- Email: [email protected]
Disinfection of human enteric viruses on fomites
FEMS Microbiology Letters 156 (1997) 107^111
Disinfection of human enteric viruses on fomites F. Xavier Abad, Rosa M. Pintoè, Albert Bosch * Department of Microbiology, University of Barcelona, Av. Diagonal 645, 08028 Barcelona, Spain
Received 24 June 1997; revised 20 August 1997; accepted 1 September 1997
Abstract
The virucidal action of several commercially available disinfectant preparations was assayed against hepatitis A virus and human rotavirus dried on polystyrene. Overall, the level of virus disinfection achieved was very poor, usually inducing less than 3 log titre reduction. Suspension tests performed with the same disinfectants showed different virus inactivation rates, thus failing to provide a reliable indication of the actual virus disinfection on fomites. In our studies, bacteriophages of Bacteroides fragilis proved to be a simple, cheap and reliable screening tool for the evaluation of virus disinfection on non-porous surfaces. The same conclusion cannot be drawn for poliovirus. Keywords :
Virus persistence; Rotavirus; Poliovirus; Hepatitis A; Indicator; Bacteriophage
1. Introduction
Outbreaks of hepatitis A or acute gastroenteritis are a major public health issue, particularly in institutions such as day-care centres, hospitals, nurseries, schools and military quarters [1,2]. The aetiological agents of these diseases are excreted in high numbers in the faeces of infected individuals, and are able to persist for extended periods of time once in the environment [3,4]. In many outbreaks caused by enteric viruses, vehicular transmission of the agents apparently occurs through faecally contaminated environmental surfaces [5]. Disinfection of inanimate surfaces with chemicals should inhibit the surface-tohuman transmission of enteric virus infections. For simplicity, the virucidal action of disinfectants is usually assayed in suspension tests, rather than in carrier * Corresponding author. Tel.: +34 (3) 402 1485; Fax: +34 (3) 411 0592; E-mail: [email protected]
tests, and usually using poliovirus as the prototypic enteric virus. However, information on the e¤cacy of commercially available products on virus-infected fomites is scarce [6,7]. In this work we compared the results generated in carrier and suspension tests of the removal of the infectivity of rotavirus and hepatitis A virus by several commercially available disinfectant preparations. The validity of Bacteroides fragilis bacteriophages [8] as indicators in studies of virus disinfection in carrier tests was ascertained.
2. Materials and methods
2.1. Viruses and cells
Poliovirus 1, strain LSc 2ab (PV), and human rotavirus (HRV) Itor p13 (G3;P?) were propagated and assayed in BGM and MA-104 cells, respectively,
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Table 1 Information on the disinfectant products employed in this study Active compound Working dilutiona pHb Ethanol 70% 7.43 Chlorhexidine digluconate 0.05% 7.75 Sodium hypochlorite 0.125% 9.56 Phenol 1.41% 8.21 +Sodium phenate 0.24% Diethylenetriamine 0.0192% 11.86 Sodium chlorite 30% 1.85 a Working dilution as recommended by the manufacturer. Tap water was used as diluent. b pH of the working dilution.
as previously described [9]. FRhK-4 cell cultures were used to propagate and assay the cytopathogenic HM-175 (courtesy of T. Cromeans, Centers for Disease Control, Atlanta, GA) strain of hepatitis A virus (HAV) [10]. Bacteriophage B40-8 of Bacteroides fragilis was assayed as previously described [11]. The preparation of viruses used in these studies was deliberately not puri¢ed so that preparations as natural as possible were presented, as also recommended by other authors [12]. Viral enumerations were performed by calculating the most probable number of cytopathogenic units per ml (MPNCU/ml) by infecting cell monolayers grown in 96-well microtitre plates [16]. Eight wells were infected for each dilution, and 10 ml of inoculum was added to each well. All experiments were
performed in triplicate. All samples from a given experiment were assayed concurrently and titrated at least in duplicate. 2.2. Disinfectants
Several commercially available disinfectant preparations were tested for their ability to remove the infectivity of the viral strains: ethanol, Hibitane (chlorhexidine digluconate; ICI Farma, Porrinìo, Pontevedra, Spain), bleach (sodium hypochlorite), phenolic disinfectant (phenol and sodium phenate; Inibsa, Barcelona, Spain), Instrunet IP (diethylenetriamine; Inibsa) and Instrunet HD (sodium chlorite; Inibsa). The disinfectants are listed in Table 1, together with their formulations, working dilutions and pH. When necessary, tap water was used as diluent following the manufacturer's recommendations. Free chlorine (FC) concentrations in household bleach were determined by the N,N-diethyl-pphenylenediamine (DPD) method [13] using a test kit (Aquamerck 11735, Merck, Darmstadt, Germany). 2.3. Disinfectant test procedure
Viruses were subjected to the action of disinfectants suspended in PBS or in a 20% faecal suspension (FS). Faeces from a healthy man were previously mixed with PBS, autoclaved, vortexed and clari¢ed by centrifugation at 1000Ug. In the suspen-
