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Comparative in vitro and in vivo study of nine alcohol-based handrubs
Comparative in vitro and in vivo study of nine alcohol-based handrubs Ste´phanie Rochon-Edouard,a Jean-Louis Pons, PhD,a Benoıˆt Veber, MD,b Martine Larkin,a Serge Vassal, PhD,a and Jean-Franc¸ois Lemeland, MDa Rouen, France
Background: Hygienic hand disinfection using alcohol-based handrubs (AHRs) is an alternative method to conventional handwashing in hospital wards. Because a documented choice of such products would consider data from in-care evaluation, we designed a comparative study of 9 AHRs both in vitro and in vivo in actual care conditions. Methods: The bactericidal activity of AHRs was first measured in vitro against 4 hospital strains exhibiting multiple antibiotic resistance: Acinetobacter baumannii, Staphylococcus aureus, Pseudomonas aeruginosa, and Enterobacter aerogenes. In a second time, AHRs were tested in an intensive care unit for antibacterial activity against the cutaneous flora of personnel hands and for acceptance by the care personnel. Results: The 9 AHRs could be classified in 3 groups on the basis of their in vitro activity: products of the first group showed a bactericidal activity higher than 4 log10 against the 4 strains. Only 1 of these products presented simultaneously an effective antibacterial activity against the cutaneous flora of care personnel hands and a good acceptance by the care personnel. Conclusion: The in vitro study allowed the differentiation of the AHRs tested on the basis of bactericidal activity, but evaluation in an in-care situation allowed further discrimination through both antibacterial activity and acceptance. Thus, the combination of in vitro and in vivo evaluations should provide helpful arguments in the choice of AHRs. (Am J Infect Control 2004;32:200-4.)
The transmission of microorganisms by hands of hospital personnel is the main cause of cross-infection in hospital wards and is responsible for the majority of nosocomial infections.1 Indeed, hands represent the major form of bacteria spread,2 mainly in services with high numbers of hospital infections and selective pressure of antibiotics. Handwashing and hand disinfection are among the most important and effective infection control measures to prevent nosocomial transmission of bacteria.3,4 However, an insufficient compliance with handwashing is reported in the majority of studies.5,6 Indeed, work overload, insufficient handwashing equipment, and cutaneous intolerance to hand-cleaning agents are many factors explaining this weak compliance with handwashing procedures. Handrubbing has proved to be an effective alternative method to conventional handwashing.7,8 Alcoholbased handrubs (AHRs) have several advantages, such Department of Microbiologya and Surgical Intensive Care Unit,b Centre Hospitalier Universitaire Charles Nicolle. Reprint requests: Jean-Louis Pons, Centre Hospitalier Universitaire de Rouen, Laboratoire de Bacte´riologie (Pr. Lemeland), 1 rue de Germont, 76031 Rouen cedex, France. 0196-6553/$30.00 Copyright ª 2004 by the Association for Professionals in Infection Control and Epidemiology, Inc. doi:10.1016/j.ajic.2003.08.003
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as easy and fast use on clean and dry hands.9 Several studies have shown the impact of AHRs use for improvement of handwashing compliance by health care workers6,10,11 and the efficacy of handwashing with AHRs as compared with conventional handwashing with antiseptic soap.12 Numerous studies have compared the antibacterial activity of hand-disinfecting agents by either in vitro13 or in vivo studies.14–18 However, the antibacterial activity of AHRs devoted to hygienic hand disinfection was never evaluated through both in vitro and in vivo studies, especially in in-care situations. Hospital wards may appreciate, for a documented choice of such products, to consider data from in-care evaluation. Therefore, we designed a comparative study of AHRs in 2 ways: first a comparison of in vitro bactericidal activity against 4 hospital strains exhibiting multiply antibiotic resistance, second an in vivo evaluation of antibacterial activity and acceptance by the hospital personnel in actual use conditions of an intensive care unit (ICU).
METHODS Products, media, and reagents The 9 AHRs tested in vitro and in vivo are described in Table 1. Tryptic-soy (TS) agar and TS agar supplemented with 5% horse blood (Bio-Rad, Marnesla-Coquette, France) were used as culture media. The neutralization solution used contained tween 80 (3%
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Table 1. AHRs tested AHR tested Sterillium (Bode Chemie, Hamburg, D)
Hibisprint (AstraZeneca, Rueil Malmaison, F) Spitaderm (Paragerm, Issy Les Moulineaux, F)
Phagobiol (Phagoge`ne DEC, Carros, F) Phisomain (Anios, Lille-Hellemmes, F)
Spitacid (Paragerm, Issy Les Moulineaux, F)
Manugel (Anios, Lille-Hellemmes, F) Clinogel (Asta Medica, Merignac, F) Gel hydro-Alcoolique (Gilbert, Herouville St Clair, F)
Composition 0.2% (w/w) mecetronium ethyl-sulphate 30% (w/w) 1-propanol 45% (w/w) isopropyl alcohol 0.5% (w/v) chlorhexidine digluconate 60% (w/v) isopropyl alcohol 0.5% (w/v) chlorhexidine gluconate 0.45% (w/v) hydrogen peroxide 70% (w/v) isopropyl alcohol 0.1% (w/v) chlorhexidine digluconate 89% (w/v) isopropyl alcohol / ethanol 0.1% (w/v) octenidine chlorhydrate 40% (w/w) 1-propanol 30% (w/w) isopropyl alcohol 46% (w/v) ethanol 27% (w/v) isopropyl alcohol 1% (w/v) benzyl alcohol 60% (v/v) isopropyl alcohol 0.05% phenoxy-ethanol 0.5% (w/v) triclosan 55% (v/v) isopropyl alcohol 0.3% (w/v) triclosan 70% (v/v) ethanol
Abbreviation 0.2% MEC
0.5% CHX 0.5% CHX–0.45% HP
0.1% CHX 0.1% OCT
46% ETH
60% ISO 0.5% TRI 0.3% TRI
AHR, Alcohol-based handrubs; V, volume; W, weight.
wt/vol), lecithin (0.3% wt/vol), and saponin (3% wt/vol) in distilled water.
