Josai International University Bulletin Vol. 31, No. 7, March 2023 1-11

〈Research Ａｒ
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Changes in Skin Adenosine Triphosphate after Antisepsis
with and without Skin Wiping
Motoko Kitada1），Minoru Kabashima1），Satoko Hoshino1），
Kazuko Tateno1），Yayoi Saito2）
1）Faculty of Nursing, Josai International University
2）Faculty of Nursing, Shumei University

Abstract
Optimization of skin antisepsis is essential for preventing infection related to a peripheral venous
catheter (PVC). Alcohol is a widely used disinfectant and its efficacy is influenced by organic substanceson the skin. PVCs are most often inserted in the forearm, which is believed to be covered with organic
substances because this area has frequent contact with environmental surfaces. Wiping off non-visibleorganic substances prior to antisepsis may serve to optimize skin cleanliness. In this study, experimentalsites on the forearm were disinfected with alcohol, preceded by skin wiping (two-step procedure) orno skin wiping (one-step procedure). Transparent dressings were applied to the sites immediately afterantisepsis and kept in place for 72 hours. Adenosine triphosphate (ATP) bioluminescence using the total
adenylate test (A3: ATP + adenosine diphosphate [ADP] + adenosine monophosphate[AMP]) and pH
were measured before and after intervention to evaluate skin cleanliness. We analyzed the data of 23
participants. ATP was dramatically decreased immediately after antisepsis in both procedures; however,
ATP was significantly lower in the two-step procedure than in the one-step procedure (p<.0001).
At 72 hours after antisepsis, ATP values were below 1,000 relative light unit (RLUs) in bothprocedures, with no significant difference. No difference in pH was found. Considering the threshold of
2,000 RLU in the evaluation of hand cleanliness, wiping the skin before antisepsis at the time of catheter
insertion is unlikely to contribute to the prevention of catheter-related infection.
Key words: adenosine triphosphate, alcohol, bed baths, catheter-associated infection, skin4

1 . Introduction
Peripheral venous catheters (PVC) are the most frequently used invasive devices in hospitals. It isestimated that up to 70% of patients require a peripheral venous line during their hospital stay (Zingg &－ 1－

Pittet, 2009). A frequent complication with PVCs is infection, and skin florae are believed to be a main
source of pathogens in catheter-related infections (Mermel, 2011). Skin preparation with an antiseptic(70% alcohol, tincture of iodine, or an iodophor or chlorhexidine gluconate) before PVC insertion isrecommended for the prevention of infections (O’Grady et al., 2011). Alcohol is widely used to disinfectthe PVC insertion site in clinical settings throughout Japan.
Alcohol denatures protein and is known to modulate lipid bilayer properties (Ingólfsson &Andersen, 2011). Therefore, the efficacy of alcohol in reducing bacterial counts is not only influenced
by proteinaceous material on the skin (Boyce & Pittet, 2002), but also affected by the presence of lipids.
It is considered that when the skin is visibly soiled or contaminated, the skin should be cleaned before
application of an antiseptic solution (Boyce & Pittet, 2002).
A PVC is commonly inserted in the forearm, which has frequent contact with environmental
surfaces, and the dorsal side of the forearm is rich in hairs, pores, and sebaceous glands. This means that
forearm skin is covered with organic substances, some of which are invisible, and these can interferewith the efficacy of alcohol.
Bed baths are often used to clean the skin of patients, especially for those who are bedridden. It isreported that bed baths can reduce sebum as well as adenosine triphosphate (ATP) on the skin (Hashimoto& Saeki, 2003), making bed baths an effective way to clear organic residues as well as microorganisms
from the skin. Although US Centers for Disease Control and Prevention guidelines do not outline a
procedure for skin cleaning before the disinfection of skin that is not visibly soiled, as part of skinpreparation before PVC insertion. Japanese nurses often perform bed baths before starting a PVC, withthe expectation that this can help to reduce the risk of infection, especially in patients who are bedridden.
Although the antimicrobial activity of alcohol has been widely studied (Harrington & Walker, 1903;
Larson & Morton, 1991; Price, 1939), to our knowledge, there are no studies examining whether skincleaning prior to the application of alcohol—even on skin that is not visibly soiled—can be beneficial
to skin cleanliness. We therefore conducted the present study to compare skin cleanliness between two
experimental sites; one site was disinfected with 83% alcohol preceded by skin wiping and the other was
disinfected with alcohol only. In the present study, we used ATP as an indicator to evaluate skin cleanliness.
The presence of ATP indicates the presence of biological secretions, microorganisms, and organic
residue. ATP bioluminescence testing is based on a reaction that occurs naturally in the North American
firefly, Photinus pyralis. Catalyzed by the enzyme luciferase, this reaction uses chemical energy
contained in the ATP molecule to drive the oxidative decarboxilation of luciferin, with the resultant
production of light. Light output, and ATP content, are measured in relative light units (RLUs) using aluminometer (Aycicek et al., 2006). The ATP level in this test is not always correlated with the viablebacteria count. However, Aycicek et al. (2006) and Bakke et al. (2018) showed that low levels of ATP
were correlated with the number of viable bacteria, although they found that low viable bacteria counts,－ 2－

