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Hygienic hand disinfection tests in three laboratories
Journal
of Hospital
Infection
Hygienic
(1990)
hand
16, 141-149
disinfection laboratories
tests in three
G. A. J. Ayliffe,* J. R. Babb,* J. G. Davies,* S. W. B. Newsom,f C. Rowland,? J. H. Platt$ and B. Masonj: *Hospital Infection Research Laboratory, Dudley Road Hospital, Birmingham, JfPapworth Hospital, Cambridge and TICI Pharmaceuticals, Cheshire Accepted for publication
26 April
1990
Summary: A comparative study was made in three laboratories of a test for hygienic hand disinfection. Staphylococcus aureus was applied to the fingertips of a total of 74 volunteers (49 female and 25 male) and the effect of washing with three chlorhexidine preparations and one non-medicated soap was assessed after one and five applications. Fingertip inoculation is convenient and is a realistic representation of the in-use situation. Although significant differences were obtained between log,, reductions in test organisms using the same formulation in different centres, and different periods in the same centre, the maximum differences after a single application of a preparation were small, e.g. between centres 0.39 and between periods in the same centre 0.55, and after multiple applications the maximum difference between centres was 0.42 and between periods in the same centre it was 0.51. The differences between preparations were similar in all centres. This test compares well with other similar tests and products can be placed in rank order of effectiveness. It is concluded that this test, if carried out under the controlled conditions described, is sufficiently reproducible between laboratories and repeatable within laboratories to be used as a standard test. Keywords
: Hand
disinfection;
tests;
standardization.
Introduction
Tests for hygienic hand disinfection, i.e. killing and removal of transient organisms, have become of increasing interest in recent years, but the relevance of these tests to practical hand disinfection remains uncertain. Nevertheless, such tests are useful for comparing the efficacy of different agents (Ayliffe et al., 1988). Much emphasis is currently placed on the development of internationally accepted tests, which could be performed by manufacturers and reference centres alike, when developing and selecting suitable agents for hand disinfection. (Ayliffe, 1989). Correspondence 019556701/90/060141+09
to: Professor
G. A. J. Ayliffe.
$03.00/O
0 1990 The Hospital
141
Infecrxon
Society
142
G. A. J. Ayliffe
et al.
One commonly used test was described by Rotter and his colleagues in Vienna (Rotter, Mittermayer & Kundi, 1974; Rotter, Koller & Kundi, 1977; Rotter, 1988), and this has been shown to be reasonably reproducible when carried out in three different test laboratories (Rotter et al., 1986). A similar test was described by the Birmingham Group (Ayliffe, Babb & Quoraishi, 1978) and modified versions of it have been widely used in the UK. The main differences between it and the Vienna test are that, in Birmingham, the test organism is rubbed onto the fingertips only, and the test formulation is applied for 30 s, whereas in the Vienna model, the hands are immersed to the mid-carpal joint in a culture of Escherichia coli, and the agents are applied for 1 min. An alcohol standard only is used in the Vienna test, whereas alcohol and soap and water are used as standards in the Birmingham test. A possible criticism of both tests is that E. coli is not the most appropriate organism because of its rapid death on drying, and that it is not representative of organisms commonly causing cross-infection in hospitals. Staphylococcus aweus (Ayliffe & Babb, 1979), Klebsiella aerogenes (Casewell, Law & Desai, 1988) and Serratia mat-cescens(Bartzokas, Corkhill & Makin, 1987) have been proposed as more suitable alternatives. In this study, Staphylococcus aureus was used as the test organism in a modified version of the Birmingham test. The immediate and cumulative effects of three chlorhexidine preparations and a non-medicated soap were measured in three laboratories. The repeatability within, and reproducibility between, laboratories was assessed.
Materials
and
methods
Subjects The number of subjects taking part in the trial was 30 in Centre 1, 24 in Centre 2 and 20 in Centre 3. They were in good health, with undamaged skin, and their ages ranged between 18 and 65 years. No antiseptics were used during the trial and none of the subjects was on antibiotic therapy. Test preparations These were as follows: A=4% chlorhexidine detergent (‘Hibiscrub’ ICI Pharmaceuticals, Macclesfield); B = trial product, foam containing 4% chlorhexidine; C = trial product, foam containing 2.5% chlorhexidine; D = non-medicated soap (Avona). Organism The test strain was Staphylococcus aureus NCTC 4163, which has been used in skin tests for many years without evidence of infection in volunteers. The organism was grown in 10 ml tryptone soya broth at 37°C for 18 h.
