This randomized controlled study, which was multicentre, comparative, investigator blinded, and open label, was approved by the ethics committee of the principal clinical trial centre (Military Hospital of Tunis, Tunisia) on 22 September 2016. The study was conducted in accordance with the principles of the Declaration of Helsinki 2013, Good Clinical Practice, and European Union Directive 2001/20/EC.
The entire study took place in two clinical trial facilities in Tunis (Tunisia), specialized in treatment of skin and nail disorders. Recruitment was performed by the Principal Investigator (dermatologist) of each trial centre and continued from 26 October 2016 (first patient, first visit) to 26 August 2017 (last patient, last visit). The trial was registered at ClinicalTrials.gov with the following code: NCT03382717.
Inclusion and Exclusion Criteria
Patients (> 18 years) were included after confirmed diagnosis of superficial or light-to-moderate disto-lateral onychomycosis (without matrix involvement, the infected area being smaller than 2/3 of the nail surface) on at least one great toe nail. Potassium hydroxide (KOH) staining was used to confirm the diagnosis . Fungal culture was performed on samples of KOH-positive subjects to characterize dermatophyte infection. However, the outcome of these fungal cultures did not restrict subject inclusion since false-negative results regularly occur in clinically confirmed cases .
Beside positive diagnosis, patients must have stopped any systemic and/or topical antifungal treatment for at least 6 and 3 months, respectively, before inclusion. Finally, female subjects of childbearing potential needed to use an accepted contraceptive regimen at least 12 weeks prior to study start, during the study, and at least 1 month after study end.
Exclusion criteria were: non-compliance with the protocol, enrolment in another clinical trial during the test period, pregnant (or planning to be) or nursing women, known allergy to one of the ingredients of both products, patients suffering from serious or progressive diseases (uncontrolled diabetes, peripheral circulatory disease, HIV, psoriasis, lichen planus, immunosuppressive disorders), and patients with other skin diseases in the studied zone.
Sample Size, Informed Consent, Randomization, and Baseline Data
Based on previous available clinical data, a sample size of 100 subjects was calculated to allow correct statistical comparison between the test product and reference. Based on this information, 102 eligible subjects were recruited by the study staff and randomly allocated to two groups. Prior to the study, a randomization list was calculated by an external statistician using SAS software (version 9.4). For this purpose, block randomization with a block size of two was used.
Each subject received oral and written information concerning the studied product, its nature, and the duration and conditions of the study. Written consent was obtained before any study-specific procedures were performed in accordance with the Helsinki Declaration.
Following this informed consent, a patient screening number was assigned to each patient by the responsible investigator. A randomization list was provided prior to the start of the study. A unique randomization number attributed each included patient to one of the treatment groups. Baseline demographic data were collected on gender, age, height, weight, blood pressure parameters, and medication use.
Discernible differences in the product properties (e.g. different bottle, odour) and in the administration process allowed patients to recognize both trial products. Therefore, blinding and unbiased evaluation was guaranteed by making digitalized macro-photographs of the toe nail, which were in turn analysed by two blinded evaluators. The detailed procedure is described below in “Evaluation of clinical efficacy”.
Study Medication, Dosage and Administration
The test product was supplied in glass bottles (with a brush applicator) by Oystershell Laboratories (Ghent, Belgium). This product consists of acetic acid (active ingredient), a peelable film-forming polymer (polyurethane), water, peppermint oil (penetration enhancer, solvent, perfume), an anti-oxidant (octyl gallate), preservatives, acetylated lanolin alcohols, and biotin.
The amorolfine nail lacquer reference (Loceryl®; available in a glass bottle) was provided by the Principal Investigator. This medicated nail lacquer contains 5% amorolfine (as amorolfine hydrochloride in ethanol, triacetin, butyl acetate, ethyl acetate, and ammonium methacrylate polymer).
The test product was applied once daily with the brush, covering the complete (cleaned) nail. After 24 h, the water-resistant film was removed by stripping and a new layer was applied. If new growth appeared, the nail was trimmed using a nail clipper.
Amorolfine was applied once a week with a reusable spatula (supplied with the product). Prior to use, the nail was filed and cleaned with isopropanol wipes.