Table 2 Virus persistencea after disinfection on carrier tests HRVc BFBd Disinfectant HAVb PBSe FSf PBS FS PBS FS Ethanol 31.36 ( þ 0.25) 31.33 ( þ 0.24) 32.34 ( þ 0.65) 30.95 ( þ 0.56) 30.19 ( þ 0.12) 30.62 ( þ 0.64) Chlorhexidine digluconate 30.95 ( þ 0.47) 30.52 ( þ 0.40) 30.82 ( þ 0.49) 30.45 ( þ 0.58) 30.22 ( þ 0.12) 30.71 ( þ 0.08) Sodium hypochlorite 32.58 ( þ 1.06) 31.12 ( þ 0.53) 32.76 ( þ 1.14) 31.62 ( þ 0.94) 31.25 ( þ 0.27) 30.67 ( þ 0.05) Phenol+Sodium phenate 32.09 ( þ 0.77) 31.66 ( þ 0.78) 30.87 ( þ 0.83) 30.19 ( þ 0.12) 30.54 ( þ 0.21) 30.22( þ 0.03) Diethylenetriamine 32.61 ( þ 0.55) 31.67 ( þ 0.68) 31.93 ( þ 0.65) 31.32 ( þ 0.63) 32.29 ( þ 1.51) 30.33 ( þ 0.06) Sodium chlorite 3 ( þ 1.31) 32.91 ( þ 0.18) 32.96 ( þ 0.89) 31.41 ( þ 0.64) 3 ( þ 0.15) 3 ( þ 0.84) Bold ¢gures represent titre reductions equal to or greater than 3 log10 . a Mean (þ S.D.) of log10 (Nd /N0 ). Experiments were performed in triplicate, and samples from a given experiment titrated at least in duplicate. b HAV: hepatitis A virus. c HRV: human rotavirus (Ito strain). d BFB: Bacteroides fragilis bacteriophages. e PBS: phosphate bu¡ered saline. f FS: faecal suspension (20% in PBS). 3.71
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Table 3 Virus persistencea after disinfection in suspension tests HRVc PVd Disinfectant HAVb PBSe FSf PBS FS PBS FS Ethanol 30.39 (þ 0.06) 30.43 ( þ 0.27) 3 ( 0.77) 3 ( 0.76) 6 3 30.33 ( þ 0.06) Chlorhexidine digluconate 0.11 (þ 0.40) 30.21 ( þ 0.49) 30.09 ( þ 0.47) 0.11 ( þ 0.58) 0.08 ( þ 0.03) 0.24 ( þ 0.12) Sodium hypochlorite 3 ( 0.89) 6 3 63 32.17 ( þ 0.55) 6 3 63 Phenol+Sodium phenate 0.11 (þ 0.24) 30.43 ( þ 0.50) 30.42 ( þ 0.49) 30.24 ( þ 0.12) 30.23 ( þ 0.12) 30.13 ( þ 0.12) Diethylenetriamine 3 ( 0.89) 3 ( 0.15) 3 ( 0.89) 32.99 ( þ 0.77) 6 3 63 Sodium chlorite 0.33 (þ 0.12) 32.07 ( þ 0.64) 32.17 ( þ 0.18) 32.41 ( þ 0.64) 32.99 ( þ 0.55) 30.09 ( þ 0.12) Bold ¢gures represent titre reductions equal to or greater than 3 log10 . a Mean (þ S.D.) of log10 (Nd /N0 ). Experiments were performed in triplicate, and samples from a given experiment titrated at least in duplicate. b HAV: hepatitis A virus. c HRV: human rotavirus. d PV: poliovirus. e PBS: phosphate bu¡ered saline. f FS: faecal suspension (20% in PBS). 3.11
3.57
þ
3.11
þ
4
3.81
þ
3.11
þ
4
þ
sion tests, 100 Wl of the virus suspensions (around 106 MPNCU) were mixed with 900 Wl of each disinfectant. Control samples received 900 Wl of PBS. After a contact time of 1 min [14] at 28 þ 1³C, a 100Wl aliquot of the virus-disinfectant mixture was removed and immediately diluted 100-fold with PBS to stop the disinfectant action. Samples were stored at 380³C until assayed for viruses. In the carrier tests, 24-well polystyrene plates were pretreated with 0.1% Tween 80, rinsed in sterile distilled water, and air dried [15]. 100 Wl of the virus suspensions (around 106 MPNCU) was placed at the centre of the wells and allowed to dry for 2^3 h inside a vertical laminar £ow hood (30þ 1³C and 55% relative humidity). For disinfection, 150 Wl of each formulation was added onto the dried virus suspensions. Control wells received the same amount of PBS. After a contact time of 10 min, the overlaying disinfectant was removed, and adsorbed viruses were eluted with 850 Wl of a solution of 3% beef extract (BE) in saline at pH 7.5 which after 10-min contact time were vigorously pipetted 20 times to recover the sample. The eluates were stored at 380³C until assayed, except an aliquot for phage assay that was kept at 4³C. Virus persistence was determined by calculating the log10 (Nd /N0 ) where N0 is the titre of viruses recovered from control samples and Nd is the titre after disinfection. Data were processed with a MPN computer program [17]. The analysis of variance (ANOVA) test [18] was performed in the log-transformed data to determine signi¢cant di¡erences gen-
3.11
þ
4
4
4
4
4
erated by the disinfection conditions and/or virus strain. 3. Results and discussion
The results of the carrier tests for the disinfection of experimentally contaminated polystyrene are summarised in Table 2. In previous studies, we veri¢ed that polystyrene may be used as a model fomite to investigate the behaviour of enteric viruses on nonporous environmental surfaces, such as aluminium, china, glazed tile and latex [15]. Poliovirus, the prototypic enteric virus which has been extensively used as a model strain, was not included in the carrier tests since it is very susceptible to desiccation (data not shown) and consequently it should not be employed in studies of disinfection of environmental surfaces [15]. A disinfectant formulation may be considered e¡ective if it is capable of inducing a 3 log10 (99.9%) or greater reduction in the virus titre [6,14]. The level of virus disinfection achieved by the tested disinfectants is considered to be very poor, with the exception of sodium chlorite which is close to inducing a 3 log titre reduction of HAV and HRV in most scenarios. In previous studies, chlorhexidine, phenolic and chlorine based disinfectants have been reported to be e¤cient bactericidal agents [19]. No previous data exist on the virus inactivating capacity of the phenolic and diethylenetriamine preparations employed in our study. In the present work, at the
