Journal of Hospital Infection 86 (2014) 64e67

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Journal of Hospital Infection

journal homepage: www.elsevierhealth.com/journals/jhin

In vivo comparative efficacy of three surgical hand

preparation agents in reducing bacterial count
P. Barbadoro a, b, E. Martini a, S. Savini a, A. Marigliano b, E. Ponzio b,
E. Prospero a, b, *, M.M. DErrico a, b
a

Hospital Hygiene Service, Ospedali Riuniti, Ancona, Italy

Department of Biomedical Sciences and Public Health, Unit of Hygiene, Preventive Medicine and Public Health,
Universita` Politecnica delle Marche, Ancona, Italy

A R T I C L E

I N F O

Article history:
Received 18 June 2013
Accepted 23 September 2013
Available online 16 October
2013
Keywords:
Alcohol-based hand rub
Antiseptics
Efficacy
Surgical hand hygiene

S U M M A R Y

Background: Besides objective efficacy, the choice between an antiseptic-based liquid

soap, or an alcohol-based hand rub for surgical hand preparation technique is based on
personal preference. Glycerol is often added to the formulations in order to enhance
tolerability; however, it has been recently reported as a factor reducing the sustained
effect of surgical hand rubs.
Aim: To compare the efficacies of three commercial products for hand decontamination.
Methods: The in vivo efficacy of an alcohol-based hand rub (isopropyl alcohol 40%; Npropyl alcohol 25%; glycerin 1.74%; triethanolamine salt of carbomer <1%) was compared
with other widely used products in surgical hand antisepsis (chlorhexidine and povidoneiodine). All products were used according to the manufacturers instructions.
Findings: The best results were achieved with the alcohol-based hand rub and these were
sustained for a period of 3 h. Some volunteers experienced skin peeling off the hands when
using alcohol-based hand rub; in this group of participants, the bacterial count was
reduced only by 0.91
1.67 log10 compared with 2.86
1.22 log10 in the group who did not
show this phenomenon.
Conclusion: Besides confirming the importance of alcohol-based hand rubs for surgical
hand decontamination, the results suggest the value of assessing the characteristics, and
response of healthcare workers skin, that may contribute to the development of skin
peeling, and the subsequent possibility of a paradoxical overcolonization of hands after
surgical preparation with alcohol-based hand rub.
2013 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Introduction
Surgical site infections (SSIs) are still among the most
common hospital-acquired infections worldwide despite
* Corresponding author. Address: Dipartimento di Scienze Biomediche e Sanita
` Pubblica, Universita
` Politecnica delle Marche, Via
Tronto 10/a, 60020 Torrette di Ancona, Italy. Tel.: 39 0712206030;
fax: 39 0712206032.
E-mail address: e.prospero@univpm.it (E. Prospero).

significant developments in surgical technique.1,2 Disinfection

can be performed using a surgical hand wash with an antiseptic
liquid soap, or with an alcohol-based hand rub.3,4 Products for
surgical hand disinfection should pass two European standards
for bactericidal efficacy: European Norm (EN) 12054, which is a
suspension test using four different test bacteria to determine
a general bactericidal activity; and EN 12791, which is a test
used to determine the bactericidal efficacy in vivo.5e7 However, recently there has been a growing interest in challenging
surgical handwashing procedure in real working settings, and

0195-6701/$ e see front matter 2013 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jhin.2013.09.013

P. Barbadoro et al. / Journal of Hospital Infection 86 (2014) 64e67

the formulations recommended by the World Health Organization have been discussed.8e10 Moreover, the glycerol
component of alcohol-based hand rub has been recently evaluated as a factor reducing the sustained effect of surgical hand
rubs.11
The objective of this work was to compare the in vivo efficacy of an alcohol formulation with respect to other widely
used products in surgical hand antisepsis.

Methods
Products tested
The tested products were based on the following formulations: (i) chlorhexidine (chlorhexidine gluconate 4%; propan-2ol 1e5%; lauryldimethylamine oxide 1e5%; glycerol 1e5%); (ii)
povidone-iodine (7.5%); and (iii) an alcohol formulation (isopropyl alcohol 40%; N-propyl alcohol 25%; glycerin 1.74%;
triethanolamine salt of carbomer <1%). The following neutralizers were used: polysorbate 80 (3%), saponin (0.3%), histidine (0.1%) and cysteine (0.1%). The in vivo bactericidal
efficacy of the three products was assessed in 20 healthy volunteers aged 27e50 years. The skin of the volunteers was free
from cuts or abrasions and no other skin disorders were present. Nails were short and clean. In three distinct cross-over
experiments, each formulation containing one of the three
products was tested. A washout period of one week was
allowed between each test run. At the end of the four experiments, each volunteer had used each formulation once. Volunteers participated after having expressed a written informed
consent.

