International Journal of

Environmental Research
and Public Health

Article
Efficacy of the Treatment of Plantar Warts Using 1064 nm Laser
and Cooling
Elena de Planell-Mas 1 , Blanca Martínez-Garriga 2 , Miguel Viñas 2 and Antonio J. Zalacain-Vicuña 1, *

1 Department of Clinical Sciences, School of Podiatry, Faculty of Medicine and Health Sciences,
University of Barcelona, L’Hospitalet de Llobregat, 08907 Barcelona, Spain; [email protected]
2 Department of Pathology & Experimental therapeutics, Faculty of Medicine and Health Sciences,
University of Barcelona, IDIBELL, L’Hospitalet de Llobregat, 08907 Barcelona, Spain;
[email protected] (B.M.-G.); [email protected] (M.V.)
* Correspondence: [email protected]

Abstract: Cutaneous plantar warts may be treated using several optional methods, with the use
of laser surgery having increased in the last few years. This work examined the efficacy of laser
treatment combined with simple cooling to reduce pain. The cure rate was approximately 84%. There
were no significant differences in the efficacy of treatment for different viral genotypes. The laser
parameters were 500 msec pulses, 30 W of power, and a fluence of 212 J/cm2 delivered in up to four
sessions. Successful treatment was achieved after an average of 3.6 sessions.

Keywords: plantar warts; laser treatment; novel technique; cooling; viral genotype





 1. Introduction
Citation: de Planell-Mas, E.; Cutaneous warts are non-malignant, intra-epidermal tumors that include common
Martínez-Garriga, B.; Viñas, M.; warts (verrucae vulgaris), plantar and palmar warts (verrucae plantares et palmares), flat
Zalacain-Vicuña, A.J. Efficacy of the warts (verrucae planae), and others [1]. Among them, plantar and palmar warts have the
Treatment of Plantar Warts Using highest prevalence [2]. Currently, warts are diagnosed based on clinical findings. While
1064 nm Laser and Cooling. Int. J.
they occasionally disappear spontaneously or remain asymptomatic for long periods of
Environ. Res. Public Health 2022, 19,
time [3,4], in most cases, treatment is sought.
801. https://doi.org/10.3390/
Warts are most commonly caused by the human papilloma virus (HPV) [5], with
ijerph19020801
cutaneous warts typically caused by HPV-2, -3, -7, -10, -27, and -57 (genus α), HPV-4, -60,
Academic Editor: Jaeho Cho and -65 (genus γ), and HPV-1 and -63 (genus µ) [2,6]. Of these, HPV-2, -27, -57, and HPV-1
are associated with plantar warts [6–8].
Received: 3 December 2021
The treatment of plantar warts is a challenge for podiatrists. Current therapeutic
Accepted: 10 January 2022
Published: 12 January 2022
strategies include chemotherapy (salicylic acid, glutaraldehyde, silver nitrate) and cyto-
statics (fluoracyl or bleomycin sulfate), but also cryotherapy, photodynamic therapy, and
Publisher’s Note: MDPI stays neutral surgery [9–11]. Moreover, laser therapy has also been proposed and has become an in-
with regard to jurisdictional claims in dispensable therapeutic modality since its introduction in dermatology, along with the
published maps and institutional affil-
development of selective photothermolysis (use of light energy to selectively treat elements
iations.
of the skin) [12].
Four general groups of visible-wavelength lasers are used in dermatology: pulsed
lasers in the green and yellow millisecond domain, used to treat vascular malformations,
hemangiomas, scars, photo-aging, proliferative lesions, and epidermal pigmented lesions;
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
pulsed lasers in the red and near-infrared millisecond domain, used for the treatment
This article is an open access article
of hypertrichosis and pigmented and deep venous lesions; low-power continuous green
distributed under the terms and or yellow lasers, used for the treatment of superficial telangiectasias and for the photo-
conditions of the Creative Commons coagulation of lesions; and Q-switch lasers, used to remove tattoos and treat dermal
Attribution (CC BY) license (https:// melanocytosis, drug hyperpigmentation, and many pigmented lesions [13].
creativecommons.org/licenses/by/ The photothermal effect is the best known. The wavelength determines the penetration
4.0/). into the tissues based on the absorption and scattering of light. Temperature increases