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dilution and conditions recommended by the manufacturer, only sodium chlorite proved to be e¡ective towards HAV in PBS, and BFB in either PBS or FS. In studies performed by other authors [12], sodium chlorite employed at a 0.275% concentration was ine¡ective against HAV. In our work, increasing the disinfectant concentration up to 30% provided a much better removal of HAV infectivity. Ethanol was used as a 70% aqueous dilution since the presence of water has been reported to be essential for its antimicrobial activity [19]. However, 70% ethanol was unable to e¤ciently remove the infectivity of the assayed enteric viruses. Similar conclusions have been reported by Mbithi et al. [12] and Lloyd-Evans et al. [6] for HAV and HRV, respectively. In the latter work [6], chlorhexidine digluconate was assayed against the Wa strain of HRV (G1;P8), inducing a reduction in the titre greater than 3 logs. In our studies employing the Ito strain of HRV, the virus decay induced by the same disinfectant at the same concentration was lower than 1 log. These data suggest the possibility of a di¡erential behaviour of di¡erent strains of HRV. The results of the suspension tests performed with the same disinfectant preparations are depicted in Table 3. Only diethylenetriamine and sodium chlorite proved to be e¤cient virus disinfectants in suspension tests. A comparison of results in suspension tests with those from the carrier tests (Table 2) shows that disinfection of viruses from contaminated surfaces gives signi¢cantly di¡erent data from suspension tests with the same viruses and chemical preparations. This agrees with the ¢ndings of other workers with di¡erent viruses [6,20]. Additionally, virus inactivation rates in suspension tests did not signi¢cantly (P 6 0.05) increase when the contact time was extended up to 10 min (data not shown). Suspension tests fail to provide a reliable indication of the e¤ciency of virus disinfection on fomites. B. fragilis bacteriophages [8] have been proposed as promising candidates to be used as substitutes of human viruses on fomites, particularly on non-porous surfaces [15]. In that study, the survival of BFB on a variety of environmental surfaces consistently exceeded that of PV and enteric adenovirus and sometimes equalled that of HAV and HRV [15]. In the present work, the behaviour of BFB in carrier tests was not signi¢cantly (P 6 0.05) di¡erent
from that of HAV or HRV after chemical disinfection, except when sodium chlorite was employed (Table 2). In this situation, BFB persisted signi¢cantly (P 6 0.05) less than HAV or HRV. However, BFB appear a cheaper, simpler and more reliable choice than PV for use as model virus in studies of virus disinfection on inanimate surfaces. Environmental contamination by enteric viruses generally occurs in the presence of body excretions, and a product should only be regarded as reliable if it can rapidly inactivate virus on a contaminated surface in the presence of organic and inorganic matter. In this study, fomite disinfection was also conducted with viruses suspended in faeces in order to mimic the actual natural conditions. In previous studies [15], the protective e¡ect of faecal material on the survival of viruses on fomites was unclear. In the present work, the presence of organic matter did not signi¢cantly (P 6 0.05) increase virus persistence after disinfection in suspension tests. In carrier tests, most chemical preparations reduced their disinfection e¤ciency in the presence of faeces, although not to a signi¢cant degree (P 6 0.05) (Table 2). Evidence gathered in some institutional outbreaks of enteric diseases suggests that surfaces may act as vehicles for the spread of the infection [21,22]. Some enteric viruses have been shown in a previous study to survive on inert surfaces long enough to represent a source of secondary transmission of disease [15]. Most studies of enteric virus disinfection have so far been conducted in suspension tests and using poliovirus as the model enteric virus strain [23,24]. Although such tests are relatively simple to perform, there is a need to evaluate the disinfectants on contaminated surfaces as well, and employing viruses showing greater persistence than poliovirus. As an alternative, bacteriophages are promising indicators of viruses because their handling is simple, inexpensive, and does not require specialized personnel or sophisticated facilities. BFB, which have been successfully employed as virus indicators for the evaluation of antiseptics and disinfectants in suspension tests [25], may act as a screening tool for assessment of surface disinfection of human enteric viruses. Proper sanitation and disinfection measures, especially in high-risk settings such as day-care centres, hospital wards or restaurants, are of seminal importance in public health.
