Rotavirus and Escherichia coli

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1989, p Vol. 55, No /89/ $02.00/0 Copyright C) 1989, American Society for Microbiology In Vivo Protocol for Testing Efficacy of Hand-Washing Agents against Viruses and Bacteria: Experiments with Rotavirus and Escherichia coli SHAMIM A. ANSARI,1 SYED A. SATTAR,1* V. SUSAN SPRINGTHORPE,1 GEORGE A. WELLS,2 AND WALTER TOSTOWARYK2 Department of Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada KIH 8M5,1 and Division of Biometrics, Laboratory Centre for Disease Control, Health and Welfare Canada, Ottawa, Ontario, Canada KIA OL22 Received 13 June 1989/Accepted 8 September 1989 Ten antiseptic formulations, an unmedicated liquid soap, and tap water alone were compared for their capacities to eliminate human rotavirus from the finger pads of adult volunteers; three of the antiseptics, the soap, and the tap water alone were also tested against Escherichia coli. A fecal suspension of virus or bacterium was placed on each finger pad and air dried. The contaminated site was exposed to the test product for 10 s, rinsed in tap water, and dried on a paper towel. The residual virus or bacterium was then eluted. Selected agents were also tested by an analogous whole-hand method by which the entire palm surfaces of both hands were contaminated. Alcohols (70%) alone or with Savlon reduced the virus titer by >99%, whereas the reductions by Proviodine, Dettol, and Hibisol ranged from 95 to 97%. Aqueous solutions of chlorhexidine gluconate were significantly less effective for virus removal or inactivation than 70% alcohol solutions. Furthermore, Savlon in water (1:200) was found to be much less effective in eliminating the virus (80.6%) than the bacterium (98.9%). The tap water alone and the soap reduced the virus titers by 83.6 and 72.5% and the bacterial titers by 90 and 68.7%, respectively. The results of the whole-hand method agreed well with those of the finger pad protocol. We conclude that the finger pad method is a suitable model for testing the in vivo efficacy of hand-washing agents and emphasize the need for using appropriate test viruses and bacteria. Hands are among the principal vehicles for transfer of infectious agents in the community in general and in institutional settings in particular (4, 23, 25, 32). Regular and proper hand washing is, therefore, considered an important step in preventing the spread of infectious diseases (15, 20), and guidelines for this purpose are available for health care workers (12). The continued occurrence of institutional outbreaks of many enteric infections, particularly those caused by rotaviruses (11, 20), is, however, believed to be due to the lack of compliance with hand-washing guidelines rather than the inadequacy of the hand-washing agents (16). Earlier studies using the suspension test have demonstrated that rotaviruses are not readily inactivated by many types of commonly used chemical disinfectants (30); these viruses proved to be even more resistant to disinfection when they were dried on nonporous inanimate surfaces (18). Since products found to be ineffective in these in vitro studies included certain commonly used antiseptics, we considered it important to examine them and other similar products for their abilities to eliminate rotaviruses from human hands. Even though the testing of hand-washing agents against viruses on experimentally contaminated human hands has been studied before (8, 13, 14, 28), at present there is no standardized protocol available for such testing. So far, in vivo testing of such products has been conducted almost exclusively with bacteria (3, 7, 19, 24). This study was, therefore, undertaken to design and test a simple in vivo protocol using finger pads for testing the rotavirus-eliminating efficiency of hand-washing agents. The protocol was then applied to compare the rotavirus-eliminating efficien- * Corresponding author. cies of the hand-washing agents with their abilities to remove or inactivate Escherichia coli. Finally, experiments were conducted with selected hand-washing agents to demonstrate that the results with the finger pad protocol are representative of those obtained when the whole hand is experimentally contaminated with the virus or the bacterium and washed in the normal manner. MATERIALS AND METHODS Cells. The