Use of Essential Oils as Natural Therapies Against Dermatophytosis and New Diagnostic Techniques
Phase: PHASE3
Conditions: Dermatophytoses, Skin Lesion
Sponsor: University of Extremadura

  
 
 
 
Date: November 27, 2023 
Title: Clinical trial in skin conditions of the foot using a cream enriched with Melaleuca alternifolia 
essential oil. 
Description of document: Materials and Methods (Study Protocol: description of the clinical study, 
including objective and design. Statistical Analysis Plan (description of the statistical considerations for 
analyzing the data collected in the study) results of the study (Results obtained in the study) and 
Supplementary data (Annexes). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

  
 
 
 
1. Material and method 
Type of study, permits, and inclusion criteria 
A randomized, double-blind, multicenter clinical trial was designed, developed at the facilities of the 
University Center of Plasencia (University of Extremadura, Spain) and at the Escola Superior de Saúde 
Dr. Lopes Dias (Polytechnic Institute of Castelo Branco, Portugal), from March to May 2023. 
The trial was authorized by the Bioethics Committee of the University of Extremadura (Register No.: 
55/2017, Annex 1) and was conducted in accordance with the ethical principles established in the 
Declaration of Helsinki in accordance with current legislation on biomedical research (Law 14/2007, of 
3 June). In addition, all participants joined voluntarily, after signing the informed consent. 
The inclusion criteria established were that the participants were free of cosmetic and pharmacological 
substances in their feet and nails, that they did not have any type of diagnosed allergy to the 
components of the cream or to the EO of choice and that they maintained correct foot hygiene. 
Participants who had a condition that compromised their health and had repercussions on the foot or 
who were under medical treatment incompatible with their participation in the trial were excluded 
from the study. 
Sample 
The initial number of participants was 65, of which 9 were excluded because they did not meet the 
inclusion criteria or because they did not sign the informed consent form (Figure 1). Of the remaining 
56 participants, 7 were excluded due to lack of adherence, as they did not attend the review, so the 
final number of participants was 49 (Figure 1). 
 
Figure 1.  Flowchart according to CONSORT standards of the process of inclusion of participants in the 
study.  
The sample consisted of 36 women and 13 men, divided into two age groups: young people between 
18 and 30 years old (22,438±3,898) and adults between 31 and 64 years old (50,212±9,633). In terms 
of origin, 23 were residents of Plasencia (Spain) and 26 in Castelo Branco (Portugal). The descriptive 
characteristics of the sample are detailed in Table 1. 

  
 
 
 
Table 1. Descriptive characteristics of the sample. 
VARIABLES 
Total 
n=49 
Origin 
Young Men 
(mean±SD) 
Adult Men 
(mean±SD) 
Young Women 
(mean±SD) 
Adult women 
(mean±SD) 
AGE 
(years) 
41,143±
15,485 
Spain 
20±2,828 
46.333±13.317 
24,111±3,723 
49±9,367 
Portugal 
26±NP 
46,857±10,495 
19±2 
53.50±8.609 
WEIGHT 
(kg) 
70,545±
12,705 
Spain 
80±14,142 
87,333±17,926 
68,667±13,684 
64,411±7,193 
Portugal 
69±NP 
80,714±11,572 
66,750±8,995 
66.857±11.016 
HEIGHT 
(m) 
1,664±0
,085 
Spain 
1.82±0.113 
1.733±0.025 
1,652±0.074 
1.618±0.068 
Portugal 
1.7±NP 
1.757±0.060 
1.657±0.049 
1,616±0.057 
IMC 
(kg/m2) 
25,404±
3,626 
Spain 
24,026±1,280 
29,170±6,725 
25,069±4,334 
24,611±2,350 
Portugal 
23,875±np 
26,148±3,488 
24,372±3,837 
25,552±3,548 
n: sample size; SD, standard deviation; NP: Not applicable; kg: kilogram; M: Metro; BMI, body mass 
index; M2: Square meter. 
 
