Influence of Applied Volume on Efficacy of 3-Minute Surgical Reference Disinfection Method prEN 12791

For assessment of the efficacy of surgical hand disinfection,European reference method prEN 12791 prescribes that the handsmust be kept wet with the reference alcohol for 3 min regardlessof the applied volume. The aim of this study was to determinewhether the applied volume of the reference disinfectant n-propanol(60%, vol/vol) influences the effect on the resident hand flora.Ten experiments with 200 reference disinfections were analyzed.Hands were washed for 1 min with soap. The bacterial prevaluewas obtained by rubbing fingertips in tryptic soy broth for1 min. After this, each subject treated the hands with n-propanol(60%, vol/vol) by using as many portions as necessary to keephands wet for a total of 3 min. Bacterial postvalues (immediateeffect) were obtained for one hand, and the other hand was glovedfor 3 h. After the gloves were taken off, a second postvaluewas obtained (sustained effect). Most surgical reference disinfections(73%) were achieved with 9 ml of the reference alcohol, followedby 12 ml (24%) and 6 ml (3%). There was no significant differencebetween the mean log₁₀ reduction values for the three treatmentgroups, both in terms of the immediate effect (P = 0.333, asdetermined by analysis of variance) and in terms of the sustainedeffect (P = 0.442). A higher number of portions did not correlatewith a higher reduction factor (for immediate effect, Pearson'scorrelation coefficient = –0.028 [P = 0.689]; for sustainedeffect, Pearson's correlation coefficient = 0.059 [P = 0.404]).If the hands were kept wet with the reference alcohol for thetotal application time, the applied volume could vary, but thisdid not alter the efficacy.

INTRODUCTION

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Introduction

With the publication of the new Centers for Disease Controland Prevention guideline on hand hygiene, new interest on thistopic has emerged worldwide. For postcontamination treatmentof hands a clear recommendation was given in favor of alcohol-basedhand rubs (3). For preoperative treatment of hands, however,no clear recommendation was provided in the United States guideline(3), although remarkable differences in the effects on residenthand bacteria were described for different agents (6). In Europe,however, alcohol-based hand rubs are thought to have betterefficacy and to result in better dermal tolerance and are thereforepreferred to antimicrobial soaps (15). There are two principaloptions for preoperative treatment of hands: use of an alcohol-basedhand rub (surgical hand disinfection) or use of an antimicrobialliquid soap (surgical hand scrub). Evaluation of the efficacyof preparations for surgical hand antisepsis is based on differenttest methods. In the United States, in 1994 a method was publishedwhich was designed to test preparations for surgical hand scrubbingbut which is difficult to apply to hand rub formulations (1).No reference treatment is included in the test method. In theEuropean reference test method prEN 12791 (4), a preparation,regardless of its type (hand rub or hand scrub), is always comparedto a reference treatment, which is the application of a variablenumber of 3-ml portions of 60% (vol/vol) n-propanol for at least3 min (4). The hands must be kept wet with the alcohol for thetotal application time, but a specific volume is not recommendedand the volume may vary from 6 to 12 ml or even more (4). Thistreatment was chosen as the treatment with the greatest effecton resident skin bacteria (18). The test principle of includinga reference treatment has been used successfully in variousstudies in order to evaluate the comparative efficacies of variouspreparations for surgical hand disinfection (5, 8, 16). Thatis why we investigated whether the applied volume has any effecton the efficacy of the reference alcohol applied for surgicalhand disinfection.

MATERIALS AND METHODS

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Materials and Methods

Test principle and prerequisites.
A total of 10 experiments were carried out by using prEN 12791.The in vivo bactericidal efficacy of the reference alcohol n-propanol(60%, vol/vol) was assessed with 20 healthy volunteers per experiment(4). The volunteers were office workers and laboratory staffand were not the same for the different experiments. No skinbreaks, such as cuts or abrasions, and no other skin disorderswere present. The nails were short and clean. The volunteersdid not use any substances with antibacterial activity or antibacterialsoaps starting 1 week prior to testing. Between experimentsthere was a rest period of at least 1 week, which allowed reconstitutionof the normal skin flora.

Wash phase.
To remove transient bacterial flora and any foreign particles,volunteers' hands were washed with a nonmedicated soap (sapokalinus). Five milliliters of the soft soap was poured intothe cupped dry hands and rubbed vigorously onto the skin upto the wrists for 1 min in accordance with the standard procedurein order to ensure total coverage of the hands. The hands werethen rinsed with running tap water and dried with a paper towel.

