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Rapid inactivation of vaccinia virus in suspension and dried on surfaces
Journal of Hospital Infection (2004) 57, 73–79
www.elsevierhealth.com/journals/jhin
Rapid inactivation of vaccinia virus in suspension and dried on surfaces A. Ferrier, D. Garin*, J.M. Crance ´ de Virologie, Centre de Recherches du Service de Sante ´ des Arme ´es (CRSSA) Emile Parde ´, Unite ´sivaudan F-38702 Grenoble, France 24 avenue des Maquis du Gre Received 4 September 2003; accepted 12 January 2004
KEYWORDS Disinfectants; Virucidal activity; Pox virus; Suspension tests; Surface tests; Organic matter
Summary A bioterrorist attack with smallpox virus would be disastrous with a 30% disease fatality rate. Such an outbreak would require biomedical laboratories for diagnosis and analyses and extensive use of clinical care facilities for patient quarantine. Safe decontamination procedures will have to be in place in order to limit the spread of the disease. In order to fulfil this need, Sanytexw, a new non-corrosive commercial solution containing quaternary ammonium, aldehydes, alcohol and detergent, was tested with a view to using it in decontamination procedures. Vaccinia virus was used in this investigation as a model for smallpox virus. We determined exposure time and the concentration of Sanytex required to inactivate the virus in suspension and dried on surfaces in the presence of protein (up to 70 mg/mL). After 3 min incubation, Sanytex at a concentration of 3% led to a complete inactivation (virus titre reduction . 104-fold of vaccinia virus in suspension containing protein up to 30 mg/mL. A virus suspension containing 70 mg protein/mL, simulating biological fluids, was decontaminated with 10% Sanytex after 3 min. After 10 min, Sanytex at a concentration of 30%, applied on to a dried vaccinia virus contaminated surface in the presence of protein (10 mg/mL before desiccation), led to complete decontamination of the surface. Thirty minutes exposure with 30% Sanytex was necessary for a virus titre reduction of . 104-fold on a surface contaminated with a dried suspension of vaccinia virus in the presence of protein at 70 mg/mL. Sanytex is not corrosive, not toxic to environment and stable for up to three months even diluted. Its virucidal effect was preserved when used under pressure in a fire-hose nozzle. These results support the use of Sanytex for decontamination of biological fluids and surfaces contaminated by the smallpox virus. Q 2004 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.
Introduction *Corresponding author. Tel.: þ 33-476-63-68-44; fax: þ 33476-63-69-06. E-mail address: [email protected]
Since the deliberate anthrax contamination in the USA, the eventuality of bioterrorism must be
0195-6701/$ - see front matter Q 2004 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jhin.2004.01.012
74
actively addressed. Smallpox is a particularly dangerous biological weapon threat because of its clinical and epidemiological properties: it is easily delivered as an infectious aerosol, its transmissibility between humans is high1 and its fatality rate is up to 30%.2 The smallpox virus belongs to the family of Poxviridae, genus Orthopoxvirus.3 This virus is assigned to biological safety level 4 (BSL 4) and the last strain libraries are maintained only in Atlanta (USA) and Novosibirsk (Federation of Russia). The vaccination of the general population against smallpox virus was stopped at the beginning of the 1980s after eradication of the disease; therefore at present a high proportion of the population is non-immunized and vulnerable to infection.4 Moreover, there is no efficient antiviral treatment against smallpox.5 The effect of smallpox virus released among the population could be disastrous if efficacious measures were not taken. The vaccine is active if it is administered in the first four days afterexposure, but only a limited number of doses is available worldwide.6 In addition to vaccination, quarantine of symptomatic people is critical to contain the epidemic. Smallpox virus is able to survive in the environment for weeks,7 depending on the ambient temperature, the air humidity and ultraviolet light exposure. A safe decontamination procedure is needed in order to decontaminate rapidly biological infectious fluids and surfaces both in medical biology laboratories and in clinical care facilities. Some disinfectants are active against orthopoxviruses, particularly sodium hypochlorite (bleach). However, this chemical is not an ideal disinfectant because chlorine solutions are unstable, inactivated by protein containing substances,8 and cannot be used on stainless steel equipment because it causes damage. In this report, the commercially available new disinfectant Sanytexw was tested on vaccinia virus, an established surrogate virus of smallpox virus. In order to test its potential efficacy, we carried out both a suspension and a surface test, taking into account the presence of increasing concentrations of proteins (up to 70 mg/mL). In addition, the stability of different dilutions of this disinfectant was also studied for up to three months.
Materials and methods Disinfectants The disinfectant used was Sanytex (Rochex
A. Ferrier et al.
Laboratories, Annemasse, France). Its composition is listed in Table I. Different dilutions were made in sterile distilled water. This disinfectant is bactericidal [French standards NF T 72: 150 (suspensions), 170 (with organic matter) and 190 (on to surfaces)], fungicidal (French standard NF T 72-200) and virucidal [French standard NF T 72-180 (suspension)] at 1% (v/v). Sodium hypochlorite solution was used as a comparative disinfectant (Bleach, La Croix Colgate-Palmolive, Courbevoie, France).
Preparation of virus The Copenhagen strain of vaccinia virus (provided by R. Drillien, INSERM, Strasbourg, France) was adapted to Vero cells (ATCC CCL81) by multiple passages and cultivated in M199 medium with 2% of foetal calf serum (FCS), without antibiotics, at 37 8C and 5% of CO2. A virus stock was prepared in Vero cells infected at a multiplicity of infection of 0.01 and incubated at 37 8C until a cytopathic effect was observed. The vaccinia virus stock was obtained by three freeze – thaw cycles of infected cells and clarification using low-speed centrifugation. The initial virus stock titre was determined to be 108.9 TCID50/mL.