Randomization, masking, and experimental design 
Participants were randomly assigned to distinct groups using free software Epidat 4.0 (2011, Galicia, 
Spain). The blinding was double-blind, so that neither the investigators nor the participants knew the 
group assigned to the participant until the completion of the study and statistical analysis. 
The total number of participants was divided into three groups: a control group (T0) (n=12), in which 
no participants had a skin infection, and two different treatment groups (T1 and T2) (n=37), with diverse 
levels of EC. The rest of the participants were included in these two treatment groups, including both 
infected (n=32) and healthy (n=5) participants. Thus, group T1 (n=25) had 23 participants with infection 
and 2 healthy participants, and group T2 (n=12) had 9 participants with infection and 3 healthy 
participants. 
The clinical evaluation of the participants was carried out before the start of treatment and, 
subsequently, on days 15, 30, 45 and 60 of treatment, except for cultures, both mycological and 
bacterial, which were performed at baseline, at 30 days and at 60 days of treatment. All reviews, 
sampling, and data collected were performed by the same observer. 
Choice of EO, base cream, and preparation of treatments 
After reviewing the available literature, the essential oil (EO) of M. alternifolia was chosen for its 
antibacterial and antifungal characteristics (16–19). There were 3 types of commercial M. alternifolia 
EO from the brands Marnys® (Cartagena, Spain), Naissance® (Neath, United Kingdom) and Esencias 
Lozano® (Murcia, Spain). However, the choice of the specific commercial EO depended on the results 
obtained from the analysis of its composition. This analysis was performed using gas chromatography 
mass spectrometry (CG-MS) and the results obtained were contrasted with those available in the online 
database SpectraBase (WILEY, John Wiley & Sons). 
The cream used as a base for the incorporation of the different concentrations of EO tested was a hand 
and nail cream (La Chinata®, Plasencia, Spain), in which Extra Virgin Olive Oil is used. This cream was 
chosen after verifying that none of its components were allergens for the skin of the participants 
according to Regulation 1223/2009(1). In addition, it was the treatment received by patients in the T0 
group, in whom no type of EC was added. 
To test the possible efficacy of the chosen M. alternifolia EO as a treatment against infections, the base 
cream was enriched with different concentrations of EO. Specifically, 1% for the T1 treatment group 

  
 
 
 
and 0.5% for the T2 treatment group. The emulsion of the EO and the base cream was carried out by 
means of a magnetic stirrer (Agimatic-N, Barcelona, Spain) under sterile conditions (laminar flow 
cabinet, Telstar AV-100), adding the necessary volume of EC until the desired two concentrations were 
achieved. 
Treatments were administered to participants in the form of prefilled 15ml syringes under sterile 
conditions with enough for 15 days of treatment. Along with the treatment, all the necessary 
information was provided on the hygienic habits to follow (washing the feet properly with soap and 
water and drying them properly with a towel for exclusive use), the correct way of applying the 
treatment, the daily volume of treatment for each foot (0.5ml/2 times a day). 
Exploration, sampling, and diagnostic tests 
Sociodemographic data (age and sex), medical and podiatric history, allergies and pharmacological 
treatments prescribed, as well as hygiene habits, were obtained through the completion of a Google 
Forms survey (Annex 2). 
Scans and measurements were performed in a temperature-controlled room (20-24°C) and 
participants aired their bare feet for 10 minutes before starting the procedures, to normalize the 
temperature. In the first session they were weighed and measured (SECA 701, Vogel & Halke Hamburg, 
Germany), calculating the BMI(2). After normalizing the temperature, the physical examination was 
carried out, samples were taken, and data were obtained. 
The Corneometer CM 825® (Electronic Courage-Khazaka, Cologne, Germany) with a measuring surface 
of 49 mm, a frequency of 0.9–1.2 MHz and an accuracy of ±3%, was used to assess the superficial 
hydration of the skin (3). Five reference points were measured, both at the dorsal level and at the 
plantar level (Figure 2), obtaining three measurements from each point to later calculate the average 
hydration of each location analyzed. 
1. 
 
Figure 2. Location of the 5 reference points on the feet for assessment of superficial hydration. 
(A) Dorsor foot: (1) dorso-distal point, above the central metatarsal heads. (B) Sole of the foot: 
(2) interdigital-plantar point, between 3rd and 4th digital space, (3) central point of the internal 
arch, (4) central point of the external arch, and (5) central point of the calcaneus. 
Regarding the diagnosis of possible infections present in the feet, the following tests were performed: 
- 
Wood's Light Examination 
The feet of the participants were examined with a Wood L1004® lamp (Weelco, Barcelona, Spain) at 
maximum power (41) and at a distance of 10-15 cm from the surface to be examined. Long-wave 
ultraviolet radiation allows visual observation of the fluorescence emitted by Pseudomonas 
aeruginosa, Microsporum audouinii, Microsporum canis, C. minutissimum, T. schoenleinii,(4) among 

  
 
 
 