In vitro study of bactericidal activity Four hospital strains exhibiting multiple resistance to antibiotics were used as test organisms: a thirdgeneration cephalosporin-resistant Acinetobacter baumannii strain, a methicillin-resistant Staphylococcus aureus strain, a ticarcillin- and imipenem-resistant Pseudomonas aeruginosa strain, and an extendedspectrum b-lactamase-producing Enterobacter aerogenes strain. Strains were stored frozen in glycerol broth at -808C and subcultured to ensure purity before testing. Bacterial inocula were prepared from 24-hour cultures in peptone-buffered water (peptone 10 g/L, sodium chloride 5 g/L, disodium hydrogenophosphate 3.5 g/L, and potassium dihydrogenophosphate 1.5 g/L, in distilled water) to obtain suspensions ranging from 108 to 5.108 colony-forming units (CFUs) per milliliter. CFU numbers of inocula were determined by plating 0.1 mL aliquots of logarithmic dilutions of bacterial suspensions onto duplicate TS blood agar plates and incubating for 48 hours at 378C. Bactericidal activity was measured by a dilution neutralization method as follows. AHRs were exposed to bacterial inocula according to European Norm (EN) 1040 standard.19 After introduction of 8 mL of the AHR into a test tube, 1 mL of sterile water, and 1 mL of bacterial inoculum were added. After contact of 1
minute, 1 mL of the well-homogenized mixture was transferred into a tube containing 8 mL of neutralizing solution and 1 mL of sterile water and incubated for 5 minutes. The number of surviving bacteria was determined by the method proposed by Eliopoulos20 for counting surviving bacteria in bactericidal activity measurements of antibiotics: 25 mL of the undiluted mixture and 10-1, 10-2, and 10-3 dilutions of the mixture were plated onto TS blood agar plates and incubated for 48 hours at 378C. Neutralization of each AHR was validated with each test strain.
Assessment of AHRs in a surgical ICU Each AHR was tested during 8 days in a surgical ICU. The antibacterial activity of each AHR was evaluated with 10 members of the medical and nonmedical staff of this care unit. For each individual testing of a given AHR, a first sample was collected immediately after nursing or medical care by carrying out fingerprints on TS agar plates containing neutralizing solution. This first sample was followed by handrubbing with the AHR. Then 2 other fingerprint samples were performed immediately after and 5 minutes after this hand disinfection. All plates were incubated for 48 hours at 378C. The difference between the 2 values obtained before and immediately after handrubbing established the RF1 logarithmic reduction factor and measured the immediate antibacterial activity of the AHR tested. The difference between the 2 values obtained before and 5 minutes after handrubbing established the RF2
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Table 2. In vitro bactericidal activity (expressed in logarithmic reduction) of 9 AHRs against Acinetobacter baumannii, Staphylococcus aureus, Pseudomonas aeruginosa, and Enterobacter aerogenes strains A baumannii Dilution of AHR
1/10
1/25
Control ÿ0.28 0.01 0.2% MEC 3.19 —* 0.5% CHX [ 5.00 [ 4.82 0.5% CHX–0.45% HP [ 5.00 [ 4.82 0.1% CHX [ 5.00 3.19 0.1% OCT [ 5.00 [ 4.82 46% ETH ÿ0.05 — 60% ISO ÿ0.02 — 0.5% TRI [ 5.00 [ 4.82 0.3% TRI 0.28 —
1/50
S aureus 1/100
ÿ0.20 ÿ0.20 — — [ 4.60 4.96 [ 4.60 [ 4.96 0.27 — [ 4.60 4.03 — — — — [ 4.60 2.20 — —
1/10
1/25
P aeruginosa
1/50
1/100
ÿ0.15 ÿ0.15 0.04 0.07 0.87 — — — [ 4.75 3.97 3.31 1.80 [ 4.75 [ 4.75 [ 4.68 [ 4.53 2.71 — — — [ 4.75 [ 4.75 [ 4.68 [ 4.53 ÿ0.08 — — — ÿ0.07 — — — [ 4.75 [ 4.75 [ 4.68 [ 4.53 ÿ0.04 — — —
1/10
1/25
1/50
E aerogenes 1/10
1/25
1/50
1/100
ÿ0.21 ÿ0.21 ÿ0.10 0.08 0.08 ÿ0.32 0.26 0.03 — — 1.00 — — — [ 4.61 [ 4.61 4.84 [ 4.76 [ 4.76 [ 4.25 [ 4.61 [ 4.61 [ 4.61 [ 5.14 [ 4.76 [ 4.76 [ 4.25 [ 4.61 [ 4.61 2.41 — 3.70 — — — [ 4.61 3.18 — [ 4.76 [ 4.76 [ 4.25 [ 4.61 0.72 — — 0.04 — — — ÿ0.09 — — 0.10 — — — 0.24 — — 0.17 0.43 — — 0.01 — — 0.22 — — —
AHR, Alcohol-based handrubs; CHX; chlorhexidine digluconate; ETH, ethanol; ISO, isopropyl alcohol; MEC, mecetronium ethyl-sulphate; OCT, octenidine chlorhydrate; TRI, triclosan. *Not measured.
logarithmic reduction factor and measured the AHR residual effect. In addition, each of the 10 participants performed an anonymous evaluation of the AHR used: 4 criteria (consistency, drying time, physical remanence, and ease of use of conditioning) were scored 0, 1, 2, or 3 (for nonsatisfactory, moderately satisfactory, satisfactory, and highly satisfactory, respectively) by each participant, and a mean score was then calculated for each criterion over the 10 participants. Finally, a total acceptance score for the 4 criteria (ranging from 012) was calculated for each AHR.