Suspension test Four Sanytex concentrations (1, 3, 5 and 10%) were assessed for virucidal activity. Tests were performed with the addition of protein [final bovine serum albumin (BSA) concentration: 1; 3; 10; 30 or 70 mg/mL]. Viral stock containing an appropriate BSA concentration (450 mL) was mixed with 50 mL Sanytex solution prepared at 10-fold concentration and then incubated for 3 min at room temperature (22 – 24 8C). Then, interruption of disinfectant activity was achieved by simple dilution.9 The disinfectant activity was neutralized by adding M199 medium with 2% FCS at 4 8C in order to obtain a final non-virucidal concentration of 0.01% Sanytex, which proved to be non-cytotoxic to Vero cells. Controls were treated in the same way as the tests Table I Composition of Sanytex Compound Lauryl dimethyl benzalmonium salt N-alkyl amido betaine Lauric ethoxyl alcohol Formaldehyde Glyoxal EDTA Diphosphoric hydroacetic acid
Content (%) 7.5 0.6 4.0 1.3 1.3 1.0 0.6
Rapid inactivation of vaccinia virus in suspension and dried on surfaces
but with sterile distilled water instead of Sanytex. The experiments were carried out in triplicate. Each sample was assayed for its virus titre. The virucidal efficacy of the disinfectant solution was calculated as the difference between the virus titres after mixing with the control medium and the virus titres after mixing with the disinfectant solution, and expressed as the log10 reduction factor of virus titres. The criterion used for virucidal activity was a log10 reduction factor of 4 in virus titre.10 Assays for virucidal activity of sodium hypochlorite were achieved following the same protocol. For sodium hypochlorite neutralization, a 1:10 dilution in M199 medium containing 2% FCS and 1.1% sodium thiosulphate was used. Three concentrations of active chlorine were tested: 0.525, 0.0525 and 0.00525%.8
Surface test Three disinfectant concentrations (3, 10 and 30%) were assessed for virucidal activity. A 30 mL drop of virus suspension, containing 10 or 70 protein/mL was placed on the bottom of a well of a six-well cell culture plate and allowed to dry for approximately 1 h in a biosafety cabinet at room temperature. A 30 mL drop of each prepared Sanytex solution was placed on the spot of dried virus, covering it entirely. After an adequate exposure time, 8 mL of M199 with 70 mg BSA/mL were added to obtain a final concentration of Sanytex of , 0.1%, corresponding to the neutralizing dilution of Sanytex in the presence of a BSA concentration of 70 mg/mL. The virus was resuspended by scraping. This suspension was removed and diluted in a final volume of 90 mL of M199 to obtain a final concentration of Sanytex of , 0.01% (corresponding to the non-cytotoxic concentration). Controls were treated in the same way as the tests but with sterile distilled water instead of Sanytex. The experiments were carried out in triplicate. Each sample was assayed for its virus titre.
Assay Viral infectivity of the mixtures was determined in Vero cells cultured in 96-well tissue culture plates. Ten four-fold dilutions were prepared from each treated sample in M199 medium. Fifty microlitres of each dilution was then dispensed into the microtitre plate (eight replicates per dilution), which was incubated under 5% CO2 at 37 8C. After seven days of incubation the wells were observed microscopically for cytopathic effect and the titres (expressed
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in terms of TCID50/mL) were calculated by the method of Reed and Muench.11
Stability of Sanytex solutions The stability of 3% and 30% Sanytex was studied. These solutions were stored at room temperature or at 30 8C for three months. After this storage period both Sanytex solutions were tested for virucidal activity on vaccinia virus at a 1% final concentration using the suspension test described above. The virucidal effect was compared with that of 1% Sanytex prepared from the non-diluted fresh Sanytex as the control.
Results Suspension tests The virucidal effect of 1% and 3% Sanytex on vaccinia virus suspension in the presence of different concentrations of protein is listed in Table II. After a 3 min time exposure, the 1% Sanytex solution effectively inactivated (i.e. a log reduction . 4) the virus suspensions containing up to 3 mg protein/mL, but was inefficient in the presence of higher concentrations of BSA. The 3% Sanytex solution was still active in the presence of protein up to 30 mg/mL, but the log10 reduction factor in virus titre was limited to 1.3 in the presence of protein at a concentration of 70 mg/ mL. With this protein content, vaccinia virus was effectively inactivated by a 10% Sanytex solution after 3 min. The virucidal effect of sodium hypochlorite on vaccinia virus suspension is listed in Table III. After a 3 min contact time, only the highest chlorine concentration is active on the vaccinia virus suspension containing 10 mg protein/ mL, but this activity is incomplete if the solution contains 70 mg protein/mL.
Surface tests In the set conditions, 100% recuperation recovery rate was obtained after vaccinia virus desiccation and resuspension (data not shown). Table IV gives the reductions in titre of vaccinia virus after treatment with different concentrations of Sanytex in the surface test. When the virus was dried from a suspension containing BSA at a concentration of 10 mg/mL, the 3% solution was not effective, even if the exposure time was prolonged to 15 min. The virus was effectively inactivated after 15 min by 10% Sanytex and after 10 min by a 30% concentration of
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A. Ferrier et al.
Table II Virucidal effect of Sanytex solutions on vaccinia virus suspension with different protein concentrations after a 3 min exposure Sanytex concentration
Virus titre (log10 reduction factor) BSA concentration
1% 3% 5% 10%
1 mg/mL
3 mg/mL
10 mg/mL
30 mg/mL
70 mg/mL
.4.4 .4.4 .4.4 .4.4
.4.4 .4.4 .4.4 .4.4
3.2 .4.4 .4.4 .4.4
0.8 .4.4 .4.4 .4.4
0.4 1.3 2.7 .4.4
The data represent average values for three experiments. The average log10 TCID50/50 mL titre of the controls was 4.4 log. Neutralization of the virucidal effect after the exposure time was checked.