MA-104 line of rhesus monkey kidney cells was used for the growth and quantitation of the rotavirus. The methods used for the cultivation, maintenance, and passage of these cells have been described in detail elsewhere (26). For the virus plaque assay, cell monolayers were prepared in 12-well plastic plates (Costar, Cambridge, Mass.) with Eagle minimal essential medium (Flow Laboratories, Inc., McLean, Va.) containing 5% fetal bovine serum (GIBCO Laboratories, Grand Island, N.Y.) and gentamicin (Cidomycin; Roussel, Montreal, Quebec, Canada) at a final concentration of 50,ug/ml. The seeded plates were sealed in plastic bags (Philips, Toronto, Ontario, Canada) and incubated at 37 C for 48 h for monolayer formation. Fecal sample. A sample of formed feces was obtained from a healthy 1-year-old child. A 10% (wt/vol) suspension of the sample was prepared in normal saline. The suspension was centrifuged at 1,000 x g for 15 min to remove coarse particulate matter, and the supernatant was collected for storage at -20 C. It was found to be nontoxic to MA-104 cells, noninhibitory to the rotavirus, and free from any agents that could produce cytopathic effects or plaques in the cell line (2). Virus. A cell culture-adapted strain (Wa) of human rotavirus obtained from R. G. Wyatt (National Institutes of 3113

2 3114 ANSARI ET AL. APPL. ENVIRON. MICROBIOL. TABLE 1. Hand-washing agents Active ingredient(s) % Concn Dilution Product (manufacturer) Isopropanol 70.0 (vol/vol) None NAa Ethanol 70.0 (vol/vol) None NA" Chlorhexidine gluconate and 1.5 (wt/vol) and 15.0 (wt/vol) 1:30 in 70% isopropanol Savlon (Ayerst, Montreal, Quebec, cetrimide Canada) Chlorhexidine gluconate and 1.5 (wt/vol) and 15.0 (wt/vol) 1:30 in 70% ethanol Savlon cetrimide Chlorhexidine gluconate and 1.5 (wt/vol) and 15.0 (wt/vol) 1:30 and 1:200 in tap water Savlon cetrimide Chlorhexidine gluconate and 0.5 (wt/vol) and 70.0 (wt/vol) None Hibisol (Ayerst) isopropanol Chlorhexidine gluconate and 2.0 (wt/vol) and 4.0 (wt/vol) None Cida-Stat (Huntington, Bramalea, isopropanol Ontario, Canada) Povidone-iodine 10.0 (wt/vol) (1% free 12) None Proviodine (Rougier, Montreal, Quebec, Canada) PCMX 4.8 (wt/vol) and 9.4 (vol/vol) None Dettol (Reckitt & Colman, Lachine, Quebec, Canada) Liquid soap 100 1:10 in tap water Ivory (Procter & Gamble, Toronto, Ontario, Canada) Tap water a NA, Not applicable. Health, Bethesda, Md.) was used in this study. The procedures for the preparation of the virus pools and plaque assay have been described earlier (2). For contamination of the finger pads or hands, the virus suspension was prepared by diluting it 10-fold in the fecal sample. Bacterium. The strain of E. coli selected for this study was isolated from the fecal sample used for suspending the rotavirus. For the initial isolation and quantitation of E. coli, mfc agar (Difco Laboratories, Detroit, Mich.) was used. The test suspension of the bacterium was prepared by mixing an 18-h broth culture with the fecal sample at a 1:10 ratio. Volunteers. Permission of the Ethics Committee of the University of Ottawa was obtained for the use of human volunteers; one adult male and two adult females participated in this study. Hand-washing agents. Ten antiseptic formulations, an unmedicated liquid soap, and tap water were tested for their efficacy in removing the rotavirus from experimentally contaminated finger pads. Details of the hand-washing agents tested are given in Table 1. Isopropanol (70%), Savlon in 70% ethanol (1:30), Savlon in tap water (1:200), liquid soap, and tap water alone were also tested against E. coli. Paper towels. Rolls of ordinary unbleached paper towels (Boudreault, Inc., Hull, Quebec, Canada) were purchased locally and used for drying treated finger pads or hands as specified in the protocols. Finger pad protocol. A modification of the protocol developed earlier in our laboratory to study the survival of rotaviruses on human hands was used (2). The details of this procedure are given in Fig. 1, but only the results for the method which included paper towel drying are reported here. The same basic procedure was also used with the bacterium, but E. coli was quantitated as CFU on mfc agar plates. Elution fluid for both the rotavirus and E. coli was Earle balanced salt solution containing 20% (vol/vol) tryptose phosphate broth, and