Hygienic
hand
disinfection
143
Test for baseline counts These were established during the first week of the trial. The hands of volunteers were contaminated with the bacterial culture as described below to determine the number of recoverable organisms. Tests were performed on two separate occasions with an interval of at least 48 h between them. Hands were washed with non-medicated soap for 15 s, rinsed and dried with paper towels, then approximately 5 ml of 70% ethanol was applied and rubbed over the hands until dry. This was done in an attempt to reduce the number of resident skin organisms. When the hands were completely dry, the palmar surfaces of the distal phalanges of one hand were each inoculated with 0.02 ml of the prepared culture of S. aureus and opposing fingers and thumbs rubbed together for 40 s and allowed to dry for 80 s. Fingers of each hand were than sampled in separate bowls for 1 min, by rubbing them vigorously on 50 ml of glass beads (3-5 mm diameter) in 100 ml neutralizer broth (nutrient broth containing 0.6% azolectin and 4% Tween 80). The hands were thoroughly disinfected with 70% ethanol after sampling. An aliquot of fluid was removed from each bowl and tenfold dilutions made in tryptone soya broth containing 0.75% azolectin and 5% Tween 80. Five 0.02 ml drops (0.1 ml) of each dilution and the undiluted sample were transferred to the surface of well-dried tryptone soya agar plates containing 0.3% azolectin and 2% Tween 80, and spread over the surface with either a standard glass spreader whiclh was sterilized by flaming after immersion in ethanol, or in one of the laboratories with a spiral plater. Each dilution and undiluted sample was plated in duplicate, as a precaution against error or contamination, thus giving two sets of plates from each hand. The plates were incubated at 37°C for 48 h to allow the golden pigment of the S. aureus to develop, so distinguishing them from the resident skin flora. Surviving test organisms were counted from the appropriate plate and the arithmetic mean of both hands was calculated and transposed to log,, values. This was the baseline or pre-treatment count from which the post-treatment count was subtracted. A viable count was also performed to establish the number of organisms present in the broth culture deposited on the hands. Subsequently, no pre-treatment counts were performed, since the number of organisms deposited on the fingertips during each test day was monitored from a viable count of the bacterial culture used. Volunteers were instructed in the handwashing technique during this period. Tests with the formulations These were randomly allocated to each volunteer, on a particular day, using a computer generated randomization scheme, such that each volunteer used each of the test formulations once only. The design was such that at least 48 h elapsed between each application to ensure that persistent effects from a prior formulation were not carried over. At the start of the study, single (immediate) or repeated (cumulative) washes were randomly allocated to
144
G. A. J. Ayliffe
et al.
the right or left hand of each volunteer, and thereafter the same hand was used for all test preparations. A chart was prepared to indicate the volunteers’ names, dates and times of arrival, scheme of products to be used and hand allocation. Immediate test The hands were washed with soap and water (product D) and treated with 70% ethanol as before. The test organisms were applied and dried as previously described, then after wetting the hands, the appropriate hand washing treatment was used as follows: 5 ml of preparation A was dispensed by a calibrated pump and the ‘standard handwash’ carried out twice in 1 min; 2.5 ml of preparations B and C, or enough soap (preparation D) to obtain a good lather, were applied and the ‘standard handwash’ carried out for 15 s. The hands were then rinsed under running water for 15 s and dried with paper towels. The ‘standard handwash’, which ensures total coverage of the hands, comprises of five strokes backwards and forwards; palm to palm, right palm over left dorsum, left palm over right dorsum, palm to palm with fingers interlaced, back of fingers to opposing palms with fingers interlocked, rotational rubbing of right thumb clasped in left palm and left thumb clasped in right palm, rotational rubbing with clasped fingers of right hand in palm of left hand and clasped fingers of left hand in palm of right hand. The hands and wrists are rubbed in this way until the end of the appropriate time period. The hands were then rinsed under running water for 15 s and dried with paper towels. When the first treatment was completed, the designated hand was sampled for 1 min in 100 ml recovery broth with glass beads as described previously, rinsed with water to remove any neutralizer and dried with paper towels by an assistant. Cumulative test Five minutes after immediate sampling, the fingertips were recontaminated, washed and dried as before, and this process was repeated until five procedures had been completed. At the end of the five treatments the other hand was sampled for 1 min in 100 ml recovery broth as before. Hands of volunteers were thoroughly disinfected with 70% ethanol at the end of the procedure. Aliquots of both samples were diluted and plated in duplicate as previously described. The mean of the counts obtained from the duplicate plates were used to calculate the log,, colony forming units (cfu) per ml of the sampling fluid. This calculation was made by taking the arithmetical mean of the duplicate plates and, after making the necessary allowance for the step in the serial dilution counted, the figure was transposed to a log,, value. If no cfu were recovered, even from the undiluted samples, then the value of 0.6 log,, cfu ml-’ was given. Where one of a set of duplicate plates
Hygienic
hand
disinfection
was uncountable for any reason, then the count from was used in the calculations. The mean log,, reduction calculated for each product.
145
the remaining plate in test bacteria was
Statistical evaluation In this study it was intended that product A (‘Hibiscrub’) should be compared with B (4% foamed chlorhexidine), and product C (2% foamed chlorhexidine) with D (non-Imedicated soap). It was estimated that at 90% power and 5% significance,, a minimum of (i) 24 volunteers would be required to detect a difference of 0.3 logi, cfu per hand between products A and B and (ii) 16 volunteers would be required to detect a difference of 0.35 log,, cfu per hand between products C and D. Analysis of variance methods were applied to the immediate and cumulative antimicrobial effects separately. Factors for group (treatment order), centre, volunteers within groups, visit and treatment were included in the analysis. Tests were also made for carryover effects (residual treatment from the previous visit) and the interactions of treatment with centre, period with centre, group with centre, and treatment with period. Statistical significance was assessed at the 5% level.