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Acknowledgments
This work was supported in part by Grant 1995SGR from the Generalitat de Catalunya. F.X. Abad was the recipient of a F.I. fellowship from the Generalitat de Catalunya. References [1] Bern, C., Martines, J., de Zoysa, I. and Glass, R.I. (1992) The magnitude of the global problem of diarrhoeal disease: a tenyear update. WHO Bull. 70, 705^714. [2] Dickinson, C.J. (1992) Hepatitis A ^ New information on an old virus. Hepatology 16, 1099^1101. [3] Flewett, T.H., Beards, G.M., Brown, D.W.G. and Sanders, R.C. (1987) The diagnostic gap in diarrhoeal aetiology. In: Novel Diarrhoea Viruses, Ciba Foundation Symposium 128 (Bock, G. and Whelan, J., Eds.), pp. 238^249. Wiley, Chichester. [4] Sobsey, M.D., Shields, P.A., Hauchman, F.S., Davis, A.L., Rullman, V.A. and Bosch, A. Survival and persistence of hepatitis A virus in environmental samples. In: Viral Hepatitis and Liver Disease (Zuckerman, A.Z., Ed.), pp. 121^124. Alan R. Liss, New York. [5] Sattar, S.A., Lloyd-Evans, N. and Springthorpe, V.S. (1986) Institutional outbreaks of rotavirus diarrhoea: potential role of fomites and environmental surfaces as vehicles for virus transmission. J. Hyg. Camb. 96, 277^289. [6] Lloyd-Evans, N., Springthorpe, V.S. and Sattar, S.A. (1986) Chemical disinfection of human rotavirus-contaminated inanimate surfaces. J. Hyg. Camb. 97, 163^173. [7] Ward, R.L., Bernstein, D.I., Knowlton, D.R., Sherwood, J.R., Young, E.C., Cusack, T.M., Rubino, J.R. and Schi¡, G.M. (1991) Prevention of surface-to-human transmission of rotaviruses by treatment with disinfectant spray. J. Clin. Microbiol. 29, 1991^1996. [8] Tartera, C. and Jofre, J. (1987) Bacteriophages active against Bacteroides fragilis in sewage-polluted waters. Appl. Environ. Microbiol. 53, 1632^1637. [9] Bosch, A., Lucena, F., Diez, J.M., Gajardo, R., Blasi, M. and Jofre, J. (1991) Waterborne viruses associated with an hepatitis A outbreak. J. Am. Water Works Assoc. 83, 80^83. [10] Cromeans, T., Sobsey, M.D. and Fields, H.A. (1987) Development of a plaque assay for a cytopathic rapidly replicating isolate of hepatitis A virus. J. Med. Virol. 22, 45^56. [11] Tartera, C., Araujo, R., Michel, T. and Jofre, J. (1992) Culture and decontamination methods a¡ecting enumeration of phages infecting Bacteroides fragilis in sewage. Appl. Environ. Microbiol. 58, 2670^2673.
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[12] Mbithi, J.N., Springthorpe, V.S. and Sattar, S.A. (1990) Chemical disinfection of hepatitis A virus on environmental surfaces. Appl. Environ. Microbiol. 56, 3601^3604. [13] American Public Health Association (1995) Standard Methods for the Examination of Water and Wastewater, 19th edn. American Public Health Association, American Water Works Association, Water Pollution Control Federation, Washington, DC. [14] Springthorpe, V.S., Grenier, J.L., Lloyd-Evans, N. and Sattar, S.A. (1986) Chemical disinfection of human rotaviruses: e¤cacy of commercial-available products in suspension test. J. Hyg. Camb. 97, 139^161. [15] Abad, F.X., Pintoè, R.M. and Bosch, A. (1994) Survival of enteric viruses on environmental fomites. Appl. Environ. Microbiol. 60, 3704^3710. [16] Pintoè, R.M., Diez, J.M. and Bosch, A. (1994) Use of the colonic carcinoma cell line CaCo-2 for in vivo ampli¢cation and detection of enteric viruses. J. Med. Virol. 44, 310^315. [17] Hurley, M.A. and Roscoe, M.E. (1983) Automated statistical analysis of microbial enumeration by dilution series. J. Appl. Bacteriol. 55, 159^164. [18] Sokal, R.R. and Rohlf, E.J. (1973) Introduction to Biostatistics. W.H. Freeman, San Francisco, CA. [19] Rusell, A.D., Hugo, W.B. and Ayli¡e, G.A.J. (1982) Principles and Practice of Disinfection, Preservation and Sterilization. Blackwell Scienti¢c Publications, Oxford. [20] Schurmann, W. and Eggers, H.J. (1983) Antiviral activity of an alcoholic hand disinfectant. Comparison of the in vitro suspension test with in vivo experiments on hands, and on individual ¢ngertips. Antiviral Res. 3, 25^41. [21] Ryder, R.W., McGowan, J.E., Hatch, M.H. and Palmer, E.L. (1977) Reovirus-like agent as a cause of nosocomial diarrhoea in infants. J. Pediatr. 90, 698^702. [22] Halvorsrud, J. and Orstavick, I. (1980) An epidemic of rotavirus-associated gastroenteritis in a nursing home for the elderly. Scand. J. Infect. Dis. 12, 161^164. [23] Payment, P., Tremblay, M. and Trudel, M. (1985) Relative resistance to chlorine of poliovirus and coxsackievirus isolates from environmental sources and drinking water. Appl. Environ. Microbiol. 49, 981^983. [24] Bailly, J.L., Chamboin, M., Peigue-Lafeuille, Laveran, H., DeChamps, C. and Beytout, D. (1991) Activity of glutaraldehyde at low concentrations ( 6 2%) against poliovirus and its relevance to gastrointestinal endoscope disinfection procedures. Appl. Environ. Microbiol. 57, 1156^1160. [25] Pintoè, R.M., Abad, F.X., Roca Riera, J.M. and Bosch, A. (1991) The use of bacteriophages of Bacteroides fragilis as indicators of the e¤ciency of virucidal products. FEMS Microbiol. Lett. 82, 61^66.