quantitation of the organisms was performed immediately after elution. At the end of each experiment, the contaminated finger pads of the volunteers were thoroughly rinsed with 70% ethanol and then washed with soap and water. Whole-hand protocol. The following whole-hand washing protocol was used to ascertain that the results obtained with our finger pad protocol were representative of the normal hand-washing situation. The volunteers did not wash or decontaminate their hands before the start of the experiment. A 0.5-ml portion of the fecal suspension of the virus or the bacterium was placed on the palm surface of one hand. The volunteer was then required to spread the inoculum over the entire palm surfaces of both hands by gently rubbing them together, and the inoculum was allowed to dry for 20 min under ambient conditions. To obtain the base titer of the I hands washed with tap water only (10 sec), rinsed with ethanol and air dried I iingerpads marked with vial rim fingerpads of one hand were each inoculated with tool nirs suspension and atlowed to dry ton 20 min thumb other fingers FIG. 1. Procedure for in vivo testing of hand-washing agents.

3 VOL. 55, 1989 HAND-WASHING AGENTS AGAINST VIRUSES AND BACTERIA 3115 TABLE 2. In vivo efficacy of hand-washing agents against human rotavirus in finger pad protocola Hand-washing agent % Reduction Tukey (concn) (mean + SD) grouping Isopropanol (70%) 99.8 ± 0.18 A Ethanol (70%) 99.8 ± 0.19 A Savlon in 70% isopropanol (1:30) 99.4 ± 0.84 A Savlon in 70% ethanol (1:30) 99.1 ± 0.50 A Proviodine 96.5 ± 2.65 A, B Dettol 96.2 ± 3.62 A, B, C Hibisol 95.3 ± 2.15 A, B, C Liquid soap 86.9 ± 2.42 D, B, C Savlon in water (1:30) 86.4 ± 4.07 D, C Tap water 83.6 ± 3.49 D Cida-Stat 80.3 ± 3.51 D Savlon in water (1:200) 78.3 ± 5.55 D a Agents with the same Tukey groupings do not have significantly different percentages of reduction (a = 0.05). test organism present at the end of this drying period, 20 ml of the eluent (20% tryptose phosphate broth in Earle balanced salt solution) was poured on the hands while they were being rubbed together over a plastic funnel 27 cm in diameter in order to bring the eluent in contact with the entire contaminated surface. Generally, more than 18 ml of the eluate was collected by this procedure. To test the efficacy of a hand-washing agent, the hands were contaminated with the test organism and dried as described above. Then 0.5 ml of the test agent was placed on the palm surface of one hand, and the volunteer was required to rub his or her hands for 10 s, thereby covering the entire contaminated surface. The hands were then washed by pouring over them 500 ml of tap water at 40 C and dried with a paper towel. The residual test organism was then eluted from the washed and dried hands, as described above. The eluates from the rotavirus-contaminated hands (controls and tests) were passed through separate columns of a Sephadex LH-20 gel (Pharmacia, Uppsala, Sweden) to remove any residual cytotoxicity due to antiseptic chemicals (5). The filtrates from the columns were then centrifuged at 10,000 x g to remove bacteria and fungi before the virus plaque assay. Eluates from bacterium-contaminated hands were titrated immediately on mfc agar plates. Statistical analyses. For the finger pad protocol, the testing of each hand-washing agent with each microorganism was conducted at least three times by using two finger pads for treatment with the test agent in each trial. Therefore, the results presented in Table 2 are the mean of six observations for each hand-washing agent with one volunteer, and those given in Table 3 are based on the mean of six observations TABLE 4. Comparison of whole-hand and finger pad protocolsa Hand-washing agent % Reduction (mean + SD) and protocolb E. coli Rotavirus 70% Isopropanol WH FP >99.9 ± ± ± Savlon in 70% ethanol (1:30) WH >99.9 ± ± 0.6 FP 98.7 ± Savlon in tap water (1:200) WH 96.6 ± FP 99.6 ± ± 5.6 Liquid soap (1:10) WH 94.5 ± ± 3.9 FP 60.8 ± ± 2.4 Tap water WH 95.1 ± ± 12.0 FP 90.0 ± ± 1.4 a Differences between whole-hand and finger pad protocols were not significant for any agents tested. b WH, Whole hand; FP, finger pad. with three volunteers. For the whole-hand washing protocol (Table 4), each experiment was repeated three times and the volunteer used was the same as in experiments summarized in