Results The number of volunteers participating in the study, their mean age (range) and the sex ratio (M/F) was as follows: Centre 1, 30, 35 years (21-SO), 13/l 7; Centre 2, 24, 34 years (19%SS), 4/20; Centre 3, 20, 36 years (1%62), 7/l 3. The mean baseline log counts were 5.59 (N= 148) for the left hand and 5.51 (N= 148) for the right ‘hand and mean baseline log counts from the three centres were 5.59 (120 tests) for Centre 1, 5.24 (96 tests) for Centre 2 and 5.71 (80 tests) for Centre 3. An analysis of variance showed a significant difference between centres but not between hands. Immediate efSect of disinfection or washing The mean log reduction for each treatment in the centres is shown in Table I. In the results from one volunteer in centre 1, period 4 was an outlier and was omitted from the analysis of variance. No evidence of a carryover effect was detected. Differences between centres were significant but were not consistent over the four periods (Table II). However, comparisons of treatment by centre and period by centre were not significantly different, allowing comparisons to be made between the products. The overall mean log reduc.tions for the four preparations were as follows: A = 2.66, B = 2.24, C = 2.09 and D = 2-05. Preparation A was significantly more effective than the other products (P= O.OOOl), preparation B was more effective than D (P= 0.004) but C was not more effective than D (P= 0.58).
146
G. A. J. Ayliffe
et al.
Cumulative effects The mean treatment reductions for each preparation obtained at each centre are shown in Table I I I. The results show a greater microbial reduction than obtained in the immediate test apart from that of preparation D (non-medicated soap). There was no evidence of carryover between tests. The statistical associations were generally similar to those obtained in the immediate test. There was again a lack of consistency between the centres (Table IV) but the treatment by centre interaction was just significant, although the rank order remained similar in the three centres. All preparations were significantly more effective than soap, but in one centre, preparation A was not significantly more effective than B.
Discussion
The value of a disinfectant test to the manufacturer and to the user depends on its safety, simplicity, cost, repeatability within laboratories and reproducibility between laboratories, and on its relevance to the clinical situation. The test described showed similar results to the Vienna test model but S. aweus was used as the test organism instead of E. coEi. Although statistically significant differences were obtained with the results between centres and in different periods in the same centre, the mean differences were low. In the immediate test the maximum difference in mean counts between centres was log 0.39 and log 0.42 in the cumulative test. The maximum differences between the mean reduction in different periods in the same centre was 0.55 in the immediate test and 0.51 in the cumulative test. The differences between preparations were similar in the three centres apart from a failure in one centre to show a significant difference between preparations A and B in the cumulative test. The test carried out under the conditions described would seem to be sufficiently reproducible to be accepted as a standard test. On the basis of these results a difference of log 0.5-0.6 would probably be necessary to demonstrate a statistically significant difference in activity between products. It is important that the details of the test are accurately followed. The individual volunteers can have a major influence on the results and it would be preferable to use the same volunteers for testing each product. Sufficient numbers are required to obtain a meaningful result and a control test should be carried out at the same time (Ayliffe et al., 1988; Rotter, 1988). The use of S. aweus rather than E. coli did not seem to have a major influence on the test results, although a direct comparison was not made. In a previous study preparation A gave an immediate log reduction with E. coli of 2.9 compared to a log reduction of 2.7 in this stud.y (Ayliffe et al., 1988). Some care is necessary to ensure that S. aweus is not present as part of the
Hygienic Table
I. Mean
log reductions
hand
in microbial counts centres-immediate
disinfection
147
following test
four
dzyerent
treatments
in three
Centre
Preparation A B C D
1
2
3
(N*=30)
(N=24)
(N=20)
2.66 2.38 2.11 2.30 *N=
Table
Number
II.
Mean
All
(N=74)
2.59 2.30 2.00 1.97
2.66 1.99 2.09 1.91
centres
2.66 2.24 2.09 2.05
of volunteers.
log reductions
in microbial
counts in three test
in thefour
centres
periods-immediate
Period Centre 1 2 3 *N=
Table
Number
III.
Mean
1
2
3
4
All
2.26 2.27 2.25
2.28 1.85 2.20
2.27 2.11 2.21
2.53 2.40 2.19
periods 2.34 2.16 2.21
(N*)
(119) (96) (80)
of volunteers.
log reductions
in microbial counts centres-cumulative
following test
four
dagerent
treatments
in three
Centre
Preparation
1
2
3
(N*=30)
(N=24)
(N=20)
All
centres
(N=74)
A :
3.63 3.13 3.35
3.87 3.20 3.40
3.99 2.97 3.48
3.81 3.12 3.42
D
1.97
1.55
1.66
1.76
*N=
Table
Number
IV.
Mean
of volunteers.
log microbial
reductions
in three
centres
in the four
periods-cumulative
test
Period Centre 1 2 3 *N=
Xumber
of volunteers.
