Vol. 59, No. 10APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1993, p. 3463-34690099-2240/93/103463-07$02.00/0Copyright ©) 1993, American Society for Microbiology

Comparative In Vivo Efficiencies of Hand-Washing Agents againstHepatitis A Virus (HM-175) and Poliovirus Type 1 (Sabin)

JOHN N. MBITHI, V. SUSAN SPRINGTHORPE, AND SYED A. SATTAR*Department ofMicrobiology and Immunology, Faculty of Medicine,

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

Received 10 May 1993/Accepted 27 July 1993

The abilities of 10 hygienic hand-washing agents and tap water (containing approximately 0.5 ppm of freechlorine) to eliminate strain HM-175 of hepatitis A virus (HAV) and poliovirus (PV) type 1 (Sabin) were

compared by using finger pad and whole-hand protocols with three adult volunteers. A mixture of the twoviruses was prepared in a 10%X suspension of feces, and 10 R1 of the mixture was placed on each finger pad.The inoculum was allowed to dry for 20 min, and the contaminated area was exposed to a hand-washing agentfor 10 s, rinsed in tap water, and dried with a paper towel. In the whole-hand protocol, the hands were

contaminated with 0.5 ml of the virus mixture, exposed for 10 s to a hand-washing agent, washed, and driedas described above. Tryptose phosphate broth was used to elute any virus remaining on the finger pads orhands. One part of the eluate was assayed directly for PV with FRhK-4 cells, while the other part was firsttreated with a PV-neutralizing serum and then assayed for HAV with the same cell line. The results are

reported as mean percentages of reduction in PFU compared with the amount of infectious virus detectableafter initial drying. In the finger pad protocol, an unmedicated liquid soap was the least effective agent (77.96%+ 7.17% reduction) for removing HAV, and a medicated liquid soap was the most effective agent (92.04%4.02% reduction) for reducing the HAV titer, the smallest reduction in PV titer was obtained with tap water(85.22% 2.91% reduction), and the same medicated soap was the most effective agent against PV (98.39%o+ 1.98% reduction). Significant differences were found between HAV removal and PV removal with 9 of the11 agents tested (F = 102.14; P < 0.05). There was good correspondence between the results of the finger padprotocol and the results of the whole-hand method. In additional experiments, we attempted to transferinfectious virus remaining on finger pads treated with various hand-washing agents by having the finger padscontact clean metal disks at a pressure of about 1 kg/cm2 for 10 s. Detectable amounts of both of the viruseswere transferred after washing with anl of the agents except a 1:30 dilution of a mixture containing 1.5%chlorhexidine gluconate and 15% cetrimide in 70% ethanol, 70%o ethanol alone, and a foam containing 62%ethanol. More work is needed to determine the health implications of residual infectious viruses on washedhands, but our findings reinforce the need to determine the virus-eliminating efficiencies of hand-washingproducts, particularly those used in health care settings, day-care centers, and food establishments.

Regular and proper washing of hands, especially by caregivers (36) and food handlers (11), is universally recognizedas crucial for infection control (14, 29). The results of studiesperformed in clinical settings have reinforced the importanceof hand washing for controlling outbreaks of bacterial gas-troenteritis (7) and viral infections (18, 19). However, thereis a lack of information concerning the virus-eliminatingabilities of commonly used hand-washing agents. Limitedstudies have shown that such agents vary greatly in theability to rid experimentally contaminated hands of viruses(3, 13) and that, in general, they do not eliminate viruses aswell as they remove bacteria (3).

Hepatitis A virus (HAV) causes outbreaks of disease ininstitutions such as hospitals (6, 20, 28, 30) and day-carecenters (17). Outbreaks have also been associated with foodhandlers in eating establishments (22, 27). The portal of virusentry is generally the mouth, and human hands are believedto play an important role in HAV spread (20, 30, 33). Theincidence of HAV infections in many industrialized coun-tries appears to be increasing (10, 26).HAV is relatively resistant to inactivation by many liquid

chemicals used as disinfectants for environmental surfaces(24). We have shown that this virus can survive on environ-

* Corresponding author.

mental surfaces for several days under ambient conditions(25) and on human hands for several hours (23); HAV-contaminated hands can readily transfer infectious virus toinanimate surfaces or other hands through casual contact(23). In view of this, proper hand washing would be expectedto reduce the risk of HAV spread directly by hands and alsoto minimize the contamination of surfaces and objects con-tacted by hands.