Statistical analysis The Newman-Keuls test was used to distinguish the antibacterial activity of AHRs both in vitro and in vivo. For the assessment of acceptance in the surgical ICU, data were analyzed using Epi Info version 6 (CDC, Atlanta, Ga).21
RESULTS In vitro study of bactericidal activity Bactericidal activity of the 9 AHRs tested against the 4 hospital strains is presented in Table 2. Three groups could be distinguished on the basis of bactericidal activity: the first group concerned AHRs exhibiting at least a 4 log10 inoculum reduction against the 4 test strains at test dilution 1/10 (0.5% CHX, 0.5% CHX– 0.45% HP, 0.1% OCT); the second group concerned AHRs exhibiting an inoculum reduction ranging from 1 to 4 log10 against at least 1 of the test strains (0.2% MEC, 0.1% CHX, 0.5% TRI); and the third group concerned AHRs exhibiting an inoculum reduction lower than 1 log10 (46% ETH, 60% ISO, 0.3% TRI). The Newman-Keuls test showed significant differences among these 3 groups (P \ 10-6).
Table 3. Antibacterial activity of 9 AHRs against cutaneous hands’ flora of hospital personnel in a surgical ICU AHR 0.2% MEC 0.5% CHX 0.5% CHX–0.45% HP 0.1% CHX 0.1% OCT 46% ETH 60% ISO 0.5% TRI 0.3% TRI
Mean RF1 (6 SD) $1.27 $1.43 $1.50 $1.11 $1.48 $1.61 $1.19 $0.98 $0.94
(60.93) (60.81) (60.49) (60.58) (60.83) (60.34) (60.74) (60.55) (60.59)
Mean RF2 (6 SD) 1.01 1.05 $0.93 0.36 0.66 0.81 0.46 0.59 0.83
(61.01) (60.68) (60.58) (60.58) (60.67) (60.86) (60.65) (60.51) (60.66)
AHR, Alcohol-based handrubs; CHX, chlorhexidine digluconate; ETH, ethanol; ICU, intensive care unit; ISO, isopropyl alcohol; OCT, octenidine chlorhydrate; MEC, mecetronium ethyl-sulphate; RF1, log10 reduction factor immediately after hand disinfection; RF2, log10 reduction factor 5 minutes after hand disinfection; SD, standard deviation; TRI, triclosan.
Assessment in a surgical ICU The antibacterial activity of AHRs in actual use conditions is reflected in Table 3. Two groups were tentatively distinguished on the basis of the logarithmic reduction factor RF1. The first group concerned AHRs exhibiting an RF1 of at least 1.4 log10 (46% ETH, 0.5% CHX–0.45% HP, 0.1% OCT, 0.5% CHX), and the second group concerned AHRs exhibiting an RF1 lower than 1.4 log10 (0.2% MEC, 60% ISO, 0.1% CHX, 0.5% TRI, 0.3% TRI). However, the Newman-Keuls test showed no significant difference among these 2 groups. The results of acceptance assessments are presented in Table 4. The 2 products exhibiting the highest global score (ie, best accepted by the hospital personnel) were 0.1% OCT and 0.2% MEC.
DISCUSSION Handwashing is considered one of the most important ways to prevent nosocomial infections3,22,23
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Table 4. Assessment of acceptance of AHRs by the hospital personnel of a surgical ICU AHR 0.2% MEC 0.5% CHX 0.5% CHX–0.45% HP 0.1% CHX 0.1% OCT 46% ETH 60% ISO 0.5% TRI 0.3% TRI
Consistency* 2.5 2.0 1.9 2.0 2.5 2.1 2.2 1.9 1.5
Drying time*
Physical remanence*
2.1 1.4 1.6 2.5 2.1 1.9 2.1 1.6 1.6
1.9 0.7 1.4 1.9 2.2 1.4 2.0 0.7 0.9
Conditioning (ease of use)* 2.3 2.1 2.0 1.8 2.3 2.0 1.9 2.0 1.5
Total acceptance score* (maximum = 12) 8.8 6.2 6.9 8.2 9.1 7.4 8.2 6.2 5.5
AHR, Alcohol-based handrubs; CHX, chlorhexidine digluconate; ETH, ethanol; ICU, intensive care unit; ISO, isopropyl alcohol; OCT, octenidine chlorhydrate; MEC, mecetronium ethyl-sulphate; TRI, triclosan. *Mean score established from the evaluation by 10 participants (individual score ranging from 0, nonsatisfactory, to 3, highly satisfactory).