1
2
3
4
2.99 3.23 2.76
2.92 2.98 2.91
3.04 2.86 3.17
3.13 2.94 3.27
All
periods 3.02 3.00 3.03
(120) (96) (80)
(N*)
148
G. A. J. Ayliffe
et al.
resident flora on the hands of volunteers. This problem was not found in the study and the test strain of S. aureu~ showed good specificity. However, care is necessary in the choice of ‘test organism’ since they may vary in sensitivity to individual disinfectants, e.g. chlorhexidine detergent gave a log reduction factor of 3.8 with S. saprophyticus as the test organism (Ayliffe & Babb, 1979). It may also be necessary to include one or more viruses in the test. Variation in sensitivity to disinfectants may be a greater problem as some viruses are relatively resistant to alcohols (Kurtz, Lee & Parsons, 1980; Tyler, Ayliffe & Bradley, 1990). Alcohol as the sole standard would therefore be inappropriate. The selection of suitable viruses, which are safe for application to the skin presents a further difficulty. The clinical relevance of the immediate test has been discussed elsewhere (Ayliffe et al., 1988; Ayliffe, 1989), but the test is clearly useful for ranking agents in order of efficacy. The cumulative test after five washes showed a log reduction of 1.O greater than obtained in the immediate test. This may have some clinical relevance, particularly in specific instances, e.g. during outbreaks of staphylococcal infection. The results of the present test compare well with the Vienna test and may be preferred for all or some of the following reasons: (a) the fingertips only are inoculated with test organisms in the Birmingham test as they are convenient to sample and are the areas most likely to become contaminated with transient micro-organisms; (b) unlike whole hand immersion, fingertip inoculation is less likely to contaminate the surrounding environment and reduces the likelihood of accidental infection through broken or damaged areas of the skin; (c) this test can be used to assessthe cumulative as well as the immediate effect of antiseptics, whereas other tests may only assessthe immediate effect. Unlike alcoholic formations which destroy microorganisms present on the skin, aqueous formulations also remove them. The handwash procedure has therefore two components; physical remova& whereby transient and resident flora are removed, and an antimicrobial component whereby surviving bacteria are destroyed or reduced. It would therefore seem appropriate that physical removal, i.e. with unmedicated soap and water, would be an appropriate standard for assessing the antimicrobial effect. A plain alcohol standard would be more appropriate for assessing alcohol hand rubs, especially those with other antimicrobial additives such as chlorhexidine, povidone-iodine or triclosan. However, it may be reasonable to include both soap and water, and an alcohol preparation as standards in a routine test. In this study, S. aUreUSwas used as the test organism. It occurs frequently as a transient, survives well on the skin and multiply resistant strains constitute a major cross-infection problem. It would therefore appear a wise choice of organism, provided that an avirulent and easily distinguishable strain is available. The necessity for a realistic inoculum size was discussed
Hygienic
hand
disinfection
149
by Casewell, Law & Desai (1988). In a recent study (Ayliffe et al., 1988) S. auYeuS was recovered from 29.4% of nurses’ hands in a general hospital, with a median count of 3-8 x 1O3 and a maximum count of 2.4 x 107, and from 78.3 % of nurses in a skin hospital. The results obtained in the present study suggest that the baseline mean log count of 5.59 is, therefore, an appropriate challenge. It is also advisable to use both S. aUreUS and E. coli as test organisms, but further comparative studies are required. This test relates well to the clinical situation, and is repeatable, reproducible, safe and simple to use. References Ayliffe,
G. A. J. (1989). Standardization of disinfectant testing. Journal of Hospital Infection 13, 211-216. Ayliffe, G. A. J. & Babb, J. R. (1979). Hygienic hand disinfection-selection of test organisms. Hospital-Hygiene Sonderheft, September 1979, 3741. A. H. (1978). A test for hygienic hand disinfection. Ayliffe, G. A. J., Babb, J. R. & Q uoraishi, Journal of Clinical Pathology 311, 923-928. Ayliffe, G. A. J., Babb, J. R., Davies, J. G. & Lilly, H. A. (1988). Hand disinfection: A comparison of various agents in laboratory and ward studies. Journal of Hospital Infection 11, 226-243. Bartzokas, C. A., Corkill, J. E. & Makin, I. (1987). Evaluation of the skin disinfectant activity and cumulative effect of chlorhexidine and triclosan handwash preparations on hands artificially contaminated with Serratia marcescens. Infection Control 8, 163-l 68. Casewell, M. W., Law, M. M. & Desai, N. (1988). A laboratory model for testing agents for hygienic hand disinfection: handwashing and chlorhexidine for the removal of Klebsiella. Journal of Hospital Infection 12, 163-175. of alcohols on rotavirus, Kurtz, J. B., Lee, T. W. & Parsons, A. J. (1980). Th e action astrovirus and enterovirus. Journal of Hospital Infection 1, 321-325. Rotter, M. (1988). Are models useful for testing hand disinfection? Journal of Hospital Infection 11 (Suppl. A), 236-243. Rotter, M., Keller, W. & Kuntdi, M. (1977). Eignung dreier alkohole fur eine Standard-Desinfektionsmethode in der Wertbestimmung von Verfahren fur die Hygienische Hiindesinfektion. Zentrablatt fiir Bakteriologie und Hygiene (I. Abteilung Originale Reihe B) 164, 428438. Rotter, M., Mittermayer, H. & Kundi, M. (1974). Untersuchungen zum model1 der kuntslich kontaminierten Hand-Vorschlag fiir eine Priifmethode. Zentralblatt ftir Bakteriologie und Hygiene (I. Abteilung Originale Reihe B) 159, 580-581. Rotter, M., Koller, W., Wewalka, G., Werner, H. P., Ayliffe, G. A. J. & Babb, J. R. (1986). Evaluation of procedures for hygienic hand-disinfection, controlled parallel experiments on the Vienna test model. Journal of Hygiene (Cambridge) 96, 27-37. Tyler, R., Ayliffe, G. A. J. & Bradley, C. R. (1990). Virucidal activity of disinfectants: studies with the poliovirus. Journal of Hospitl Infection 15, 339-345.
