Contact inactivation of orthopoxviruses by household disinfectants

Transcription

1 Journal of Applied Microbiology 2005, 99, doi: /j x Contact inactivation of orthopoxviruses by household disinfectants W. Butcher and D. Ulaeto Department of Biomedical Sciences, Dstl Porton Down, Porton Down, Salisbury, UK 2004/1323: received 16 November 2004, revised 27 January 2005 and accepted 27 January 2005 ABSTRACT W. B U T C H E R A N D D. U LAETO Aims: The aim of this study is to identify common household disinfectants that combine significant activity against the type orthopoxvirus, vaccinia virus with minimal impact in terms of potential toxicity and/or damage to household or personal items. Methods and Results: Laboratory scale experiments assessed common disinfectants containing anionic and nonionic detergents, oxygen-based bleach, potassium peroxomonosulfate, chloroxylenol or halogenated phenols. Disinfectants were assessed for their ability to inactivate the virus on contact or after a short incubation period in the presence and absence of foetal bovine serum as a potential interferant. Significant differences were observed ranging from negligible effect of detergents to complete inactivation on contact with chloroxylenol. Conclusions: At least one chloroxylenol-based household disinfectant is available, which inactivates vaccinia virus on contact. Significance and Impact of the Study: In the event of a release or major outbreak of a pathogenic orthopoxvirus there is likely to be significant public demand for disinfectants with activity against these viruses. The identification of common household disinfectants with such activity obviates any requirement to stockpile or distribute laboratory/industrial disinfectants for this purpose. Keywords: disinfection, monkeypox, orthopoxvirus, Reed Muench, vaccinia, variola. INTRODUCTION The increased mobility and concentration of human populations in recent history is likely to have enhanced the transmission of respiratory pathogens and increases the difficulty of interrupting transmission in epidemics (Morrell et al. 1975; Hall 1981; Meiklejohn 1983; Monto 2004). In the event of a bioterror (BT) or biowarfare (BW) release of variola virus (VARV), the causative agent of smallpox, the efficient control of smallpox in a largely unvaccinated population may be enhanced by disinfection of contaminated and potentially contaminated items and areas in households and public buildings (Grossgebauer et al. 1975). The ability to disinfect such items and areas in an emergency situation will itself be affected by the applicability of specific Correspondence to: David Ulaeto, Department of Biomedical Sciences, Dstl Porton Down, Salisbury SP4 0JQ, UK ( dulaeto@dstl.gov.uk). disinfectants to this task (Fenner 1949; Grossgebauer et al. 1975; Mahnel 1979; Jonczy et al. 2000; van Engelenburg et al. 2002; Ferrier et al. 2004), and their availability in large quantities at short notice over a possibly wide distribution. For both VARV and the closely related monkeypox virus (MPXV), inhalation of respiratory droplets is probably the most significant mechanism for person-to-person transmission in an outbreak situation. However, the possibility of transmission via fomites should not be ignored, and would probably be an issue of great concern for householders and building managers where infected persons live or have spent time (Dixon 1962; Fenner et al. 1988). Various disinfectants are used for inactivation of orthopoxviruses (OPV) in laboratory environments (Fenner 1949). However, these are generally not suitable for use in home environments, either because they pose their own health and environmental hazards, or because they may ª 2005 Crown copyright