Therefore, the main objectives of this study were (i) to testthe HAV-eliminating abilities of various types of hygienichand-washing agents commonly used in healthcare settingsand (ii) to determine whether infectious viruses remaining onwashed and dried hands could be transferred to hard envi-ronmental surfaces on contact. Poliovirus (PV) type 1(Sabin) was included in this study because it is often used asa surrogate for testing the virucidal activity of chemicalgermicides (9) and hand-washing agents (13). The fecallysuspended mixture of the two viruses used as the inoculumpermitted us to make direct comparisons of the relativeefficacies of hand-washing agents in dealing with the viruses.

MATERIALS AND METHODS

Cells and viruses. A seed culture of FRhK-4 cells andstrain HM-175 of HAV were kindly provided by M. D.Sobsey, University of North Carolina, Chapel Hill. PV was

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TABLE 1. Relevant information on the hygienic hand-washing agents tested

Agent Active ingredient(s) Dilution Manufacturer

Alcare 62% (vol/vol) Emolliented ethanol foam None Calgon Vestal, Bramalea, Ontario,Canada

Aquaress None None DEB, Waterford, Ontario, CanadaBacti-Stat soap 0.3% Triclosan None Huntington, Bramalea, Ontario, CanadaBioprep soap 0.1% Chlorhexidine gluconate, 0.50% None JC Corporation, Bolton, Ontario, Canada

didecyl dimethyl ammoniumchloride, 5% isopropanol

Dettol 4.8% 4-Chloro-3,5-xylenol, 9.4% None Reckitt-Colman, Lachine, Quebec,isopropanol Canada

Ethanol 70% (vol/vol) Ethanol None BDH, Toronto, Ontario, CanadaSavlon 1.5% Chlorhexidine gluconate, 15% 1:30 in 70% Ayerst, Montreal, Quebec, Canada

cetrimide ethanolScrub Stat IV 4% Chlorhexidine gluconate, 4% None Huntington, Huntington, Pa.

isopropanolSeptisol 0.75% Hexachlorophene 1:2 KVL, Cambridge, Ontario, CanadaTap water Approx 0.5 ppm of free chlorine None NoneTriclosan hand soap Triclosan, 0.5% None G. H. Wood, Toronto, Ontario, Canada

received from the Laboratory Center for Disease Control,Ottawa, Ontario, Canada. The methods used for cultivationand maintenance of the cells and preparation of virus poolshave been described previously (23, 25). The virus poolswhich we used were unconcentrated cell harvests.

Plaque assay. Both viruses were plaque assayed by usingmonolayers of FRhK-4 cells in 12-well cluster plates (Costar,Cambridge, Mass.). Three wells were used for each virusdilution tested, and each well was inoculated with 0.1 ml ofthe inoculum. Virus was adsorbed for 90 min at 37°C. Thecomplete formulation of the overlay used has been describedby Mbithi et al. (25). The plates containing the overlaidmonolayers were sealed in clear plastic bags (Dazey Corp.,Industrial Airport, Kans.) and incubated for 8 days (forHAV) or for 48 h (for PV) at 37°C. The procedure used to fixand stain the monolayers before the plaques were countedhas been described previously (32). The limit of detection ofthis technique was 3 to 4 PFU for both viruses.Virus suspension medium. The fecal sample used in this

study was obtained from a healthy 5-month-old baby. A 10%(wt/vol) suspension of this sample was prepared in normalsaline; gross particulate matter was removed by centrifuga-tion at 1,000 x g for 10 min, and the suspension was passedthrough a 0.2-,um-pore-size membrane filter (Nalge Co.,Rochester, N.Y.). The filtrate was found to be free ofcytotoxicity and endogenous viruses when it was tested withFRhK-4 cells by the procedure described previously (21).When HAV or PV was diluted 1:10 in this fecal suspensionand the preparation was kept at room temperature for 4 h, noloss of virus infectivity was observed.