of Hospital
Infection
Hygienic
(1990)
hand
16, 141-149
disinfection laboratories
tests in three
G. A. J. Ayliffe,* J. R. Babb,* J. G. Davies,* S. W. B. Newsom,f C. Rowland,? J. H. Platt$ and B. Masonj: *Hospital Infection Research Laboratory, Dudley Road Hospital, Birmingham, JfPapworth Hospital, Cambridge and TICI Pharmaceuticals, Cheshire Accepted for publication
26 April
1990
Summary: A comparative study was made in three laboratories of a test for hygienic hand disinfection. Staphylococcus aureus was applied to the fingertips of a total of 74 volunteers (49 female and 25 male) and the effect of washing with three chlorhexidine preparations and one non-medicated soap was assessed after one and five applications. Fingertip inoculation is convenient and is a realistic representation of the in-use situation. Although significant differences were obtained between log,, reductions in test organisms using the same formulation in different centres, and different periods in the same centre, the maximum differences after a single application of a preparation were small, e.g. between centres 0.39 and between periods in the same centre 0.55, and after multiple applications the maximum difference between centres was 0.42 and between periods in the same centre it was 0.51. The differences between preparations were similar in all centres. This test compares well with other similar tests and products can be placed in rank order of effectiveness. It is concluded that this test, if carried out under the controlled conditions described, is sufficiently reproducible between laboratories and repeatable within laboratories to be used as a standard test. Keywords
: Hand
disinfection;
tests;
standardization.
Introduction
Tests for hygienic hand disinfection, i.e. killing and removal of transient organisms, have become of increasing interest in recent years, but the relevance of these tests to practical hand disinfection remains uncertain. Nevertheless, such tests are useful for comparing the efficacy of different agents (Ayliffe et al., 1988). Much emphasis is currently placed on the development of internationally accepted tests, which could be performed by manufacturers and reference centres alike, when developing and selecting suitable agents for hand disinfection. (Ayliffe, 1989). Correspondence 019556701/90/060141+09
to: Professor
G. A. J. Ayliffe.
$03.00/O
0 1990 The Hospital
141
Infecrxon
Society
142
G. A. J. Ayliffe
et al.
One commonly used test was described by Rotter and his colleagues in Vienna (Rotter, Mittermayer & Kundi, 1974; Rotter, Koller & Kundi, 1977; Rotter, 1988), and this has been shown to be reasonably reproducible when carried out in three different test laboratories (Rotter et al., 1986). A similar test was described by the Birmingham Group (Ayliffe, Babb & Quoraishi, 1978) and modified versions of it have been widely used in the UK. The main differences between it and the Vienna test are that, in Birmingham, the test organism is rubbed onto the fingertips only, and the test formulation is applied for 30 s, whereas in the Vienna model, the hands are immersed to the mid-carpal joint in a culture of Escherichia coli, and the agents are applied for 1 min. An alcohol standard only is used in the Vienna test, whereas alcohol and soap and water are used as standards in the Birmingham test. A possible criticism of both tests is that E. coli is not the most appropriate organism because of its rapid death on drying, and that it is not representative of organisms commonly causing cross-infection in hospitals. Staphylococcus aweus (Ayliffe & Babb, 1979), Klebsiella aerogenes (Casewell, Law & Desai, 1988) and Serratia mat-cescens(Bartzokas, Corkhill & Makin, 1987) have been proposed as more suitable alternatives. In this study, Staphylococcus aureus was used as the test organism in a modified version of the Birmingham test. The immediate and cumulative effects of three chlorhexidine preparations and a non-medicated soap were measured in three laboratories. The repeatability within, and reproducibility between, laboratories was assessed.
Materials
and
methods
Subjects The number of subjects taking part in the trial was 30 in Centre 1, 24 in Centre 2 and 20 in Centre 3. They were in good health, with undamaged skin, and their ages ranged between 18 and 65 years. No antiseptics were used during the trial and none of the subjects was on antibiotic therapy. Test preparations These were as follows: A=4% chlorhexidine detergent (‘Hibiscrub’ ICI Pharmaceuticals, Macclesfield); B = trial product, foam containing 4% chlorhexidine; C = trial product, foam containing 2.5% chlorhexidine; D = non-medicated soap (Avona). Organism The test strain was Staphylococcus aureus NCTC 4163, which has been used in skin tests for many years without evidence of infection in volunteers. The organism was grown in 10 ml tryptone soya broth at 37°C for 18 h.