others, whose color varies depending on the pathogen. The areas that fluoresced were recorded along 
with the color of the fluorescence in each review conducted from the beginning of the study to the day 
of its completion. 
- 
Potassium hydroxide (KOH) test and mycological culture 
A skin sample was taken following the standard procedure described for the diagnosis of mycosis (5). 
The epithelial surface was disinfected with 70° alcohol and a sample was collected from the affected 
area by scalpel scraping. Subsequently, the sample was analyzed using two different procedures. The 
30% KOH test (35.3g/100ml distilled water) (Labbox, Barcelona, Spain)  followed by conventional light 
microscopy observation to identify the infective fungal agent, and  microbiological culture on 
Sabouraud dextrose agar with chloramphenicol and cycloheximide (Condalab, Torrejón de Ardoz, 
Madrid), at a constant temperature (between 25°C and 28°C) for 2 to 4 weeks, in order to inhibit molds 
and bacteria and observe the growth of theinfective fungal agent. Participants with infection were 
considered to be those who had positive results in both tests, excluding cases in which only one test 
was positive. 
- 
Bacteriological culture 
A skin sample was taken following the standardized procedure for microbiological analysis (6). The skin 
was disinfected with 70° alcohol and a sample was taken by smear with a sterile swab from the affected 
surface. Again, the analysis was carried out using two distinct and independent procedures. A Gram 
stain was performed to identify bacteria and the sample was inoculated with Sabouraud dextrose broth 
(SDA) (Merck, Germany), at a constant temperature (between 27°C and 31°C) for 1 to 3 days. 
Participants with infection were considered to be those who had positive results in both tests, excluding 
cases in which only one test was positive. 
Variables and statistical analysis 
The following variables were analyzed: sociodemographic (sex and age), anthropometric (weight, 
height, and BMI), hygiene-related habits (frequency of foot washing, use of flip-flops in showers/pools, 
type of footwear and socks and frequency of sock change), presence or absence and type of infection, 
degree of hydration and concentration of EC group T0 (0%), group T1 (1%) and group T2 (0.5%). Of 
these variables, age, weight, height, BMI, and hydration were considered quantitative compared to the 
rest that were analyzed as qualitative. 
Statistical analysis was performed using the statistical analysis program IBM-SPSS Statistics for 
Windows version 25 (IBM Corp. Armonk, NY, USA). Some figures have been obtained using the Python 
programming language, using the Seaborn library (Waskom, M. L., (2021). Seaborn: statistical data 
visualization. Journal of Open-Source Software, 6(60), 3021, https://doi.org/10.21105/joss.03021). 
Normality was assessed using the Shapiro-Wilk test. To evaluate the influence of different variables on 
infection remission, a Cox regression model was used, and survival curves of significant factors were 
plotted. To compare the efficacy of different EC concentrations, survival curves were compared using 
the Log-rank test.  The Mann-Whitney U test was used to assess hydration averages according to sex. 
To check if there were improvements in hydration due to the use of the cream with different 
concentrations of EC (T1 and T2) compared to the control group (T0), hydration levels were compared 
using Kruskal-Wallis tests, using the Mann-Whitney U test with correction of p-values by the Bonferroni 
method for multiple comparisons. 
In all statistical tests, significance was considered when p-value<0.05 and high significance when p-
value<0.01. 
 

  
 
 
 
2. Results 
Analysis of the Elemental Composition of Commercial S. S. 
The determination of the specific composition of the 3 types of EO made it possible to quantify the 
exact concentration of the different components of each of the EO, choosing the EO with the highest 
concentration of components with antimicrobial activity and the lowest concentration of components 
susceptible to interaction with other drugs or to produce allergies in the participant, such as 
terpinolene.  alpha-terpinene and sabinene and the aged components of EO(7–10).Specifically, the 
main component in the 3 EO samples was terpinen-4-ol, with a concentration between 28.47% and 
29.15%, followed by gamma-terpinene and alpha-terpinene (Table 2). These three components 
accounted for approximately 65% of the total EO in the 3 samples analyzed. On the other hand, alpha-
terpineol and eucalyptol were found to be the fourth and fifth most concentrated components, with 
percentages between 3.85%-5.85% and 4.12%-5.81%, respectively. These two, along with m-cymene, 
alpha-pinene, and terpinolene, made up about 24% of the total EO. The rest of the EO was made up of 
up to 22 different compounds with minority percentages, considered together as traces. For all the 
above results, the EO of M. alternifolia of the brand Esencias Lozano® was used, since it was the EO 
that presented the highest percentage of the components of terpinen-4-ol, eucalyptol and alpha-
terpineol, whose antimicrobial activity has been described in the bibliography(7,11–14). 
Table 2.  Percentages of the different components according to the EO studied in the CG-MS. 
 
Trademarks of EO 
Components 
Naissance® 
Marnys® 
Esencias Lozano® 
Terpinen-4-ol 
28.47% 
29.10% 
29.15% 
Gamma-terpinene 
21.92% 
20.42% 
22.86% 
Alpha-Terpinene 
13.10% 
11.23% 
13.58% 
Alpha-terpineol 
3.85% 
4.03% 
5.85% 
Eucalyptol 
4.12% 
4.82% 
5.81% 
M-cymene 
3.40% 
5.28% 
4.96% 
Alpha-pinene 
3.18% 
3.15% 
5.12% 
Terpinolene 
4.65% 
4.26% 
3.72% 
Traces 
17.31% 
17.71% 
8.95% 
Total 
100% 
100% 
100% 
®: trademark; %: percentage of the component in the sample. 
Test results for microbiological diagnostics 
After reading the tests performed for microbiological diagnosis, it was determined that, of the 49 total 
participants, 32 (65.3%) were positive for skin infections. Of these 32 participants, 4 (12.5%) had fungal 
infection, 19 (59.4%) showed bacterial infection, and 9 (28.1%) had mixed fungal and bacterial 
infections simultaneously. 
Of the participants who developed skin infections, 23 were women and 9 were men. The prevalence 
of skin infections by sex is shown in Table 3. 
Table 3.  Prevalence of infections as a function of sex. 
Sex   
Infections     
Fungal  
Bacterial 
Mixed 
Total 
Women   
3(9.3%)   
13 (40.6%)   
7 (21.8%)   
23 (71.7%)   
Men   
1 (3.1%)   
6 (18.8%)   
2 (6.4%)   
9 (28.3%)   
Total   
4 (12.4%)   
19 (59.4%)   
9 (28.2%)   
32 (100%) 
%: Percentage. 