FEMSLE 7838 10-11-97
Disinfection of human enteric viruses on fomites F. Xavier Abad, Rosa M. Pintoè, Albert Bosch * Department of Microbiology, University of Barcelona, Av. Diagonal 645, 08028 Barcelona, Spain
Received 24 June 1997; revised 20 August 1997; accepted 1 September 1997
Abstract
The virucidal action of several commercially available disinfectant preparations was assayed against hepatitis A virus and human rotavirus dried on polystyrene. Overall, the level of virus disinfection achieved was very poor, usually inducing less than 3 log titre reduction. Suspension tests performed with the same disinfectants showed different virus inactivation rates, thus failing to provide a reliable indication of the actual virus disinfection on fomites. In our studies, bacteriophages of Bacteroides fragilis proved to be a simple, cheap and reliable screening tool for the evaluation of virus disinfection on non-porous surfaces. The same conclusion cannot be drawn for poliovirus. Keywords :
Virus persistence; Rotavirus; Poliovirus; Hepatitis A; Indicator; Bacteriophage
1. Introduction
Outbreaks of hepatitis A or acute gastroenteritis are a major public health issue, particularly in institutions such as day-care centres, hospitals, nurseries, schools and military quarters [1,2]. The aetiological agents of these diseases are excreted in high numbers in the faeces of infected individuals, and are able to persist for extended periods of time once in the environment [3,4]. In many outbreaks caused by enteric viruses, vehicular transmission of the agents apparently occurs through faecally contaminated environmental surfaces [5]. Disinfection of inanimate surfaces with chemicals should inhibit the surface-tohuman transmission of enteric virus infections. For simplicity, the virucidal action of disinfectants is usually assayed in suspension tests, rather than in carrier * Corresponding author. Tel.: +34 (3) 402 1485; Fax: +34 (3) 411 0592; E-mail: [email protected]
tests, and usually using poliovirus as the prototypic enteric virus. However, information on the e¤cacy of commercially available products on virus-infected fomites is scarce [6,7]. In this work we compared the results generated in carrier and suspension tests of the removal of the infectivity of rotavirus and hepatitis A virus by several commercially available disinfectant preparations. The validity of Bacteroides fragilis bacteriophages [8] as indicators in studies of virus disinfection in carrier tests was ascertained.
2. Materials and methods
2.1. Viruses and cells
Poliovirus 1, strain LSc 2ab (PV), and human rotavirus (HRV) Itor p13 (G3;P?) were propagated and assayed in BGM and MA-104 cells, respectively,
0378-1097 / 97 / $17.00 ß 1997 Published by Elsevier Science B.V. All rights reserved. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 4 1 0 - 2
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Table 1 Information on the disinfectant products employed in this study Active compound Working dilutiona pHb Ethanol 70% 7.43 Chlorhexidine digluconate 0.05% 7.75 Sodium hypochlorite 0.125% 9.56 Phenol 1.41% 8.21 +Sodium phenate 0.24% Diethylenetriamine 0.0192% 11.86 Sodium chlorite 30% 1.85 a Working dilution as recommended by the manufacturer. Tap water was used as diluent. b pH of the working dilution.
as previously described [9]. FRhK-4 cell cultures were used to propagate and assay the cytopathogenic HM-175 (courtesy of T. Cromeans, Centers for Disease Control, Atlanta, GA) strain of hepatitis A virus (HAV) [10]. Bacteriophage B40-8 of Bacteroides fragilis was assayed as previously described [11]. The preparation of viruses used in these studies was deliberately not puri¢ed so that preparations as natural as possible were presented, as also recommended by other authors [12]. Viral enumerations were performed by calculating the most probable number of cytopathogenic units per ml (MPNCU/ml) by infecting cell monolayers grown in 96-well microtitre plates [16]. Eight wells were infected for each dilution, and 10 ml of inoculum was added to each well. All experiments were
performed in triplicate. All samples from a given experiment were assayed concurrently and titrated at least in duplicate. 2.2. Disinfectants
Several commercially available disinfectant preparations were tested for their ability to remove the infectivity of the viral strains: ethanol, Hibitane (chlorhexidine digluconate; ICI Farma, Porrinìo, Pontevedra, Spain), bleach (sodium hypochlorite), phenolic disinfectant (phenol and sodium phenate; Inibsa, Barcelona, Spain), Instrunet IP (diethylenetriamine; Inibsa) and Instrunet HD (sodium chlorite; Inibsa). The disinfectants are listed in Table 1, together with their formulations, working dilutions and pH. When necessary, tap water was used as diluent following the manufacturer's recommendations. Free chlorine (FC) concentrations in household bleach were determined by the N,N-diethyl-pphenylenediamine (DPD) method [13] using a test kit (Aquamerck 11735, Merck, Darmstadt, Germany). 2.3. Disinfectant test procedure