because of its effectiveness in removing from the hands of hospital personnel transient microbial flora acquired by contact with infected or colonized patients or with medical devices and environmental sources. This study sought to compare the antibacterial activity and acceptability of several AHRs in actual care situations to facilitate the choice of effective hand disinfection products that could reduce the risk of nosocomial infections in an ICU. The choice of AHRs for hand disinfection may be documented by a comparative study of their antibacterial activity both in vitro and in vivo, because results recorded in vitro cannot be extrapolated to estimate the in vivo activity of hand disinfection agents.24 The antibacterial activity of hand disinfection agents that agree with European standards19,25 is rarely evaluated against nosocomial bacterial strains exhibiting multiple resistance to antibiotics. However, some studies have demonstrated decreased activity of handdisinfecting agents on multiple-resistant bacteria (eg, in methicillin-resistant S aureus).26 Therefore the in vitro antibacterial activity of the various AHRs tested in the current work was assessed against 4 hospital multiple-resistant strains. The method proposed by Eliopoulos20 for counting surviving bacteria in the assessment of bactericidal activity of antibiotics was found well suited to the simultaneous study of various AHRs. The first step of the work characterizes a group of 3 products exhibiting major bactericidal activity ($4 log inoculum reduction) that contain either chlorhexidine digluconate (0.5% CHX, 0.5 % CHX–0.45 % HP) or octenidine chlorhydrate (0.1% OCT). Although AHRs are intended to be applied without dilution on dry skin, a range of AHR dilutions was introduced to better evaluate bactericidal activity and thus to differentiate among AHRs more accurately. For example, 0.5% CHX–0.45% HP and 0.1% OCT exhibited more than 4 log inoculum reduction after 1/100 dilution against A baumannii, S aureus, and E aerogenes. The difference between the highest dilution allowing a bactericidal
activity in vitro and actual use conditions allows a safety activity margin against hospital strains.27 The various AHRs were then tested in actual use conditions in a surgical ICU. The current evaluation allowed us to take into consideration the contamination of the nursing and medical staff hands, using a sampling method of finger pulp imprints. This fingerprinting method has been used in recent works studying AHRs.12,28 We also chose this method in the current work because it is easy to perform in a care service, unless other methods such as the EN 1500 standard25 or the glove-juice test,29 which are not adapted to an evaluation in care situation. A reduction of the bacterial flora higher than 1.4 log10 was observed in the in vivo study with 46% ETH, 0.5% CHX–0.45% HP, 0.1% OCT, and 0.5% CHX, these last 3 AHRs being the most active in the in vitro study; thus the in vitro and in vivo evaluations of antibacterial activities were fairly concordant. It is well-known that if studies involving a limited number of volunteers can assess the activity of a given product, they may be insufficient to reveal significant differences among various products, since the inter- and intra-individual variability remains a major problem in spite of standardization efforts.14,17 This part of the study allowed us also to evaluate the rate of recolonization of hands after use of AHRs: several AHRs exhibited a poorly remanent antibacterial effect, as revealed by bacterial counts from hands performed 5 minutes after friction. This information should be also taken into account in the choice of an AHR. Acceptance by the hospital personnel is an important criterion in the choice of an AHR, because it conditions compliance to hygienic hand disinfection. In the current work, combining results of both antibacterial activity and acceptance allowed a further discrimination of AHRs, especially among products exhibiting a similar antibacterial activity. Among the 3 in vitro most active AHRs, 1 product (0.1% OCT) was
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differentiated by a better acceptance. Alcohol-based hand gels have been proposed as alternative pharmaceutical formulations well suited to rapid and efficient hand disinfection. In our study, the 3 gels analyzed were found less active than the other formulations, as was already reported,30,31 and were poorly accepted by ICU personnel.
CONCLUSION This study provides information to help health care professionals choose effective AHRs that may reduce the risk of nosocomial infections. The combination of in vitro and in vivo data allowed for a good discrimination among the AHRs tested. A long period of observation would be required to assess the clinical consequences of the use of these solutions on the rate of nosocomial infections. A decrease in the rate of nosocomial infections and in the acquisition of multiple-resistant bacteria was noticed in a study that confirmed the efficiency of AHR use to improve the compliance rate of handwashing by health care workers.8,32 Because AHRs are efficient handwashing agents, they can be safely used in special situations—in the ICU, during periods of work overload, where access to ward sinks is difficult—and should be largely encouraged in various care units. The authors thank the nursing and medical staff of the surgical ICU of the Centre Hospitalier Universitaire de Rouen for participation in the in vivo study.
References 1. Weinstein RA. Epidemiology and control of nosocomial infections in adult intensive care units. Am J Med 1991;10:S179-84. 2. Reybrouck G. Handwashing and hand disinfection. J Hosp Infect 1986;8:5-23. 3. Steere AC, Mallison GF. Handwashing practices for the prevention of nosocomial infections. Ann Intern Med 1975;83:683-90. 4. Larson EL. A causal link between handwashing and risk of infection?: Examination of an evidence. Infect Control Hosp Epidemiol 1988;9: 28-36. 5. Pittet D, Mourouga P, Perneger TV. Compliance with handwashing in a teaching hospital: infection control program. Ann Intern Med 1999; 130:126-30. 6. Voss A, Widner AF. No time for handwashing? Handwashing versus alcoholic rub: Can we afford 100% compliance? Infect Control Hosp Epidemiol 1997;18:205-8. 7. Pittet D, Hugonnet S, Harbarth S, Mourouga P, Sauvan V, Touveneau S, et al. Effectiveness of a hospital-wide programme to improve compliance with hand hygiene. Lancet 2000;356:1307-12. 8. Girard R, Amazian K, Fabry J. Better compliance and better tolerance in relation to a well-conducted introduction to rub-in hand disinfection. J Hosp Infect 2001;47:131-7. 9. Rotter ML. Arguments for alcoholic hand disinfection. J Hosp Infect 2001;48:S4-8. 10. Maury E, Alzien M, Baudel JL, Haram N, Barbut F, Guidet B, et al. Availability of an alcohol solution can improve hand disinfection compliance in an intensive care unit. Am J Resp Crit Care Med 2000;162:324-7. 11. Hugonnet S, Perneger T, Pittet D. Alcohol-based handrub improves compliance with hand hygiene in intensive care units. Arch Intern Med 2002;162:1037-43.