Int. J. Environ. Res. Public Health 2022, 19, 801. https://doi.org/10.3390/ijerph19020801 https://www.mdpi.com/journal/ijerph
Int. J. Environ. Res. Public Health 2022, 19, 801 2 of 9

when the power density increases, thus causing different tissue effects. As the temperature
rises, the effects may begin with a transient hyperthermia, and subsequently, desiccation,
protein denaturation and coagulation, tissue coagulation fusion, then tissue vaporization,
and finally, its charring [14].
The 1064 nm laser seems to be suitable for wart treatment, thanks to the generation
of a hyperthermic environment that has been shown to be effective in fighting HPV [15].
Histological studies have shown the coagulation and destruction of blood vessels in the
papillary dermis in the wart region after laser irradiation [11].
Here, we report the clinical effectiveness of a procedure based on the use of laser at
a wavelength of 1064 nm, in combination with cooling in order to reduce pain. We also
consider the relation between the genotype of the etiologic virus and the effectiveness of
laser treatment.

2. Materials and Methods

2.1. Design of the Work and Ethical Aspects
This was a prospective, observational, open study carried out at our center and without
a control group. The main purpose was to investigate the efficacy of the 1064 nm laser in
the treatment of plantar warts.
The study was performed in accordance with the recommendations of the Declaration
of Helsinki. The protocol was previously approved by the Bioethics Committee of the
University of Barcelona (IRB00003099). Informed consent was obtained from all patients
who participated in the study.

2.2. Patients
Thirty-two patients clinically diagnosed with plantar warts were recruited in the laser
unit of the Hospital de Podología de la Universidad de Barcelona, Spain. The clinical signs
considered were the presence of divergent skin lines, small dark lesions, and hyperkeratosis.
Wart tissue does not retain dermatoglyphics, and the application of compression laterally
(pinch) produces pain [16]. The morphological characteristics of the papilloma lesions
were carefully observed by dermatoscopy [17]. Inclusion criteria were male and female
patients who had been clinically diagnosed with plantar warts not treated topically in the
previous 3 months. Patients with neuropathy and vascular compromise were excluded.
The recorded data included sex and age—with the patients classified into three groups
(4–11 years, 12–20 years, and >21 years); the location of the warts on the right or left foot,
heel (internal or external aspect), midfoot (internal or external aspect), forefoot (first, second,
third, fourth, or fifth radius), and toes (first, second, third, fourth, or fifth); the time elapsed
between the moment when the patient first became aware of the wart’s presence and when
medical assistance was sought (≤6 months, 6–12 months, >12 months, or unknown); and
the number of warts/patient.

2.3. Laser Instrumentation

The laser instrument was a 1064 nm laserS30 PODYLASTM (INTERmedic), Barcelona
(Spain), and the parameters were as follows: pulse 500 msec, pause 1.0 sec, potency 30 W,
and fluence 212 J/cm2 . The minimal effective energy was 120 J, and the exposure time was
4 sec. These parameters were established after experimental work had been performed
on cadaver specimens to evaluate the effect of vaporization produced by the laser on the
area treated. The test method consisted of mimicking the therapeutic irradiation of a spot
that was 4.0 mm in diameter, located in regions where warts usually appear (metatarsal
and heel). A 1 cm3 sample of the vaporized region was taken, cut longitudinally, and
the depth of vaporization was measured, with a depth between 6 and 8 mm consistently
determined [18,19].
 2.4. Cutaneous Warts Treatment
The treatment protocol is summarized in a flow diagram shown in Figure 1. Initially
Int. J. Environ. Res. Public Health 2022, 19, 801 3 of 9
(t 0), patients diagnosed with plantar warts completed a questionnaire, and their data
were registered. The region surrounding the warts was disinfected using 1% chlorhexi-
dine, and the hyperkeratinic surface was removed using a scalpel.
2.4. Cutaneous Wartspainted
The wart was Treatment
with India ink for targeted laser absorption [20]. Before the
wart was irradiated, the area
The treatment protocol is was cooled to reduce
summarized pain.
in a flow Cooling
diagram was achieved
shown in Figureby
1. placing
Initially
a(t0),
frozen
patients diagnosed with plantar warts completed a questionnaire, and their dataHun-
non-colored gel (Gel UltrasonidoTransparente, Versus Medico Kft Budapest were
gary) on a glass
registered. slide, which
The region was then
surrounding thecovered with
warts was a coverslip
disinfected and 1%
using frozen using an ethyl
chlorhexidine, and
chloride spray. The surface
the hyperkeratinic frozen slide was placed
was removed on the
using wart prior to its irradiation (Figure 2).
a scalpel.