Determination of the prevalues.
The distal phalanges of the right and left hands, includingthe thumbs, were rubbed separately for 1 min onto two petridishes (diameter, 9 cm) containing 10 ml of tryptic soy broth(TSB) as described in prEN 12791 (4). This is the same samplingtechnique that is used in other European test methods whichare used for preparations for hygienic hand disinfection (EN1500) and hygienic hand washing (EN 1499) (9, 10). A 1:10 dilutionof the sampling fluid obtained from each hand was prepared inTSB. Aliquots were taken from the sampling fluid (1 and 0.1ml) and the dilution (0.1 ml) and spread onto tryptic soy agardishes with a sterile glass spatula. No more than 30 min elapsedbetween the time of sampling and the time of seeding. The disheswere incubated for a total of 48 h at 36 ± 1°C, andthe number of CFU counted ranged from 15 to 300 colonies perplate.

Disinfection phase.
Each of the volunteers was treated with a reference product,60% (vol/vol) n-propanol, for at least 3 min. The number of3-ml portions which was necessary to keep the hands wet withthe reference alcohol was counted for each application.

Determination of postvalues.
After disinfection, each volunteer rubbed the distal phalangesof one hand (randomly selected) for 1 min in a petri dish containing10 ml of TSB supplemented with neutralizers (immediate effect).The following neutralizers were used: 3% Tween 80, 3% saponin,0.1% histidine, and 0.1% cysteine (7). The other hand was glovedfor 3 h for assessment of the sustained effect. After the glovewas taken off, sampling was done by using the same method thatwas used for the immediate effect. A 1:10 dilution in TSB wasprepared from the sampling fluid obtained from each hand. Aliquotsof the sampling fluid (1 and 0.1 ml) and the dilution step (0.1ml) were removed and spread onto tryptic soy agar dishes witha sterile glass spatula. The dishes were incubated at 36 ±1°C for a total of 48 h, and the number of CFU counted rangedfrom 15 to 300 colonies per plate.

For each dilution the mean number of CFU was calculated. Themean was then multiplied by the dilution factor in order toobtain the number of CFU per milliliter of sampling liquid.

All pre- and postvalues were expressed as log₁₀ values. Forcalculation purposes, values of 0 (log 0 = – ${infty}$ ) were resetto 1 (log 1 = 0). If values in the range that could be enteredinto calculations were obtained from more than one dilution,the mean was used as the final logarithm value. For each volunteerthe logarithmic reduction factor (RF) was obtained by determiningthe difference between the log₁₀ prevalue and log₁₀ postvalues.

Statistical analysis of multiple means was performed by usingthe analysis of variance (ANOVA) model (SPSS for Windows, version11.5.1; SPSS Inc., Chicago, Ill.). Pearson's correlation coefficientwas determined to evaluate a possible correlation between thenumber of 3-ml disinfectant portions used for the referencedisinfection and the mean RF (SPSS for Windows, version 11.5.1,SPSS Inc.).

RESULTS

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Results

A total of 10 experiments were performed, resulting in a totalof 200 surgical reference disinfections. The mean prevalue forthe resident hand bacteria before application of the referencealcohol was 4.44 ± 0.77 log₁₀ CFU (range, 4.26 ±1.10 to 4.52 ± 0.68 log₁₀ CFU) (Table 1). The 10 meanprevalues were not significantly different (P = 0.994, as determinedby ANOVA). The mean log₁₀ RF immediately after the referencedisinfection (zero hour value) was 2.70 ± 1.15 (range,2.20 ± 1.32 to 3.03 ± 1.09) (Table 1). The 10mean RFs were not significantly different (P = 0.271, as determinedby ANOVA). The mean log₁₀ RF 3 h after the reference disinfection(3-h value) was 2.20 ± 1.10 (range, 1.53 ± 1.09to 2.59 ± 0.72) (Table 1), and the 10 mean RFs were significantlydifferent (P = 0.016, as determined by ANOVA).

