
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Place 50 ml of germicide on 3/5 test carriers; place an equivalent volume of EBSS on each of at least 3 control carriers; hold carriers for required contact time ¡/ Add 950 ml of EBSS, with/without a neutralizer, to each disc carrier ¡/ Elute the inoculum from the carriers ¡/ Subject eluates to 10-fold dilutions in EBSS as necessary ¡/ Inoculate samples onto cell monolayers and incubate for virus adsorption ¡/ Examine the cultures and determine the log10 reduction in the viability titer of the test organism in relation to control carriers ¡/ Determine if the test formulation meets the specified performance criterion
5/15
2
5/10 10
70/100
30/60
10
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