Figure 1. Flux diagram for the diagnosis and laser treatment of plantar warts.
Figure 1. Flux diagram for the diagnosis and laser treatment of plantar warts.
The wart was painted with India ink for targeted laser absorption [20]. Before the wart
was irradiated, the area was cooled to reduce pain. Cooling was achieved by placing a
frozen non-colored gel (Gel UltrasonidoTransparente, Versus Medico Kft Budapest Hungary)
on a glass slide, which was then covered with a coverslip and frozen using an ethyl chloride
spray. The frozen slide was placed on the wart prior to its irradiation (Figure 2).
In addition to the anti-pain effect, the use of the slide improves the uniformity of the
wart surface. The laser beam is directed at an angle of 90◦ , which allows for an optimal,
uniform focal distance. The hand piece of the laser had a spot diameter of 4 mm. For a
wart surface >4 mm in diameter, the area was divided to cover the full surface (Figure 3).
After laser treatment, the region was carefully observed using a dermatoscope. In the case
 Int. J. Environ. Res. Public Health 2022, 19, 801 4 of 9

of persistent papillae, irradiation was repeated until the subcutaneous tissue was visible.
Int. J. Environ. Res. Public Health 2022,
If a19, x FORexudate
serous PEER REVIEW
developed due to vaporization, antiseptic solution (povidone iodine)
and a protective bandage were applied. Finally, if the plantar wart was in a pressure area, a
felted offloading device was applied to prevent overload and support.

Figure 2. Wart treatment using a heel-targeted laser and in situ cooling.

In addition to the anti-pain effect, the use of the slide improves the unifo
wart surface. The laser beam is directed at an angle of 90°, which allows for
uniform focal distance. The hand piece of the laser had a spot diameter of
wart surface >4 mm in diameter, the area was divided to cover the full surfac
After laser treatment, the region was carefully observed using a dermatoscope
of persistent papillae, irradiation was repeated until the subcutaneous tissue
If a serous exudate developed due to vaporization, antiseptic solution (povid
and a protective bandage were applied. Finally, if the plantar wart was in a pr
aFigure
felted
Figure
offloading
2. Wart
2. Wart
device
treatment
treatment using ausing
was applied
a heel-targeted
heel-targeted
to situ
prevent
laser and inlaser
overload and support.
and in situ cooling.
cooling.

In addition to the anti-pain effect, the use of the slide improves the uniform
wart surface. The laser beam is directed at an angle of 90°, which allows for an
uniform focal distance. The hand piece of the laser had a spot diameter of 4 m
wart surface >4 mm in diameter, the area was divided to cover the full surface (F
After laser treatment, the region was carefully observed using a dermatoscope. In
of persistent papillae, irradiation was repeated until the subcutaneous tissue wa
If a serous exudate developed due to vaporization, antiseptic solution (povidon
and a protective bandage were applied. Finally, if the plantar wart was in a press
a felted offloading device was applied to prevent overload and support.

Figure
Figure 3. Plantar
3. Plantar wart
wart with with a diameter
a diameter >4 mm.ofSimulation
>4 mm. Simulation ofofthe
the distribution thedistribution of the las
laser application
zones
zones ensuring
ensuring coverage
coverage of the
of the whole whole lesion.
lesion.