  
 
 
 
The colorations observed with Wood's light in the interdigital spaces were coral red and opaque blue 
(Figure 3). The coral-red coloration corresponds to a superficial bacterial infection of the skin caused 
by C. minutissimum, known as erythrasma, and the dull blue coloration corresponds to a fungal 
infection caused by the dermatophyte Trichophyton schoenleinii. 
 
Figure 3. Fluorescence of different color emitted by pathogens under Wood's light in interdigital 
spaces of the feet. (Aand B) erythrasma with coral-red coloration (*) and (B) suspected Trichophyton 
schoenleinii with bluish coloration (↑). 
Mycological cultures were used to determine the causative agents of fungal infections. The 
dermatophytes and yeasts that were identified were: Trichophyton schoenleinii, Trichophyton 
verrucosum, Trichophyton tonsurans and Candida albicans. On the other hand, the following bacteria 
were identified in bacterial cultures: Corinebacterium minutissimum, staphylococcus haemolyticus, 
staphylococcus epidermidis, staphylococcus lugdunensis, micrococcus luteus and micrococcus sp. 
Efficacy of treatments  
To test whether the EC-enriched cream was effective against fungal infections, the cumulative survival 
curves of the two treatments, T1 and T2, were plotted (Figure 4). 100% of fungal infections resolved 
within 30 days with bothtreatments, although the EC curve at 1% was higher than 0.5%.  
 
 
Figure 4.  Cumulative survival curves as a function of the two treatments for fungal infections. 
In fact, the mean time to achieve remission in the T1 group (1%) was 11.61 days, compared to 14.00 
days inthe T2 group (Table 4). However, according to the Lo g-ra nk test, the differences between the 
two groups were not statistically significant (p-value=0.611). Therefore, both concentrations have 
similar efficacy in terms of the time required to achieve remission of infection. 
1. Cumulative survival 
Moment (days) 
1. Survival function 
Group 
Treatment 1% 
Treatment 0.05% 

  
 
 
 
Table 4. Mean and median survival time (in days) of remission of fungal infection for the two EC 
concentrations. 
  
Media 
Median 
Group 
East. 
Err. East 
IC 95% 
East. 
Err. East 
IC 95% 
Lim. Inf. Lim. Sup. 
Lim. Inf. Lim. Sup. 
Treatment 1% 
11.61 
2.68 
6.36 
16.86 
 - 
- 
- 
- 
Treatment 0.5% 
14.00 
4.04 
6.09 
21.91 
 - 
- 
- 
- 
Est: statistical survival test; Err. Est: statistician error; CI, confidence interval; Lim. Inf: lower bound; Lim. 
Sup: upper limit; "-": data not available. 
Similarly, to evaluate whether the EO-enriched cream was effective against bacterial infections, the 
cumulative survival curves for both treatments were plotted (Figure 5). In this case, the treatment curve 
at 0.5% concentration showed better results than the 1% treatment curve. 
 
Figure 5.  Cumulative survival curves as a function of the two treatments for bacterial infections. 
Thus, the mean time to achieve remission in the T1 group (1%) was 49.35 days, compared to 44.82 days 
in the T2 group (0.5%) (Table 5). This indicated that the treatment of the T2 group was better, since the 
infection remitted 5 days earlier on average, although when comparing both treatments using the 
Long-rank test, no statistically significant differences were found (p-value=0.130). Median survival was  
60.00 days for treatment at 0.5%, so half of the  participants in the T2 group achieved remission at the 
end of the study. This value could not be computed for the 1% treatment, indicating that less than half 
of the subjects in this group experienced resolution of the infection at the end of the trial (Table 5). 
Table 5. Mean and mediansurvival time (in days) of remission of bacterial infection for the two EC 
concentrations. 
  