Hygienic
hand
disinfection
143
Test for baseline counts These were established during the first week of the trial. The hands of volunteers were contaminated with the bacterial culture as described below to determine the number of recoverable organisms. Tests were performed on two separate occasions with an interval of at least 48 h between them. Hands were washed with non-medicated soap for 15 s, rinsed and dried with paper towels, then approximately 5 ml of 70% ethanol was applied and rubbed over the hands until dry. This was done in an attempt to reduce the number of resident skin organisms. When the hands were completely dry, the palmar surfaces of the distal phalanges of one hand were each inoculated with 0.02 ml of the prepared culture of S. aureus and opposing fingers and thumbs rubbed together for 40 s and allowed to dry for 80 s. Fingers of each hand were than sampled in separate bowls for 1 min, by rubbing them vigorously on 50 ml of glass beads (3-5 mm diameter) in 100 ml neutralizer broth (nutrient broth containing 0.6% azolectin and 4% Tween 80). The hands were thoroughly disinfected with 70% ethanol after sampling. An aliquot of fluid was removed from each bowl and tenfold dilutions made in tryptone soya broth containing 0.75% azolectin and 5% Tween 80. Five 0.02 ml drops (0.1 ml) of each dilution and the undiluted sample were transferred to the surface of well-dried tryptone soya agar plates containing 0.3% azolectin and 2% Tween 80, and spread over the surface with either a standard glass spreader whiclh was sterilized by flaming after immersion in ethanol, or in one of the laboratories with a spiral plater. Each dilution and undiluted sample was plated in duplicate, as a precaution against error or contamination, thus giving two sets of plates from each hand. The plates were incubated at 37°C for 48 h to allow the golden pigment of the S. aureus to develop, so distinguishing them from the resident skin flora. Surviving test organisms were counted from the appropriate plate and the arithmetic mean of both hands was calculated and transposed to log,, values. This was the baseline or pre-treatment count from which the post-treatment count was subtracted. A viable count was also performed to establish the number of organisms present in the broth culture deposited on the hands. Subsequently, no pre-treatment counts were performed, since the number of organisms deposited on the fingertips during each test day was monitored from a viable count of the bacterial culture used. Volunteers were instructed in the handwashing technique during this period. Tests with the formulations These were randomly allocated to each volunteer, on a particular day, using a computer generated randomization scheme, such that each volunteer used each of the test formulations once only. The design was such that at least 48 h elapsed between each application to ensure that persistent effects from a prior formulation were not carried over. At the start of the study, single (immediate) or repeated (cumulative) washes were randomly allocated to
144
G. A. J. Ayliffe
et al.
the right or left hand of each volunteer, and thereafter the same hand was used for all test preparations. A chart was prepared to indicate the volunteers’ names, dates and times of arrival, scheme of products to be used and hand allocation. Immediate test The hands were washed with soap and water (product D) and treated with 70% ethanol as before. The test organisms were applied and dried as previously described, then after wetting the hands, the appropriate hand washing treatment was used as follows: 5 ml of preparation A was dispensed by a calibrated pump and the ‘standard handwash’ carried out twice in 1 min; 2.5 ml of preparations B and C, or enough soap (preparation D) to obtain a good lather, were applied and the ‘standard handwash’ carried out for 15 s. The hands were then rinsed under running water for 15 s and dried with paper towels. The ‘standard handwash’, which ensures total coverage of the hands, comprises of five strokes backwards and forwards; palm to palm, right palm over left dorsum, left palm over right dorsum, palm to palm with fingers interlaced, back of fingers to opposing palms with fingers interlocked, rotational rubbing of right thumb clasped in left palm and left thumb clasped in right palm, rotational rubbing with clasped fingers of right hand in palm of left hand and clasped fingers of left hand in palm of right hand. The hands and wrists are rubbed in this way until the end of the appropriate time period. The hands were then rinsed under running water for 15 s and dried with paper towels. When the first treatment was completed, the designated hand was sampled for 1 min in 100 ml recovery broth with glass beads as described previously, rinsed with water to remove any neutralizer and dried with paper towels by an assistant. Cumulative test Five minutes after immediate sampling, the fingertips were recontaminated, washed and dried as before, and this process was repeated until five procedures had been completed. At the end of the five treatments the other hand was sampled for 1 min in 100 ml recovery broth as before. Hands of volunteers were thoroughly disinfected with 70% ethanol at the end of the procedure. Aliquots of both samples were diluted and plated in duplicate as previously described. The mean of the counts obtained from the duplicate plates were used to calculate the log,, colony forming units (cfu) per ml of the sampling fluid. This calculation was made by taking the arithmetical mean of the duplicate plates and, after making the necessary allowance for the step in the serial dilution counted, the figure was transposed to a log,, value. If no cfu were recovered, even from the undiluted samples, then the value of 0.6 log,, cfu ml-’ was given. Where one of a set of duplicate plates
Hygienic
hand
disinfection
was uncountable for any reason, then the count from was used in the calculations. The mean log,, reduction calculated for each product.
145
the remaining plate in test bacteria was
Statistical evaluation In this study it was intended that product A (‘Hibiscrub’) should be compared with B (4% foamed chlorhexidine), and product C (2% foamed chlorhexidine) with D (non-Imedicated soap). It was estimated that at 90% power and 5% significance,, a minimum of (i) 24 volunteers would be required to detect a difference of 0.3 logi, cfu per hand between products A and B and (ii) 16 volunteers would be required to detect a difference of 0.35 log,, cfu per hand between products C and D. Analysis of variance methods were applied to the immediate and cumulative antimicrobial effects separately. Factors for group (treatment order), centre, volunteers within groups, visit and treatment were included in the analysis. Tests were also made for carryover effects (residual treatment from the previous visit) and the interactions of treatment with centre, period with centre, group with centre, and treatment with period. Statistical significance was assessed at the 5% level.
