Epidem. Inf. (1989), 102, 493-505 493Printed in (reat Britaiin

Chemical disinfection of non-porous inanimate surfacesexperimentally contaminated with four human pathogenic viruses

S. A. SATTAR, V. S. SPRINGTHORPE, Y. KARIM AND P. LORODepartment of Microbiology and Immunology, Faculty of Health Sciences,

University of Ottawa, Ottawa, Ontario, Canada KJH 8M5

(Accepted 27 January 1989)

SUMNIARY

The chemical disinfection of virus-contaminated non-porous inanimate surfaceswas investigated using coxsackievirus B3, adenovirus type 5, parainfluenzavirustype 3 and coronavirus 229E as representatives of important nosocomial viralpathogens. A 10 ,ul amount of the test virus, suspended in either faeces or mucin,was placed onto each stainless steel disk (about 1 cm in diameter) and theinoculum allowed to dry for 1 h under ambient conditions. Sixteen disinfectantformulations were selected for this study based on the findings of an earlierinvestigation with a human rotavirus. After 1 min exposure to 20,u1 of thedisinfectant, the virus from the disks was immediately eluted into tryptosephosphate broth and plaque assayed. Using an efficacy criterion of a 3 log10 orgreater reduction in virus infectivity titre and irrespective of the virus suspendingmedium, only the following five disinfectants proved to be effective against all thefour viruses tested: (1) 2% glutaraldehyde normally used as an instrument soak,(2) a strongly alkaline mixture of 05% sodium o-benzyl-p-chlorophenate and0-6% sodium lauryl sulphate, generally used as a domestic disinfectant cleaner forhard surfaces, (3) a 0 04% solution of a quaternary ammonium compoundcontaining 7% hydrochloric acid, which is the basis of many toilet bowl cleaners.(4) chloramine T at a minimum free chlorine level of 3000 p.p.m. and (5) sodiumhypochlorite at a minimum free chlorine concentration of 5000 p.p.m. Of thosechemicals suitable for use as topical antiseptics, 70% ethanol alone or productscontaining at least 70% ethanol were ineffective only against coxsackievirus B3.These results emphasize the care needed in selecting chemical disinfectants forroutine use in infection control.

INTROD)UCTION

Outbreaks of viral infections in institutional settings arc quite common andl itis not unusual for more than one virus to be circulating simultaneously within a

given institution (Meissner et al. 1984; Payne, Grilli & Smith, 1984). The exactmeans of spread of viral agents in many such outbreaks still remain unclear, but,for many viral pathogens, multiple vehicles mav be involved. Evidence suggeststhat virus-contaminated surfaces may play a role (Pattison et al. 1974; Maynard,1976; Hall, Douglas & Geiman, 1980; England, 1982; Pancic, Carpenter & Came,

494 S. A. SATTAR AND OTIIERS

1980; Hutto et al. 1986; Sattar et al. 1986; Sattar & Springthorpe. 19877; Anisariet al. 1988a). Even when aerosolization of infeetious virus oceurs. settling of coarseparticles results in the contamination of surfaces. Tfherefore. chemical (lisinfectionof environmnental surfaces as well as hand antisepsis are routinely )ractised in anieffort to )revent anid control the spread of infectious diseases, atnd may l)ebparticularly important in the case of viral infections, where the inimlitnal infectivedose is often very low (Westwood & Sattar, 1976; Graham. I)ufour & Estes. 1987:WVard et al. 1986).Although many commercial disi nfectants are teste(d for their l)acteri idal

efficacyv, their virucidal potential is rarely examiine(l. \Ve have previouslvdemonstrate(d that inan v commercial products are ineffective for the disinfectionof rotavirus-contaminated inanimate surfaces (Lloyd-Evans. Springthorpe &Sattar. 1986) and skiin (Ansari et al. 1988b). In this investigation a selected Irantlgeof disinfectants was tested to determine whether the trends observed withrotaviruses coul(i be extend(ed to other types of hutmain pathogenic viruises knownto cause outbreaks in institutions.