Sanytex. When the concentration of protein in the initial suspension was 70 mg/mL an exposure time of 30 min was necessary to inactivate completely the vaccinia virus.
Stability of Sanytex solutions Table V shows the virucidal effect of two solutions of Sanytex (3 and 30%) after a three-month storage period at 30 8C and at room temperature. There was no evidence of loss of stability of these Sanytex solutions stored at room temperature or at 30 8C. Indeed, the virus titre reductions assayed with final 1% solutions prepared from these stored solutions and with the fresh disinfectant were similar (t-test, P . 0:05).
Discussion This study was performed to assess the activity of a new disinfectant formulation, Sanytex, against the
Table III Virucidal effect of sodium hypochlorite solutions on vaccinia virus suspension with different protein concentrations after a 3 min exposure Hypochlorite sodium (%)
Log10 reduction factor of virus titre BSA concentration
0.525 0.0525 0.00525
10 mg/mL
70 mg/mL
.4.4 1.8 0.3
3.8 0.2 0.2
The data represent average values for three experiments. The average titre of the controls was 104.4 TCID50/50 mL. Neutralization of the virucidal effect after the exposure time was checked.
vaccinia virus, used as a surrogate virus of the variola virus, both in suspension and dried on surfaces. These experiments were conducted in the presence of proteins, which are known to be one of the main factors affecting the efficacy of the disinfectants.12 Sanytex at a concentration of 1% proved to be an effective disinfectant, with a rapid virucidal effect. However, this efficacy was observed in the virus suspension containing a protein concentration of , 3 mg/mL. In our study, tests were also carried out with higher protein concentrations to mimic the usual conditions of contamination with biological fluids in the laboratory. Indeed, organic matter interacts with several disinfectants and neutralizes their virucidal effects.8,13 – 16 Several studies have been performed to determine the virucidal activity of disinfectants on poliovirus in blood8 or serum,13, 17 but there are very few reported studies on poxviruses and those reported have been carried out with protein concentrations of up to 30 mg/mL only.17 In our study, in the presence of protein concentrations . 3 mg/mL, higher concentrations of Sanytex were required to completely inactivate vaccinia virus after a short exposure time. Incubation in the presence of 30 and 70 mg/mL protein required concentrations of 3 and 10% Sanytex, respectively, to obtain a log10 reduction factor of more than 4 after 3 min exposure. As expected, the effect of Sanytex on vaccinia virus spread on surfaces was less rapid: it took a 15 min incubation with 10% Sanytex to completely inactivate dried vaccinia virus when the initial suspension (before desiccation) contained 10 mg protein/mL. With a protein concentration of 70 mg/mL, simulating body fluids, it took a 30 min incubation with a 30% Sanytex solution for virus inactivation. Sodium hypochlorite has been tested as a comparative agent of Sanytex at chlorine concentrations previously used on poliovirus.8 These concentrations include the concentration of
Rapid inactivation of vaccinia virus in suspension and dried on surfaces
77
Table IV Virucidal effect of Sanytex solutions on dried virus suspension containing 10 and 70 mg BSA/mL Exposure time (min)
Reduction factor of virus titre (log10) BSA concentration 10 mg/mL
3 5 10 15 30
70 mg/mL
Sanytex 3%
Sanytex 10%
Sanytex 30%
Sanytex 30%
1.3 1.6 2.0 3.0 –
1.3 2.6 3.8 .4.2 –
1.5 3.0 .4.2 .4.2 –
– – 1.7 2.0 .4.2
The data represent average values for three experiments. The average titre of the controls was 104.2 TCID50/50 mL. Neutralization of the virucidal effect after the exposure time was checked. Virus suspension containing BSA 10 or 70 mg/mL was dried on a plastic surface.
0.0525% of active chlorine selected by the Centers of Disease Control and Prevention (CDC) to decontaminate HIV suspensions in blood spills18 and concentration of 0.036% active chlorine choose by French standard NF T 72-180 on virucidal activity.19 In this study, the highest concentration of active chlorine (0.525%) did not prove to be completely efficient on vaccinia virus suspension in presence of 70 mg protein/mL after 3 min exposure. Higher sodium hypochlorite concentrations might be efficient, but are highly corrosive and toxic compared with 10% Sanytex used to effectively inactivate the same suspension of vaccinia virus. The effect of the disinfectant Gigaseptw has been determined on vaccinia virus suspension containing 30 mg/mL proteins (40% calf serum). This dialdehyde, at a concentration of 5% achieved a log10 reduction factor of only 3 after 30 min contact time17 compared with 3% Sanytex allowing an efficient decontamination (104-fold reduction) of the same suspension in 3 min.
The French standard criteria19 (NF T 72-180) for assessment of virucidal effect of a disinfectant do not require the tests to be carried out in the presence of high concentrations of proteins and do not advise an exposure time of , 15 min or surface tests. The American Society for Testing and Materials20 (ASTM) mentions the addition of FCS at a final concentration of only 5% (protein concentration: 3.5 mg/mL) and the DVV21 (German association for the control of virus diseases) specified the presence of 10% FCS (approximately 7 mg/mL). However, it is important to take into account the high protein concentration of some biological samples such as body fluids, particularly serum or plasma, and this is why we carried out experiments in the presence of proteins at the concentration of 70 mg/mL, mimicking serum protein concentration. Surface tests have been described in literature,22 – 26 but have to be taken into account in European standards (CEN/TC 216) to simulate real conditions of use. Virucidal activity evaluated by
Table V Virucidal effect of Sanytex solutions on vaccinia virus suspension containing BSA 10 mg/mL for 3 min, after three months storage of the Sanytex solutions Storage conditionsa Sanytex concentration (%) 3 30 Pure fresh disinfectantd
Reduction factor of virus titreb (log10) Storage temperature (8C) RTc 30 RTc 30 –
3.0 3.1 2.9 3.1 3.0
The data represent average values for three experiments. The average titre of the controls was 104.3 TCID50/50 mL. Neutralization of the virucidal effect after the exposure time was checked. a The two solutions of Sanytex were stored at 30 8C or at room temperature for three months. b The different solutions of Sanytex and the fresh Sanytex were diluted to obtain a final concentration of 1%, then suspension test was achieved. c Room temperature (20–24 8C). d Pure Sanytex was used. Virus titre reduction achieved with a final 1% dilution of Sanytex performed immediately before the test.