V'irus mixtures. The two viruses were first diluted 1:10separately in the fecal suspension. Equal volumes of the twosuspensions were then mixed together. The mean titers ofHAV and PV in the mixture were about 8.0 x 104 and 5.0 x105 PFU/10 ,ul, respectively. The mixtures or eluates did notrequire any additional manipulations for the PV assay be-cause PV could form countable plaques within 48 h, whileHAV required nearly 8 days to form countable plaques.However, for HAV titrations, the samples were first treatedwith a hyperimmune rabbit serum to neutralize PV. Theserum was kindly supplied by P. Payment, Institut Armand-Frappier, Laval, Quebec, Canada. It had a 50% plaqueneutralization titer of 1:30,000, and it was diluted in Earlebalanced salt solution to a level (final dilution, 1:100) at

which it could completely neutralize at least 106 PFU of PVafter 30 min of incubation at room temperature.

Disks. Stainless steel disks (diameter, 1 cm) were punchedout of locally purchased no. 4 finish polished sheets (thick-ness, 0.75 mm), and these disks were used to represent hardinanimate surfaces. The procedures used for decontamina-tion, cleaning, and sterilization of the disks before reusehave been described previously (21).

Volunteers. Permission to place the viruses on the hands ofadult volunteers was first obtained from the University ofOttawa Ethics Committee. Any individual with cuts orabrasions on his or her hands was automatically excludedfrom the study. Each volunteer was then thoroughly briefedon the experimental protocol and the risks involved beforebeing asked to sign a consent form. The ages of the partici-pants ranged from 26 to 45 years.At the end of the finger pad protocol, each volunteer was

asked to gently press his or her experimentally contaminatedfinger pads on a piece of paper towel soaked in a 6% solutionof sodium hypochlorite (Colgate-Palmolive, Inc., Toronto,Ontario, Canada) for 3 min for decontamination purposes.The hands were then washed thoroughly with ordinary soapand running tap water and dried with a paper towel. After thewhole-hand method, about 0.5 ml of the chlorine solutionwas placed on a palm and the two hands were rubbedtogether for nearly 3 min before washing and drying asdescribed above.

Hygienic hand-washing agents tested. Tap water (contain-ing about 0.5 ppm of free chlorine) and 10 other hand-washing agents were tested; 7 of the 10 commercial productsused are currently used at a 530-bed general hospital inOttawa, Ontario, Canada, and the others were preparationsrecently acquired from the local market for use in this study.Information about the agents used is shown in Table 1. Anydilution of a product was in accordance with the directionson its label. In our laboratory, all products to be tested werestored at room temperature and they were accessible only toauthorized personnel.

Finger pad protocol. The participating volunteers wereasked to wash their hands in lukewarm (about 40°C) runningtap water for 30 s and to apply 70% (vol/vol) ethanol to theirhands after drying them with an ordinary paper towel(Kimberly-Clark, Inc., Mississauga, Ontario, Canada). Tospread the alcohol, the hands were gently rubbed against

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each other for 15 s; the hands were then allowed to air dry.The procedure used to test the hand-washing agents was theprocedure described previously (3). Briefly, a small area on

each finger pad was demarcated by pressing the finger padhard on the mouth (inside diameter, 8 mm) of an emptyplastic vial (catalog no. 72.694.006; Sarstedt, Inc., St. Lau-rent, Quebec, Canada). A 10-,ul inoculum consisting of virussuspended in 10% feces was placed at the center of thedemarcated area on each of three finger pads of one hand andthen eluted (see below) immediately with 990 p,l of tryptosephosphate broth (TPB).The five fingers of the other hand were similarly contam-