or no viable bacteria—were detected in some samples showing high ATP levels. ATP bioluminescencetesting has been widely used in sanitation and hygiene monitoring (Tršan et al., 2020; Sogin et al., 2020;
Bakke & Suzuki, 2018) and for the evaluation of skin cleanliness (Ishii et al., 2019; Konya et al., 2020).
Peripheral catheters are normally kept under the dressing and are not replaced at least for 72
hours unless clinically indicated (O'Grady, 2011). In this study, we compared skin cleanliness, with andwithout skin wiping, immediately after antisepsis and at 72 hours after antisepsis, once the skin had
been covered with the dressing. We hypothesized that wiping the skin before antisepsis would maximize
the effect of alcohol and result in cleaner skin with reduced proteinaceous material in comparison with
antisepsis alone and that skin cleanliness could be maintained for 72 hours under a dressing, which
would contribute to the inhibition of bacterial growth and catheter-related infection.

2 . Methods
2 . 1 Study design and participants
In this study, we used a within-participant, cross-sectional, quasi-experimental design. Healthy adults aged
18 years and older were recruited from an educational institute in Chiba, Japan. Those who were allergic
to alcohol, sensitive to adhesive medical tape, or who had skin abnormalities at the experimental site were
excluded from the study. This study was approved by the ethics review board of the authors’ affiliateduniversity (reference no. 04X200014, 2 December 2020). Written informed consent was obtained fromall participants. Upon study inclusion, participants were instructed to take a bath before midnight and torefrain from shaving the left forearm on the day before the intervention. Participants were also asked not
to take a bath or apply any substances to the experimental site on the day of the intervention.
2 . 2 Procedures
We selected two experimental areas the size of the dressing that was applied later (6×7 cm) on theleft side of the forearm in each participant. Each site was disinfected with 83% (v/v) ethanol precededby skin wiping (two-step procedure) or with no skin wiping (one-step procedure). We alternated theprocedure (one- or two-step) between the central and distal sites among participants (Figure 1). Allprocedures were performed by one trained researcher to standardize the procedure.
In the one-step procedure, the researcher disinfected the site with 83% (v/v) alcohol(Alwety®one2 ethanol, Osaki Medical, Japan) by wiping in a single direction, followed by air drying. In
the two-step procedure, the site was first cleaned twice with a disposable rayon and polyester washcloth
saturated with warm (40℃) water, with scrubbing in a single direction and a different washcloth surface
used each time; the site was then disinfected with 83% (v/v) alcohol. After outcome measurement(described below), the sites were immediately dressed with a semi-permeable transparent dressing－3－

(9534HP, Tegaderm™, 3M, MN, USA), which was kept in place for 72 hours. Participants were required
to adhere to certain restrictions during the following 72 hours: the experimental sites were to be kept
away from water and covered with a waterproof dressing before showering, and participants were torefrain from performing any exercise or activities that might cause perspiration.
2 . 3 Outcome measures
The total adenylate test (A3; ATP + adenosine diphosphate [ADP] + adenosine monophosphate[AMP])
was used to evaluate cleanliness of the skin. ATP, which is the universal energy-carrying molecule
produced in living cells, can be unstable and decomposes into ADP and AMP via enzymatic reaction,
heating, and pH. In the A3 monitoring test, ADP and AMP are converted back into ATP by pyruvatekinase and pyruvate orthophosphate dikinase; ATP levels are determined using the firefly luciferase–
luciferin reaction (Sakakibara et al., 2003). The A3 test has higher detection sensitivities than theconventional ATP test for organic substances associated with health care settings (Bakke et al., 2019).
In addition to total adenylate, skin pH was measured as an environmental factor that influences
microbial growth. The skin surface normally has a mildly acidic pH (4.5–5.75), with functions in
cutaneous antimicrobial defense (Schmid-Wendtner & Korting, 2006). Most bacteria prefer ambient pHthat is neutral to slightly alkaline for bacterial adhesion, growth, and invasion, and microbial metabolites
further increase the skin pH (Rippke et al., 2018).
To collect samples for testing of skin total adenylate, the skin surface in a specific area at theexperimental site (Figure 1) was wiped three times using a dedicated swab, following our previous study(Kitada et al., 2022). ATP was reacted as a reagent, and RLUs were quantified using the Lumitester