78
suspension tests cannot validate efficient surfaces disinfection (virus titre reduction of 104-fold) for which the concentration of disinfectant used27 or time exposure must be increased (see Sanytex results). Diluted Sanytex solution proved to be stable up to three months when stored between 20 and 30 8C. Moreover, trials have been achieved with the local fire brigade, in order to verify the stability of Sanytex under pressure in a fire hose nozzle. This study has shown that Sanytex is a powerful disinfectant for complete inactivation of vaccinia virus contaminated suspension and surfaces. This disinfectant should also be active on smallpox virus, and could be used in a case of bioterrorism involving a smallpox aerosolization attack. Indeed, vaccinia virus and smallpox viruses are very close viruses belonging to the genus Orthopoxvirus. Their genome homology is approximately of 95%.28 Moreover a previous study showed that there is no essential difference in terms of disinfectant sensitivity between smallpox virus and vaccinia virus, 29 suggesting that the decontamination procedures proposed in the present study could be used to decontaminate biological fluids or surfaces contaminated with smallpox virus. Sanytex was previously tested on several other viruses. It was shown to be efficient on adenovirus suspension at the 0.5% concentration (French standard AFNOR NF T 72-180). At this same concentration, human immunodeficiency virus (HIV) was effectively inactivated after a 3 min exposure (Barre ´-Sinoussi F., unpublished data). Moreover, in our laboratory, 1% Sanytex proved to be effective against yellow fever virus (the surrogate virus of hepatitis C virus), Semliki Forest virus and Rift Valley fever virus (data not shown) after a 3 min contact time in a suspension containing 10 mg protein/mL. The standard procedure for the complete decontamination of the laboratory uses gaseous formaldehyde, which has been shown to be efficient on vaccinia virus (data not shown). However, this method is time-consuming (12 h) and could not be applied routinely in the case of a major smallpox bioterrorism crisis when medical biology laboratories would need to be continuously operational. In such a situation, Sanytex could find an application in medical biology laboratories, hospitals or contaminated zones in the conditions described in this study. Indeed biological safety in such dramatic circumstances is achieved not only by anti-infectious treatment or vaccination, but also by a safe decontamination of laboratories, clinical facilities and contaminated locations, especially with the present risk of bioterrorism with the smallpox virus.
A. Ferrier et al.
Acknowledgements We thank Danielle Gratier and Henri Blancquaert for their excellent technical assistance and Bernard Souberbielle for critically reviewing the manuscript. This work was supported by research grants from the ‘Service de Sante ´ des Arme ´es’.
References 1. Committee on the Assessment of Future Needs for Variola Virus, Epidemiology. Assessement of future scientific needs for live Variola virus. Institute of Medicine, Washington: National Academy Press; 1999. p. 33—35. 2. Henderson DA, Inglesby TV, Bartlett JG, et al. Smallpox as a biological weapon: medical and public health management. Working Group on Civilian Biodefense. J Am Med Assoc 1999; 281:2127—2137. 3. Fenner F. Poxviruses. In: Fields BN, Knipe DM, Howley PM, et al., editors. Fields virology, 3rd ed. Philadelphia: Lippincott/Raven Publishers; 1996. p. 2673—2702. 4. Cohen J. Bioterrorism. Smallpox vaccinations: how much protection remains? Science 2001;294:985. 5. Berche P. The threat of smallpox and bioterrorism. Trends Microbiol 2001;9:15—18. 6. Breman JG, Henderson DA. Poxvirus dilemmas—monkeypox, smallpox, and biologic terrorism. N Engl J Med 1998;339: 556—559. 7. Harper GJ. Airborne micro-organisms: survival tests with four viruses. J Hyg 1961;59:479—486. 8. Weber DJ, Barbee SL, Sobsey MD, Rutala WA. The effect of blood on the antiviral activity of sodium hypochlorite, a phenolic, and a quaternary ammonium compound. Infect Control Hosp Epidemiol 1999;20:821—827. 9. Garrigue G, Hengy C, Leguenedal R, Bartoli M, Pignon D. In vitro virucidal activity of antiseptics and disinfectants. II. Simple dilution technic. Pathol Biol Paris 1984;32:643—646. 10. Garrigue G. In vitro virucidal activity of antiseptics and disinfectants. I. Draft of the AFNOR virucidal activity standard. Pathol Biol Paris 1984;32:640—642. 11. Reed LJ, Muench LH. A simple method of estimating fifty per cent end points. Am J Hyg 1938;27:493—497. 12. Chambon M, Bailly J, Peigue-Lafeuille H. Antiseptiques, de ´ sinfectants chimiques et virus en secteur medical. Virologie 1999;3:367—379. 13. Wallbank AM, Drulak M, Poffenroth L, Barnes C, Kay C, Lebtag I. Wescodyne: lack of activity against poliovirus in the presence of organic matter. Health Lab Sci 1978;15: 133—137. 14. Hanson PJ, Gor D, Jeffries DJ, Collins JV. Chemical inactivation of HIV on surfaces (see comments). BMJ 1989; 298:862—864. 15. Maris P. Virucidal efficacy of eight disinfectants against pneumovirus, coronavirus and parvovirus. Ann Rech Vet 1990;21:275—279. 16. Noda M, Matsuda S, Kobayashi M. Virucidal activity of disinfectants. Influence of the serum protein upon the virucidal activity of disinfectants. Kansenshogaku Zasshi 2000;74:664—669. 17. Thraenhart O, Kuwert E. Virucidal activity of the disinfectant gigasept against different enveloped and nonenveloped RNA- and DNA-viruses, pathogenic for men.
