Study design. This study was conducted using a randomized cross-over design with participation of 16 healthy volunteers, 9 female and 7 male healthy individuals of Caucasian background and an average age of 31.3 years (range: 22 to 74 years). Two different skin antiseptics (CHG-ALC or PVP-I-ALC) and two different contact times for each antiseptic (2.5 min or 30 min) were applied on day 1 and 4, based on the assumption that a period of 3 days is sufficient for complete re-colonization of the skin. The application was carried out on both shoulders, using two separate shoulder areas for sampling (immediate and 3-h values) on the antero-lateral site of each shoulder on each day (Fig. 1). Participants were randomized by drawing opaque folded paper tickets from a container, such that two different antiseptic treatments per day were represented in a cross-over design, and each volunteer completed four different treatments. The Ethics Committee of the University of Greifswald approved the study (Reg. No. BB 109/10).
Volunteers. Inclusion criteria were age > 18 years, legal capacity, informed consent after the study procedure was explained, as well as willingness and ability to comply with the test protocol. Exclusion criteria were macroscopically visible lesions of the skin except acne juvenilis, infections of the shoulder areas, dermatosis except acne, participation in another study within the last 30 days, pregnancy, lactation, thyroid disorders, intolerance to CHG or iodine, age under 18, therapy with radioactive iodine, antiseptic procedures on the designated areas within in the last 7 days, and systemic antibiotics within the last 7 days. Two female volunteers suffered from active acne juvenilis at the shoulder areas that was less pronounced in the face; this was confirmed dermatologically.
Tested compounds and application modes. Skin antisepsis was performed using the following commercially available products:
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ChloraPrep® (CHG 2% w/v, IPA 55% w/v [70% v/v], coloured; CareFusion, Leawood, USA): vigorous rubbing using the 26 ml applicator for 30 s, afterwards keeping the treated area wet for 2 min with the antiseptic.
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Chlorhexidine 2% alcoholic (CHG 2% w/v, IPA 55% w/v [70% v/v]; B. Braun Medical, Sempach, CH): vigorous rubbing for 30 s using sterile forceps and gauze for 30 s, afterwards keeping the area wet with the antiseptic using a soaked sterile dressing (Zetuvit®, Hartmann, Heidenheim, Germany, 20 × 40 cm) for 29.5 min.
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Betaseptic® (3.24% w/v PVP-I, 38.9% w/v IPA, 37.3% w/v ethanol; Mundipharma, Limburg, Germany): vigorous rubbing for 30 s by using sterile forceps and gauze for 30 s, afterward keeping the area wet with the antiseptic by using forceps and gauze for 2 min or a soaked sterile dressing (Zetuvit®, 20 × 40 cm) for 29.5 min.
For the two CHG-ALC products, the IPA percentage was converted to 55% w/v from the manufacturers’ stated 70% v/v for the purpose of uniformity of measurements.
Sampling. Microbial skin counts were obtained before antiseptic application (pre-values), after application and air-drying of the sampling site (immediate post-values), and 3 h after treatment (3-h post-values). The cup-scrub technique according to ASTM E1874–14 [23] was used on 2.5 cm2 of skin, with 1 mL of sampling solution.
A first 10− 1 dilution was prepared by adding 0.4 mL of sampling solution to 3.6 mL of neutralizer solution. For CHG-containing antiseptics, this was Lipofundin MCT 20% (B. Braun, Melsungen, Germany), for PVP-I-containing antiseptics, this was 3% Tween 80 (BioChemica, AppliChem, Darmstadt, Germany), 0.3% lecithin (AppliChem), 0.1% L-histidine (Roth, Nürnberg, Germany) and 0.5% sodium thiosulfate (Merck, Darmstadt, Germany).
Concurrently with the retrieval of the immediate post-values, another skin area of 4 × 4 cm was covered with a sterile dressing (Hydrofilm® transparent dressing 12 × 25 cm, Hartmann, Heidenheim, Germany) to protect a skin area where the 3-h samples were to be collected later.
Microbiological techniques. After 5 min neutralization in the first 10− 1 dilution, further dilutions of 10− 2 and 10− 3 were prepared in the respective neutralization solution, and 0.1 mL of each dilution was plated onto Columbia agar with 5% sheep blood (Becton Dickinson, Heidelberg, Germany) for aerobic incubation (37 °C, 48 h) and onto Schaedler agar (BioMérieux, Nürtingen, Germany) for anaerobic incubation (37 °C, 7 days). The anaerobic atmosphere was generated in anaerobic jars using Anaerocult A sachets (Merck, Darmstadt, Germany). After aerobic incubation, the colony forming units (CFU) were counted and a representative sample of colonies was picked for identification, such that at least one colony of each morphologically different colony type was tested. Isolates were subjected to simple phenotypic identification, including Gram stain, catalase and coagulase tests, followed by VITEK® Cards (BioMérieux). The anaerobic CFUs were counted after 7 days incubation, and again representative colonies were analysed by Gram stain and VITEK® Cards.