inated with the virus mixture as described above for theinput controls, and the inocula were allowed to dry for 20min by keeping the hands in a laminar flow hood with theblower on. After drying, the inoculum from one finger padwas eluted to determine the loss of virus infectivity causedby drying. Three finger pads were used to assess the virus-eliminating efficacy of each agent tested. To do this, thecontaminated area of a finger pad was placed on the mouth ofa plastic vial containing 1 ml of the test agent. The vial wasinverted, and the test agent was brought into contact withthe contaminated area for 10 s. The treated area was thenrinsed in 15 ml of tap water contained in a plastic tube; thetube, with the finger pad pressed on its mouth, was invertedthree times within 3 s. After the water rinse, the treatedfinger pad was dried by pressing and holding it on a paper

towel for 3 s. The virus remaining on the finger pad was

eluted.Virus elution procedure. To recover virus from a finger

pad, the contaminated area was placed on the mouth of a vialidentical to the vial used for finger pad demarcation butcontaining 990 ,ul of TPB. The vial was inverted, with thefinger pad still on it, and held in that position for 5 s. Thiswas followed by 20 full inversions of the vial with the vialstill in place and then an additional 5 s of soaking and 20more inversions. The surface of the finger pad was thenscraped on the inside rim of the vial to recover as much ofthe fluid as possible. This elution procedure could recovernearly 80% of the infectious virus placed on finger pads.Virus transfer. The fifth finger was treated with a test

agent, washed with 15 ml of tap water, and dried with a

paper towel, and the remaining virus was transferred to a

stainless steel disk by placing the finger pad on the disk at a

pressure of 1 kg/cm without friction (23). To recover thevirus transferred, each disk was placed in 1 ml of TPB, thevials were sonicated in a bath (Bransonic Ultrasonics Corp.,Danbury, Conn.) for 10 min at room temperature, and theeluates were plaque assayed.Whole-hand protocol. The participating volunteer was not

required to wash his or her hands prior to experimentation inorder to reflect field practice, in which hand-washing prep-arations are used without prewashing. Thus, the resultsobtained should have closely simulated what may occur innature. These results were also used to cross-check theresults obtained with the finger pad method.Equal amounts of HAV and PV were suspended in a 10%

fecal suspension to give approximately 3.0 x 106 and 3.0 x

107 PFU/ml, respectively. To estimate the amounts of vi-ruses recoverable from the hands after drying, a control wasset up as follows. A 0.5-ml portion of the virus inoculum wasplaced in the middle of the palm of one hand of thevolunteer, and the two palms were rubbed together tocontaminate them evenly. They were then allowed to air dryas described above. With the help of an assistant, the virusesthat survived drying were eluted by slowly pouring 20 ml of

TABLE 2. Comparative efficacies of hygienic hand-washingagents against HAV and PV as determined by the finger

pad protocol

% Reduction in PFU TukeyHand-washing (mean + SD)' groupb

agentHAV PV HAV PV

Alcare 89.27 + 4.38 97.52 ± 3.59 B AAquaress 77.96 + 7.17 89.01 ± 4.18 B ABacti-Stat soap 92.04 ± 4.02 98.39 ± 1.98 B ABioprep hand soap 83.35 ± 2.76 90.93 + 2.86 B ADettol 88.63 + 5.38 93.77 + 4.14 B AEthanol (70%) 87.40 ± 4.59 95.47 ± 4.06 B ASavlon 90.91 + 5.08 98.13 + 1.58 B AScrub Stat IV 89.57 + 6.70 91.45 ± 3.49 A ASeptisol soap 88.60 ± 5.36 94.37 ± 4.34 B ATap water 79.74 + 4.80 85.22 + 2.91 B ATriclosan hand soap 91.29 ± 4.47 94.80 ± 3.76 A A

a Mean levels of reduction in PFU were determined by comparison with theamount of infectious virus detectable on untreated finger pads after 20 min ofdrying.

b The percentages of reduction for agents in the same Tukey group are notsignificantly different (a = 0.05).