Results The number of volunteers participating in the study, their mean age (range) and the sex ratio (M/F) was as follows: Centre 1, 30, 35 years (21-SO), 13/l 7; Centre 2, 24, 34 years (19%SS), 4/20; Centre 3, 20, 36 years (1%62), 7/l 3. The mean baseline log counts were 5.59 (N= 148) for the left hand and 5.51 (N= 148) for the right ‘hand and mean baseline log counts from the three centres were 5.59 (120 tests) for Centre 1, 5.24 (96 tests) for Centre 2 and 5.71 (80 tests) for Centre 3. An analysis of variance showed a significant difference between centres but not between hands. Immediate efSect of disinfection or washing The mean log reduction for each treatment in the centres is shown in Table I. In the results from one volunteer in centre 1, period 4 was an outlier and was omitted from the analysis of variance. No evidence of a carryover effect was detected. Differences between centres were significant but were not consistent over the four periods (Table II). However, comparisons of treatment by centre and period by centre were not significantly different, allowing comparisons to be made between the products. The overall mean log reduc.tions for the four preparations were as follows: A = 2.66, B = 2.24, C = 2.09 and D = 2-05. Preparation A was significantly more effective than the other products (P= O.OOOl), preparation B was more effective than D (P= 0.004) but C was not more effective than D (P= 0.58).
146
G. A. J. Ayliffe
et al.
Cumulative effects The mean treatment reductions for each preparation obtained at each centre are shown in Table I I I. The results show a greater microbial reduction than obtained in the immediate test apart from that of preparation D (non-medicated soap). There was no evidence of carryover between tests. The statistical associations were generally similar to those obtained in the immediate test. There was again a lack of consistency between the centres (Table IV) but the treatment by centre interaction was just significant, although the rank order remained similar in the three centres. All preparations were significantly more effective than soap, but in one centre, preparation A was not significantly more effective than B.
Discussion
The value of a disinfectant test to the manufacturer and to the user depends on its safety, simplicity, cost, repeatability within laboratories and reproducibility between laboratories, and on its relevance to the clinical situation. The test described showed similar results to the Vienna test model but S. aweus was used as the test organism instead of E. coEi. Although statistically significant differences were obtained with the results between centres and in different periods in the same centre, the mean differences were low. In the immediate test the maximum difference in mean counts between centres was log 0.39 and log 0.42 in the cumulative test. The maximum differences between the mean reduction in different periods in the same centre was 0.55 in the immediate test and 0.51 in the cumulative test. The differences between preparations were similar in the three centres apart from a failure in one centre to show a significant difference between preparations A and B in the cumulative test. The test carried out under the conditions described would seem to be sufficiently reproducible to be accepted as a standard test. On the basis of these results a difference of log 0.5-0.6 would probably be necessary to demonstrate a statistically significant difference in activity between products. It is important that the details of the test are accurately followed. The individual volunteers can have a major influence on the results and it would be preferable to use the same volunteers for testing each product. Sufficient numbers are required to obtain a meaningful result and a control test should be carried out at the same time (Ayliffe et al., 1988; Rotter, 1988). The use of S. aweus rather than E. coli did not seem to have a major influence on the test results, although a direct comparison was not made. In a previous study preparation A gave an immediate log reduction with E. coli of 2.9 compared to a log reduction of 2.7 in this stud.y (Ayliffe et al., 1988). Some care is necessary to ensure that S. aweus is not present as part of the
Hygienic Table
I. Mean
log reductions
hand
in microbial counts centres-immediate
disinfection
147
following test
four
dzyerent
treatments
in three
Centre
Preparation A B C D
1
2
3
(N*=30)
(N=24)
(N=20)
2.66 2.38 2.11 2.30 *N=
Table
Number
II.
Mean
All
(N=74)
2.59 2.30 2.00 1.97
2.66 1.99 2.09 1.91
centres
2.66 2.24 2.09 2.05
of volunteers.
log reductions
in microbial
counts in three test
in thefour
centres
periods-immediate
Period Centre 1 2 3 *N=
Table
Number
III.
Mean
1
2
3
4
All
2.26 2.27 2.25
2.28 1.85 2.20
2.27 2.11 2.21
2.53 2.40 2.19
periods 2.34 2.16 2.21
(N*)
(119) (96) (80)
of volunteers.
log reductions
in microbial counts centres-cumulative
following test
four
dagerent
treatments
in three
Centre
Preparation
1
2
3
(N*=30)
(N=24)
(N=20)
All
centres
(N=74)
A :
3.63 3.13 3.35
3.87 3.20 3.40
3.99 2.97 3.48
3.81 3.12 3.42
D
1.97
1.55
1.66
1.76
*N=
Table
Number
IV.
Mean
of volunteers.
log microbial
reductions
in three
centres
in the four
periods-cumulative
test
Period Centre 1 2 3 *N=
Xumber
of volunteers.
1
2
3
4
2.99 3.23 2.76
2.92 2.98 2.91
3.04 2.86 3.17
3.13 2.94 3.27
All
periods 3.02 3.00 3.03
(120) (96) (80)
(N*)
148
G. A. J. Ayliffe
et al.