Rochon-Edouard et al 12. Girou E, Loyeau S, Legrand P, Oppein F, Brun-Buisson C. Efficacy of handrubbing with alcohol based solution versus standard handwashing with antiseptic soap: Randomised clinical trial. Brit Med J 2002; 325:362-5. 13. Mahwachi M, Attiso M, Druilles J. Etude comparative de l’activite´ bacte´ricide in vitro de 9 antiseptiques vis-a`-vis de souches re´fe´rence´es et de souches isole´es en milieu hospitalier. Me´d Mal Infect 1992;22: 732-7. 14. Savage C, Leclerc H, Reverdy ME, Fleurette J, Guiraud-Dauriac H, Cremieux A, et al. Etude multicentrique de l’activite´ antimicrobienne des antiseptiques sur les mains. Pathol Biol 1984;32:581-4. 15. Ayliff GA, Babb JR, Davies JG, Lilly HA. Hand disinfection: a comparison of various agents in laboratory and ward studies. J Hosp Infect 1988;11:226-43. 16. Cardoso CL, Pereira HH, Zequim JC, Guilhermetti M. Effectiveness of hand-cleansing agents for removing Acinetobacter baumannii strain from contaminated hands. Am J Infect Control 1999;27:327-31. 17. Cre´mieux A, Reverdy ME, Pons J-L, Savage C, Chevalier J, Fleurette J, et al. Standardized method for evaluation of hand disinfection by surgical scrub formulations. Appl Environ Microbiol 1989;55:2944-8. 18. Guilhermetti M, Hernandes SED, Fukushigue Y, Garcia LB, Cardoso CL. Effectiveness of hand-cleansing agents for removing methicillinresistant Staphylococcus aureus from contaminated hands. Infect Control Hosp Epidemiol 2001;22:105-8. 19. AFNOR (Association Franc¸aise de Normalisation). NF EN 1040. Antiseptiques et de´sinfectants chimiques: Aactivite´ bacte´ricide de base—me´thode d’essai et prescription (phase 1). April 1997. 20. Eliopoulos GM, Moellering RC. Antimicrobial combinations. In: Lorian V, editor. Antibiotics in laboratory medicine. 4th ed. Baltimore (MD): Williams & Wilkins; 1996. p. 330-96. 21. Dean AG, Dean JA, Coulombier D. Epi Info version 6: A word processing, database, and statistics programs for public health on microcomputers. Atlanta (GA): Centers for Disease Control and Prevention. 22. Larson EL. APIC guideline for handwashing and antisepsis in health care settings. Am J Infect Control 1995;23:251-69. 23. Boyce JM, Pittet D. Guideline for hand hygiene in health-care settings. Recommendations of the healthcare infection control practices advisory committee and the hicpac/shea/apic/idsa hand hygiene task force. Am J Infect Control 2002;30:S1-46. 24. Fleurette J, Freney J, Reverdy ME. Antisepsie et de´sinfection. Paris: Eska; 1995. 25. AFNOR (Association Franc¸aise de Normalisation). NF EN 1500. Antiseptiques et de´sinfectants chimiques: traitement hygie´nique des mains par frictions—me´thode d’essai et prescription (phase 2/e´tape 2). September 1997. 26. Kampf G, Jarosch R, Ru¨den H. Limited effectiveness of chlorhexidine based hand disinfectants against methicillin-resistant Staphylococcus aureus. J Hosp Infect 1998;38:297-303. 27. Dusseau JY, Chapalain JC, Rouby Y, Reverdy ME, Bartoli M. Evaluation par une microme´thode de l’activite´ bacte´ricide de cinq de´sinfectants sur 108 souches hospitalie`res. Pathol Biol 1993;41:349-57. 28. Lucet J-C, Rigaud M-P, Mentre F, Kassis N, Deblangy C, Andremont A, et al. Hand contamination before and after different hand hygiene techniques: a randomized clinical trial. J Hosp Infect 2002;50:276-80. 29. Gaschen M. Etude comparative de deux me´thodes destine´es au pre´le`vement de la flore microbienne des mains. Helv Chir Acta 1968;3:372-7. 30. Pietsch H. Hand antiseptics: rubs versus scrubs, alcoholic solutions versus alcoholic gels. J Hosp Infect 2001;48:S23-6. 31. Kramer A, Rudolph P, Kampf G, Pittet D. Limited efficacy of alcoholbased hand gels. Lancet 2002;359:1489-90. 32. Doebbeling BN, Stanley GL, Sheetz CT, Pfaller MA, Houston AK, Annis L, et al. Comparative efficacy of alternative handwashing agents in reducing nosocomial infections in intensive care units. N Engl J Med 1992;327:88-93.
Background: Hygienic hand disinfection using alcohol-based handrubs (AHRs) is an alternative method to conventional handwashing in hospital wards. Because a documented choice of such products would consider data from in-care evaluation, we designed a comparative study of 9 AHRs both in vitro and in vivo in actual care conditions. Methods: The bactericidal activity of AHRs was first measured in vitro against 4 hospital strains exhibiting multiple antibiotic resistance: Acinetobacter baumannii, Staphylococcus aureus, Pseudomonas aeruginosa, and Enterobacter aerogenes. In a second time, AHRs were tested in an intensive care unit for antibacterial activity against the cutaneous flora of personnel hands and for acceptance by the care personnel. Results: The 9 AHRs could be classified in 3 groups on the basis of their in vitro activity: products of the first group showed a bactericidal activity higher than 4 log10 against the 4 strains. Only 1 of these products presented simultaneously an effective antibacterial activity against the cutaneous flora of care personnel hands and a good acceptance by the care personnel. Conclusion: The in vitro study allowed the differentiation of the AHRs tested on the basis of bactericidal activity, but evaluation in an in-care situation allowed further discrimination through both antibacterial activity and acceptance. Thus, the combination of in vitro and in vivo evaluations should provide helpful arguments in the choice of AHRs. (Am J Infect Control 2004;32:200-4.)
The transmission of microorganisms by hands of hospital personnel is the main cause of cross-infection in hospital wards and is responsible for the majority of nosocomial infections.1 Indeed, hands represent the major form of bacteria spread,2 mainly in services with high numbers of hospital infections and selective pressure of antibiotics. Handwashing and hand disinfection are among the most important and effective infection control measures to prevent nosocomial transmission of bacteria.3,4 However, an insufficient compliance with handwashing is reported in the majority of studies.5,6 Indeed, work overload, insufficient handwashing equipment, and cutaneous intolerance to hand-cleaning agents are many factors explaining this weak compliance with handwashing procedures. Handrubbing has proved to be an effective alternative method to conventional handwashing.7,8 Alcoholbased handrubs (AHRs) have several advantages, such Department of Microbiologya and Surgical Intensive Care Unit,b Centre Hospitalier Universitaire Charles Nicolle. Reprint requests: Jean-Louis Pons, Centre Hospitalier Universitaire de Rouen, Laboratoire de Bacte´riologie (Pr. Lemeland), 1 rue de Germont, 76031 Rouen cedex, France. 0196-6553/$30.00 Copyright ª 2004 by the Association for Professionals in Infection Control and Epidemiology, Inc. doi:10.1016/j.ajic.2003.08.003
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as easy and fast use on clean and dry hands.9 Several studies have shown the impact of AHRs use for improvement of handwashing compliance by health care workers6,10,11 and the efficacy of handwashing with AHRs as compared with conventional handwashing with antiseptic soap.12 Numerous studies have compared the antibacterial activity of hand-disinfecting agents by either in vitro13 or in vivo studies.14–18 However, the antibacterial activity of AHRs devoted to hygienic hand disinfection was never evaluated through both in vitro and in vivo studies, especially in in-care situations. Hospital wards may appreciate, for a documented choice of such products, to consider data from in-care evaluation. Therefore, we designed a comparative study of AHRs in 2 ways: first a comparison of in vitro bactericidal activity against 4 hospital strains exhibiting multiply antibiotic resistance, second an in vivo evaluation of antibacterial activity and acceptance by the hospital personnel in actual use conditions of an intensive care unit (ICU).