Wash phase (pre-values)

To remove transient bacterial flora and foreign agents,
volunteers hands were washed with a plain soap with the
following procedure: 10 mL of the soap was poured into the
cupped dry hands and rubbed vigorously on to the skin up to the
wrists in accordance with the standard procedure to ensure
total coverage of the hands, which were then rinsed in running
tap water and dried with a sterile paper towel.
For the determination of the pre-values of colony-forming
units (cfu), the distal phalanges of the right and left hand
were rubbed separately, including thumbs, for 1 min on to two
9 cm Petri dishes containing 10 mL tryptic soy broth (TSB). A
0.1 mL aliquot, as well as the same volume of 1:10 and 1:100
dilutions, were seeded in TSB. Sampling fluids were spread over
tryptic soy agar dishes with a sterile glass spatula. Two dishes
were used for each dilution. No more than 5 min elapsed between sampling and seeding. Dishes were incubated for 24 h at
37
2  C. After an initial count of the cfu, Petri dishes were
incubated for another 24 h to detect slow-growing colonies.5

Surgical preparation phase

Each volunteer used the test products at least on a weekly
basis, in order to allow reconstitution of participants skin
flora. All products were used according to the manufacturers
instructions. After surgical hygiene, hands were rinsed with
running tap water for 15 s and dried with a sterile cotton towel.

65

Determination of post values

After hand preparation, one hand was randomly selected to
obtain the post-value (immediate effect). The other hand was
allowed to dry and thereafter gloved (sterile surgical glove) for
3 h for assessment of the sustained effect, obtained after
removal of the glove. In order to obtain the post-value, TSB
with neutralizers was used. The neutralizers were 3% Tween80, 3% saponin, 0.1% histidine and 0.1% cysteine. Sampling
was done in a similar way to the immediate effect.
Moreover, participants were asked to report eventual personal notation about the effects of the different products on
their skin (such as: dusty, sticky sensations).

Data analysis
For each dilution the mean number of cfu scored in duplicate
dishes was calculated. This was multiplied by the dilution factor
in order to obtain the number of cfu per millilitre of sampling
liquid. Pre- and post-values were expressed as log10 values. For
calculation purposes values of 0 were reset to 1, whereas values
uncountable in the Petri dish were considered as 1,000,000 cfu
(with log10 6). If countable values of cfu were obtained from
more than one dilution their mean was used to calculate the
final logarithm value. For each volunteer the reduction factor
(RF) was obtained as the difference between log10 post-values
and the log10 pre-value. The mean of the log10 values (RF) of
each product were compared with the corresponding values for
a paired analysis of the immediate and sustained effect. Paired
t-test was used to compare immediate and sustained effect
globally for each product. Difference between mean RFs of
different products was performed with analysis of variance
(ANOVA) with Bonferroni correction for multiple comparisons; a
post-hoc analysis was performed with Tukeys honestly significant difference (HSD) test. All analysis were two-tailed, with
level of significance set at P < 0.05. Analyses were performed by
using Stata 9.0 software (Stata Corp., College Station, TX, USA).

Results
Alcohol-based product had an immediate mean RF significantly higher than the other agents (Figure 1); in particular, the
alcohol formulation showed a mean 2.27
1.64 log10 reduction, followed by chlorhexidine, with 0.94
1.11 log10 reduction, and povidone-iodine 0.16
0.42.
Comparison of mean RFs using an ANOVA model revealed a
significant difference between the products (F 17.03;
P < 0.0001). In order to clarify the results, we report pair-wise
comparisons between each couple of tested products (Table I).
The post-hoc analysis revealed that the alcohol-based product
was significantly more effective compared with the other
tested products (P < 0.0001; Tukeys HSD). After 3 h (Figure 2)
the situation was similar to that registered immediately. In
particular, after 3 h the alcohol formulation showed a mean
1.91
1.52 log10 reduction, followed by chlorhexidine, with
0.82
1.16 log10 reduction, and povidone iodine 0.52
0.92.
ANOVA tests showed a significant difference between sustained effects for all the products (F 7.12; P < 0.01); details
for pairwise comparisons of product are reported in Table II.
The post hoc analysis revealed that the alcohol-based product
was significantly more effective compared with the other
tested products (P < 0.0001; Tukeys HSD).