Sin(ce the structure and composition of a given virus determninie the degree of itssusceptibility to various classes of chemical disinfectants, four rel)resentatixehuman pathogenic viruses were choseni according to the following criteria: (I)should belong to a virus group which is known to cause outbreaks of disease ininstitutional settings such as hospitals. nursing hotnes anid day-care centres, (2)should grow to relatively high titres in cell culture an(1 permnit (uantitation byplaque assay and (3) should be capable of surviving onI fomites long einough toallow virus transmission, and (4) should not require a high level of biohazardcontainment. On this basis. coxsackievirus 133 (C13-3) was selected to represent theenteroviruses which are conisi(lered to be relatively resistant to chemnicaldisinfection, and adenovirus type 5 (AD-5) was used to rep)resent adlenoviruseswhich have intermediate susceptibility to disinfectants.Even though enveloped viruses are known to be more rea(lilv inactivate(l b)v

chemical disinfcctants than non-enveloped viruses, their susceptibility may l)eoverestimated when they are dried onto surfaces in their natural organic loa(l.Two different types of enveloped virus, parainfluenzavirus ty)pe 3 (HPIV1\-3) aindhuman coronavirus 229-E (HCV-229E). were chosen. As a cause of lowerrespiratory tract infections in young children, parainfluenzaviruses are onlysecond in importance to RSNV, and their ability to spread in iiistitutionalcommunities is also well documented (Mufson, Alocega & Krause, 1973: I)eFabritis et al. 1979; Aleissner et al. 1984; WHO. 1985; Ford-Jones. 1987).Inistitutional outbreaks of respiratory (Kaye. Alarsh & IDowdle. 1971 ; Wenzl et atl.1974). and possibly enteric (Vaucher et at. 1982). coronavirus infections have alsobeen recorded.

MATERIALS ANI) METHOI)S

Viruses and cellsA field isolate of C'13-3 and a laboratory strain of HPIV-3 were grown an(l

plaque-assayed in the MA-104 line of rhesus monkey kidney cells. Cultivation,maintenance and passage of these cells have been described in (letail elsewhere(Sattar et al. 1984). AD-5 w\as obtained from Dr L. Previc, McAlaster University.

Disinfection of virus-contaminated surfacesHamilton, Ontario, Canada. This virus was propagated and plaque assayed inHeLa cells. HCV-229E was grown and plaque-assayed in L-132 cells.For virus assays, cell monolayers were prepared in 12-well plastic cell culture

plates (Costar, Cambridge, MA, USA) with minimal essential medium (MEM;Autopow; Flow Laboratories, Inc., Rockville, MD, USA) containing 5% fetalbovine serum (FBS) and 50,tg/ml gentamicin (Cidomycin; Roussel, Montreal,Quebec, Canada). The seeded plates were sealed in plastic bags (Philips, Toronto,Ontario, Canada) and incubated for 48 h at 37 °C for monolayer formation.

Plaque assaysAliquots (0-1 ml) of tenfold virus dilutions in Earle's balanced salt solution

(EBSS) were allowed to adsorb to cell monolayers for 1 h at 37 °C; not less thanthree wells of a 12-well cell culture plate were inoculated for each virus dilution.The overlay media were as follows: for CB-3 and HPIV-3, MEM with 0-6 % (w/v)agarose (Sigma Chemical Co., St Louis, MO, USA; type II, product numberA6877) and 2% FBS (Gibco); for AD-5, MEM with 0-8 % (w/v) agarose (Sigma),0.1 % (w/v) yeast extract (Difco; BDH Inc., Toronto, Ontario, Canada), 5 % horseserum (Flow Laboratories, Inc.) and 20 mm magnesium chloride; for HCV-229E,M-199 (Gibco) with 0-6% (w/v) Oxoid No. 1 agar (Oxoid Canada Ltd, Ottawa,Ontario, Canada), 2% (v/v) FBS, 0005% (w/v) 5-bromodeoxyuridine (Sigma;product number B-5002) and 0-02% (w/v) DEAE-dextran (Sigma; productnumber D9885). All overlays contained gentamicin at 50 jtg/ml.