Figure 1 Experimental areas and measurements－ 4－

Smart (Kikkoman Biochemifa Company, Japan). ATP levels were measured three times: before theprocedure (baseline), immediately after antisepsis, and 72 hours after antisepsis. Skin pH was measured
before the procedure and 72 hours later. Skin pH was measured using the Skin-pH-meter PH 905(Courage+Khazaka electronic GmbH, Koln, Germany) at a specific area on the skin (Figure 1) in an airconditioned room (21.6±1.7℃, 49.5%±7.1% relative humidity). Skin pH measurement was performed intriplicate, and the average of three measurements was used for the analysis.
2 . 4 Statistical analysis
The data are expressed as mean with standard deviation (SD) when normally distributed. In the case ofa skewed distribution, median with interquartile range are reported. To assess statistical significance, weused the paired-samples t-test or Wilcoxon signed-rank test. A p-value ≤.05 was considered statisticallysignificant. All statistical analyses were performed using JMP®11 (SAS Institute Japan Ltd., Tokyo, Japan).

3 . Results
Between January and April 2021, we included 24 women aged 19–22 years in this study. Participants
followed all instructions and restrictions on the day prior to, on the day of, and 72 hours after theexperimental procedures. All experimental sites were visibly clean and free of any visible changes tothe skin, and no loosening nor breaks in the transparent dressing were observed during the study. We
excluded one participant with outlier data at baseline and analyzed the data of 23 participants.
Time-course ATP changes are presented in Figure 2. In the one-step procedure, ATP levels atbaseline, immediately after antisepsis, and 72 hours after antisepsis were 2223 (1130) RLU, 424 (338–Figure 2 Time- course ATP changes
ATP (RLU) at baseline, immediately after antisepsis, and 72 hours after antisepsis in the one-step andtwo-step procedure (n=23)*** p<.0001, Wilcoxon signed-rank.
ATP, adenosine triphosphate; RLU, relative light unit.

－5－691) RLU, and 772 (563–1926) RLU, respectively. In the two-step procedure, ATP levels were 2169
(971) RLU, 222 (173–394) RLU, and 978 (554–1610) RLU, respectively. Baseline ATP levels showedno significant difference between procedures (p=.82). Although ATP levels were dramatically decreased
immediately after antisepsis in both procedures, the ATP level was significantly lower in the two-stepprocedure (p<.0001). ATP increased over time after antisepsis in both procedures and there was no
significant difference between the procedures at 72 hours.
Time-course pH changes are shown in Table 1. No significant differences were found between
procedures both at baseline and at 72 hours after antisepsis (p=.59, p=.89, respectively). Skin pH wassignificantly decreased at 72 hours after antisepsis compared with the pH at baseline in both procedures(p<.0001).
Table 1 Time-course pH changes†
One-step procedureTwo-step procedurep*Baseline, mean (SD)5.64 (0.42)

5.62 (0.41)

0.59

72 hours after antisepsis, mean (SD)4.80 (0.18)

4.80 (0.22)

0.89† pH at baseline and 72 hours after antisepsis in one-step and two-step procedure (n=23).
* Paired-samples t-test.
SD, standard deviation.