2 280 W. BUTCHER AND D. ULAETO damage the items which are to be disinfected. Additionally, with the exception of household bleach laboratory disinfectants are not readily available to the public and may not be stockpiled in sufficient quantity to satisfy demand in the event of a confirmed smallpox attack. We have examined a panel of readily available cleaning agents and disinfectants for their activity against vaccinia virus (VACV). The compounds were all purchased at a local supermarket and are commonly found in UK supermarket outlets. The agents were assessed for their ability to inactivate VACV on contact and after a short incubation period, either as partially purified virus preparations, or preparations spiked with foetal bovine serum (FBS) as an interferant. MATERIALS AND METHODS Virus production All experiments used the IHD-J strain of VACV. Virus was isolated from infected RK13 rabbit kidney cells by manual homogenization in a stainless steel homogenizer. Homogenates were clarified by centrifugation at 900 g for 10 min, and the virus-containing supernates were ultracentrifuged through a cushion of 36% sucrose at g for 80 min. Virus pellets were resuspended in H 2 O and dispersed in a duall homogenizer prior to titration by limiting dilution and storage at )80 C. Culture medium was Dulbecco s modified Eagle s medium (DMEM) (Sigma) supplemented with 2% FBS (Sigma), 3 mmol l )1 L-glutamine (Sigma) and 100 units ml )1 of penicillin and streptomycin (Sigma). Virus cultures were grown at 37 C in a 7% CO 2, humidified atmosphere. Disinfection assays For disinfection experiments, virus was used at the highest achievable titre in suspension. A 5-ll volume of virus suspension was placed in the bottom of a 1Æ5-ml capacity screw cap vial. Five microlitres of disinfectant was added to the virus suspension and mixed by pipetting three times, after which 1 ml of sterile water was immediately added, and the suspension mixed by pipetting up-and-down once. The virus was then pelleted by centrifugation at maximum speed (at least 00 g) in a microcentrifuge for 10 min. The supernate was discarded and the pellet resuspended in 1Æ5 ml of culture medium and transferred to a sterile polystyrene bijou vial prior to sonication in a low power (80 VA, 40 khz) water bath sonicator for 1 min. The virus suspension was then dispensed in 175-ll volumes in each well of the first column of a sterile tissue-culture 96-well microtitre plate. The remaining columns were filled with 200 ll per well of culture medium. The virus aliquots were serially diluted across the remaining columns of the plate in steps of 1 : 3, transferring 100-ll volumes. A 50-ll volume of each serial dilution was transferred to a replica plate containing freshly confluent monolayers of RK13 cells, and incubated at 37 C, 7% CO 2 for 1 h, after which an additional 50 ll of culture medium was added to each well. Cultures were maintained at 37 C in a 7% CO 2, humidified atmosphere for 6 7 d before fixing with 1% formaldehyde and staining with crystal violet. The 50% tissue culture infectious dose (TCID 50 ) was calculated by Reed Muench analysis of virus positive wells. Disinfection by this protocol is regarded as disinfection on contact. In some cases, virus and disinfectant were incubated at room temperature for 30 min before diluting the disinfectant. When FBS was added as an interferant, virus was used in 2Æ5-ll volumes, to which 2Æ5 ll of neat FBS was added prior to addition of disinfectant in a 5-ll volume. The immediate washing step after addition of disinfectants gave a 100-fold dilution of the disinfectants, followed by further dilution when the virus pellet was resuspended in 1Æ5 ml of culture medium. Mock samples containing no virus but treated with disinfectant had no adverse effect on cell monolayers used for virus titration (not given). The washing step was thus held to have removed the disinfectants prior to virus titration and no additional steps to neutralize disinfectants were taken. The disinfectants used are listed in Table 1. For Reed Muench analysis, a microsoft excel spreadsheet was generated to calculate virus titre as TCID 50 from the numbers of virus positive wells in each column of the titration plate. This spreadsheet is available by on request to the corresponding author. RESULTS Our first assays examined ÔFlashÕ, the mildest compound in the panel, comprising a detergent cleaning fluid containing no specific disinfectants. Used neat, Flash reduced the titre of virus in the sample by approximately two orders of magnitude on contact, in the presence and absence of FBS. Only minimal disinfection was seen if the concentration of Flash was reduced to 10% (Fig. 1a). When the virus was incubated with neat Flash for 30 min, the titre was reduced by only three orders of magnitude in both the presence and absence of FBS. In the positive control for disinfection, our standard laboratory disinfectant for poxviruses, a solution of 5% Virkon Ò, completely inactivated the virus in the sample on contact in both the presence and absence of FBS (Fig. 1b). Further experiments examined additional compounds in the form of ÔCif Oxy-Gel Ò Õ, a cleaning agent that may be regarded as similar to Flash, but containing in addition <5% oxygen-based bleach, and ÔDettol Ò Õ, a household disinfectant containing 4Æ8% chloroxylenol. Cif Oxy-Gel Ò gave a result