Table 2. Results were statistically analyzed by using two-way analysis of variance based on the arcsine transformation of the raw data, i.e., the percentage of organisms removed. The arcsine transformation was utilized in order to stabilize the variance. Comparisons of the means of results obtained with the various hand-washing agents were made by the Tukey w procedure (31). This procedure allows for multiple comparisons of means while controlling for the error rate at the desired level. RESULTS The challenge titers of the test organisms in the finger pad experiments were ( ) x 104 and ( ) x 104 for the rotavirus and E. coli, respectively. These titers were measured at the end of a 20-min drying period, the minimum time for a visually dry appearance of the inoculum under ambient conditions, and they represent 57 and 7% of the original inoculum for rotavirus and E. coli, respectively. In the whole-hand experiments, after 20 min of drying, ( ) X 104 and ( ) x 104 were the challenge titers left TABLE 3. Comparison of in vivo efficacy of hand-washing agents against human rotavirus and E. coli in finger pad protocol Mean % reduction in infectivity titer with: Volunteer 70% Isopropanol Savlon in 70% ethanol Savlon in water Liquid soap Tap water (1:30) (1:200) Rotavirus E. coli Rotavirus E. coli Rotavirus E. coli Rotavirus E. coli Rotavirus E. coli Overall mean ± ± ± 1.9 ± SD

4 3116 ANSARI ET AL. on the hands. These amount to 22.9 ± 6.0 and 1.4 ± 1.3% of the original inoculum for the rotavirus and E. coli, respectively. The results of testing hand-washing agents and tap water against the rotavirus are shown in Table 2. Seventy percent isopropanol, 70% ethanol, and Savlon containing 70% of either one of the alcohols were each able to reduce the infectivity titer of the virus by >99.0%. Hibisol, a waterless product containing chlorhexidine gluconate and 70% isopropanol, eliminated 95.0% of the virus. Dettol and Proviodine achieved 96.2 and 96.5% virus removal, respectively. Cida- Stat and Savlon in water (1:30 and 1:200) produced the lowest levels of virus removal of all the antiseptics tested, as they were able to reduce the virus titer by only 80.3, 86.4, and 78.3%, respectively. Surprisingly, in terms of the mean percentages of virus removal, the liquid soap (86.9%) and tap water alone (83.6%) were as good as, if not better than, Cida-Stat and Savlon in water (1:200). The percentages of virus removal by the various handwashing agents were statistically compared by using the Tukey w procedure. Results of the multiple comparisons made by the w procedure of the means of the 12 hand-washing agents are displayed in Table 2. Those means designated by the same letter under the Tukey grouping were considered not significantly different from each other at the ot = 0.05 level. No Jifferences were seen among the hand-washing agents represented by the letter A. Likewise, no differences were demonstrated among those with the letter D. Group A, primarily the alcohol-containing formulations and those solutions with high concentrations of iodine or p-chloro-mxylenol (PCMX), was significantly different from group D, which included the aqueous chlorhexidine salts, soap and water, and water alone. However, multiple comparisons also showed the existence of intermediate groups B and C, which showed some overlap with the means for hand-washing agents in groups A and D. Despite these overlaps, the alcohols or alcohol-containing formulations were clearly superior to water and the aqueous solutions of chlorhexidine salts in reducing the levels of contaminating virus. In a separate set of experiments using three volunteers, four hand-washing agents and tap water alone were compared for their abilities to remove the rotavirus and E. coli from experimentally contaminated finger pads (Table 3). The differences in the mean percent removals for the virus and the bacterium were not statistically significant for the alcohols, the alcoholic solution of Savlon, the soap, or the tap water alone. However, there was a statistically significant difference (P = <0.0001) between the mean percent removals of E. coli (98.9%) and the rotavirus (80.6%) by Savlon in water (1:200). Statistical analyses of the data summarized in Table 3 also showed that the person-to-person variation in mean percent reductions by a hand-washing