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TABLE 1. Evaluation of 10 surgical reference disinfection experiments with 20 volunteers involving application of 60% (vol/vol) n-propanol for at least 3 min

Most surgical reference disinfections were achieved with 9 mlof the reference alcohol (n = 146; 73%), followed by 12 ml (n= 48; 24%) and 6 ml (n = 6; 3%) (Table 1). The mean log₁₀ RFswith 6 ml of reference alcohol were 2.18 ± 0.88 (immediateeffect) and 1.65 ± 0.79 (sustained effect). Use of 9ml of reference alcohol yielded mean log₁₀ RFs of 2.76 ±1.11 (immediate effect) and 2.20 ± 1.11 (sustained effect).Application of 12 ml of reference alcohol resulted in mean log₁₀RFs of 2.57 ± 1.27 (immediate effect) and 2.26 ±1.09 (sustained effect) (Table 2). The mean log₁₀ RFs for thethree treatment groups were not significantly different, bothfor the immediate effect (P = 0.333, as determined by ANOVA)and for the sustained effect (P = 0.442). A higher number ofportions did not correlate with a higher log₁₀ RF, both forthe immediate effect (Pearson's correlation coefficient, –0.028;P = 0.689) and for the sustained effect (Pearson's correlationcoefficient, 0.059; P = 0.404).

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TABLE 2. Evaluation of 10 surgical reference disinfection experiments with 60% (vol/vol) n-propanol and 3-min treatments

DISCUSSION

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Discussion

For the first time, we were able to show that in surgical handdisinfection with a standard 3-min rub-in procedure the appliedvolume of the formulation has no impact on the reduction ofresident skin bacteria. This result is not a surprise, becauseas long as the skin is covered with the alcohol, an effect onthe resident hand bacteria can be expected regardless of thevolume that is applied. This result is in line with data fromanother study, in which two 3-ml portions of a propanol-basedhand rub were applied in one part of the study for a total of60 s and in another part of the study for 90 s. The same doseled to sufficient efficacy compared with the reference treatmentafter 90 s but not after 60 s, indicating that the applicationtime has the main impact on the efficacy (11).

Further evaluation of the reference disinfection revealed thatthe prevalues for 10 experiments were highly reproducible (variancecoefficient, 0.17). Although there was no artificial contaminationof the hands and although the people were not the same in theexperiments, the volunteers nevertheless provided a standardamount of resident hand bacteria prior to application of thereference alcohol. The reproducibility of the number of residenthand bacteria is similar to that observed for artificial contaminationwith a specific test organism (9, 10). It can therefore be concludedthat the resident hand flora is suitable for studying the efficacyof a preparation for preoperative treatment of hands. The immediateeffect on the resident skin bacteria also yielded good reproducibility(variance coefficient, 0.43), but it was lower than the reproducibilityof the prevalues. The sustained effect showed a significantdifference for all 10 experiments (P = 0.016, as determinedby ANOVA) and had the highest variance coefficient (0.50). Thisfinding may have various explanations. After 3 h in a surgicalglove the microbial density on the skin depends on various factors,like the distribution and abundance of skin bacteria (14) andthe number and activity of sweat and sebaceous glands, whichmay bring up more resident skin bacteria out of the ducts ontothe stratum corneum (2, 17). Although the sweat glands do notcontain any bacteria themselves (19), the ducts of the sweatglands do. Increased sweat production may bring up the bacteriaof the ducts to the skin. The activity of the sweat glands alsodepends on the physical activity of the volunteer. Currently,what a volunteer does or does not do during the 3 h is not standardized.In addition, microorganisms may also recolonize the skin fromthe hair follicles. Also, variations in microbial density after3 h may also be explained by the specific flora of a volunteer,which may recover faster or slower for different volunteersdepending on the composition of different species (12, 13).If all these factors are taken into account, it becomes quiteevident that the variation is likely to be greatest after 3h despite a standard treatment.

Overall, reference disinfection with 60% (vol/vol) n-propanolfor 3 min for evaluation of the efficacy of a preparation forsurgical hand disinfection is an accepted principle in Europe.If hands are kept wet with the disinfectant for the total applicationtime, the applied volume may vary, but this does not alter theefficacy.

FOOTNOTES

* Corresponding author. Mailing address: BODE Chemie GmbH & Co., Scientific Affairs, Melanchthonstrasse 27, 22525 Hamburg, Germany. Phone: 49 (0) 40 54006-0. Fax: 49 (0) 40 54006-128. E-mail: guenter.kampf{at}bode-chemie.de.

REFERENCES

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