METHODS Products, media, and reagents The 9 AHRs tested in vitro and in vivo are described in Table 1. Tryptic-soy (TS) agar and TS agar supplemented with 5% horse blood (Bio-Rad, Marnesla-Coquette, France) were used as culture media. The neutralization solution used contained tween 80 (3%
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Table 1. AHRs tested AHR tested Sterillium (Bode Chemie, Hamburg, D)
Hibisprint (AstraZeneca, Rueil Malmaison, F) Spitaderm (Paragerm, Issy Les Moulineaux, F)
Phagobiol (Phagoge`ne DEC, Carros, F) Phisomain (Anios, Lille-Hellemmes, F)
Spitacid (Paragerm, Issy Les Moulineaux, F)
Manugel (Anios, Lille-Hellemmes, F) Clinogel (Asta Medica, Merignac, F) Gel hydro-Alcoolique (Gilbert, Herouville St Clair, F)
Composition 0.2% (w/w) mecetronium ethyl-sulphate 30% (w/w) 1-propanol 45% (w/w) isopropyl alcohol 0.5% (w/v) chlorhexidine digluconate 60% (w/v) isopropyl alcohol 0.5% (w/v) chlorhexidine gluconate 0.45% (w/v) hydrogen peroxide 70% (w/v) isopropyl alcohol 0.1% (w/v) chlorhexidine digluconate 89% (w/v) isopropyl alcohol / ethanol 0.1% (w/v) octenidine chlorhydrate 40% (w/w) 1-propanol 30% (w/w) isopropyl alcohol 46% (w/v) ethanol 27% (w/v) isopropyl alcohol 1% (w/v) benzyl alcohol 60% (v/v) isopropyl alcohol 0.05% phenoxy-ethanol 0.5% (w/v) triclosan 55% (v/v) isopropyl alcohol 0.3% (w/v) triclosan 70% (v/v) ethanol
Abbreviation 0.2% MEC
0.5% CHX 0.5% CHX–0.45% HP
0.1% CHX 0.1% OCT
46% ETH
60% ISO 0.5% TRI 0.3% TRI
AHR, Alcohol-based handrubs; V, volume; W, weight.
wt/vol), lecithin (0.3% wt/vol), and saponin (3% wt/vol) in distilled water.
In vitro study of bactericidal activity Four hospital strains exhibiting multiple resistance to antibiotics were used as test organisms: a thirdgeneration cephalosporin-resistant Acinetobacter baumannii strain, a methicillin-resistant Staphylococcus aureus strain, a ticarcillin- and imipenem-resistant Pseudomonas aeruginosa strain, and an extendedspectrum b-lactamase-producing Enterobacter aerogenes strain. Strains were stored frozen in glycerol broth at -808C and subcultured to ensure purity before testing. Bacterial inocula were prepared from 24-hour cultures in peptone-buffered water (peptone 10 g/L, sodium chloride 5 g/L, disodium hydrogenophosphate 3.5 g/L, and potassium dihydrogenophosphate 1.5 g/L, in distilled water) to obtain suspensions ranging from 108 to 5.108 colony-forming units (CFUs) per milliliter. CFU numbers of inocula were determined by plating 0.1 mL aliquots of logarithmic dilutions of bacterial suspensions onto duplicate TS blood agar plates and incubating for 48 hours at 378C. Bactericidal activity was measured by a dilution neutralization method as follows. AHRs were exposed to bacterial inocula according to European Norm (EN) 1040 standard.19 After introduction of 8 mL of the AHR into a test tube, 1 mL of sterile water, and 1 mL of bacterial inoculum were added. After contact of 1
minute, 1 mL of the well-homogenized mixture was transferred into a tube containing 8 mL of neutralizing solution and 1 mL of sterile water and incubated for 5 minutes. The number of surviving bacteria was determined by the method proposed by Eliopoulos20 for counting surviving bacteria in bactericidal activity measurements of antibiotics: 25 mL of the undiluted mixture and 10-1, 10-2, and 10-3 dilutions of the mixture were plated onto TS blood agar plates and incubated for 48 hours at 378C. Neutralization of each AHR was validated with each test strain.