TPB over the hands while they were being rubbed together.The eluents were collected in a clean, sterile plastic bagabout 30 cm in diameter and 60 cm deep; the volumes ofeluent recovered ranged from 17 to 19.5 ml. The efficiency ofvirus elution was more than 70%.To test a hand-washing preparation, 0.5 ml of it was placed

at the center of one of the virus-contaminated palms, and thematerial was spread evenly over both hands by rubbing themtogether for 10 s. The assistant was then requested to slowlypour 500 ml of lukewarm tap water over the hands while theywere being rubbed together as in normal washing, and thehands were dried with a paper towel. The viruses thatsurvived this washing, rinsing, and drying procedure wereeluted from the hands by using 20 ml of TPB as describedabove. The eluate was immediately diluted fourfold in TPBto arrest the virucidal activity of the hand-washing agent andto render it noncytotoxic. All of the eluates were then passedthrough a membrane filter (pore size, 0.2 ,um) before theplaque assay was performed.

Statistical analysis. In the finger pad protocol, at least threefinger pads were treated with each agent, and each experi-ment was repeated no less than three times with each of thethree volunteers. Therefore, the reductions in virus PFUdetermined in these experiments were based on a minimumof 27 replicates, and the mean percentages of reduction forvarious agents (Table 2) were determined by comparisonwith the amount of infectious virus detected after 20 min ofdrying. Since virus transfer from washed hands was testedby using one finger pad in each experiment in parallel withthe tests on virus elimination, the total number of replicatesfor each hand-washing agent was nine (Table 3). The per-centage of virus transferred was determined by comparisonwith the amount of infectious virus left after the finger padwas washed and dried. The whole-hand experiments wereconducted with only one volunteer, but each agent wastested at least three times.HAV and PV log1o PFU reduction or log1o PFU of virus

transferred was analyzed for each hand-washing agent usedby a two-way analysis of variance in SAS; post hoc tests(Tukey) were also performed (3).

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TABLE 3. Transfer of HAV and PV to stainless steel disks fromfinger pads washed with hygienic

hand-washing agents

% PFU transferred TukeyHand-washing (mean ± SD)' groupb

agentHAV PV HAV PV

Alcare Undetectable UndetectableAquaress 1.57 ± 0.50 0.96 ± 0.84 A BBacti-Stat soap 0.63 ± 0.13 0.62 ± 0.62 A BBioprep hand soap 0.34 ± 0.12 0.26 ± 0.06 A BDettol 0.5 ± 0.20 0.37 ± 0.09 A BEthanol (70%) Undetectable UndetectableSavlon Undetectable UndetectableScrub Stat IV 0.64 ± 0.02 0.23 ± 0.20 A BSeptisol liquid soap 0.44 ± 0.09 0.41 ± 0.10 A BTap water 3.88 ± 0.63 3.15 ± 1.32 A BTriclosan hand soap 1.74 ± 0.35 1.35 ± 0.02 A B

a Mean levels of virus PFU transferred were determined by comparisonwith the amount of infectious virus left on a finger pad after washing with thehand-washing agent.

b The percentages of virus transfer for agents in the same Tukey group arenot significantly different (a = 0.05).

RESULTS

An analysis of variance in which a general linear modelwas used revealed no significant differences among theresults obtained with the three volunteers used in this study.This analysis also revealed significant differences betweenlevels of HAV elimination and levels of PV elimination by 9of the 11 agents tested (F = 102.14; P < 0.05), as well assignificant differences between the agents (F = 17.10; P <0.05).Virus elimination from finger pads. The agents were com-

pared for their ability to remove HAV from experimentallycontaminated finger pads (Table 2). Lukewarm tap water,which was used as a control, reduced the titers of the viruson the finger pads by 79.74% + 4.80%, compared with the77.96% + 7.17% reduction in HAV titer observed for Aqua-ress (unmedicated soap); the mean differences were notsignificant. Bacti-Stat reduced the HAV titer by 92.04% +4.02%, compared with 91.29% + 4.47% reduction by Tri-closan soap; these two means were also not statisticallysignificantly different.

Savlon, Scrub Stat IV, and Biorep all contain differentlevels of chlorhexidine gluconate and alcohol; the resultsobserved for the last two agents were comparable. Savlonreduced the HAV titer 90.91% + 5.08%, while 70% ethanol,Alcare, and Dettol reduced the HAV titer 87.40% + 4.59%,89.27% ± 4.38%, and 88.63% ± 5.38%, respectively. Septi-sol reduced the HAV titer 88.60% ± 5.36%. A post hocanalysis (Tukey tests) summarized the differences betweenthe agents examined (Table 2).