Viruses were subjected to the action of disinfectants suspended in PBS or in a 20% faecal suspension (FS). Faeces from a healthy man were previously mixed with PBS, autoclaved, vortexed and clari¢ed by centrifugation at 1000Ug. In the suspen-
Table 2 Virus persistencea after disinfection on carrier tests HRVc BFBd Disinfectant HAVb PBSe FSf PBS FS PBS FS Ethanol 31.36 ( þ 0.25) 31.33 ( þ 0.24) 32.34 ( þ 0.65) 30.95 ( þ 0.56) 30.19 ( þ 0.12) 30.62 ( þ 0.64) Chlorhexidine digluconate 30.95 ( þ 0.47) 30.52 ( þ 0.40) 30.82 ( þ 0.49) 30.45 ( þ 0.58) 30.22 ( þ 0.12) 30.71 ( þ 0.08) Sodium hypochlorite 32.58 ( þ 1.06) 31.12 ( þ 0.53) 32.76 ( þ 1.14) 31.62 ( þ 0.94) 31.25 ( þ 0.27) 30.67 ( þ 0.05) Phenol+Sodium phenate 32.09 ( þ 0.77) 31.66 ( þ 0.78) 30.87 ( þ 0.83) 30.19 ( þ 0.12) 30.54 ( þ 0.21) 30.22( þ 0.03) Diethylenetriamine 32.61 ( þ 0.55) 31.67 ( þ 0.68) 31.93 ( þ 0.65) 31.32 ( þ 0.63) 32.29 ( þ 1.51) 30.33 ( þ 0.06) Sodium chlorite 3 ( þ 1.31) 32.91 ( þ 0.18) 32.96 ( þ 0.89) 31.41 ( þ 0.64) 3 ( þ 0.15) 3 ( þ 0.84) Bold ¢gures represent titre reductions equal to or greater than 3 log10 . a Mean (þ S.D.) of log10 (Nd /N0 ). Experiments were performed in triplicate, and samples from a given experiment titrated at least in duplicate. b HAV: hepatitis A virus. c HRV: human rotavirus (Ito strain). d BFB: Bacteroides fragilis bacteriophages. e PBS: phosphate bu¡ered saline. f FS: faecal suspension (20% in PBS). 3.71
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Table 3 Virus persistencea after disinfection in suspension tests HRVc PVd Disinfectant HAVb PBSe FSf PBS FS PBS FS Ethanol 30.39 (þ 0.06) 30.43 ( þ 0.27) 3 ( 0.77) 3 ( 0.76) 6 3 30.33 ( þ 0.06) Chlorhexidine digluconate 0.11 (þ 0.40) 30.21 ( þ 0.49) 30.09 ( þ 0.47) 0.11 ( þ 0.58) 0.08 ( þ 0.03) 0.24 ( þ 0.12) Sodium hypochlorite 3 ( 0.89) 6 3 63 32.17 ( þ 0.55) 6 3 63 Phenol+Sodium phenate 0.11 (þ 0.24) 30.43 ( þ 0.50) 30.42 ( þ 0.49) 30.24 ( þ 0.12) 30.23 ( þ 0.12) 30.13 ( þ 0.12) Diethylenetriamine 3 ( 0.89) 3 ( 0.15) 3 ( 0.89) 32.99 ( þ 0.77) 6 3 63 Sodium chlorite 0.33 (þ 0.12) 32.07 ( þ 0.64) 32.17 ( þ 0.18) 32.41 ( þ 0.64) 32.99 ( þ 0.55) 30.09 ( þ 0.12) Bold ¢gures represent titre reductions equal to or greater than 3 log10 . a Mean (þ S.D.) of log10 (Nd /N0 ). Experiments were performed in triplicate, and samples from a given experiment titrated at least in duplicate. b HAV: hepatitis A virus. c HRV: human rotavirus. d PV: poliovirus. e PBS: phosphate bu¡ered saline. f FS: faecal suspension (20% in PBS). 3.11
3.57
þ
3.11
þ
4
3.81
þ
3.11
þ
4
þ
sion tests, 100 Wl of the virus suspensions (around 106 MPNCU) were mixed with 900 Wl of each disinfectant. Control samples received 900 Wl of PBS. After a contact time of 1 min [14] at 28 þ 1³C, a 100Wl aliquot of the virus-disinfectant mixture was removed and immediately diluted 100-fold with PBS to stop the disinfectant action. Samples were stored at 380³C until assayed for viruses. In the carrier tests, 24-well polystyrene plates were pretreated with 0.1% Tween 80, rinsed in sterile distilled water, and air dried [15]. 100 Wl of the virus suspensions (around 106 MPNCU) was placed at the centre of the wells and allowed to dry for 2^3 h inside a vertical laminar £ow hood (30þ 1³C and 55% relative humidity). For disinfection, 150 Wl of each formulation was added onto the dried virus suspensions. Control wells received the same amount of PBS. After a contact time of 10 min, the overlaying disinfectant was removed, and adsorbed viruses were eluted with 850 Wl of a solution of 3% beef extract (BE) in saline at pH 7.5 which after 10-min contact time were vigorously pipetted 20 times to recover the sample. The eluates were stored at 380³C until assayed, except an aliquot for phage assay that was kept at 4³C. Virus persistence was determined by calculating the log10 (Nd /N0 ) where N0 is the titre of viruses recovered from control samples and Nd is the titre after disinfection. Data were processed with a MPN computer program [17]. The analysis of variance (ANOVA) test [18] was performed in the log-transformed data to determine signi¢cant di¡erences gen-
3.11
þ
4
4
4
4
4
erated by the disinfection conditions and/or virus strain. 3. Results and discussion
The results of the carrier tests for the disinfection of experimentally contaminated polystyrene are summarised in Table 2. In previous studies, we veri¢ed that polystyrene may be used as a model fomite to investigate the behaviour of enteric viruses on nonporous environmental surfaces, such as aluminium, china, glazed tile and latex [15]. Poliovirus, the prototypic enteric virus which has been extensively used as a model strain, was not included in the carrier tests since it is very susceptible to desiccation (data not shown) and consequently it should not be employed in studies of disinfection of environmental surfaces [15]. A disinfectant formulation may be considered e¡ective if it is capable of inducing a 3 log10 (99.9%) or greater reduction in the virus titre [6,14]. The level of virus disinfection achieved by the tested disinfectants is considered to be very poor, with the exception of sodium chlorite which is close to inducing a 3 log titre reduction of HAV and HRV in most scenarios. In previous studies, chlorhexidine, phenolic and chlorine based disinfectants have been reported to be e¤cient bactericidal agents [19]. No previous data exist on the virus inactivating capacity of the phenolic and diethylenetriamine preparations employed in our study. In the present work, at the