Assessment of AHRs in a surgical ICU Each AHR was tested during 8 days in a surgical ICU. The antibacterial activity of each AHR was evaluated with 10 members of the medical and nonmedical staff of this care unit. For each individual testing of a given AHR, a first sample was collected immediately after nursing or medical care by carrying out fingerprints on TS agar plates containing neutralizing solution. This first sample was followed by handrubbing with the AHR. Then 2 other fingerprint samples were performed immediately after and 5 minutes after this hand disinfection. All plates were incubated for 48 hours at 378C. The difference between the 2 values obtained before and immediately after handrubbing established the RF1 logarithmic reduction factor and measured the immediate antibacterial activity of the AHR tested. The difference between the 2 values obtained before and 5 minutes after handrubbing established the RF2
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Table 2. In vitro bactericidal activity (expressed in logarithmic reduction) of 9 AHRs against Acinetobacter baumannii, Staphylococcus aureus, Pseudomonas aeruginosa, and Enterobacter aerogenes strains A baumannii Dilution of AHR
1/10
1/25
Control ÿ0.28 0.01 0.2% MEC 3.19 —* 0.5% CHX [ 5.00 [ 4.82 0.5% CHX–0.45% HP [ 5.00 [ 4.82 0.1% CHX [ 5.00 3.19 0.1% OCT [ 5.00 [ 4.82 46% ETH ÿ0.05 — 60% ISO ÿ0.02 — 0.5% TRI [ 5.00 [ 4.82 0.3% TRI 0.28 —
1/50
S aureus 1/100
ÿ0.20 ÿ0.20 — — [ 4.60 4.96 [ 4.60 [ 4.96 0.27 — [ 4.60 4.03 — — — — [ 4.60 2.20 — —
1/10
1/25
P aeruginosa
1/50
1/100
ÿ0.15 ÿ0.15 0.04 0.07 0.87 — — — [ 4.75 3.97 3.31 1.80 [ 4.75 [ 4.75 [ 4.68 [ 4.53 2.71 — — — [ 4.75 [ 4.75 [ 4.68 [ 4.53 ÿ0.08 — — — ÿ0.07 — — — [ 4.75 [ 4.75 [ 4.68 [ 4.53 ÿ0.04 — — —
1/10
1/25
1/50
E aerogenes 1/10
1/25
1/50
1/100
ÿ0.21 ÿ0.21 ÿ0.10 0.08 0.08 ÿ0.32 0.26 0.03 — — 1.00 — — — [ 4.61 [ 4.61 4.84 [ 4.76 [ 4.76 [ 4.25 [ 4.61 [ 4.61 [ 4.61 [ 5.14 [ 4.76 [ 4.76 [ 4.25 [ 4.61 [ 4.61 2.41 — 3.70 — — — [ 4.61 3.18 — [ 4.76 [ 4.76 [ 4.25 [ 4.61 0.72 — — 0.04 — — — ÿ0.09 — — 0.10 — — — 0.24 — — 0.17 0.43 — — 0.01 — — 0.22 — — —
AHR, Alcohol-based handrubs; CHX; chlorhexidine digluconate; ETH, ethanol; ISO, isopropyl alcohol; MEC, mecetronium ethyl-sulphate; OCT, octenidine chlorhydrate; TRI, triclosan. *Not measured.
logarithmic reduction factor and measured the AHR residual effect. In addition, each of the 10 participants performed an anonymous evaluation of the AHR used: 4 criteria (consistency, drying time, physical remanence, and ease of use of conditioning) were scored 0, 1, 2, or 3 (for nonsatisfactory, moderately satisfactory, satisfactory, and highly satisfactory, respectively) by each participant, and a mean score was then calculated for each criterion over the 10 participants. Finally, a total acceptance score for the 4 criteria (ranging from 012) was calculated for each AHR.
Statistical analysis The Newman-Keuls test was used to distinguish the antibacterial activity of AHRs both in vitro and in vivo. For the assessment of acceptance in the surgical ICU, data were analyzed using Epi Info version 6 (CDC, Atlanta, Ga).21
RESULTS In vitro study of bactericidal activity Bactericidal activity of the 9 AHRs tested against the 4 hospital strains is presented in Table 2. Three groups could be distinguished on the basis of bactericidal activity: the first group concerned AHRs exhibiting at least a 4 log10 inoculum reduction against the 4 test strains at test dilution 1/10 (0.5% CHX, 0.5% CHX– 0.45% HP, 0.1% OCT); the second group concerned AHRs exhibiting an inoculum reduction ranging from 1 to 4 log10 against at least 1 of the test strains (0.2% MEC, 0.1% CHX, 0.5% TRI); and the third group concerned AHRs exhibiting an inoculum reduction lower than 1 log10 (46% ETH, 60% ISO, 0.3% TRI). The Newman-Keuls test showed significant differences among these 3 groups (P \ 10-6).
Table 3. Antibacterial activity of 9 AHRs against cutaneous hands’ flora of hospital personnel in a surgical ICU AHR 0.2% MEC 0.5% CHX 0.5% CHX–0.45% HP 0.1% CHX 0.1% OCT 46% ETH 60% ISO 0.5% TRI 0.3% TRI
Mean RF1 (6 SD) $1.27 $1.43 $1.50 $1.11 $1.48 $1.61 $1.19 $0.98 $0.94
(60.93) (60.81) (60.49) (60.58) (60.83) (60.34) (60.74) (60.55) (60.59)
Mean RF2 (6 SD) 1.01 1.05 $0.93 0.36 0.66 0.81 0.46 0.59 0.83
(61.01) (60.68) (60.58) (60.58) (60.67) (60.86) (60.65) (60.51) (60.66)
AHR, Alcohol-based handrubs; CHX, chlorhexidine digluconate; ETH, ethanol; ICU, intensive care unit; ISO, isopropyl alcohol; OCT, octenidine chlorhydrate; MEC, mecetronium ethyl-sulphate; RF1, log10 reduction factor immediately after hand disinfection; RF2, log10 reduction factor 5 minutes after hand disinfection; SD, standard deviation; TRI, triclosan.
Assessment in a surgical ICU The antibacterial activity of AHRs in actual use conditions is reflected in Table 3. Two groups were tentatively distinguished on the basis of the logarithmic reduction factor RF1. The first group concerned AHRs exhibiting an RF1 of at least 1.4 log10 (46% ETH, 0.5% CHX–0.45% HP, 0.1% OCT, 0.5% CHX), and the second group concerned AHRs exhibiting an RF1 lower than 1.4 log10 (0.2% MEC, 60% ISO, 0.1% CHX, 0.5% TRI, 0.3% TRI). However, the Newman-Keuls test showed no significant difference among these 2 groups. The results of acceptance assessments are presented in Table 4. The 2 products exhibiting the highest global score (ie, best accepted by the hospital personnel) were 0.1% OCT and 0.2% MEC.