Overlaid cultures were incubated at 37 °C except those infected with HCV-229E, which were kept at 33 'C. Incubation was continued for 2 days for CB-3, 5days for HCV-229E and HPIV-3, and 7-9 days for AD-5. Monolayers were fixedovernight in 3-7 % formaldehyde in normal saline and stained with a 0 1 %aqueous solution of crystal violet to demonstrate virus plaques.

Suspending mediaThe virus samples tested were suspended in either faeces or mucin. Normal

infant faeces, which had been previously tested and found to be free of anyindigenous viruses, were suspended 1: 10 (w/v) in normal saline and clarified oflarge clumps of faecal material by centrifugation at 1000 g for 15 min. Aliquots ofthe virus were diluted 1: 10 (v/v) in the clarified faeces before testing. Lyophilizedbovine mucin (Sigma, Product No. M-4503) was used at a concentration of5 mg/ml, which is representative of the levels found in normal human secretions(Diem & Lentner, 1970); the mucin preparation was also found to be free ofindigenous viruses. Aliquots of the virus were diluted 1: 10 (v/v) in the mucinpreparation before testing. Controls were always included to determine the plaquetitres of the inoculated virus in the suspending medium used and after drying theinoculum for 1 h on the inanimate surface; infectious virus titres of 104-106 plaqueforming units (p.f.u.)/disk were used to demonstrate a 3 log10 reduction.

Inanimate surfacesThe inanimate surfaces used for this study were stainless steel disks, approx

1 cm diameter, punched from # 4 finish polished stainless steel purchased locally.The procedures for the cleaning, decontamination and sterilization of these diskshave already been described (Lloyd-Evans, Springthorpe & Sattar, 1986).

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D)isitifectant.sThe disinfeetants used were selected on the basis of previous work wvith a human

rotaxvirus (Springthorpe et al. 1986; Llod-dEvans, Springthorpe & Sattar, 1986);they are listed in T'able 1 und(ler their active ingredients. Trade names are not givento avoid recommen(ding a particular product when other similar formulations maybe available but have not beeni tested. Furthermore, many disinfectants aremarkete(d on a local or nationial rather than an international basis, and the samnefortmulation may be sol(d under a variety of trade names in different countries. Inad(lition. some of the disinfectants listed in Table 1 are not commercialformulations, but are modifications where the nature of the modificatioil and theconcentration of additives may have be(en suggested by competing products. rhenature of the (liluent. if any, can markedly affect the virucidal activitv of adisinifectant (Sattar et al. 1983: Wallis et al. 1963). Disinfectants were (lilute(laccor(ling to the manutfacturers' instructions. using tap) water except where 70)0/ethanol is in(licated as an active inigredient and (liluent. l)eterminations ofavailable chlorine by the DI'D methodtused a commercial kit (Hach (ChemicalCoompany. Amnes. IA. UtSA).

Tes4 pJroedlreiThe test procedure was mnodified only! slightly from that used previously (Lloyd-

Evans. Springthorpe & Sattar, 1986). The virus suspension (10 ,ul) was (lde)osite(donlto the centre of each dlisk held in a horizontal position and the inoculum allowedto air dry for I h un(ler ambient, conditions. The contaminat,ed area on the cliskwas overlaid with 20I,u of the disinfectant. After 1 min of contact at rooimtemperature (22-24 °C) each disk, with the virus and( disinfectant still in place,was (lropp)e(l into a vial containing 1 ml of tryptose phosphate broth (TPlB ; L)ifco)for virus elution. Tlhis immediatelyodiluted and neutralized the disinfectant an(lallowcd efficicnt recovcrv of thc inoculated virus.

RESULTS AN) DISCUSSION

i'he restilts of this study are sumnmarizeol in Table 1. The criterion of efficacy forthe olisinfectants was ) '3 logl reduction in the number of infectious virus uilits.EIach result is basedl on ito less than three trials on each of two batchcs of thedisinifectant. If the ) 99o9% re(luction coul(l not be mnet consistently (luring thetrials. then the olisinfectant was regartded as ineffective. Since nO (liffercnce indlisinfectant effitcac was observedl when (CB-3 was suspcllded in facces or imtucin.thc results shown in TUable I (1o not specify the suspen(ling ne(lium. 'I'he rationalefor the eXperi mental protocols and( )arameters chosen has alreadyv been published(Lloyd-Evans. Springthorpe & Sattar. 1986: Sattar & Springthor)e. 1988). butt itis imp)ortant to rcstate that the (contact time of' 1 min is longer thall that oftcnuseCl fotr hard surfaoce disinfe(tants in the field. Where disinfc(tants are use(l in