Patients were instructed to wash and dry the lesion and to apply antiseptic (povidone
iodine) Patients
daily usingwere instructed
a dressing in case antoexudate
wash and dry the lesion and to apply antisept
developed.
iodine) dailyexamination
A follow-up using a dressing in case
was performed onean exudate
week developed.
after treatment to clinically evalu-
ate the lesion (Figure 4), remove surface hyperkeratosis,
A follow-up examination was performed one week after and monitor the eventual reap- to cli
treatment
pearance of dermatoglyphics. Treatment was considered to be successfully finalized when
uate the
papillae werelesion (Figure
no longer visible4), remove
under surface Ifhyperkeratosis,
dermatoscopy. and monitor
small ulcers were observed, treat- the ev
pearance
ment aimed atoftissue
dermatoglyphics. Treatment
regeneration was prescribed, was
and the considered
patients wouldtobebe successfully fin
re-examined
weekly until were
papillae a complete cure wasvisible
no longer achieved. For those
under with remaining papillae,
dermatoscopy. If smalla ulcers
new round were obs
of laser treatment was administered. A maximum of five laser treatment sessions were
ment aimed at tissue regeneration was prescribed, and the patients would be r
Figure 3. Plantar
performed. wart
If the warts with a diameter
persisted, >4treatment
alternative mm. Simulation of the distribution
was prescribed of the laser a
for the sixth visit.
weekly until coverage
zones ensuring a complete of thecure was
whole achieved. For those with remaining pap
lesion.
2.5. Viral Genotypes
round of laser treatment was administered. A maximum of five laser treatm
The
were HPV genotypes
performed.
Patients of thewarts
wereIfinstructed
the treated warts were
topersisted,
wash also
dry determined.
andalternative The to
tissue
treatment
the lesion and obtained
was
apply prescribed
antiseptic (p
after hyperkeratosis removal was used for DNA extraction following a previously de-
visit.
iodine) daily using a dressing in case an exudate developed.
A follow-up examination was performed one week after treatment to clinic
uate the lesion (Figure 4), remove surface hyperkeratosis, and monitor the event
pearance of dermatoglyphics. Treatment was considered to be successfully finaliz
 Int. J. Environ. Res. Public Health 2022, 19, 801 5 of 9

scribed phenol-chloroform-isoamyl alcohol method (Green and Sambrook, 2012). The

concentration and purity of the DNA were determined spectrophotometrically (NanoDrop
Spectrophotometer ND-1000). Thereafter, the DNA was analyzed using the SK-polymerase
chain reaction (SKPCR) method introduced by Sasagawa and Mitsuishi (2012), which de-
Int. J. Environ. Res. Public Health 2022, 19, x FOR PEER REVIEW
tects most of the common HPV types causing cutaneous warts. After DNA sequencing, the
HPV types were identified using the BLAST server (https://blast.ncbi.nlm.nih.gov/blast
(accessed on 12 August 2019) [8].

Figure
Figure 4. A4.plantar
A plantar
wart >4wart >4diameter
mm in mm inonediameter one
week after week after
treatment. treatment.
2.6. Statistics
2.5.AViral Genotypes
descriptive statistical analysis was conducted for all of the variables. Continuous
variablesThe
wereHPV
reported according of
genotypes to the
thenumber of valid
treated wartscases and the
were alsomean and standardThe tiss
determined.
deviation (SD). Categorical variables were described according to the absolute and relative
after hyperkeratosis removal was used for DNA extraction following a pr
frequencies of each category compared to the total number of relevant values (N). For
scribed
absent phenol-chloroform-isoamyl
values, their number was described by group.alcohol methodwere
Comparisons (Green
carriedand Sambrook
out using
anconcentration and statistical
ANOVA and a bilateral purity significance
of the DNA wereAll determined
level of 0.05. spectropho
analyses were carried
out on the data set, using all available information, in accordance
(NanoDrop Spectrophotometer ND-1000). Thereafter, the DNA was with the intention to treatanalyze
criteria. The statistical analyses were performed using the SAS program, version 9.2.3.
SK-polymerase chain reaction (SKPCR) method introduced by Sasagawa an
3.(2012),
Results which detects most of the common HPV types causing cutaneous
DNA sequencing,
This section the into
may be divided HPV types It should
subheadings. were provide
identified
a conciseusing the BLA
and precise
description of the experimental results and their interpretation.
(https://blast.ncbi.nlm.nih.gov/blast (accessed on 12 August 2019) [8].
The 32 patients included in the study had an average age of 36.19 (±17.77) years, and
65.6% were female. A single wart was treated in 84.4% of the patients, two warts in 9.4%,
2.6.three
and Statistics
or more warts in 6.2%, for a total of 41 warts. The genotypes identified in the
41 samples were HPV-1 (14.63%), HPV-2 (19.51%), HPV-27 (17.07%), HPV-57 (41.46%), and
A descriptive statistical analysis was conducted for all of the variables.
HPV-65 (7.32%).
variables were reported
The effectiveness of the laseraccording
treatment ofto the number
plantar of valid
warts is reported in cases
Table 1,and the mean
which
ard deviation
shows (SD).
the healing rate Categorical
according variables
to the viral genotype.were
Among described according
the different genotypes,to the a
the cure rate
relative differed and of
frequencies was the lowest
each for patients
category comparedwith to
warts
thecaused by the HPV-2
total number of relevan
genotype (p = 0.0315). The differences in the rates of change over the course of the treatment
For absent values, their number was described by group. Comparisons were
were small.
using
CureanwasANOVA
achieved inand
85.5%a bilateral statistical
of the laser-treated significance
plantar warts (Tablelevel of 0.05.
2). There were All an
carried in
differences outtheon the of
healing data
wartsset, using all
at different available
locations; information,
up to 100% in in
cure for warts accordance
the heel wi
area, followed by those located on the toes (92.3%), midfoot (75%), and forefoot
tion to treat criteria. The statistical analyses were performed using the SAS p (69.2%).
An analysis of the warts according to their diameter and response to treatment is
sion
shown in 9.2.3.
Table 3. Cure was achieved in 83.3% of the warts with a diameter ≤4 mm. For
larger warts, the cure rate was quite similar at 87%.
3. Results
This section may be divided into subheadings. It should provide a conc
cise description of the experimental results and their interpretation.
Int. J. Environ. Res. Public Health 2022, 19, 801 6 of 9