Media 
Median 
Group 
East. 
Err. east. 
IC 95% 
East. 
Err. east. 
IC 95% 
Lim. Inf. 
Lim. Sup. 
Lim. Inf. 
Lim. Sup. 
Treatment 1% 
49.35 
2.38 
44.68 
54.01 
- 
- 
- 
- 
Treatment 0.5% 
44.82 
4.38 
36.2 
53.40 
60.0 
6.37 
47.51 
72.49 
Est: statistical survival test; Err. Est: statistician error; CI, confidence interval; Lim. Inf: lower bound; Lim. 
Sup: upper limit; "-": data not available. 
 
1. Cumulative survival 
Moment (days) 
1. Survival function 
Treatment 1% 
Treatment 0.05% 
Group 

  
 
 
 
Relationship between infections and the rest of the variables analyzed 
In relation to hygiene habits, it was found that most of the participants washed their feet daily, used 
flip-flops in swimming pools or showers, wore breathable footwear and cotton socks, and changed 
their socks daily (Table 6), although it is true that these habits did not show a statistically significant 
relationship with respect to the presence or absence of skin infections (p-value≥0.103) (Table 7). 
Table 6.  Contingency table between hygiene habits and skin infections. 
Category  
Fungal infection  
Total  
Bacterial infection  
Total  
No 
Yes 
No 
Yes 
Frequency of foot washing  
1 time every 2 days 
2 (4.88%)  
-  
2 (4.08%)  
2 (6.90%)  
-  
2 (4.08%)  
Daily 33 (80.49%)  8 (100.00%)  41 (83.67%)  23 (79.31%) 18 (90.00%)  41 (83.67%) 
2 times a day 6 (14.63%)  
-  
6 (12.24%)  
4 (13.79%)  
2 (10.00%)  
6 (12.24%)  
Total 41 (83.67%)  
8 (16.33%)  
49  
29 (59.18%) 20 (40.82%)  
49  
Flip flops in the shower  
No 7 (17.07%)  
2 (25.00%)  
9 (18.37%)  
8 (27.59%)  
1 (5.00%)  
9 (18.37%)  
Yes 34 (82.93%)  
6 (75.00%)  
40 (81.63%)  21 (72.41%) 19 (95.00%)  40 (81.63%) 
Total 41 (83.67%)  
8 (16.33%)  
49  
29 (59.18%) 20 (40.82%)  
49  
Type of footwear  
Non-breathable 18 (43.90%)  
4 (50.00%)  
22 (44.90%)  12 (41.38%) 10 (50.00%)  22 (44.90%) 
Breathable 23 (56.10%)  
4 (50.00%)  
27 (55.10%)  17 (58.62%) 10 (50.00%)  27 (55.10%) 
Total 41 (83.67%)  
8 (16.33%)  
49  
29 (59.18%) 20 (40.82%)  
49  
Sock Type  
Cotton 21 (51.22%)  
6 (75.00%)  
27 (55.10%)  17 (58.62%) 10 (50.00%)  27 (55.10%) 
Polyester 10 (24.39%)  
-  
10 (20.41%)  6 (20.69%)  
4 (20.00%)  10 (20.41%) 
I don't know 10 (24.39%)  
2 (25.00%)  
12 (24.49%)  6 (20.69%)  
6 (30.00%)  12 (24.49%) 
Total 41 (83.67%)  
8 (16.33%)  
49  
29 (59.18%) 20 (40.82%)  
49  
Daily sock change  
   
1 time every 2 days 5 (12.20%)  
-  
5 (10.20%)  
1 (3.45%)  
4 (20.00%)  
5 (10.20%)  
Daily 32 (78.05%)  8 (100.00%)  40 (81.63%)  26 (89.66%) 14 (70.00%)  40 (81.63%) 
2 times a day 
4 (9.76%)  
-  
4 (8.16%)  
2 (6.90%)  
2 (10.00%)  
4 (8.16%)  
Total 41 (83.67%)  
8 (16.33%)  
49  
29 (59.18%) 20 (40.82%)  
49 
"-": data not available, %: percentage. 
Table 7. Relationship between hygiene habits and skin infections. 
Variables  
Fungal infection  
Bacterial infection  
X2 
p-value 
X2 
p-value 
Frequency of foot washing  
1.87  
0.393  
1.68  
0.432  
Flip flops in the shower  
0.00  
0.976  
2.66  
0.103  
Type of footwear  
0.00  
1.000  
0.09  
0.761  
Sock Type  
2.64  
0.267  
0.58  
0.748  
Daily sock change  
2.15  
0.542  
4.08  
0.252  
X2: Chi-Square 
On the other hand, the possible relationship between anthropometric and sociodemographic variables 
and treatment efficacy for fungal infections was examined, where no statistically significant association 
was observed (p-values ≥0.145) (Table 8). Therefore, for this sample, anthropometric and 
sociodemographic variables did not seem to be critical factors in the efficacy of treatment for fungal 
infections of the foot. 