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18.
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I. Investigation in the suspension test. Zentralbl Bakteriol Orig B 1975;161:209—232. Centers for Disease Control and Prevention. Guidelines for prevention of transmission of human immunodeficiency virus and hepatitis B virus to health-care and public-safety workers. MMWR 1989;38(Suppl.6):1—37. AFNOR. De ´termination de l’activite ´ virucide vis-a `-vis des virus de verte ´bre ´s. Antiseptiques et de ´sinfectants utilise ´s a ` l’e ¸aise de ´tat liquide, miscibles a ` l’eau, Association Franc Normalisation; 1989. p. NF T 72-180. ASTM. Standard Test Method for efficacy of Antimicrobial Agents against Viruses in suspension. Annual Book of ASTM Standards; 2001. West Conshohocken, p. E1052-96. DVV. Guidelines of Bundesgesundheitsamt (BGA; German Federal Health Office) and Deutsche Vereinigung zur Bekampfung der Viruskrankheiten e.V (DVV; German Association for the Control of Virus Diseases) for testing the effectiveness of chemical disinfectants against viruses. Zentralbl Hyg Umweltmed 1990;189:554—556. Lloyd Evans N, Springthorpe VS, Sattar SA. Chemical disinfection of human rotavirus-contaminated inanimate surfaces. J Hyg Lond 1986;97:163—173.
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23. Tyler R, Ayliffe GA. A surface test for virucidal activity of disinfectants: preliminary study with herpes virus. J Hosp Infect 1987;9:22—29. 24. Sattar SA, Springthorpe VS, Karim Y, Loro P. Chemical disinfection of non-porous inanimate surfaces experimentally contaminated with four human pathogenic viruses. Epidemiol Infect 1989;102:493—505. 25. Tyler R, Ayliffe GA, Bradley C. Virucidal activity of disinfectants: studies with the poliovirus. J Hosp Infect 1990;15:339—345. 26. Mbithi JN, Springthorpe VS, Sattar SA. Chemical disinfection of hepatitis A virus on environmental surfaces. Appl Environ Microbiol 1990;56:3601—3604. 27. Bellamy K. A review of the test methods used to establish virucidal activity. J Hosp Infect 1995;(Suppl. 30):389—396. 28. Shchelkunov SN, Totmenin AV, Loparev VN, et al. Alastrim smallpox variola minor virus genome DNA sequences. Virology 2000;266:361—386. 29. Tanabe I, Hotta S. Effect of disinfectants on variola virus in cell culture. Appl Environ Microbiol 1976;32:209—212.
www.elsevierhealth.com/journals/jhin
Rapid inactivation of vaccinia virus in suspension and dried on surfaces A. Ferrier, D. Garin*, J.M. Crance ´ de Virologie, Centre de Recherches du Service de Sante ´ des Arme ´es (CRSSA) Emile Parde ´, Unite ´sivaudan F-38702 Grenoble, France 24 avenue des Maquis du Gre Received 4 September 2003; accepted 12 January 2004
KEYWORDS Disinfectants; Virucidal activity; Pox virus; Suspension tests; Surface tests; Organic matter
Summary A bioterrorist attack with smallpox virus would be disastrous with a 30% disease fatality rate. Such an outbreak would require biomedical laboratories for diagnosis and analyses and extensive use of clinical care facilities for patient quarantine. Safe decontamination procedures will have to be in place in order to limit the spread of the disease. In order to fulfil this need, Sanytexw, a new non-corrosive commercial solution containing quaternary ammonium, aldehydes, alcohol and detergent, was tested with a view to using it in decontamination procedures. Vaccinia virus was used in this investigation as a model for smallpox virus. We determined exposure time and the concentration of Sanytex required to inactivate the virus in suspension and dried on surfaces in the presence of protein (up to 70 mg/mL). After 3 min incubation, Sanytex at a concentration of 3% led to a complete inactivation (virus titre reduction . 104-fold of vaccinia virus in suspension containing protein up to 30 mg/mL. A virus suspension containing 70 mg protein/mL, simulating biological fluids, was decontaminated with 10% Sanytex after 3 min. After 10 min, Sanytex at a concentration of 30%, applied on to a dried vaccinia virus contaminated surface in the presence of protein (10 mg/mL before desiccation), led to complete decontamination of the surface. Thirty minutes exposure with 30% Sanytex was necessary for a virus titre reduction of . 104-fold on a surface contaminated with a dried suspension of vaccinia virus in the presence of protein at 70 mg/mL. Sanytex is not corrosive, not toxic to environment and stable for up to three months even diluted. Its virucidal effect was preserved when used under pressure in a fire-hose nozzle. These results support the use of Sanytex for decontamination of biological fluids and surfaces contaminated by the smallpox virus. Q 2004 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.