3 DISINFECTION OF VACCINIA VIRUS 281 Table 1 Cleaning/disinfection products. Cif Oxy-Gel Ò, Dettol Ò, TCP Ò and Virkon Ò are registered trademarks Product Active ingredients Formulation Source Manufacturer Flash Cif Oxy-Gel Ò Dettol Ò TCP Ò Virkon Ò <5% anionic surfactants, 5 15% nonionic surfactants <5% oxygen-based bleaching agent, 5 15% nonionic surfactants 4Æ8% w/v chloroxylenol, also contains isopropanol and castor oil soap 0Æ68% w/v halogenated phenols, 0Æ175% w/v phenol 50% w/w potassium peroxomonosulfate, 5% sulfamic acid, 15% sodium alkylbenzene sulfonate Liquid Supermarket Proctor & Gamble Liquid Supermarket Unilever Liquid Supermarket Reckitt Benckiser Liquid Supermarket Pfizer Dry powder Fisher Scientific Antec International very similar to that of Flash, reducing the virus titre in the sample between two and three orders of magnitude. Dettol Ò performed much better, reducing the virus titre by seven orders of magnitude on contact, and by almost six orders of magnitude on contact in the presence of 50% FBS (Fig. 2a). Only 7Æ5 TCID 50 were recovered from the sample treated with Dettol Ò in the absence of FBS. This assay was repeated several times, including experiments using a different lot of virus at a slightly lower titre in which complete inactivation of virus by Dettol Ò was observed (Fig. 2b). Unlike Flash and Cif Oxy-Gel Ò, Dettol Ò is marketed as a household disinfectant rather than as a cleaning agent. The activity of this compound against VACV led us to examine it in greater depth, alongside an additional household disinfectant, ÔTCP Ò Õ (active ingredients 0Æ68% halogenated phenols, 0Æ175% phenol). Both compounds were assessed as a solution of 10% of neat, with an extended incubation period of 30 min. The efficacy of Dettol Ò was reduced, with virus titres depleted by >3 orders of magnitude. The TCP Ò had minimal effect on virus titre under these conditions (Fig. 3a). The experiments were repeated using neat Dettol Ò or neat TCP Ò with a 30-min incubation period. In these experiments, Dettol Ò achieved complete inactivation of VACV even in the presence of 50% FBS. TCP Ò achieved a reduction in titre of two orders of magnitude (Fig. 3b). DISCUSSION In the unlikely but much feared event of a BT or BW attack with VARV, there is likely to be a heavy demand by both the public and the emergency services for disinfectants with confirmed activity against poxviruses. The laboratory disinfectants that have known activity against poxviruses present significant problems for domestic use. Some disinfectants such as formaldehyde, glutaraldehyde and paraformaldehdye will present hazards to the user in domestic situations. Others such as Virkon Ò have to be reconstituted at or close to point-of-use and degrade rapidly thereafter. It is likely that none is stockpiled in quantity sufficient to meet probable household demand in the case of a confirmed attack or outbreak, and none is easily obtainable for household use. Our work has looked at purified virus preparations in the presence and absence of a protein-based interferant. In the unlikely event of an outbreak of smallpox, patients with the disease will at some point shed virus embedded within proteinaceous scabs. This may be expected to physically protect the virus from chemical disinfectants, and it is known that OPV embedded in scabs are more resistant to disinfection with formaldehyde vapour, requiring the exposure time to be doubled (Grossgebauer et al. 1975). However, direct application of liquid disinfectant is likely to result in greater penetration of scabs and thus more effective disinfection than occurs with vapour deposition. In addition, it is thought that VARV in scabs presents a greatly reduced infection hazard relative to VARV in respiratory droplets, body fluids or nonscabbed lesions. We have examined a panel of common household cleaning/disinfection products for virucidal activity against VACV, the type OPV. Cleaning agents consisting of anionic and/or nonionic surfactants have little activity against this virus either on contact or after a 30-min incubation. This is true even in the case of Cif Oxy-Gel Ò, which has an oxygen-based bleach included in the formulation. The poor activity of Cif Oxy-Gel Ò is in contrast to the activity of Virkon Ò, the standard disinfectant in use in our laboratory for surface and liquid disinfection of poxviruses. Virkon Ò includes a high concentration of potassium peroxomonosulfate and we assumed that Cif Oxy-Gel Ò would mimic some of its activity. The disparity