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dilution and conditions recommended by the manufacturer, only sodium chlorite proved to be e¡ective towards HAV in PBS, and BFB in either PBS or FS. In studies performed by other authors [12], sodium chlorite employed at a 0.275% concentration was ine¡ective against HAV. In our work, increasing the disinfectant concentration up to 30% provided a much better removal of HAV infectivity. Ethanol was used as a 70% aqueous dilution since the presence of water has been reported to be essential for its antimicrobial activity [19]. However, 70% ethanol was unable to e¤ciently remove the infectivity of the assayed enteric viruses. Similar conclusions have been reported by Mbithi et al. [12] and Lloyd-Evans et al. [6] for HAV and HRV, respectively. In the latter work [6], chlorhexidine digluconate was assayed against the Wa strain of HRV (G1;P8), inducing a reduction in the titre greater than 3 logs. In our studies employing the Ito strain of HRV, the virus decay induced by the same disinfectant at the same concentration was lower than 1 log. These data suggest the possibility of a di¡erential behaviour of di¡erent strains of HRV. The results of the suspension tests performed with the same disinfectant preparations are depicted in Table 3. Only diethylenetriamine and sodium chlorite proved to be e¤cient virus disinfectants in suspension tests. A comparison of results in suspension tests with those from the carrier tests (Table 2) shows that disinfection of viruses from contaminated surfaces gives signi¢cantly di¡erent data from suspension tests with the same viruses and chemical preparations. This agrees with the ¢ndings of other workers with di¡erent viruses [6,20]. Additionally, virus inactivation rates in suspension tests did not signi¢cantly (P 6 0.05) increase when the contact time was extended up to 10 min (data not shown). Suspension tests fail to provide a reliable indication of the e¤ciency of virus disinfection on fomites. B. fragilis bacteriophages [8] have been proposed as promising candidates to be used as substitutes of human viruses on fomites, particularly on non-porous surfaces [15]. In that study, the survival of BFB on a variety of environmental surfaces consistently exceeded that of PV and enteric adenovirus and sometimes equalled that of HAV and HRV [15]. In the present work, the behaviour of BFB in carrier tests was not signi¢cantly (P 6 0.05) di¡erent
from that of HAV or HRV after chemical disinfection, except when sodium chlorite was employed (Table 2). In this situation, BFB persisted signi¢cantly (P 6 0.05) less than HAV or HRV. However, BFB appear a cheaper, simpler and more reliable choice than PV for use as model virus in studies of virus disinfection on inanimate surfaces. Environmental contamination by enteric viruses generally occurs in the presence of body excretions, and a product should only be regarded as reliable if it can rapidly inactivate virus on a contaminated surface in the presence of organic and inorganic matter. In this study, fomite disinfection was also conducted with viruses suspended in faeces in order to mimic the actual natural conditions. In previous studies [15], the protective e¡ect of faecal material on the survival of viruses on fomites was unclear. In the present work, the presence of organic matter did not signi¢cantly (P 6 0.05) increase virus persistence after disinfection in suspension tests. In carrier tests, most chemical preparations reduced their disinfection e¤ciency in the presence of faeces, although not to a signi¢cant degree (P 6 0.05) (Table 2). Evidence gathered in some institutional outbreaks of enteric diseases suggests that surfaces may act as vehicles for the spread of the infection [21,22]. Some enteric viruses have been shown in a previous study to survive on inert surfaces long enough to represent a source of secondary transmission of disease [15]. Most studies of enteric virus disinfection have so far been conducted in suspension tests and using poliovirus as the model enteric virus strain [23,24]. Although such tests are relatively simple to perform, there is a need to evaluate the disinfectants on contaminated surfaces as well, and employing viruses showing greater persistence than poliovirus. As an alternative, bacteriophages are promising indicators of viruses because their handling is simple, inexpensive, and does not require specialized personnel or sophisticated facilities. BFB, which have been successfully employed as virus indicators for the evaluation of antiseptics and disinfectants in suspension tests [25], may act as a screening tool for assessment of surface disinfection of human enteric viruses. Proper sanitation and disinfection measures, especially in high-risk settings such as day-care centres, hospital wards or restaurants, are of seminal importance in public health.