For uniformity of measurements, we converted the numbers of colonies counted to CFU per 5 cm2 of skin and expressed these as log10 values. Then, we calculated the reduction factors (RFs) as the differences between the log10 pre-values and the log10 post-values. To calculate the reduction factors and transform to log10, plates without growth were set to a value of 1.
Sampling and validation of neutralization. Skin bacteria from five volunteers were collected by the cup-scrub technique [23] and pooled. Using the methodology of ASTM E1054-08 [24], pooled bacteria were used to verify the effectiveness of Lipofundin to inactivate CHG, of sodium thiosulfate to diminish the oxidizing agent iodine, and to ensure that the inactivation solutions did not significantly influence the bacterial counts, quantitatively and qualitatively. Final concentrations of 0.4% CHG and 0.6% PVP-I in 1 ml 0.9% NaCl were tested for neutralizer effectiveness. The concentration of the active agent was calculated using the treated skin area of 300 cm² (17.5 cm x 17.5 cm) with 17 mL of antiseptic solution from the applicator and 3 mL of additional antiseptic solution, which arises from the equilibrium of the soaked dressing with the antiseptic liquid film on the skin. Finally, an area of 2.5 cm² served as the basis for the microbiological examinations after scrubbing.
Validation of neutralization was conducted according to the methodology of ASTM E1054-08 [24]. The suitable neutralizers, lecithin for inactivating biguanides and thiosulfate for quenching iodine, were derived from Table 1 in ASTM E1054 [24] and Annex B in EN 1040 [25] and EN 13727 [26]. Lipofundin containing 1.2% egg yolk lecithin inactivated 0.4% CHG without any inhibitory effect on growth of pooled skin bacteria after aerobic and anaerobic incubation, and sodium thiosulfate, the quenching agent for iodine in the neutralization mixture, was effective for 0.6% PVP-I without influencing bacterial counts. Similar results were obtained in tests of neutralizer effectiveness, neutralizer toxicity and organism viability under aerobic and anaerobic culture conditions, using test solutions containing the residual antimicrobial agent which were derived from volunteers after skin antisepsis. Therefore, it was ascertained that CHG or PVP-I were effectively inactivated by the respective neutralization solutions without influencing bacterial growth after aerobic and anaerobic incubation.
Table 1
Efficacy of chlorhexidine-alcohol (CHG-ALC) versus povidone-iodine-alcohol (PVP-I-ALC) against aerobic flora at 2.5 and 30 min contact time, immediate and 3-h values
Preparation | Contact time (min) | Pre-values (log10) | Immediate effect | 3-h effect |
| | | RFa | n (0 cfu)b | RFa | n (0 cfu)b |
CHG-ALC | 2.5 | 2.26±0.93 | 1.94±0.91 | 13 | 1.74±1.08 | 10 |
30 | 2.17±0.73 | 2.17±0.73 | 16 | 1.93±0.92 | 13 |
PVP-I-ALC | 2.5 | 2.55±0.75 | 2.55±0.75 | 16 | 2.25±1.05 | 13 |
30 | 2.20±0.87 | 2.11±0.93 | 15 | 1.94±1.11 | 13 |
aMean reduction factor (RF) and standard deviation. |
bNumber of volunteers with “0” cfu counts. |
Statistical analysis. The analysis of the raw data was performed using Graphpad Prism (GraphPad, La Jolla, CA, USA) and SPSS (IBM, Armonk, NY, USA) software. Mann-Whitney- and Wilcoxon-tests were calculated. A p-value < 0.05 was considered to indicate a statistically significant difference. Presence of carry-over effects was tested using linear mixed models, including treatment, sequence, period and treatment x period interaction effects (MIXED procedure in SPSS). Sample size calculations for testing differences in RFs were based on two-sided Wilcoxon signed-rank tests for matched pairs at p = 0.05. Effect sizes (i.e. differences in RFs divided by the standard deviation of the differences) of 0.75 and 1.0 were assumed. Based on a required power of 0.80, results of sample size calculation indicated that a sample between n = 10 and n = 16 cases was required (G*Power 3.1).
Skin tolerability. All volunteers received a questionnaire for self-assessment of skin tolerability to evaluate the following parameters on an analogue scale from 1 to 10. Items “redness”, “burning”, “pruritus”, “scaliness”, and “pain” were assessed. In case of skin irritation, volunteers were asked to contact the investigators to have the nature of the irritation evaluated, and if necessary, to obtain treatment.