4 282 W. BUTCHER AND D. ULAETO (a) (a) (b) (b) Fig. 1 Treatment of VACV samples with Flash cleaning fluid or Virkon Ò disinfectant. Flash was used either as neat (final concentration in assay 50%) or as a 10% v/v solution (final concentration in assay 5%). Virkon Ò was used as a 5% w/v solution (final concentration in assay 2Æ5%) made fresh on the day of use. Neat Flash, contact (a, ); neat Flash + 50% serum, contact (a, j); 10% Flash, contact (a, ); 10% Flash + 50% serum, contact (a, j); neat Flash, 30 min incubation (b, ); neat Flash + 50% serum, 30 min incubation (b, j); 5% Virkon Ò, contact (b, ); 5% Virkon Ò + 50% serum, contact (b, j); mock treated virus (a, b, ). Starting titre for virus preparation, 1Æ8 0 TCID 50 per ml between the two suggests that the lower concentration of oxygen-based bleach in Cif Oxy-Gel Ò is simply insufficient to the task. Thus household disinfectants need to be properly evaluated for decontamination of poxviruses even Fig. 2 Treatment of VACV samples with Cif Oxy-Gel Ò cleaning fluid (a), or Dettol Ò (a, b) or Virkon Ò (b) disinfectants. Cif Oxy-Gel Ò and Dettol Ò were used neat (final concentration in assay 50%). Virkon Ò was used as a 5% w/v solution (final concentration in assay 2Æ5%) made fresh on the day of use. Neat Cif Oxy-Gel Ò, contact (a, ); neat Cif Oxy-Gel Ò + 50% serum, contact (a, j); Dettol Ò, contact (a, b, ); Dettol Ò + 50% serum, contact (a, b, j); 5% Virkon Ò, contact (b, ); 5% Virkon Ò + 50% serum, contact (b, j); mock treated virus (a, b, ). Starting titre for virus preparations, 1Æ8 0 TCID 50 per ml (a), and 4Æ9 TCID 50 per ml (b) if the active or a similar ingredient is known to have activity in nonhousehold or industrial products. The poor activity of TCP Ò is not entirely surprising, given that phenol was frequently added to smallpox vaccine during manufacture in order to reduce levels of bacterial contamination without

5 DISINFECTION OF VACCINIA VIRUS 283 (a) (b) very well known and very readily obtainable. Dettol Ò was able to achieve complete or near complete inactivation of VACV on contact. After a 30-min incubation, it achieved complete inactivation even in the presence of 50% FBS as an interferant. In our experiments, VACV was used at the highest concentrations available, and the disinfectant added to the sample in an equal volume, thus being instantly diluted to 50% when used neat. It is highly unlikely that virus will be present at such high concentrations on any domestically contaminated surface, or in any clinical waste or spillage. It is thus unlikely that virus will be present at concentrations approaching 1 0 TCID 50 per ml, which is the virus concentration at which the contact disinfection by Dettol Ò appears to wane slightly. For disinfection of surfaces, there will be no dilution of the disinfectant upon application to the surface, and there will be an immediate and large dilution of the virus and any potential interferants. This is likely to ensure that any application of neat Dettol Ò for surface decontamination will automatically fall within the window of its efficacy. For decontamination of large quantities of liquid or liquid spills containing high concentrations of potential interferants (e.g. body fluids) one must consider where this is likely to occur. If it occurs in a hospital environment, industrial disinfectants such as Virkon Ò may be more readily available and there is no reason why they should not be used in accordance with protocols that should already be in place for dealing with potentially infectious clinical material. In household environments, again anticipating that any potentially contaminated body fluids will harbour virus at only low titres, it should be sufficient to apply an equal volume of Dettol Ò to the spill and leave it for 30 min or longer before attempting to clean up. Fig. 3 Treatment of VACV samples with TCP Ò or Dettol Ò disinfectants. TCP Ò and Dettol Ò were used either as a 10% v/v solution (final concentration in assay 5%) (a) or neat (final concentration in assay 50%) (b), with a 30 min incubation period. TCP Ò ( ); TCP Ò + 50% serum (j); Dettol Ò ( ); Dettol Ò + 50% serum (j); mock treated virus ( ). Starting titre for virus preparation, 4Æ9 TCID 50 per ml inactivating the vaccine. TCP Ò was included because it is well known and readily available. In the event of an outbreak, it will be important to know that TCP Ò is not suitable for disinfection of poxvirus contaminated surfaces or materials. The product with the most significant activity against VACV in this study was Dettol Ò. Like TCP Ò this is both REFERENCES Dixon, C.W. (1962) Smallpox. London, UK: J. & A. Churchill Ltd. van Engelenburg, F.A., Terpstra, F.G., Schuitemaker, H. and Moorer, W.R. (2002) The virucidal spectrum of a high concentration alcohol mixture. J Hosp Infect 51, Fenner, F. (1949) Mousepox (Infectious Ectromelia of Mice): A Review. J Immunol 63, Fenner, F., Henderson, D.A., Arita, I., Jezek, Z. and Ladnyi, I.D. (1988) Smallpox and Its Eradication. Geneva: World Heath Organization. Ferrier, A., Garin, D. and Crance, J.M. (2004) Rapid inactivation of Vaccinia virus in suspension and dried on surfaces. J Hosp Infect 57, Grossgebauer, K., Spicher, G., Peters, J., Kuwert, E., Pohle, H.D. and Kerner, H. (1975) Experiments on terminal disinfection by formaldehyde vapour in the case of smallpox. J Clin Microbiol 2,

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