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Acknowledgments
This work was supported in part by Grant 1995SGR from the Generalitat de Catalunya. F.X. Abad was the recipient of a F.I. fellowship from the Generalitat de Catalunya. References [1] Bern, C., Martines, J., de Zoysa, I. and Glass, R.I. (1992) The magnitude of the global problem of diarrhoeal disease: a tenyear update. WHO Bull. 70, 705^714. [2] Dickinson, C.J. (1992) Hepatitis A ^ New information on an old virus. Hepatology 16, 1099^1101. [3] Flewett, T.H., Beards, G.M., Brown, D.W.G. and Sanders, R.C. (1987) The diagnostic gap in diarrhoeal aetiology. In: Novel Diarrhoea Viruses, Ciba Foundation Symposium 128 (Bock, G. and Whelan, J., Eds.), pp. 238^249. Wiley, Chichester. [4] Sobsey, M.D., Shields, P.A., Hauchman, F.S., Davis, A.L., Rullman, V.A. and Bosch, A. Survival and persistence of hepatitis A virus in environmental samples. In: Viral Hepatitis and Liver Disease (Zuckerman, A.Z., Ed.), pp. 121^124. Alan R. Liss, New York. [5] Sattar, S.A., Lloyd-Evans, N. and Springthorpe, V.S. (1986) Institutional outbreaks of rotavirus diarrhoea: potential role of fomites and environmental surfaces as vehicles for virus transmission. J. Hyg. Camb. 96, 277^289. [6] Lloyd-Evans, N., Springthorpe, V.S. and Sattar, S.A. (1986) Chemical disinfection of human rotavirus-contaminated inanimate surfaces. J. Hyg. Camb. 97, 163^173. [7] Ward, R.L., Bernstein, D.I., Knowlton, D.R., Sherwood, J.R., Young, E.C., Cusack, T.M., Rubino, J.R. and Schi¡, G.M. (1991) Prevention of surface-to-human transmission of rotaviruses by treatment with disinfectant spray. J. Clin. Microbiol. 29, 1991^1996. [8] Tartera, C. and Jofre, J. (1987) Bacteriophages active against Bacteroides fragilis in sewage-polluted waters. Appl. Environ. Microbiol. 53, 1632^1637. [9] Bosch, A., Lucena, F., Diez, J.M., Gajardo, R., Blasi, M. and Jofre, J. (1991) Waterborne viruses associated with an hepatitis A outbreak. J. Am. Water Works Assoc. 83, 80^83. [10] Cromeans, T., Sobsey, M.D. and Fields, H.A. (1987) Development of a plaque assay for a cytopathic rapidly replicating isolate of hepatitis A virus. J. Med. Virol. 22, 45^56. [11] Tartera, C., Araujo, R., Michel, T. and Jofre, J. (1992) Culture and decontamination methods a¡ecting enumeration of phages infecting Bacteroides fragilis in sewage. Appl. Environ. Microbiol. 58, 2670^2673.
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[12] Mbithi, J.N., Springthorpe, V.S. and Sattar, S.A. (1990) Chemical disinfection of hepatitis A virus on environmental surfaces. Appl. Environ. Microbiol. 56, 3601^3604. [13] American Public Health Association (1995) Standard Methods for the Examination of Water and Wastewater, 19th edn. American Public Health Association, American Water Works Association, Water Pollution Control Federation, Washington, DC. [14] Springthorpe, V.S., Grenier, J.L., Lloyd-Evans, N. and Sattar, S.A. (1986) Chemical disinfection of human rotaviruses: e¤cacy of commercial-available products in suspension test. J. Hyg. Camb. 97, 139^161. [15] Abad, F.X., Pintoè, R.M. and Bosch, A. (1994) Survival of enteric viruses on environmental fomites. Appl. Environ. Microbiol. 60, 3704^3710. [16] Pintoè, R.M., Diez, J.M. and Bosch, A. (1994) Use of the colonic carcinoma cell line CaCo-2 for in vivo ampli¢cation and detection of enteric viruses. J. Med. Virol. 44, 310^315. [17] Hurley, M.A. and Roscoe, M.E. (1983) Automated statistical analysis of microbial enumeration by dilution series. J. Appl. Bacteriol. 55, 159^164. [18] Sokal, R.R. and Rohlf, E.J. (1973) Introduction to Biostatistics. W.H. Freeman, San Francisco, CA. [19] Rusell, A.D., Hugo, W.B. and Ayli¡e, G.A.J. (1982) Principles and Practice of Disinfection, Preservation and Sterilization. Blackwell Scienti¢c Publications, Oxford. [20] Schurmann, W. and Eggers, H.J. (1983) Antiviral activity of an alcoholic hand disinfectant. Comparison of the in vitro suspension test with in vivo experiments on hands, and on individual ¢ngertips. Antiviral Res. 3, 25^41. [21] Ryder, R.W., McGowan, J.E., Hatch, M.H. and Palmer, E.L. (1977) Reovirus-like agent as a cause of nosocomial diarrhoea in infants. J. Pediatr. 90, 698^702. [22] Halvorsrud, J. and Orstavick, I. (1980) An epidemic of rotavirus-associated gastroenteritis in a nursing home for the elderly. Scand. J. Infect. Dis. 12, 161^164. [23] Payment, P., Tremblay, M. and Trudel, M. (1985) Relative resistance to chlorine of poliovirus and coxsackievirus isolates from environmental sources and drinking water. Appl. Environ. Microbiol. 49, 981^983. [24] Bailly, J.L., Chamboin, M., Peigue-Lafeuille, Laveran, H., DeChamps, C. and Beytout, D. (1991) Activity of glutaraldehyde at low concentrations ( 6 2%) against poliovirus and its relevance to gastrointestinal endoscope disinfection procedures. Appl. Environ. Microbiol. 57, 1156^1160. [25] Pintoè, R.M., Abad, F.X., Roca Riera, J.M. and Bosch, A. (1991) The use of bacteriophages of Bacteroides fragilis as indicators of the e¤ciency of virucidal products. FEMS Microbiol. Lett. 82, 61^66.
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