inore (criti(al a)pli(ations as instrumnenit soaks with longem' contact timnes in thefiel(l. thenr the test conditions of 1 mim enisurc a safety margin for forrtulationiswhich are (leeme(l effective.The enveloped viruses, H1IPIV-:3 ancl H(V'-229E. were imore readilx inactivate(l

499

S. A. SATTAR AND OTHERSby all the disinfectants tested than were the non-enveloped viruses, CB-3 andAD-5. This is not surprising, and is in agreement with the other studies wherecomparisons have been made between the susceptibility to disinfectants ofenveloped and non-enveloped viruses (Klein & Deforest, 1963; Mahnel, 1974,1979; Brown, 1981; Klein & Deforest, 1983; Schurmann & Eggers, 1983). In spiteof this, not all the disinfectants tested at their recommended in-use dilutions wereable to inactivate the enveloped viruses dried onto surfaces.The different types of chlorine-based disinfectants tested all reliably inactivated

the enveloped viruses at 1000 p.p.m. free chlorine. Interestingly, at this freechlorine concentration, the action of sodium hypochlorite against CB-3 was muchweaker (a reduction of about 1 log1o) than for either the organochlorine or themixed halide (a reduction of 2-3 log1o). All chlorine-based disinfectants aremarkedly inhibited by organic material, and these results suggest that, subject toany toxicity constraints, organic chlorine compounds which act as demand-typedisinfectants, may be more effective than solutions of sodium hypochlorite in thefield. Mixed halogens have been reported to be more effective than chlorine alone(Cheremisinoff, Cheremisinoff & Trattner, 1981) although they are not widely usedas disinfectants, and the mechanism of their increased efficacy is not completelyunderstood.

Increasing the free chlorine concentration to 3000 and 5000 p.p.m. for theorganochlorine and hypochlorite products, respectively, inactivated all the virusestested. Our previous studies (Lloyd-Evans, Springthorpe & Sattar 1986) suggestthat even 5000 p.p.m. free chlorine as sodium hypochlorite may be insufficient toinactivate human rotavirus suspended in faeces and dried onto inanimate surfaceswhereas chloramine T, providing a similar concentration of free chlorine, couldreadily inactivate the virus under the same conditions. It is possible that at leastsome component of the disinfecting power of concentrated hypochlorite solutionsis due to their content of sodium hydroxide; the pH of hypochlorite solutions inthe 5000-10000 p.p.m. range is in excess of 11-0, whereas the organochlorinetested here had a pH of approximately 8-5.

In the field, free chlorine concentrations between 25 and 500 p.p.m. aregenerally used for disinfection of surfaces. On the other hand, the guidelinesgenerally used for disposal of virus-cohtaminated material or coping with spills ofvirus-contaminated liquids recomnmend the use of sodium hypochlorite at a freechlorine concentration of 5000-10000 p.p.m. The results obtained here andpreviously (Lloyd-Evans, Springthorpe & Sattar., 1986) support the use of thesehigh concentrations when virus contamination is suspected.The results with chloramine T at 0 01 % (100 p.p.m.) show an apparent

difference in sensitivity to chlorine between HCV-220E and HPIV-3. Thisdifference may be related to the degree of cell association of the virus; HPIV-3 washarvested from infected MA104 cells as a cell free supernatant, whereas HCV-229Ewas harvested after cell degeneration and is known to be more closely associatedwith cellular material.The high in-use concentration of available iodine (1 %) in the iodophore failed

to inactivate either of the non-enveloped viruses in this study, although it haddemonstrated previously (Lloyd-Evans, Springthorpe & Sattar, 1986) a muchgreater efficacy against human rotavirus than other iodophores tested under

500

Disinfection of virus-contaminated surfacesidentical conditions. This iodophore is designed for topical application as apreoperative skin preparation: the pH is approximately 3 0. Other, more acidic,iodophores may have greater activity against the non-enveloped viruses and bothiodine and acid activity may be augmented if additional non-ionic surfactant ispresent (Jordan & Nassar, 1973).