Introduction *Corresponding author. Tel.: þ 33-476-63-68-44; fax: þ 33476-63-69-06. E-mail address: [email protected]
Since the deliberate anthrax contamination in the USA, the eventuality of bioterrorism must be
0195-6701/$ - see front matter Q 2004 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jhin.2004.01.012
74
actively addressed. Smallpox is a particularly dangerous biological weapon threat because of its clinical and epidemiological properties: it is easily delivered as an infectious aerosol, its transmissibility between humans is high1 and its fatality rate is up to 30%.2 The smallpox virus belongs to the family of Poxviridae, genus Orthopoxvirus.3 This virus is assigned to biological safety level 4 (BSL 4) and the last strain libraries are maintained only in Atlanta (USA) and Novosibirsk (Federation of Russia). The vaccination of the general population against smallpox virus was stopped at the beginning of the 1980s after eradication of the disease; therefore at present a high proportion of the population is non-immunized and vulnerable to infection.4 Moreover, there is no efficient antiviral treatment against smallpox.5 The effect of smallpox virus released among the population could be disastrous if efficacious measures were not taken. The vaccine is active if it is administered in the first four days afterexposure, but only a limited number of doses is available worldwide.6 In addition to vaccination, quarantine of symptomatic people is critical to contain the epidemic. Smallpox virus is able to survive in the environment for weeks,7 depending on the ambient temperature, the air humidity and ultraviolet light exposure. A safe decontamination procedure is needed in order to decontaminate rapidly biological infectious fluids and surfaces both in medical biology laboratories and in clinical care facilities. Some disinfectants are active against orthopoxviruses, particularly sodium hypochlorite (bleach). However, this chemical is not an ideal disinfectant because chlorine solutions are unstable, inactivated by protein containing substances,8 and cannot be used on stainless steel equipment because it causes damage. In this report, the commercially available new disinfectant Sanytexw was tested on vaccinia virus, an established surrogate virus of smallpox virus. In order to test its potential efficacy, we carried out both a suspension and a surface test, taking into account the presence of increasing concentrations of proteins (up to 70 mg/mL). In addition, the stability of different dilutions of this disinfectant was also studied for up to three months.
Materials and methods Disinfectants The disinfectant used was Sanytex (Rochex
A. Ferrier et al.
Laboratories, Annemasse, France). Its composition is listed in Table I. Different dilutions were made in sterile distilled water. This disinfectant is bactericidal [French standards NF T 72: 150 (suspensions), 170 (with organic matter) and 190 (on to surfaces)], fungicidal (French standard NF T 72-200) and virucidal [French standard NF T 72-180 (suspension)] at 1% (v/v). Sodium hypochlorite solution was used as a comparative disinfectant (Bleach, La Croix Colgate-Palmolive, Courbevoie, France).
Preparation of virus The Copenhagen strain of vaccinia virus (provided by R. Drillien, INSERM, Strasbourg, France) was adapted to Vero cells (ATCC CCL81) by multiple passages and cultivated in M199 medium with 2% of foetal calf serum (FCS), without antibiotics, at 37 8C and 5% of CO2. A virus stock was prepared in Vero cells infected at a multiplicity of infection of 0.01 and incubated at 37 8C until a cytopathic effect was observed. The vaccinia virus stock was obtained by three freeze – thaw cycles of infected cells and clarification using low-speed centrifugation. The initial virus stock titre was determined to be 108.9 TCID50/mL.
Suspension test Four Sanytex concentrations (1, 3, 5 and 10%) were assessed for virucidal activity. Tests were performed with the addition of protein [final bovine serum albumin (BSA) concentration: 1; 3; 10; 30 or 70 mg/mL]. Viral stock containing an appropriate BSA concentration (450 mL) was mixed with 50 mL Sanytex solution prepared at 10-fold concentration and then incubated for 3 min at room temperature (22 – 24 8C). Then, interruption of disinfectant activity was achieved by simple dilution.9 The disinfectant activity was neutralized by adding M199 medium with 2% FCS at 4 8C in order to obtain a final non-virucidal concentration of 0.01% Sanytex, which proved to be non-cytotoxic to Vero cells. Controls were treated in the same way as the tests Table I Composition of Sanytex Compound Lauryl dimethyl benzalmonium salt N-alkyl amido betaine Lauric ethoxyl alcohol Formaldehyde Glyoxal EDTA Diphosphoric hydroacetic acid
Content (%) 7.5 0.6 4.0 1.3 1.3 1.0 0.6
Rapid inactivation of vaccinia virus in suspension and dried on surfaces
but with sterile distilled water instead of Sanytex. The experiments were carried out in triplicate. Each sample was assayed for its virus titre. The virucidal efficacy of the disinfectant solution was calculated as the difference between the virus titres after mixing with the control medium and the virus titres after mixing with the disinfectant solution, and expressed as the log10 reduction factor of virus titres. The criterion used for virucidal activity was a log10 reduction factor of 4 in virus titre.10 Assays for virucidal activity of sodium hypochlorite were achieved following the same protocol. For sodium hypochlorite neutralization, a 1:10 dilution in M199 medium containing 2% FCS and 1.1% sodium thiosulphate was used. Three concentrations of active chlorine were tested: 0.525, 0.0525 and 0.00525%.8
Surface test Three disinfectant concentrations (3, 10 and 30%) were assessed for virucidal activity. A 30 mL drop of virus suspension, containing 10 or 70 protein/mL was placed on the bottom of a well of a six-well cell culture plate and allowed to dry for approximately 1 h in a biosafety cabinet at room temperature. A 30 mL drop of each prepared Sanytex solution was placed on the spot of dried virus, covering it entirely. After an adequate exposure time, 8 mL of M199 with 70 mg BSA/mL were added to obtain a final concentration of Sanytex of , 0.1%, corresponding to the neutralizing dilution of Sanytex in the presence of a BSA concentration of 70 mg/mL. The virus was resuspended by scraping. This suspension was removed and diluted in a final volume of 90 mL of M199 to obtain a final concentration of Sanytex of , 0.01% (corresponding to the non-cytotoxic concentration). Controls were treated in the same way as the tests but with sterile distilled water instead of Sanytex. The experiments were carried out in triplicate. Each sample was assayed for its virus titre.