agent against a given organism was not significant (P = 0.14). In order to determine that the finger pad protocol was representative of the normal hand-washing practice, selected hand-washing agents and tap water alone were also tested by the whole-hand protocol for their abilities to remove the two test organisms. The results of these experiments are summarized in Table 4. Isopropanol (70%) and the alcohol solution of Savlon reduced the titers of both the organisms by at least 98.0% in both the protocols tested. Even though the overall removal of the virus and the bacterium by the other hand-washing agents and tap water alone was not as high as that seen with the alcohol solutions, there was no significant difference between the results obtained with the APPL. ENVIRON. MICROBIOL. whole-hand washing and the finger pad protocols. The wide variations observed between the finger pad and whole-hand experiments with liquid soap and E. coli were not found to be significant at the at = 0.05 level. For Savlon in water (1:200), the results with the whole-hand experiments were similar to those with the finger pad protocol; this solution was found to be significantly less effective in the elimination of the virus (84.8%) than of the bacterium (96.6%). DISCUSSION Hand washing is regarded as the single most important procedure for preventing the transmission of infections (23). However, the impact of hand washing depends not only on the regularity and thoroughness of the procedures used but also on the type of hand-washing agent selected. The continued occurrence of disease outbreaks, such as rotaviral gastroenteritis (9, 20), in health care settings suggests poor compliance with hand-washing guidelines (1, 10). However, a high degree of compliance alone may not be sufficient for proper infection control if the hand-washing agent is not effective in the removal or inactivation of important nosocomial pathogens. The findings of this study indicate that some commonly used hand-washing agents may be limited in their abilities to deal effectively with viruses which are known to cause disease outbreaks in institutions. Most of the in vivo evaluation of hand-washing agents has been conducted with bacteria; there are very few published reports in which the agents have been tested against viruses on experimentally contaminated hands (8, 13, 14, 28, 29). Furthermore, there is no standard in vivo protocol to test the virucidal properties of hand-washing agents. Whereas in commonly used suspension and carrier testing of germicides a disinfectant is required to reduce the infectious titer of the test organism by at least 99.9% to be considered effective, there is no such generally recognized criterion of efficacy for the in vivo testing of antiseptics for viruses. Schurmann and Eggers (28) and Carter et al. (6) reported that antiseptics were more effective in vitro than in vivo. In view of this, it is considered necessary to test other formulations and additional representative viruses and bacteria before deciding on a reasonable criterion for efficacy for antiseptics tested by in vivo protocols. Although surveys on health care personnel have shown that the mean duration for total hand washing is about 8 s (21), in this study, the 10-s exposure of the contaminated area to the agent being tested was based on the handwashing guidelines of the U.S. Centers for Disease Control (12). This relatively short exposure time is also considered more suitable for testing antiseptics used for hygienic hand disinfection (21, 23). The rinsing of hands may be an important step in hand antisepsis; tap water is normally used in the field, and we followed this practice in the testing protocol. Although it is acknowledged that the composition of tap water may vary geographically and temporally, neither distilled nor standard hard water is believed to be more predictive than tap water of the results which would be obtained under natural conditions. Moreover, the organic or inorganic content of tap water could affect the elution of the test organism from the fingers. It can be argued that the residual chlorine in tap water plays a role in the inactivation of pathogens on contaminated hands. We do not believe this to have been an important factor in this study, as the residual chlorine in our tap water was low (0.05 to 0.2 ppm [0.05 to 0.2,ug/ml]). Previous work in our laboratory has shown that rotavirus is