In general, the levels of reduction ofPV titers on the fingerpads of each volunteer were higher than the levels ofreduction of HAV titers by the same agent. For instance,Alcare reduced the PV titer by 97.52% ± 3.59%, comparedwith 89.27% ± 4.38% reduction for the HAV titer.

Alcohol-based products were generally more activeagainst PV than they were against HAV (compare the95.47% ± 4.06% reduction in PV titer by 70% ethanol withthe 87.40% ± 4.59% reduction in HAV titer by the sameproduct). Among the chlorhexidine-containing substances,Savlon was the best, with a level of reduction of the PV titerof 98.13% ± 1.58% (Table 2). Small differences between the

TABLE 4. Comparative efficacies of hygienic hand-washingagents against HAV and PV as determined

by the whole-hand protocol

Hand-washing % Reduction in PFU Tukey groupbHand-washing (mean ±SD)'agent

HAV PV HAV PV

Alcare 86.17 ± 4.28 93.39 ± 5.74 A AAquaress 91.39 ± 2.65 90.86 ± 4.03 A ABacti-Stat soap 94.56 ± 5.75 97.28 ± 2.05 A ABioprep soap 81.44 ± 1.59 88.99 ± 2.59 B ADettol 90.67 ± 2.08 97.09 ± 2.32 B AEthanol (70%) 86.92 ± 1.63 92.77 ± 2.79 B ASavlon 86.53 ± 3.44 95.81 ± 3.08 B AScrub Stat IV 81.15 ± 1.15 89.27 ± 1.41 B ASeptisol 89.20 ± 0.81 92.03 ± 6.82 A ATap water 81.57 ± 4.55 85.06 ± 3.73 A ATriclosan soap 88.98 ± 1.73 96.63 ± 2.86 B A

a Mean levels of reduction were determined by comparison with the amountof infectious virus detectable on untreated hands after 20 min of drying.

b The percentages of reduction for agents in the same Tukey group are notsignificantly different (a = 0.05).

levels of reduction of PV and HAV titers were also noticedwhen finger pads were treated with tap water or unmedicatedsoap, with greater reduction occurring with PV (compare the85.22% + 2.19% reduction in PV titer obtained with tapwater and the 89.01% + 4.18% reduction in PV titer obtainedwith Aquaress with levels of HAV titer reduction of 79.74%± 4.80% and 77.96% + 7.17% obtained with the sameagents, respectively).

Dettol, Septisol, and Bioprep were found to be moreactive against PV than they were against HAV (Table 2). Ofthe two triclosan-based products, Bacti-Stat was better forvirus removal than Triclosan soap (the levels of a reductionwere 98.39% + 1.98% for the former and 94.80% + 3.76%for the latter).Virus transfer from washed finger pads to metal disks. The

amount of HAV transferred was greatest from the fingerpads treated with the tap water alone (3.88% + 0.63%)(Table 3). Treatment with Aquaress reduced the level ofHAV transferred to 1.57% + 0.50%. Among the otherhand-washing agents, Triclosan soap resulted in the highestlevel of HAV transfer (1.74% + 0.35%). Finger pads treatedwith products containing high levels of alcohol (Alcare, 70%ethanol, or Savlon) (Table 1) transferred no HAV. All otherproducts resulted in lower levels of HAV transfer comparedwith the tap water control.