Assay Viral infectivity of the mixtures was determined in Vero cells cultured in 96-well tissue culture plates. Ten four-fold dilutions were prepared from each treated sample in M199 medium. Fifty microlitres of each dilution was then dispensed into the microtitre plate (eight replicates per dilution), which was incubated under 5% CO2 at 37 8C. After seven days of incubation the wells were observed microscopically for cytopathic effect and the titres (expressed
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in terms of TCID50/mL) were calculated by the method of Reed and Muench.11
Stability of Sanytex solutions The stability of 3% and 30% Sanytex was studied. These solutions were stored at room temperature or at 30 8C for three months. After this storage period both Sanytex solutions were tested for virucidal activity on vaccinia virus at a 1% final concentration using the suspension test described above. The virucidal effect was compared with that of 1% Sanytex prepared from the non-diluted fresh Sanytex as the control.
Results Suspension tests The virucidal effect of 1% and 3% Sanytex on vaccinia virus suspension in the presence of different concentrations of protein is listed in Table II. After a 3 min time exposure, the 1% Sanytex solution effectively inactivated (i.e. a log reduction . 4) the virus suspensions containing up to 3 mg protein/mL, but was inefficient in the presence of higher concentrations of BSA. The 3% Sanytex solution was still active in the presence of protein up to 30 mg/mL, but the log10 reduction factor in virus titre was limited to 1.3 in the presence of protein at a concentration of 70 mg/ mL. With this protein content, vaccinia virus was effectively inactivated by a 10% Sanytex solution after 3 min. The virucidal effect of sodium hypochlorite on vaccinia virus suspension is listed in Table III. After a 3 min contact time, only the highest chlorine concentration is active on the vaccinia virus suspension containing 10 mg protein/ mL, but this activity is incomplete if the solution contains 70 mg protein/mL.
Surface tests In the set conditions, 100% recuperation recovery rate was obtained after vaccinia virus desiccation and resuspension (data not shown). Table IV gives the reductions in titre of vaccinia virus after treatment with different concentrations of Sanytex in the surface test. When the virus was dried from a suspension containing BSA at a concentration of 10 mg/mL, the 3% solution was not effective, even if the exposure time was prolonged to 15 min. The virus was effectively inactivated after 15 min by 10% Sanytex and after 10 min by a 30% concentration of
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Table II Virucidal effect of Sanytex solutions on vaccinia virus suspension with different protein concentrations after a 3 min exposure Sanytex concentration
Virus titre (log10 reduction factor) BSA concentration
1% 3% 5% 10%
1 mg/mL
3 mg/mL
10 mg/mL
30 mg/mL
70 mg/mL
.4.4 .4.4 .4.4 .4.4
.4.4 .4.4 .4.4 .4.4
3.2 .4.4 .4.4 .4.4
0.8 .4.4 .4.4 .4.4
0.4 1.3 2.7 .4.4
The data represent average values for three experiments. The average log10 TCID50/50 mL titre of the controls was 4.4 log. Neutralization of the virucidal effect after the exposure time was checked.
Sanytex. When the concentration of protein in the initial suspension was 70 mg/mL an exposure time of 30 min was necessary to inactivate completely the vaccinia virus.
Stability of Sanytex solutions Table V shows the virucidal effect of two solutions of Sanytex (3 and 30%) after a three-month storage period at 30 8C and at room temperature. There was no evidence of loss of stability of these Sanytex solutions stored at room temperature or at 30 8C. Indeed, the virus titre reductions assayed with final 1% solutions prepared from these stored solutions and with the fresh disinfectant were similar (t-test, P . 0:05).
Discussion This study was performed to assess the activity of a new disinfectant formulation, Sanytex, against the
Table III Virucidal effect of sodium hypochlorite solutions on vaccinia virus suspension with different protein concentrations after a 3 min exposure Hypochlorite sodium (%)
Log10 reduction factor of virus titre BSA concentration
0.525 0.0525 0.00525
10 mg/mL
70 mg/mL
.4.4 1.8 0.3
3.8 0.2 0.2
The data represent average values for three experiments. The average titre of the controls was 104.4 TCID50/50 mL. Neutralization of the virucidal effect after the exposure time was checked.
vaccinia virus, used as a surrogate virus of the variola virus, both in suspension and dried on surfaces. These experiments were conducted in the presence of proteins, which are known to be one of the main factors affecting the efficacy of the disinfectants.12 Sanytex at a concentration of 1% proved to be an effective disinfectant, with a rapid virucidal effect. However, this efficacy was observed in the virus suspension containing a protein concentration of , 3 mg/mL. In our study, tests were also carried out with higher protein concentrations to mimic the usual conditions of contamination with biological fluids in the laboratory. Indeed, organic matter interacts with several disinfectants and neutralizes their virucidal effects.8,13 – 16 Several studies have been performed to determine the virucidal activity of disinfectants on poliovirus in blood8 or serum,13, 17 but there are very few reported studies on poxviruses and those reported have been carried out with protein concentrations of up to 30 mg/mL only.17 In our study, in the presence of protein concentrations . 3 mg/mL, higher concentrations of Sanytex were required to completely inactivate vaccinia virus after a short exposure time. Incubation in the presence of 30 and 70 mg/mL protein required concentrations of 3 and 10% Sanytex, respectively, to obtain a log10 reduction factor of more than 4 after 3 min exposure. As expected, the effect of Sanytex on vaccinia virus spread on surfaces was less rapid: it took a 15 min incubation with 10% Sanytex to completely inactivate dried vaccinia virus when the initial suspension (before desiccation) contained 10 mg protein/mL. With a protein concentration of 70 mg/mL, simulating body fluids, it took a 30 min incubation with a 30% Sanytex solution for virus inactivation. Sodium hypochlorite has been tested as a comparative agent of Sanytex at chlorine concentrations previously used on poliovirus.8 These concentrations include the concentration of
Rapid inactivation of vaccinia virus in suspension and dried on surfaces
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Table IV Virucidal effect of Sanytex solutions on dried virus suspension containing 10 and 70 mg BSA/mL Exposure time (min)
Reduction factor of virus titre (log10) BSA concentration 10 mg/mL
3 5 10 15 30
70 mg/mL
Sanytex 3%
Sanytex 10%
Sanytex 30%
Sanytex 30%
1.3 1.6 2.0 3.0 –
1.3 2.6 3.8 .4.2 –
1.5 3.0 .4.2 .4.2 –
– – 1.7 2.0 .4.2
The data represent average values for three experiments. The average titre of the controls was 104.2 TCID50/50 mL. Neutralization of the virucidal effect after the exposure time was checked. Virus suspension containing BSA 10 or 70 mg/mL was dried on a plastic surface.