5 VOL. 55, 1989 HAND-WASHING AGENTS AGAINST VIRUSES AND BACTERIA 3117 relatively resistant to chlorine in tap water (22). Furthermore, the feces used for suspending the test organisms may have interfered with the action of such small amounts of chlorine by neutralization of the chlorine or protection of the test organisms or both. However, inactivation by tap water could occur for some other viruses (8, 22) and bacteria. Therefore, we suggest that dechlorinated tap water may be a suitable rinse water for routine in vivo testing of antiseptics, but this needs further investigation. The selection of a human rotavirus for this study was based on several facts. Not only are these viruses among the major causes of acute gastroenteritis, but they are frequently implicated in disease outbreaks, even when chemical disinfection and antisepsis are regularly used for infection control (20). They are also among the very few human pathogenic viruses whose minimal infective dose, as judged from human volunteer studies (33), has been shown to be 1 cell culture infective unit. The viruses are capable of surviving on human hands for several hours (2) and on nonporous inanimate surfaces for several days (27). Infectious rotaviruses can be transferred readily between hands and inanimate objects (2), and both viable viruses (15) and their antigens (25) have been demonstrated on the hands of care givers. Considerable evidence on the relative resistance of rotaviruses to chemical disinfectants is already available (18, 30). E. coli was chosen as the representative bacterium because it is a widely accepted indicator of fecal contamination and is also a common pathogen transmitted by hands. Previous studies on antisepsis have used strains of this organism as models (3, 24). The challenge levels of both of the test organisms and the organic load were realistic for normal hand contamination in the field, although it is recognized that higher levels of challenge may exist from time to time. In a field situation, antiseptics should be suitable for the inactivation of both viral and bacterial pathogens on contaminated skin. This may be particularly true when the minimal infective dose for some viral agents is extremely low. Furthermore, some viruses may survive on human skin more readily than many bacteria. In this study, E. coli was clearly more susceptible to inactivation during drying than was the rotavirus since a smaller proportion of its initial inoculum was recovered after 20 min of drying on either finger pads or hands. Recovery of both the virus and the bacterium from the whole-hand surface after 20 min was less than when only the finger pads were contaminated. However, it is not known whether this was due to the decreased efficiency of recovery or the increased rate of inactivation of the test organisms or both. Use of the whole hand for regular testing of antiseptics against viruses is not considered feasible. Apart from the relatively large volumes of a high-titered virus pool required for each experiment, determination of the base titer of the organism and treatment of hands with the test product must be conducted at different times. For viruses, a detoxification (disinfectant removal) step is also required. Moreover, recovery of inoculated organisms from the whole hand is generally lower and more variable than from finger pads. These factors make the protocol inherently difficult to control. On the other hand, the finger pad method described here represents a simpler and better way of testing antiseptics against viruses as well as bacteria. It not only permits the use of microliter quantities of the infectious agent but also allows the inclusion of proper controls and sufficient numbers of replicates in the same test. The results obtained with this protocol were reproducible and did not show any significant person-to-person variation; they were also in agreement with the findings based on the whole-hand technique. The suitability of using fingers instead of whole hands in antisepsis experiments has also been noted by others (28). Studies in progress in our laboratory have also successfully applied the finger pad protocol for the testing of handwashing agents against rhinoviruses. Alcohols and formulations