Only CB-3 was not inactivated by either ethanol alone, or when ethanol wasintroduced as an addition to other disinfectants. In earlier studies on enterovirusdisinfection (Drulak, Wallbank & Lebtag, 1978; Drulak et al. 1978) ethanol hadappeared to be among the most effective disinfectants. However, these studies hadbeen conducted using suspension not carrier tests. Similarly, ethanol alone wasalso very effective against human rotavirus in suspension (Springthorpe et al.1986) but not in carrier tests (Lloyd-Evans, Springthorpe & Sattar, 1986).Nevertheless, ethanol and products containing at least 70 % ethanol were the mosteffective among antiseptics tested on rotavirus-contaminated hands (Ansari et al.1988b).At the concentration used for disinfection of fibre-optic endoscopes and other

instruments (2 %), glutaraldehyde was highly effective against all tested viruses asit was against rotavirus (Lloyd-Evans, Springthorpe & Sattar, 1986). It has alsobeen shown to be effective against many viruses in suspension tests, althoughparvoviruses were somewhat more resistant and required longer contact times(Scott, 1980; Brown, 1981). We are not aware of tests on parvovirus disinfectionon contaminated surfaces. The virucidal potential of glutaraldehyde is con-centration dependent and claims for the efficacy of formulations with lowerglutaraldehyde levels on the disinfection of naturally-contaminated surfacesshould be treated with some caution.The quaternary ammonium compound alone was virtually useless against the

test viruses dried onto surfaces; it did not even inactivate the enveloped virusesHPIV-3 and HCV-229E. A similar result was obtained previously with rotaviruses(Springthorpe et al. 1986; Lloyd-Evans, Springthorpe & Sattar, 1986). This isimportant because of the widespread use of quaternary ammonium basedformulations for general hard surface disinfection, and the usual recommendationthat they be applied to precleaned surfaces is neither safe nor practical. Studies ofvirus disinfection with quaternary ammonium compounds in suspension tests,have shown various degrees of efficacy, but non-enveloped viruses, particularlypicorna-, calici- and parvoviruses, are the most resistant (Klein & Deforest, 1963;Blackwell, 1978; Poli et al. 1978; Scott, 1980; Brown, 1981).The quaternary ammonium/HCl combination is commonly used in formulating

toilet bowl cleaners, and its efficacy is believed to be mainly due to the pH of thesolution. Similar compounds were shown to be effective against rotavirus (Lloyd-Evans, Springthorpe & Sattar, 1986) although a differential sensitivity ofrotaviruses to acids was noted (Springthorpe et al. 1986). Addition of ethanol tothe quaternary ammonium compound conferred on this combination the samevirucidal properties as for ethanol alone and a similar result was obtained whenthis combination was used against rotavirus (Lloyd-Evans. Springthorpe &Sattar, 1986). Sodium metasilicate is a highly alkaline compound in solution andis widely used alone as a disinfectant for veterinary purposes and at lowerconcentrations in quaternary ammonium disinfectant and detergent formulations.

501

502 S. A. SATTAR AND OTHERSAddition of sodium metasilicate to the quaternary ammonium compound resultedin an increased inactivation of all tested viruses exceptCBl3; a result consistentwith that obtained against human rotavirus (Springthorpe et al. 1986).

Aqueous chlorhexidine gluconate was effective only against HPIV-3. and eventhat may have been partially due to the cetrimide present. Chlorhexidine

gluconate alone is known to be a poor virucide (Derbyshire & Arkell. 1971: Bailev& Longson, 1972; Gardner & Gray. 1983; Springthorpe et al. 1986). As discussedabove, observed differences in susceptibility of HPIV-3 and HCV-299E may bedue to differences in cell association of the viruses. The alcoholic solution of

chlorhexidine, at the concentration recommended for preoperative skin prep-aration,showed the samespectrum of activity against the tested viruses as

ethanol alone and it was also effective against human rotaviruses (Lloyd-Evans.Springthorpe & Sattar, 1986). Due to the potent bactericidal properties ofchlorhexidine salts and their residual activity on the skin, their importance in

topical antisepsis.particularly in alcoholic solution, cannot be underestimate(l.The two phenolics were selected because they were among 4 of the17 phenolics

which proved to be effective against human rotavirus in suspension (Springthorpe

etal. 1986). However. on surfaces one was effective and the otherw as not (Lloyd-