Table 1. Warts healed by laser treatment.

Total HPV-1 HPV-2 HPV-27 HPV-57 HPV-65 p-Value

Variable
(n = 41) (n = 6) (n = 8) (n = 7) (n = 17) (n = 3) (1)
Yes 41 6 8 7 17 3
Warts
Total 41 6 8 7 17 3
Yes 35 6 4 6 16 3
Healed warts No 6 4 1 1
Total 41 6 8 7 17 3 0.0315
Visit 2 7 1 2 1 3
Visit 3 10 1 1 2 5 1
Visit 4 12 2 3 5 2
Healing achieved at:
Visit 5 1 1
Visit 6 5 2 1 2
Total 35 6 4 6 16 3 0.8653
Yes 4 3 1
Change of treatment No 37 6 5 7 16 3
Total 41 6 8 7 17 3 0.0608
Yes 2 1 1
Treatment No 39 6 7 6 17 3
abandonment Total 41 6 8 7 17 3 0.4525

Table 2. Healed warts according to their location.

Total Forefoot Midfoot Toes Heel

Variable p-Value (1)
(n = 41) (n = 13) (n = 4) (n = 13) (n = 11)
Total 41 13 4 13 11
Warts
Yes 41 13 4 13 11
Yes 35 9 3 12 11
Healed warts No 6 4 1 1
Total 41 13 4 13 11 0.1422
Visit 2 7 2 2 2 1
Visit 3 10 1 3 6
Visit 4 12 5 1 4 2
Healing achieved at:
Visit 5 1 1
Visit 6 5 1 2 2
Total 35 9 3 12 11 0.3636
Yes 4 2 1 1
Change of treatment No 37 11 3 12 11
Total 41 13 4 13 11 0.4275
Yes 2 2
Treatment abandonment No 39 11 4 13 11
Total 41 13 4 13 11 0.2098

Table 3. Healed warts according to their diameter.

Variable Total ≤4 mm >4 mm p-Value (1)

Warts Yes
Total 41 18 23
Yes 35 15 20
Healing warts No 6 3 3
Total 41 18 23 0.7446
Visit 2 7 4 3
Visit 3 10 4 6
Visit 4 12 6 6
Healing achieved at:
Visit 5 1 1
Visit 6 5 5
Total 35 15 20 0.2029
Yes 4 2 2
Change of treatment No 37 16 21
Total 41 18 23 0.7959
Yes 2 1 1
Treatment abandonment No 39 17 22
Total 41 18 23 0.8586
Int. J. Environ. Res. Public Health 2022, 19, 801 7 of 9