containing 70% alcohol were the most effective in reducing rotavirus and E. coli contamination of hands. This is in agreement with findings of other investigators (3, 24). In The Federal Republic of Germany, 60% isopropanol is used as an index for comparing the in vivo bactericidal efficacy of antiseptics (24). Much of the loss of infectivity of the virus and the bacterium on the skin surface due to these antiseptics may be the result of in situ inactivation. In the case of the other agents tested, it may have been predominantly due to wash-off, as discussed below. Furthermore, the addition of emollients to alcohols or alcohol solutions of chlorhexidine salts makes them less astringent and more widely acceptable (17). Although both Hibisol and Savlon contain 70% alcohol, Hibisol contains a higher concentration of chlorhexidine gluconate than Savlon does at the recommended use dilution. Despite this, Savlon performed better under the test conditions used. This may have been due to the presence in Savlon of cetrimide as an additional active ingredient. Dettol was chosen as a product containing PCMX. The concentration of PCMX in the undiluted product tested was higher than is usually found in PCMX-based hand-washing agents (17). Like Dettol, Proviodine is an antiseptic not routinely used for hygienic hand disinfection. However, it was selected because previous studies of rotavirus disinfection (30) showed it to be the most effective of the iodinebased formulations tested. Even though these products were found to be more effective than soap and aqueous chlorhexidine salts, the level of rotavirus removal by the more dilute solutions of PCMX or iodine-based products formulated for hygienic hand washing may not be as high. Aqueous solutions of chlorhexidine gluconate (Savlon and Cida-Stat) are commonly used for hand washing; the results of this study clearly demonstrate that they may not be reliable antiseptics for pathogens such as rotaviruses. In fact, virus removal by tap water alone was comparable to that achieved by both dilutions of Savlon in water. This suggests that the reduction in titer levels by this product may be due primarily to wash-off. Similar results for the action of aqueous chlorhexidine salts have been obtained in in vitro studies of rotavirus disinfection (18, 30). The routine use of plain soap and water is considered by many to be quite adequate for the decontamination of hands in infection control. This is not borne out by the results reported here. Washing of contaminated hands with almost any agent is clearly desirable, but it should be noted that use of a less effective product could actually lead to the spread of localized contamination over the entire surfaces of both hands (29). This phenomenon is under investigation in our laboratory. Regular and proper washing of hands by health care personnel may be inadequate if ineffective products relied are on. Therefore, selection of an antiseptic with demonstrated broad-spectrum efficacy is considered essential in conjunction with efforts to increase compliance with good hand-washing practice. Proper testing of hand-washing agents against both viruses and bacteria by an in vivo protocol is clearly desirable, and the finger pad protocol presented here may be a suitable model for such standard

6 3118 ANSARI ET AL. tests. Further evaluation of the finger pad protocol with other viral and bacterial pathogens is under way in our laboratory. ACKNOWLEDGMENTS Financial support for S.A.A. has been provided by a scholarship from the Ministry of Education of the Government of Pakistan. Complimentary supplies of Cidomycin were received from Roussel. We are grateful to the volunteers who agreed to participate in this study. LITERATURE CITED 1. Albert, R. K., and F. Condie Handwashing patterns in medical intensive care units. N. Engl. J. Med. 304: Ansari, S. A., S. A. Sattar, V. S. Springthorpe, G. A. Wells, and W. Tostowaryk Rotavirus survival on human hands and transfer of infectious virus to animate and nonporous inanimate surfaces. J. Clin. Microbiol. 26: Ayliffe, G. A., J. R. Babb, J. G. Davies, and H. A. Lilly Hand disinfection: a comparison of various agents in laboratory and ward studies. J. Hosp. Infect. 11: Black, R. E., A. C. Dykes, K. E. Anderson, J. G. Wells, S. P. 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