DISCUSSION Handwashing is considered one of the most important ways to prevent nosocomial infections3,22,23
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Table 4. Assessment of acceptance of AHRs by the hospital personnel of a surgical ICU AHR 0.2% MEC 0.5% CHX 0.5% CHX–0.45% HP 0.1% CHX 0.1% OCT 46% ETH 60% ISO 0.5% TRI 0.3% TRI
Consistency* 2.5 2.0 1.9 2.0 2.5 2.1 2.2 1.9 1.5
Drying time*
Physical remanence*
2.1 1.4 1.6 2.5 2.1 1.9 2.1 1.6 1.6
1.9 0.7 1.4 1.9 2.2 1.4 2.0 0.7 0.9
Conditioning (ease of use)* 2.3 2.1 2.0 1.8 2.3 2.0 1.9 2.0 1.5
Total acceptance score* (maximum = 12) 8.8 6.2 6.9 8.2 9.1 7.4 8.2 6.2 5.5
AHR, Alcohol-based handrubs; CHX, chlorhexidine digluconate; ETH, ethanol; ICU, intensive care unit; ISO, isopropyl alcohol; OCT, octenidine chlorhydrate; MEC, mecetronium ethyl-sulphate; TRI, triclosan. *Mean score established from the evaluation by 10 participants (individual score ranging from 0, nonsatisfactory, to 3, highly satisfactory).
because of its effectiveness in removing from the hands of hospital personnel transient microbial flora acquired by contact with infected or colonized patients or with medical devices and environmental sources. This study sought to compare the antibacterial activity and acceptability of several AHRs in actual care situations to facilitate the choice of effective hand disinfection products that could reduce the risk of nosocomial infections in an ICU. The choice of AHRs for hand disinfection may be documented by a comparative study of their antibacterial activity both in vitro and in vivo, because results recorded in vitro cannot be extrapolated to estimate the in vivo activity of hand disinfection agents.24 The antibacterial activity of hand disinfection agents that agree with European standards19,25 is rarely evaluated against nosocomial bacterial strains exhibiting multiple resistance to antibiotics. However, some studies have demonstrated decreased activity of handdisinfecting agents on multiple-resistant bacteria (eg, in methicillin-resistant S aureus).26 Therefore the in vitro antibacterial activity of the various AHRs tested in the current work was assessed against 4 hospital multiple-resistant strains. The method proposed by Eliopoulos20 for counting surviving bacteria in the assessment of bactericidal activity of antibiotics was found well suited to the simultaneous study of various AHRs. The first step of the work characterizes a group of 3 products exhibiting major bactericidal activity ($4 log inoculum reduction) that contain either chlorhexidine digluconate (0.5% CHX, 0.5 % CHX–0.45 % HP) or octenidine chlorhydrate (0.1% OCT). Although AHRs are intended to be applied without dilution on dry skin, a range of AHR dilutions was introduced to better evaluate bactericidal activity and thus to differentiate among AHRs more accurately. For example, 0.5% CHX–0.45% HP and 0.1% OCT exhibited more than 4 log inoculum reduction after 1/100 dilution against A baumannii, S aureus, and E aerogenes. The difference between the highest dilution allowing a bactericidal
activity in vitro and actual use conditions allows a safety activity margin against hospital strains.27 The various AHRs were then tested in actual use conditions in a surgical ICU. The current evaluation allowed us to take into consideration the contamination of the nursing and medical staff hands, using a sampling method of finger pulp imprints. This fingerprinting method has been used in recent works studying AHRs.12,28 We also chose this method in the current work because it is easy to perform in a care service, unless other methods such as the EN 1500 standard25 or the glove-juice test,29 which are not adapted to an evaluation in care situation. A reduction of the bacterial flora higher than 1.4 log10 was observed in the in vivo study with 46% ETH, 0.5% CHX–0.45% HP, 0.1% OCT, and 0.5% CHX, these last 3 AHRs being the most active in the in vitro study; thus the in vitro and in vivo evaluations of antibacterial activities were fairly concordant. It is well-known that if studies involving a limited number of volunteers can assess the activity of a given product, they may be insufficient to reveal significant differences among various products, since the inter- and intra-individual variability remains a major problem in spite of standardization efforts.14,17 This part of the study allowed us also to evaluate the rate of recolonization of hands after use of AHRs: several AHRs exhibited a poorly remanent antibacterial effect, as revealed by bacterial counts from hands performed 5 minutes after friction. This information should be also taken into account in the choice of an AHR. Acceptance by the hospital personnel is an important criterion in the choice of an AHR, because it conditions compliance to hygienic hand disinfection. In the current work, combining results of both antibacterial activity and acceptance allowed a further discrimination of AHRs, especially among products exhibiting a similar antibacterial activity. Among the 3 in vitro most active AHRs, 1 product (0.1% OCT) was
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differentiated by a better acceptance. Alcohol-based hand gels have been proposed as alternative pharmaceutical formulations well suited to rapid and efficient hand disinfection. In our study, the 3 gels analyzed were found less active than the other formulations, as was already reported,30,31 and were poorly accepted by ICU personnel.
CONCLUSION This study provides information to help health care professionals choose effective AHRs that may reduce the risk of nosocomial infections. The combination of in vitro and in vivo data allowed for a good discrimination among the AHRs tested. A long period of observation would be required to assess the clinical consequences of the use of these solutions on the rate of nosocomial infections. A decrease in the rate of nosocomial infections and in the acquisition of multiple-resistant bacteria was noticed in a study that confirmed the efficiency of AHR use to improve the compliance rate of handwashing by health care workers.8,32 Because AHRs are efficient handwashing agents, they can be safely used in special situations—in the ICU, during periods of work overload, where access to ward sinks is difficult—and should be largely encouraged in various care units. The authors thank the nursing and medical staff of the surgical ICU of the Centre Hospitalier Universitaire de Rouen for participation in the in vivo study.
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