  
 
 
 
Table 8.  Cox model for different anthropometric and sociodemographic variables and fungal infection. 
Variable  
B (coef.)  
Exp(B)  
IC 95%  
p-value   
Lim. Inf. 
Lim. Sup 
Stature 
-0.02 
0.98 
0.73 
1.30 
0.874 
Weight 
0.01 
1.01 
0.73 
1.40 
0.948 
BMI 
-0.04 
0.96 
0.39 
2.40 
0.938 
Age 
0.03 
1.03 
0.54 
1.97 
0.918 
Sex 
-0.64 
0.52 
0.20 
1.35 
0.181 
Coef: coefficient; Exp: exponent; B: correlation coefficient; CI, confidence interval; Lim. Inf: lower 
bound; Lim. Sup: upper limit; *: statistically significant. 
Regarding the possible relationship between anthropometric and sociodemographic variables and the 
efficacy of treatment for bacterial infections, it was observed that only sex showed a statistically 
significant relationship (p-value=0.028) (Table 9). 
Table 9. Cox model for different anthropometric and sociodemographic variables and bacterial 
infection. 
Variable 
B (coef.) 
Exp(B) 
IC 95% 
p-value  
Lim. Inf. 
Lim. Sup 
Stature 
0.11 
1.12 
0.82 
1.54 
0.484 
Weight 
-0.08 
0.93 
0.64 
1.34 
0.684 
BMI 
0.13 
1.14 
0.41 
3.21 
0.803 
Age 
0.55 
1.74 
0.82 
3.67 
0.146 
Sex 
1.41* 
4.11 
1.16 
14.50 
0.028* 
Coef: coefficient; Exp: exponent; B: correlation coefficient; CI, confidence interval; Lim. Inf: lower 
bound; Lim. Sup: upper limit; *: statistically significant. 
To assess the efficacy of the treatment over time according to sex, a cumulative survival curve was used 
(Figure 6) and the proportion of individuals in whom the resolution of the infection over time was 
observed was represented, observing that the healing time was shorter in women than in men and,  
therefore, a faster resolution of bacterial infection is observed in women. 
 
Figure 6.  Cumulative survival curve as a function of sex. 
Finally, in relation to skin hydration, significant differences were obtained according to sex (p-value 
≤0.010), with women having higher hydration averages (Table 10).  
1. Cumulative survival 
Moment (days) 
1. Survival function 
Treatment 1% 
Treatment 0.05% 

  
 
 
 
Table 10. Results of the Kruskal-Wallis tests for the analysis of hydration according to sex. 
 
R: right; L: Left; Sd: standard deviation; *: significant; **: Highly significant. 
On the other hand, in relation to skin  hydration, it was obtained that the  three groups had similar 
hydrations at the beginning of the study, although it is true that it was observed that the hydration of 
the T2 group  decreased throughout the treatment, being lower at the end of the treatment in all the 
measured points except for the left dorsal point, where a marked increase in hydration was observed 
after a month and a half of treatment (Figure 7).  
Figure 7.  Evolution of hydration of the different points of the foot over the study time according to the 
test group. 
Although descriptively variations in skin hydration are observed depending on the treatment applied, 
only the area of the "Outer Arch" of the right p-ie showed significant differences between groups, both 
at one and a half months of treatment (p-value=0.028) where the T1 group presented a lower 
hydration, and at its completion (p-valor=0.002), with the  T2 group having the lowest hydration (Table 
1-1). After the pairwise comparison, statistically significant differences  were obtained between the T0 
and T1 groups at one and a half months  (p-value=0.016),  observing that the control group had greater 
hydration, and between the T0 and T1 groups (p-value=0.021) and  the T0 and T2 groups (p-
value=0.003)   at the end of treatment  where, as in the month and a half, the control group had greater 
hydration (Table 1-2). 
 
 
 
 
GROUPS 
Digital plant 
Dorsal 
Inner Arc 
External Arc 
Heel 
Right Foot 
Left Foot 
No treatment (control) 
Treatment 1% 
Treatment 0.5% 

  
 
 
 
Table 11. Results of the Kruskal-Wallis tests for the analysis of hydration as a function of time and 
treatment applied. 
 
R: right; L: Left; G: group; G. T0: control group; G T1: T1 treatment group; G T2: T2 treatment group; 
Sd: standard deviation; *: significant; **: Highly significant. 
Table 12. Mann-Whitney U test with Bonferroni p-value correction for outer arch hydration data at one 
and a half months and completion of treatment. 
 
 
p-value 
Adjusted p-value 
External arch after a 
month and a half of 
treatment 
No treatment (T0) 
Treatment 1% (T1) 
0.008** 
0.023* 
No treatment (T0) 
Treatment 0.5% (T2) 
0.092 
0.276 
Treatment 1% (T1) 
Treatment 0.5% (T2) 
0.476 
1.000 
External arch at the 
end of treatment 
No treatment (T0) 
Treatment 1% (T1) 
0.008** 
0.024* 
No treatment (T0) 
Treatment 0.5% (T2) 
0.001* 
0.002** 
Treatment 1% (T1) 
Treatment 0.5% (T2) 
0.211 
0.632 
*: meaningful; **: Highly significant. 
 