4 . Discussion
In this study, we found that skin ATP was significantly decreased using the two-step procedure,
indicating that wiping the skin before antisepsis with alcohol reduced organic residues andmicroorganisms on the skin. However, ATP levels under the dressing site were not significantly different
72 hours after antisepsis, with and without skin wiping, contrary to our expectations. The manufacturer
instructions for the A3 test state that a skin sample can be considered “clean” if the ATP level is below
2,000 RLU after handwashing (Kikkoman Biochemifa Company, Operation manual, accessed 1 October2022). Considering this value, at 72 hours after antisepsis, the skin could be evaluated as equally “clean”
using both procedures because ATP levels were below 1,000 RLU at this time point. Although the ATP
increased over time after antisepsis, increased ATP does not necessarily indicate bacterial growth on theskin because ATP is produced during normal cellular activity (Bonora et al., 2012). Additionally, skin
pH was significantly decreased 72 hours after antisepsis in comparison with baseline pH, indicating thatbacterial growth on the skin under the dressing was unlikely. We presumed that acidification of the skin
was caused by the intrinsic pH of the dressing base.
ATP was significantly decreased immediately after antisepsis in the two-step procedure;
however, wiping the skin before antisepsis did not affect cleanliness of the skin at 72 hours. Thus, the－ 6－question remains as to whether the temporal reduction in skin ATP at the time of catheter insertion (i.e.,
immediately after antisepsis) serves any role in controlling infection.
Bacteria on the skin are divided into two categories, transient and resident (Price, 1938).
Transient florae colonize the superficial layers of the skin and are believed to be related to hospitalacquired infection whereas resident florae are attached to deeper layers of the skin, making them lesslikely to be associated with infection (Boyce et al., 2002). When considering infection control, it may benecessary to determine not only bacterial counts but also the types of flora targeted in antisepsis.
Maximal barrier precautions (surgical hand antisepsis and wearing a gown, cap, mask, and sterile
gloves) during central venous catheter insertion contribute to reducing the risk of serious catheter-related
infection (Lorente, 2019; Kinoshita et al., 2019; Mermel, 2000). It is considered that maximal barrier
precautions are an effective way to prevent the ingress of surrounding superficial pathogenic transientflora along the catheter at the time of insertion, resulting in reduced infection risk. However, it is possiblethat the temporal reduction in ATP shown in our two-step procedure was a result of additional reductionof organic residues and microorganisms, possibly resident flora, which are less likely to be associatedwith infection.
A previous study showed that scrubbing the skin with an antiseptic detergent before applicationof povidone iodine/ethanol or chlorhexidine/isopropyl alcohol was not associated with a significant
difference in catheter colonization and catheter-related infection among patients with a peripheral arterial
catheter or central venous catheter (Mimoz et al., 2015). Taken together, it seems reasonable that wiping
the skin before antisepsis with alcohol would cause no changes in the incidence of future catheter-related
infection.
Nurses commonly perform bed baths before PVC insertion, with the expectation that thisprocedure reduces the risk of catheter-related infection, especially in patients who are bedridden.
Considering our results, unless the skin is visibly soiled, it is unlikely that bed baths at the time ofcatheter insertion contribute to preventing catheter-related infection. Importantly, however, several
studies have indicated that daily bathing with a 2% chlorhexidine-impregnated washcloth reduces theincidence of primary bloodstream infection among patients in the intensive care unit (Climo et al., 2013,
Bleasdale et al., 2007). It should be noted that daily bed baths during infusion therapy would preventthe migration of transient flora under the dressing and contribute to reducing the risk of catheter-related
infection.

5 . Study limitations
Our study has several limitations. First, we could not measure bacterial counts on the skin using
culture methods. Although ATP bioluminescence can reflect the bacterial count (Sogin et al., 2020),－7－

culturing may have provided additional important information in this study. Second, we performed
our experiments among participants with healthy, intact skin. Therefore, our results, especially thoseregarding skin cleanliness under the dressing, are not applicable to patients with a catheter. Third,
although we hypothesized that wiping the skin before antisepsis would maximize the effect of alcoholand result in cleaner skin, we could not confirm whether the efficacy of alcohol was enhanced by wiping
the skin. The decrease in ATP with the two-step procedure might be the result of removing ATP via skinwiping but may not be owing to enhanced alcohol efficacy. Additionally, ATP samples at 72 hours afterantisepsis were collected from the skin after the dressing had been stripped off. It was impossible toprevent the dressing adhesive stripping off the stratum corneum to some degree. Thus, differences in theskin layers from which skin ATP samples were taken could affect our results. Finally, the environmentand daily activities of participants might have affected our results; however, the impact of these should
be limited because participants were required to adhere to restrictions during the experimental period
and both the one-step and two-step procedures were performed at the same time on the left arm of eachparticipant.

6 . Conclusions
Although antisepsis with 83% alcohol preceded by skin wiping significantly decreased skin ATP levels
in comparison with antisepsis only, ATP and skin pH under the dressing were not significantly different
72 hours after antisepsis. Our results showed that the effect of wiping the skin before antisepsis was
temporary and did not help in maintaining cleanliness of the skin under the dressing site. We conclude
that the temporal reduction in ATP in our study was possibly caused by the additional reduction ofresident flora and is unlikely to be associated with reduced infection risk.
Acknowledgments
The authors would like to thank all participants for their cooperation and participation in our study. We
also thank Dr. Shigeo Yamamura for his support in statistical analysis. This study was supported by aJosai International University research grant. We thank Analisa Avila, MPH, ELS, of Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.
Disclosure
The authors declare no conflicts of interest.
Contributors
M.K. designed the study, was responsible for data collection and data analysis, and wrote the manuscript.
－ 8－

M.K, S.H., and K.T. made substantial contributions to data collection and were involved in drafting themanuscript. Y.S. was involved in drafting and critically reviewing the manuscript. All authors read andapproved the final draft.

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