resident flora on the hands of volunteers. This problem was not found in the study and the test strain of S. aureu~ showed good specificity. However, care is necessary in the choice of ‘test organism’ since they may vary in sensitivity to individual disinfectants, e.g. chlorhexidine detergent gave a log reduction factor of 3.8 with S. saprophyticus as the test organism (Ayliffe & Babb, 1979). It may also be necessary to include one or more viruses in the test. Variation in sensitivity to disinfectants may be a greater problem as some viruses are relatively resistant to alcohols (Kurtz, Lee & Parsons, 1980; Tyler, Ayliffe & Bradley, 1990). Alcohol as the sole standard would therefore be inappropriate. The selection of suitable viruses, which are safe for application to the skin presents a further difficulty. The clinical relevance of the immediate test has been discussed elsewhere (Ayliffe et al., 1988; Ayliffe, 1989), but the test is clearly useful for ranking agents in order of efficacy. The cumulative test after five washes showed a log reduction of 1.O greater than obtained in the immediate test. This may have some clinical relevance, particularly in specific instances, e.g. during outbreaks of staphylococcal infection. The results of the present test compare well with the Vienna test and may be preferred for all or some of the following reasons: (a) the fingertips only are inoculated with test organisms in the Birmingham test as they are convenient to sample and are the areas most likely to become contaminated with transient micro-organisms; (b) unlike whole hand immersion, fingertip inoculation is less likely to contaminate the surrounding environment and reduces the likelihood of accidental infection through broken or damaged areas of the skin; (c) this test can be used to assessthe cumulative as well as the immediate effect of antiseptics, whereas other tests may only assessthe immediate effect. Unlike alcoholic formations which destroy microorganisms present on the skin, aqueous formulations also remove them. The handwash procedure has therefore two components; physical remova& whereby transient and resident flora are removed, and an antimicrobial component whereby surviving bacteria are destroyed or reduced. It would therefore seem appropriate that physical removal, i.e. with unmedicated soap and water, would be an appropriate standard for assessing the antimicrobial effect. A plain alcohol standard would be more appropriate for assessing alcohol hand rubs, especially those with other antimicrobial additives such as chlorhexidine, povidone-iodine or triclosan. However, it may be reasonable to include both soap and water, and an alcohol preparation as standards in a routine test. In this study, S. aUreUSwas used as the test organism. It occurs frequently as a transient, survives well on the skin and multiply resistant strains constitute a major cross-infection problem. It would therefore appear a wise choice of organism, provided that an avirulent and easily distinguishable strain is available. The necessity for a realistic inoculum size was discussed
Hygienic
hand
disinfection
149
by Casewell, Law & Desai (1988). In a recent study (Ayliffe et al., 1988) S. auYeuS was recovered from 29.4% of nurses’ hands in a general hospital, with a median count of 3-8 x 1O3 and a maximum count of 2.4 x 107, and from 78.3 % of nurses in a skin hospital. The results obtained in the present study suggest that the baseline mean log count of 5.59 is, therefore, an appropriate challenge. It is also advisable to use both S. aUreUS and E. coli as test organisms, but further comparative studies are required. This test relates well to the clinical situation, and is repeatable, reproducible, safe and simple to use. References Ayliffe,
G. A. J. (1989). Standardization of disinfectant testing. Journal of Hospital Infection 13, 211-216. Ayliffe, G. A. J. & Babb, J. R. (1979). Hygienic hand disinfection-selection of test organisms. Hospital-Hygiene Sonderheft, September 1979, 3741. A. H. (1978). A test for hygienic hand disinfection. Ayliffe, G. A. J., Babb, J. R. & Q uoraishi, Journal of Clinical Pathology 311, 923-928. Ayliffe, G. A. J., Babb, J. R., Davies, J. G. & Lilly, H. A. (1988). Hand disinfection: A comparison of various agents in laboratory and ward studies. Journal of Hospital Infection 11, 226-243. Bartzokas, C. A., Corkill, J. E. & Makin, I. (1987). Evaluation of the skin disinfectant activity and cumulative effect of chlorhexidine and triclosan handwash preparations on hands artificially contaminated with Serratia marcescens. Infection Control 8, 163-l 68. Casewell, M. W., Law, M. M. & Desai, N. (1988). A laboratory model for testing agents for hygienic hand disinfection: handwashing and chlorhexidine for the removal of Klebsiella. Journal of Hospital Infection 12, 163-175. of alcohols on rotavirus, Kurtz, J. B., Lee, T. W. & Parsons, A. J. (1980). Th e action astrovirus and enterovirus. Journal of Hospital Infection 1, 321-325. Rotter, M. (1988). Are models useful for testing hand disinfection? Journal of Hospital Infection 11 (Suppl. A), 236-243. Rotter, M., Keller, W. & Kuntdi, M. (1977). Eignung dreier alkohole fur eine Standard-Desinfektionsmethode in der Wertbestimmung von Verfahren fur die Hygienische Hiindesinfektion. Zentrablatt fiir Bakteriologie und Hygiene (I. Abteilung Originale Reihe B) 164, 428438. Rotter, M., Mittermayer, H. & Kundi, M. (1974). Untersuchungen zum model1 der kuntslich kontaminierten Hand-Vorschlag fiir eine Priifmethode. Zentralblatt ftir Bakteriologie und Hygiene (I. Abteilung Originale Reihe B) 159, 580-581. Rotter, M., Koller, W., Wewalka, G., Werner, H. P., Ayliffe, G. A. J. & Babb, J. R. (1986). Evaluation of procedures for hygienic hand-disinfection, controlled parallel experiments on the Vienna test model. Journal of Hygiene (Cambridge) 96, 27-37. Tyler, R., Ayliffe, G. A. J. & Bradley, C. R. (1990). Virucidal activity of disinfectants: studies with the poliovirus. Journal of Hospitl Infection 15, 339-345.