P. Barbadoro et al. / Journal of Hospital Infection 86 (2014) 64e67

-6

-6

-4

-4

Reduction factor
0
-2

Reduction factor
-2
0

66

Alcohol

Chlorhexidine

Povidone-iodine

Alcohol

Chlorhexidine

Povidone-iodine

Figure 1. Box plot showing immediate effect (reduction factor

with respect to plain soap) of different agents.

Figure 2. Box plot showing sustained effect (reduction factor

with respect to plain soap after 3 h) of different agents.

On the hands of 35% (7/20) of volunteers, small sticky agglomerates appeared, presumably formed by the reaction between flaking skin cells and the glycerol of the alcohol hand rub
while performing hand hygiene with alcohol-based hand rub.
We have defined this phenomenon as skin peeling. In participants experiencing this phenomenon, the RF for the sustained
effect was significantly lower than that registered for the immediate effect (1.06
0.47 log10 vs 2.48
1.24 log10 with
respect to the group who did not show this phenomenon,
P < 0.05). This difference was not significant in participants
who did not experience the skin peeling (with immediate RF of
2.16
1.82 log10 vs 2.37
1.71 log10 of RF for sustained effect,
P > 0.05).

Our main finding is that significant differences under practical conditions were observed in vivo for products currently
used in surgical hand preparation. The best results were achieved with the alcohol-based hand rub. This is not surprising as
their efficacy in the preoperative treatment of hands is well
known; nevertheless many professionals involved in surgical
procedures remain reluctant to switch from an antiseptic soap
to an alcohol-based hand rub.12 Moreover, Tanner et al. have
concluded that the quality of the sum of the evidence
regarding alcohol hand rub use for surgical hand preparation is
variable and that the effects on the outcome, when considering
surgical site infections, is mixed.1
The excellent immediate effect may also be enhanced by
the presence of excipients, that have been shown to be
important.9 However, we also note the paradoxical effect
shown in vivo in volunteers with self-reported skin peeling,

resulting in a higher number of cfu measured at 48 h compared

with volunteers without skin peeling. In our opinion, such hand
rubs may cause the formation of small agglomerates in selected
users which feel like gritty particles on the hands (skin
peeling). This effect has already been described in association
with the use of powdered gloves, but in this trial this effect was
not related to use of powdered gloves, since it was noted after
hand disinfection and before glove use.3 This finding may suggest that the substantially reduced effect of alcohol-based
hand rub, in selected participants, may be associated with
the glycerol content of the gel, as has been recently reported,
and may also be responsible for the gritty particles noted by
some participants.11 We agree with Suchomel et al., who
discuss the possible role of glycerol in producing a moister
environment under surgical gloves, thus supporting the multiplication of residual skin flora and drawing bacterial flora from
deeper skin layers. In accordance with the Guideline for Hand
Hygiene in Health-Care Settings, we may suggest switching to
an alternative product, or using hand washing after disinfection with alcohol products for these professionals.3,11 Further
studies are needed to investigate the interactions between skin
flora and the glycerol content of the alcohol hand rub. Moreover, we must underline that a modified version of the above
hand rub has been adopted in other countries, but is not
licensed in Italy, where only the above-tested product is
available.
In conclusion, our experience confirms the importance of
alcohol-based hand rubs for the surgical decontamination of
hands. Moreover, our results highlight the value of assessing the
characteristics, and response of healthcare workers skin, that
may contribute to the development of clumps which may
contribute to a paradoxical overcolonization 3 h after surgical

Table I
Difference between reduction factor, pairwise comparison of
product for immediate effect

Table II
Difference between reduction factor, pairwise comparison of
product for sustained effect

Discussion

Alcohol
Chlorhexidine
Povidone-iodine
a

1.33
2.11a

P < 0.05 (analysis of variance).

Chlorhexidine

0.78a

Alcohol
Chlorhexidine
Povidone-iodine
a

1.09
1.39a

P < 0.05 (analysis of variance).

Chlorhexidine

0.30a

P. Barbadoro et al. / Journal of Hospital Infection 86 (2014) 64e67

hand decontamination with alcohol-based hand rubs containing
glycerol. These results underline the need to obtain a more
powerful, yet tolerable, hand solution in order to overcome
barriers to utilization, side-effects, and to improve hand hygiene behaviour.
Conflict of interest statement
None declared.
Funding sources
None.

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67

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