Evaluation of Clinical Efficacy
Patients were treated with the test product or reference, respectively, for a period of 180 days. Onychomycosis evolution was evaluated at distinct time points, day (D) 30, D60, D120, and D180, and compared with D0 (baseline). The primary objectives were to assess variation in the % of healthy surface of the great toenail at the end of the study (D180) compared with baseline in both treatment groups. Diagnosis was performed using digital image analysis . For each photograph, a blinded dermatologist traced the healthy surface. Next, a second evaluator, also blinded, determined the percentage of healthy surface and assigned the following scores: 100% healthy surface, > 66.6% healthy surface, 33.3–66% healthy surface, and < 33.3% healthy surface.
Secondary objectives implied evaluation of the following parameters:
Clinical efficacy against onychomycosis of the great toenail at D30, D60, and D120.
Microbiological efficacy of the product (KOH staining method).
Impact on the quality of life (QoL) of the subjects using the NailQoL questionnaire, with assessment on D0, D60, and D180 .
Product efficacy, tolerance, and acceptability by subject’s self-assessment.
Impact on onycholysis, nail dystrophy, nail discolouration, nail thickening, and onychomycosis evolution at D30, D60, D120, and D180.
At each visit, the local and global tolerance (collection of all adverse events and subjective signs) were evaluated. In addition, all patients were asked to report adverse events in a log book. Study staff investigated all adverse events and determined the relationship to the treatment.
A subjective questionnaire was included to evaluate product usability and functionality of the test product (completion on days 30, 60, 120, and 180). Questions were related to packaging, cosmetic effects, overall satisfaction, tolerance, product application, and intention to purchase.
Clinical efficacy and safety were evaluated in the intent-to-treat (ITT) population, including all subjects eligible for the study and who received at least one application of the test product and had at least one post-dose efficacy evaluation. Briefly, continuous data were summarized by their mean, standard deviation (SD), median, and minimum and maximum. Categorical data were summarized by frequencies and percentages.
Mean absolute changes in % healthy surface from baseline (D0) at day 180 between the test product and reference were compared with an independent t test after having verified the assumptions of normality (QQ-plot) of the differences. Additionally, changes in mean % healthy surface from baseline in function of visit for subjects treated with test product versus reference product were studied in more detail using generalized linear mixed effects models.
Mean absolute changes in NailQol global score from baseline (D0) at day 180 between test product and reference were compared with an independent t-test after having verified the assumptions of normality (QQ-plot) of the differences.
To compare changes in nail dystrophy, discolouration, and nail thickening between baseline and day 180, five categories were reduced to two (none to slight versus moderate to severe). The same was done for healthy aspect of the nail. The McNemar test for paired data was used to test if there was a change in nail dystrophy, discolouration, and nail thickening between baseline and day 180.
All descriptive and statistical analyses were performed in R version 3.3.1. (R development core team, 2017). p < 0.05 was considered statistically significant. No imputation of missing data was performed. The amount of missing data is presented in the tables wherever appropriate.
Study data were collected between October 2016 and August 2017. In total, 102 subjects were randomized into the study (n = 52, test product group; n = 50, reference group). For two patients in the test product group and one patient in the reference group, post-dose efficacy and safety data were not available (lost to follow-up). In total, 99 subjects (n = 50, test product group; n = 49, reference group) were included in the intention-to-treat (ITT) population. A CONSORT flow chart is shown in Fig. 1. A summary of demographic characteristics is presented in Table 1.
Prior to product application (D0), no significant differences were found between the two treatment groups for average healthy surface, secondary clinical parameters, and average NailQoL score.
Direct detection of fungal infection with the KOH staining method was positive for all recruited subjects, except for one patient in the test product group, showing a false-negative result. Indeed, fungal culture was positive for this patient, and for this reason, the Principal Investigator decided to include this patient. Consequent fungal culture was positive for a majority of the subjects (67% test product group, 52% reference group), with Trichophyton (T.) rubrum being the most common pathogen (88.6% and 80.8% for test product and reference, respectively). Other dermatophytic fungi were also detected: T. mentagrophytes (5.7%, test product group; 3.9%, reference group) and Microsporum canis (3.9%, reference group). A few non-dermatophytic fungi were also detected: Aspergillus niger (7.7%, reference group), Aspergillus flavus (2.9%, test product group), and Candida albicans (2.9%, test product group; 3.9%, reference group), respectively.