0.0525% of active chlorine selected by the Centers of Disease Control and Prevention (CDC) to decontaminate HIV suspensions in blood spills18 and concentration of 0.036% active chlorine choose by French standard NF T 72-180 on virucidal activity.19 In this study, the highest concentration of active chlorine (0.525%) did not prove to be completely efficient on vaccinia virus suspension in presence of 70 mg protein/mL after 3 min exposure. Higher sodium hypochlorite concentrations might be efficient, but are highly corrosive and toxic compared with 10% Sanytex used to effectively inactivate the same suspension of vaccinia virus. The effect of the disinfectant Gigaseptw has been determined on vaccinia virus suspension containing 30 mg/mL proteins (40% calf serum). This dialdehyde, at a concentration of 5% achieved a log10 reduction factor of only 3 after 30 min contact time17 compared with 3% Sanytex allowing an efficient decontamination (104-fold reduction) of the same suspension in 3 min.
The French standard criteria19 (NF T 72-180) for assessment of virucidal effect of a disinfectant do not require the tests to be carried out in the presence of high concentrations of proteins and do not advise an exposure time of , 15 min or surface tests. The American Society for Testing and Materials20 (ASTM) mentions the addition of FCS at a final concentration of only 5% (protein concentration: 3.5 mg/mL) and the DVV21 (German association for the control of virus diseases) specified the presence of 10% FCS (approximately 7 mg/mL). However, it is important to take into account the high protein concentration of some biological samples such as body fluids, particularly serum or plasma, and this is why we carried out experiments in the presence of proteins at the concentration of 70 mg/mL, mimicking serum protein concentration. Surface tests have been described in literature,22 – 26 but have to be taken into account in European standards (CEN/TC 216) to simulate real conditions of use. Virucidal activity evaluated by
Table V Virucidal effect of Sanytex solutions on vaccinia virus suspension containing BSA 10 mg/mL for 3 min, after three months storage of the Sanytex solutions Storage conditionsa Sanytex concentration (%) 3 30 Pure fresh disinfectantd
Reduction factor of virus titreb (log10) Storage temperature (8C) RTc 30 RTc 30 –
3.0 3.1 2.9 3.1 3.0
The data represent average values for three experiments. The average titre of the controls was 104.3 TCID50/50 mL. Neutralization of the virucidal effect after the exposure time was checked. a The two solutions of Sanytex were stored at 30 8C or at room temperature for three months. b The different solutions of Sanytex and the fresh Sanytex were diluted to obtain a final concentration of 1%, then suspension test was achieved. c Room temperature (20–24 8C). d Pure Sanytex was used. Virus titre reduction achieved with a final 1% dilution of Sanytex performed immediately before the test.
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suspension tests cannot validate efficient surfaces disinfection (virus titre reduction of 104-fold) for which the concentration of disinfectant used27 or time exposure must be increased (see Sanytex results). Diluted Sanytex solution proved to be stable up to three months when stored between 20 and 30 8C. Moreover, trials have been achieved with the local fire brigade, in order to verify the stability of Sanytex under pressure in a fire hose nozzle. This study has shown that Sanytex is a powerful disinfectant for complete inactivation of vaccinia virus contaminated suspension and surfaces. This disinfectant should also be active on smallpox virus, and could be used in a case of bioterrorism involving a smallpox aerosolization attack. Indeed, vaccinia virus and smallpox viruses are very close viruses belonging to the genus Orthopoxvirus. Their genome homology is approximately of 95%.28 Moreover a previous study showed that there is no essential difference in terms of disinfectant sensitivity between smallpox virus and vaccinia virus, 29 suggesting that the decontamination procedures proposed in the present study could be used to decontaminate biological fluids or surfaces contaminated with smallpox virus. Sanytex was previously tested on several other viruses. It was shown to be efficient on adenovirus suspension at the 0.5% concentration (French standard AFNOR NF T 72-180). At this same concentration, human immunodeficiency virus (HIV) was effectively inactivated after a 3 min exposure (Barre ´-Sinoussi F., unpublished data). Moreover, in our laboratory, 1% Sanytex proved to be effective against yellow fever virus (the surrogate virus of hepatitis C virus), Semliki Forest virus and Rift Valley fever virus (data not shown) after a 3 min contact time in a suspension containing 10 mg protein/mL. The standard procedure for the complete decontamination of the laboratory uses gaseous formaldehyde, which has been shown to be efficient on vaccinia virus (data not shown). However, this method is time-consuming (12 h) and could not be applied routinely in the case of a major smallpox bioterrorism crisis when medical biology laboratories would need to be continuously operational. In such a situation, Sanytex could find an application in medical biology laboratories, hospitals or contaminated zones in the conditions described in this study. Indeed biological safety in such dramatic circumstances is achieved not only by anti-infectious treatment or vaccination, but also by a safe decontamination of laboratories, clinical facilities and contaminated locations, especially with the present risk of bioterrorism with the smallpox virus.
A. Ferrier et al.
Acknowledgements We thank Danielle Gratier and Henri Blancquaert for their excellent technical assistance and Bernard Souberbielle for critically reviewing the manuscript. This work was supported by research grants from the ‘Service de Sante ´ des Arme ´es’.
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