4. Discussion
The cooling method used in conjunction with laser enabled the use of a focal length
appropriate for the targeted spot on the skin surface. The optimal laser parameters were:
500 msec pulse, 30 W of power, and a fluence of 212 J/cm2 . With these settings, pulse
emission resulted in the delivery of an energy of 15 J to the tissue. With an emission energy
of 120 J, treatment was effective without causing intolerable pain to the patient, and an
average of 3.6 sessions per wart were needed.
Kimura et al. [11] and Han et al. [21] reported the results obtained using a 1064 nm
laser for the treatment of skin warts, including plantar warts. In both studies, a handpiece
with a spot diameter of 5 mm was used. In the Kimura study, the application parameters
comprised a pulse duration of 15 msec and a fluence of 150–185 J/cm2 . Six sessions were
administered, with an interval of 4 weeks between sessions. The overall cure rate was 56%,
and that of plantar warts specifically was at 39% [11]. In the study of Han et al. [21], the
pulse duration was 20 msec, and the fluence was 200 J/cm2 . Treatment was administered
in four sessions, with an interval of 4 weeks between sessions. The cure rate was 96% [21].
In both studies, pain was avoided by applying ice before treatment and topical anesthetic
ointment or 1% lidocaine infiltration. Bacelieri et al. [22] used a 585 nm pulsed dye laser
treatment and obtained global cure values of 48–93% for warts located in different skin areas.
In the study of Tan et al., using the same type of laser, the cure rate was 50% in patients
with plantar warts [23]. Neither the diameter of the warts nor their zone of appearance
seemed to influence healing. According to the literature, spontaneous remission occurs in
4–8% of cases in a period of six months [24]; thus, we expect 2 or 3 spontaneous cures in the
group studied. Our results pointed out slight differences in the rate of healing, depending
on the location of the warts. A higher proportion of unsuccessful treatment (30%) was seen
in the forefoot and midfoot (25%), while in areas submitted to higher pressures such as the
heel or toes, the treatment failed at a lower proportion (0% and 7%, respectively).
Plantar warts may be caused by different HPV genotypes, as we pointed out in the
case of the patients of our service [8]. Warts of the genotype HPV-1a of the genus µ, HPV-65
of the genus γ, and HPV-27 and -57 of the genus α were the most effectively treated using
the 1064 nm laser. However, for genotype HPV-2, also of the α-genus, cure could not be
demonstrated, either because the patients abandoned treatment or surgery was necessary
due to unfavorable healing. In our patients, plantar warts caused by the HPV-2 genotype
evolved over >6 months. Whether warts differing in their genotype respond differently to
different laser parameters has not been determined and was not considered in our study
since the number of cases were not enough to obtain conclusions. Another parameter
considered was the size of the warts. In this regard, it seems that rates of healing do not
depend upon the size of the wart (Table 3). Assuming that bigger warts should also be
older, this result suggests that the time of evolution is probably not a critical condition.

5. Conclusions
Our study showed that plantar warts can be treated using a 1064 nm laser for an
average of three to four treatment sessions, and that any resulting pain can be successfully
treated using a cooling method. The treatment parameters consisted of 500 ms pulses, 30 W
power, and 212J/cm2 fluence. This protocol was particularly effective for the treatment
of the plantar wart genotypes: HPV-1a, -27, -57, and -65. Therefore, it is possible that
the efficacy of the cure may not depend only on the treatment used but also on the wart
genotype, which should be considered.

Author Contributions: Conceptualization, M.V. and A.J.Z.-V.; methodology, A.J.Z.-V. and E.d.P.-M.;
investigation, E.d.P.-M., B.M.-G., M.V. and A.J.Z.-V.; resources, M.V. and A.J.Z.-V.; data curation,
E.d.P.-M. and B.M.-G.; original draft preparation, E.d.P.-M. and A.J.Z.-V.; review and editing, M.V.;
funding acquisition, M.V. and A.J.Z.-V. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Int. J. Environ. Res. Public Health 2022, 19, 801 8 of 9

Institutional Review Board Statement: The study was conducted according to the guidelines of
the Declaration of Helsinki and approved by Ethics Committee for Research of the University of
Barcelona (Spain) (IRB00003099).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the
study. Written informed consent has been obtained from the patients to publish this paper.
Acknowledgments: We thank the study participants. The authors give thanks to the Podiatric
Hospital (Fundació Josep Finestres), Universidad de Barcelona (Barcelona, Spain) for logistic support.
Conflicts of Interest: The authors declare no conflict of interest.

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