 
 
 
 
 
 
 
 

  
 
 
 
Bibliography 
 
1. 
Spanish Agency for Medicines and Health Products (AEMPS). Compendium of European 
standards and guidelines relating to cosmetic products for the application of the regulation 
1223/2009 [Internet]. 2019. Available from: https://www.aemps.gob.es 
2. 
Quételet ALJ. Fisica sociale ossia svolgimento delle facolta dell’uomo. 1869.  
3. 
Léveque J, Rigal J. IMPEDANCE METHODS FOR STUDYING SKIN MOISTURIZATION. SOC 
COSMETIC CHEMISTS. 1983;34(8):419–28.  
4. 
Dyer J, Foy V. Revealing the unseen a review of woodslamps in dermatology. J Clin Aesthet 
Dermatol. 2022;15(6):25–30.  
5. 
Pérez Pico AM, Mingorance Álvarez E, Pérez Luque C, Mayordomo Acevedo R. Adquisición de 
competencias para el diagnóstico de onicomicosis mediante entrenamiento práctico 
podológico preclínico y clínico. Educ Med Super. 2019;33(4):37–49.  
6. 
Burillo A, Moreno A, Salas C. Procedimientos en microbiología clínica. 2006.  
7. 
Carson CF, Hammer KA, Riley T V. Melaleuca alternifolia (tea tree) oil: A review of antimicrobial 
and other medicinal properties. Clin Microbiol Rev. 2006;19(1):50–62.  
8. 
Lee CJ, Chen LW, Chen LG, Chang TL, Huang CW, Huang MC, et al. Correlations of the 
components of tea tree oil with its antibacterial effects and skin irritation. J Food Drug Anal 
[Internet]. 2013;21(2):169–76. Available from: http://dx.doi.org/10.1016/j.jfda.2013.05.007 
9. 
Groot AC De, Schmidt E. Tea tree oil: contact allergy and chemical composition. Contact 
Dermatitis. 2016; 75:129–43.  
10. 
Rudbäck J, Bergström MA, Börje A, Nilsson U, Karlberg AT. α -Terpinene, an Antioxidant in Tea 
Tree Oil, Autoxidizes Rapidly to Skin Allergens on Air Exposure. Chem Res Toxicol. 
2012;25(3):713–21.  
11. 
Cox SD, Mann CM, Markham JL, Bell HC, Gustafson JE, Warmington JR. The mode of 
antimicrobial action of the essential oil of Melaleuca alternifolia (tea tree oil). J Chromatogr A. 
2000; 1406:170–5.  
12. 
Yang S ah, Jeon S kyung, Lee E jung, Im N kyung, Jung J young, Lee I seon. Bioactivity and 
Chemical Composition of the Essential oil of Tea Tree (Melaleuca alternifolia). Journal of Life 
Sciences. 2008;18(12):1644–50.  
13. 
Yu D, Wang J, Shao X, Xu F, Wang H. Antifungal modes of action of tea tree oil and its two 
characteristic components against Botrytis cinerea. J Appl Microbiol. 2015; 119:1253–62.  
14. 
Winkelman WJ. Aromatherapy, botanicals, and essential oils in acne. Clin Dermatol [Internet]. 
2018;36(3):299–305. Available from: https://doi.org/10.1016/j.clindermatol.2018.03.004 
 

  
 
 
 
 

VICERRECTORADO DE INVESTIGACIÓN Y TRANSFERENCIA 
 Campus Universitario 
NºRegistro: 195//2022 
 Avda de Elvas, s/n 
 06006 - BADAJOZ 
 Tel.: 924 28 93 05 
 Fax: 924 27 29 83 
D. JOÃO NUNO MEIRELES DA SILVA GONÇALVES RIBEIRO, SECRETARY OF 
THE BIOETHICS AND BIOSECURITY COMMISSION OF THE UNIVERSITY OF 
EXTREMADURA
REPORTS:That once analyzed by this Commission, in its session held on 12/19/2022, the 
request for a Thesis Project entitled "Use of essential oils as natural therapies against 
dermatophytosis and new diagnostic techniques" , whose Director is Mrs. Raquel Mayordomo 
Acevedo and whose Principal Investigator is Mrs. Julia Villar Rodríguez has unanimously 
decided to positively value the aforementioned project considering that it complies with 
essential ethical standards, complying with current regulations for this purpose.
And so that it is recorded and has the appropriate effects, I sign this report
V.º B.º
Signed.: Javier de Francisco Morcillo 
President by delegation of the Bioethics 
and Biosafety Commission
MEIRELES DA 
SILVA GONCALVES 
RIBEIRO JUAN 
NUNO - 
08844642S
Firmado digitalmente 
por MEIRELES DA SILVA 
GONCALVES RIBEIRO 
JUAN NUNO - 
08844642S 
Fecha: 2022.12.21 
18:07:37 +01'00'
DE FRANCISCO 
MORCILLO 
JAVIER - 
28946338X
Firmado digitalmente por DE 
FRANCISCO MORCILLO JAVIER - 
28946338X 
Nombre de reconocimiento (DN): 
c=ES, 
serialNumber=IDCES-28946338X, 
givenName=JAVIER, sn=DE 
FRANCISCO MORCILLO, cn=DE 
FRANCISCO MORCILLO JAVIER - 
28946338X 
Fecha: 2022.12.22 14:29:39 +01'00'