There was no PV transfer from the finger pads treated withthe alcohol solution of Savlon, 70% ethanol, and Alcare.Bacti-Stat resulted in a mean level of virus transfer of 0.62%+ 0.62%. As was the case with HAV, more PV wastransferred by finger pads treated with tap water alone(3.15% + 1.32%). Aquaress resulted in the second highestlevel of PV transfer. Triclosan soap resulted in a PV transferlevel of 1.35% ± 0.02% while the other agents resulted inmuch lower virus transfer levels.Tukey tests revealed significant differences in the amounts

of HAV and PV transferred after treatment with eachhand-washing agent (Table 3); in each case, the percentageof HAV transferred was higher than that for PV.Virus elimination by using the whole-hand protocol. Table 4

shows the results obtained with the whole-hand test whenone volunteer was used. The level of HAV titer reductionobtained with each agent was comparable to the results

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observed when the finger pad method was used (Table 2).There were no statistically significant differences betweenthe levels of HAV reduction obtained with the finger padmethod and the levels of HAV reduction obtained with thewhole-hand procedure.As was the case with HAV, the levels of PV titer reduction

at the end of the hand-washing events were comparable tothe levels observed when the finger pad procedure was used(Tables 2 and 4). There were no statistically significantdifferences between the results of the two methods.

DISCUSSION

Our results clearly show that the finger pad protocol,which was initially developed with a human rotavirus (3), issuitable for working with HAV and PV and that the virus-eliminating efficiencies of hand-washing agents as deter-mined by this protocol are consistent with the findingsobtained with the whole-hand technique. HAV was selectedfor use in this study on the basis of our previous observa-tions that this virus survives well on human hands (23) andenvironmental surfaces (25) and that it can be readily trans-ferred to and from hands and environmental surfaces (23).Our previous studies (24) also showed that HAV is resistantto many commonly available chemical disinfectants used forthe control of microbial agents in institutional settings andfood establishments. PV was chosen for comparison sincebecause of its safety, ease of handling, and relative resis-tance to disinfection, it is often recommended as a surrogateto determine the virucidal activities of liquid chemical disin-fectants (2, 9).The relative activities of the hand-washing agents against

the two viruses are directly comparable because not onlywere the viruses tested as a mixture, but also the otherconditions of the protocols were identical. The initial titer ofPV in the mixture was somewhat higher than that of HAVbecause PV is known to lose more infectivity during drying,and indeed the levels of the two viruses became quite similarafter the initial drying of the inoculum on the finger pads orhands. The activities of the agents tested against HAV andPV in the finger pad protocol were similar in the threevolunteers who participated in this study. This further rein-forces the suitability of this in vivo protocol for assessing thevirus-removing ability of hygienic hand-washing agents. Asfar as we know, no other standardized methods are currentlyavailable for this purpose. In our view, the Vienna testmodel (31) is more suitable for assessing the bactericidalactivity of surgical scrubs.Seven of the agents tested are products currently in use at

a large general hospital in Ottawa, Ontario, Canada, andthey were selected in consultation with an infection controlofficer at the hospital. Savlon, 70% ethanol, and Dettol arealso commonly used in other health care settings. Thecommercial formulations which we tested are identified bytheir trade names in Canada, and readers are urged tocarefully examine the relative proportions of the activeingredients in a given formulation (Table 1). In other places,products with identical formulations may be sold underdifferent trade names and the same trade name may be usedfor products with different formulations.

In this study, formulations with germicidal chemicals werebetter than unmedicated soap for reducing virus titers onhands; Bacti-Stat soap resulted in the highest levels ofreduction with both viruses (Table 2). However, the typesand levels of the chemicals in the medicated formulations arein themselves relatively ineffective for inactivation of non-

enveloped viruses (35), and it is likely that the higher levelsof virus reduction caused by these formulations were simplydue to more effective surface-active agents. Except when thealcohol solution of Savlon, 70% ethanol, and Alcare wereused, enough infectious virus was left on the washed handsso that virus was transferred to the metal disks (Table 3).

Previous studies have also shown that the use of hand-washing agents, even with vigorous rubbing of hands, leavesnearly 1% of the infectious virus on the hands (13). Properfield studies will be necessary to determine the significanceof the residual virus in the spread of HAV.

Liquid chemical disinfectants other than sporicides areconsidered effective against a given organism if they reduceits infectivity titer by at least 99.9% in a properly designedand conducted carrier test (9). We are not aware of such agenerally accepted albeit arbitrary criterion of efficacy forhand-washing agents. If a criterion of >99.9% reduction ofplaque titer were used, none of the agents which we testedcould meet it, and any chemical or formulation capable ofmeeting this criterion may prove to be too harsh for regularuse in hand washing. There is, therefore, an urgent need,particularly on the part of regulatory bodies, to establish arealistic standard by which the efficacy of hand-washingagents can be assessed.