Evans, Springthorpe & Sattar. 1986). The results of the present study with thetwoproducts tested alone showed the same dramatic difference; one was effective

against all the test viruses while the other was not effective against any. Therelative concentrations of thephenolics (05 %.vw/v for the effetive product

compared with 00160/0. w/v) are assumed to be responsible for this differen(cebecause addition of 0'6%SD)S to theineffective product made it effective againstHP'1V-3and HCV-299E onlv. Addition of ethanol to the ineffectiveproduct gaveit the same spectrum of efficacy against the tested viruses as ethanol alone.

D)isinfection implies that pathogen concentrations have been re(duce(d to levels

'whichno longer pose a risk to the susceptible host. Apart from specialized clinical

an(d research laboratories. field application of disinfectants is inevitably oIninanimate surfaces or skin. In view of theresults obtained here an(d in previousstudies (Kirchhoff. 1969: Nakaoet al. 1978; Klein & Deforest.1983:,Schurmann& Eggers.1983: Lloyd-Evans. Springthorpe & Sattar. 1986). it is essential thatdisinfectants ares teste(d on contarninatedl carriers, although suspension tests -naybe useful for initial screeninig of formnulations (Springthorpe et al. 1986).

r1'he known susceptibility of many disinfectant chemicals toneutralization by

organic soil suggests that testing of these chemicals should always bec(onduictedlwith a realistic challeinge. Although the organic loa(ds preseint ltirinig these

disinfectant tests were olesigned to simulate natural organic loadls. naturally-shedvirts. already embedded in an organic matrix such as faeces or mucus. may bee\ enimore resistant to inactivation than when the virus is adl(le(1 externally to thatmiatrix. 'r'hereforc. caution should be exercised in extrapolating disinfectantefficacydirectlv from the laboratory to the field. Noneotheless, laboratory tests are

capable of(listinguishing which of a range of chemical (lisinfe(tants is mnost likelyto be effe(tive in the fiel(l. and( of showing inadequate concentrations. Laboratorytests therefore examine the potential of (lisinifectant solutions but fiel(d trials arene(essary to determine their true effi(acv.No stan(lar(1 metho(ls are available for testing (lisinfectant efficacv agaiinst

502

Disinfection of virus-contaminated surfaces 503vilrlses even those in routine use for bactericidal efficacy are unId(ler review. AInnigf(ent Ineed( exists for testing prot!ocols which use clinically relevant con(litionsanid( contact timnes and( can reliably predict field efficacy. Many clinicians believethat most (lisinfectants are equally efftective in the field: wve (10 nIot think this ist'ti(e. Thew iajority of antimicrobial chemicals sol(1 in Noith America are either(ltiaternarv ainmoniium compounds (38%/n) or phenolics (20%) which behave veryloorly as surface virucidies under simulated fiel(d conditiotis. It is. however.lifficult to (lefinie what role effective disinfection could play inldisease preventionbecauise the presenit spectrum of (lisease transmnission exists where many of thedisinfectants are iniadequate. Formulations use(d in this study were carefully'selected after a systematic survey of many products; selection base(1 only ona(lvertising or sales advice is likely to be a lottery at best. Because the miniinalinifective dose for manyv viruses is extremely low. and the numbers shed in bo(dysecretions and excretions may be high. disinifectants, used routinely for controllinga I(d1)eventinlg the spread of viral inlfectioins in areas sutch as (lavare centres(Klein. 1986) and(l hospital nieoniatal anid critical care facilities shouild be (hoselnvith oreat(are.

AC(NN()WLEEI)(ENIENTS

This stu(lyr was supporte(l by a grant from the Nationial Health ResearchDevelopment Program of Health and Welfare (Canadla. \We thank Roussel (Cana(lafor the complimentary supplies of Cidomycin.

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