Anamnesis study creams 
You will then be asked a series of questions to find out your previous stage before starting the study. In 
this study, the possibility of suffering from fungal infections in the feet and its subsequent treatment 
with natural therapies such as essential oils will be evaluated. 
 
 
1. 
Reference  
 
 
2. 
  Participant's Name   
 
 
3. 
Age  
 
 
4. 
 Contact phone number  
 
 
5. 
Dedication?   
Mark only one oval. 
 
Student 
Unemployed 
Worker  
Other 
6. 
If you  answered student in the previous answer,  please tell us that you are studying: 
 
 
7. 
If you  answered worker in the previous answer,  please tell us what you work  for: 
 
 
8. 
  Foot No.   
 
 
9. 
If you have any medication/drug allergies please set it to continuation.  If you don't have anything, say 
NO. 
 
 
10. 
  If you take any medication, please put it below. If you don't take anything put NO. 
 
 
11. 
Weight  
 
 
12. 
Height  

 
 
13. 
  Have you had any of these on your feet before?  
Select all that apply. 
 
Discoloration changes  
Excessive sweating Wounds 
Cracks between the toes  
Cracks in another part of the foot 
Maceration 
None of the above 
 
14. 
 Do   you have any of these on  your feet today?  
Select all that apply. 
 
 Discoloration  changes  
Excessive sweating Wounds 
Cracks between the toes  
Cracks in another part of the foot 
Maceracion 
None of the above 
 
15. 
Have you had any of these symptoms  on your feet before?  
Select all that apply. 
 
Itching between the toes  
Itching on the bottom of the foot 
Generalized itching of the foot 
Stinging between the toes  
Stinging of the sole of the foot   
Generalized stinging of the foot 
Burning between the toes  
Burning of the sole of the foot 
Widespread burn on the foot  
None of  the above 

16. 
 Do you have any of these symptoms on your feet   today?  
Select all that apply. 
 
Itching between the toes  
Itching on the bottom of the foot 
Generalized itching of the foot 
Stinging between the toes  
Stinging of the sole of the foot   
Generalized stinging of the foot  
Burning between the toes  
Burning of the sole of the foot  
Widespread burn on the foot 
None of  the above 
 
17. 
Have you been diagnosed with fungal infections on your feet before?  
Mark only one oval. 
 
Yes
No 
18. 
Have you used drug treatment to treat fungal infections in your feet?  
Mark only one oval. 
 
Yes 
No 
19. 
  If your previous answers  were "YES",  could you please tell us  what treatment it was? 
 
20. 
 Have you   ever been examined  by a podiatrist?  
Mark only one oval. 
 
Yes
No 
 
 

21. 
 Have you  ever  had a mycological foot culture?  
Mark only one oval. 
 
Yes
No 
22. 
  If the previous answer was  "YES", please  indicate why 
 
 
23. 
How often do you wash your feet?  
Mark only one oval. 
 
Daily  
2 times a day 
Less 
24. 
 How often do you examine your feet?  
Mark only one oval. 
 
Daily  
2 times a day 
Less 
25. 
What kind of sock do you wear?  
Mark only one oval. 
 
Cotton 
Polyester  
Don’t know 
26. 
How often do you change your socks?  
Mark only one oval. 
 
Daily  
2 times a day 
Less 

27. 
Do you wear  breathable shoes?  
Mark only one oval. 
 
Yes 
No 
28. 
What kind of footwear do you usually wear on a daily basis?  
Mark only one oval. 
 
Sneakers  
Boots/Trekking Boot/ Work/Protective Boots 
Other Style 
29. 
How many times a day do you change your shoes?  
Mark only one oval. 
 
I usually wear  the same shoes  all  day  
I change shoes 2 times a day  
I change my shoes  according to the  activity  
I go every  day  with the same shoes   
30. 
Do you play any sports? If  yes,  tell us which one  and if you don't practice sport,  put "NO"  
 
 
31. 
 If you    play a sport, do you usually have  specific shoes just for it? 
Mark only one oval. 
 
Yes 
No 
32. 
Do you wear protection (flip flops) when using showers in gyms, spas, clubs Sports? 
Mark only one oval. 
 
Yes 
No