In a previous study (4), we showed that drying of washedhands further reduces the amount of residual infectiousvirus. In the present study, no attempt was made to deter-mine the proportion of infectious HAV and PV removed bywashing alone and the proportion removed by the combinedeffects of washing and drying.The levels ofHAV removal and PV removal by the agents

tested were comparable, except that the levels of PV re-moval were higher than the levels observed for HAV. Thissuggests that the capacity of hand-washing agents to removevirus from hands depends on the type of virus used. There-fore, caution must be exercised in generalizing that theresults obtained with one virus are applicable to all otherviruses. This may be particularly true with HAV because ithas proven to be more resistant to chemical disinfection thanmost other human-pathogenic viruses (24, 32).

Studies of diarrheal outbreaks in institutions have re-vealed that diarrhea incidence declines after hand-washingprocedures are imposed (7). Similarly, viral outbreaks inpediatric nurseries have also been contained by institutingstringent hand-washing procedures with antiseptics (18, 19).However, transient bacteria have been shown to persist onhands after washing, and only a few products are able toreduce the level of these bacteria significantly (31, 34, 37).The failure of hand-washing agents to inactivate or remove

microbial pathogens from human hands is confounded by alack of compliance with hand-washing guidelines, both interms of frequency (1) and in terms of the actual procedure(15, 16, 36). However, the fact that as much as 20% of theinput infectious HAV, PV, and other viruses (3) remaineddetectable on washed and dried hands and the fact thatnearly 2% of the input viruses could be transferred to othersurfaces upon contact show that careful hand washing per semay not be enough to control the horizontal dissemination ofsuch pathogens and that care must be exercised in theselection of the hand-washing agent(s).

Alcohol-containing hand-washing agents result in goodbacterial removal from the skin (31). They were, however,less effective against HAV and PV. Chlorhexidine glu-conate-containing products are widely used and are effectiveagainst bacteria (5, 8, 12, 34) but are less effective in theremoval of HAV and PV from hands, even in combination

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with alcohols. These results are consistent with those ob-tained for human rotavirus (3). The behavior of the only4-chloro-3,5-xylenol-containing product examined in thisstudy (Dettol) was similar when it was tested against bacteria(34) and rotaviruses (3). Although the two triclosan-contain-ing products were also ineffective when measured by the99.9% criterion mentioned above, one of them (Bacti-Stat)showed the highest level of reduction achieved by any of the11 agents examined for both of the test viruses. This was truefor both the finger pad and whole-hand test protocols (Tables2 and 4).Not many in vivo studies of hand washing have been

performed with either bacteria or viruses (3, 12), and cur-rently testing of hand-washing agents is based on in vitrosuspension assays. We tested commonly available hospitaland institutional hand-washing agents by using a mixture ofHAV and PV in a single step. As far as we know, this is thefirst time that an in vivo procedure has been used to testviruses in a mixed inoculum. Although the number ofvolunteers used in this investigation is small, our resultssuggest that the data are reproducible, and moreover, our

results are consistent with the findings obtained with humanrotavirus when it was tested alone in an inoculum (3).The findings of this study and other laboratory-based

studies (3) clearly show that certain types of hand-washingagents are superior to others in ridding hands of human-pathogenic viruses. However, it should be emphasized thatthese findings in themselves are not meant to recommend theacceptance or rejection of any of the products tested. Theactual selection of a given formulation for use in a particularsetting must also take into consideration other factors, suchas personal safety with repeated use, environmental toxicity,cost, availability, etc.

ACKNOWLEDGMENTS

We thank C. Oxley for her assistance in the selection of thehand-washing agents. We also thank Hoechst-Roussel, Montreal,Canada, for complimentary supplies of Cidomycin.

This work was supported in part by a Canadian InternationalDevelopment Agency-Kenya Technical Cooperation GFT/2 fellow-ship.

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