An Investigator Blinded, Clinical Trial Assessing the Efficacy of Superfine Merino Wool Base Layer Garments (SMWBG) in Children with Atopic Dermatitis (AD) Measuring SCORAD1, EASI2, POEM3 and DSA4 Scores

Atopic Dermatitis (AD) is a chronic inflammatory skin disease, which occurs most often in children compared with the adult AD population [5]. Wool is commonly considered an allergen and trigger for exacerbation of AD. However, Super Fine Merino Wool (SMW) has not been extensively studied and may show some effect in improving AD symptoms such as itch, erythema and induration. To explore this hypothesis, we performed an investigator blinded, repeated measures, self-controlled experimental design study with Super Fine Merino Base Layer Garments (SMWBG). SMWBG were worn by 29 patients with AD between the ages of 6 to 25 years over a 15 week period. We examined the tolerability and potential therapeutic effect of SMWBG (garments worn directly against the skin) in children and young adults with atopic dermatitis (AD). Response was assessed using validated scoring scales: Severity Scoring of Atopic Dermatitis (SCORAD1), Eczema Area Severty Index (EASI2), Patient-Orientated Eczema Measures (POEM3) and a non-validated Dermatitis Severity Assessment (DSA4). Wilcoxon signed-rank tests were conducted to measure the difference between garment intervention phases. Statistically significant results were observed between the pre-garment intervention phase and the with-garment interventionphases for many of the scores. Results found that SMWBG were well-tolerated in children and young adults as an adjunctive therapy for the treatment of AD. This study was conducted in Brisbane, Australia between July 2014 and October 2015 and is Human Research Ethics Committee (HREC) approved.


Introduction
Atopic Dermatitis (AD), otherwise known as eczema is a chronic inflammatory skin disease. AD occurs most often in children and the treatment options available are more complex when compared with the adult AD population [1]. The three prescribed main treatments in AD are: emollients, topical corticosteroids (TCS) and systemic therapies. TCS and diligent emollient application remain the mainstay of AD therapy. Systemic therapy and longterm TCS use have potential problems including skin atrophy and immuno suppression. There is a need for safer and better-tolerated alternatives in the management of AD [6]. There has been a wellestablished assumption that cotton, poly-cotton and silk are the preferred garment fabric choices for people with sensitive skin [7,8]. Superfine Merino Wool (SMW) has been demonstrated to have little or no skin irritancy and has superior thermal and moisture buffering properties than other fabric options and therefore, may be suitable in patients with AD [9,10]. Previous associations with wool and the prickle sensation have been further clarified as being related to a neurological response to fiber diameter irrespective of the fiber type [11,12]. The tolerability of SMWBG in adults with AD has been addressed in a previous study by [4] in 29 adult participants [4]. This study seeks to test the following Hypotheses: a) Hypothesis 1: The fabric characteristics of SMWBG will be well tolerated as an adjunct therapy in participants with AD.

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b) Hypothesis 2: When SMWBG are worn by children and young adults, they will experience a reduction in their AD symptoms and signs.

Outcome Measures
Primary outcome measures included assessing the tolerability of SMWBG measured using AD severity scores. Secondary outcome measures included assessing AD symptoms of sleep and itch through the validated measures: Patient Oriented Eczema Measurement (POEM 3 ) and Severity Scoring of Atopic Dermatitis (SCORAD 1 ). The study design incorporated use of SCORAD 1 and EASI 2 as the validated and standardized tools to evaluate the subjects' atopic dermatitis throughout the trial. POEM 3 and participants' demographics were also recorded in this study. The study included a composite score of AD intensity, labeled by the investigators as the Dermatitis Severity Assessment (DSA 4 ) score, a novel scale for rating the severity of dermatitis. This tool utilized a simple scoring system of 0 (none) to 3 (severe) each of redness, dryness, pruritus, swelling and pain with a total score range between 0 and 15.

Participants
Participants were enrolled from a cohort of clinic patients identified with AD in Brisbane, Australia. Participants were referred from local community doctors and other dermatologists. A convenience sample of thirty-one (31) participants diagnosed with AD in accordance with the criteria of Hanifin & Rajka [13], aged 6 to 25 years (M=11.86 years of age) were enrolled in this study.
Demographics were summarized by age, gender, race, ethnicity, height and weight. Thirty-one (31) participants were recruited into the study, two (2) participants chose to withdraw for personal reasons and did not complete the study and were excluded from stastistical analysis. No participants had worn SMWBG prior to commencing the study.

Study Design
The study was conducted in Brisbane, Australia. All participants had AD as defined by Hanifin and Rajka. 13 Participants were recruited between July 2014 (Winter) and January (Summer) 2016 with the last patient completing the study in April (2016). It is important to note that seasonal variations in temperature in Brisbane were minimal during this period of time [14]. This study employed a clinical trials design whereby a with-garment intervention phase (IP) (where participants wore SMWBG for 5 weeks), measuring AD parameters, was compared with pre-garment IP and postgarment IP where participant's normal clothing was worn. This study employed a repeated measures design where each patient's outcome measures were compared between pre-and post-garment, so that individual differences could be better accounted for in the error term. To minimise the effect of washing detergent bias throughout the study, all participants washed their garments with Martha Gardner Wool Mix™ laundry detergent. The participants were instructed to wear the garments for a minimum of 6 hours per day. This time interval was chosen to avoid interference with other clothing requirements for example: school uniforms. Base layer garments were chosen as they were in direct contact with the skin.  Note: gsm is a measure of fabric weight and is reported in grams per square meter

The Intervention
Commercially sourced SMWBG were chosen as the intervention for this study. SMW is defined as wool fabric with a mean fiber diameter less than or equal to 18.5 micrometers. Garments included singlets, long and short-sleeved shirts, underwear, leggings and socks. All garments comprised 100% SMW with the exception of socks, which contain 60% 19.0 micrometer merino wool and synthetic fibers. Participants were dispensed with a maximum of four different garment groups, based on the cutaneous distribution of their AD. Garment details are included in Table 1.

Investigator Blinding Procedures
The blinded team included assessors, principal investigator and sub-investigators. The investigators were not aware of the style or number of garments dispensed to each participant. At Visit 3 the unblinded team member randomly allocated garment types to participants [15]. The purpose of the randomisation procedure was to reduce scoring bias by the investigators. The unblinded team distributed and collected the garments from participants.
Participants, care providers and non-assessors were unblinded.
Participants were asked not to disclose the garment locations to the blinded team. Garments were given to participants in opaque bags and they did not wear the study garments to their visits. The blind was maintained throughout the study. Participants were allocated garment inclusion and exclusion areas as their own control.
Participants wore the garments in areas identified by the unblinded team at Visit 3. 20 participants wore lower limb garments (pants or socks), 15 wore upper limb garments (short or long-sleeved shirt), 23 wore trunk garments (singlet or shirt).

c)
Post-Garment IP: A 5-week phase comprised of two visits (V6 and V7) to measure AD severity post garment.

Measures of AD Severity
Participant's demographics were obtained at Visit 0 (Screening). They included: age, gender, frequency of AD flares per week, clothing size and information about the fabric of garments and bedding that they usually used. In addition, the demographics contained information regarding creams, powders and medications

Results
The following section details the AD Severity Scores: SCORAD 1 , EASI 2 , DSA 4 and POEM 3 for this study. Unless otherwise stated, the comparison between the IP phases was conducted using a Wilcoxon signed rank test, to determine the statistical significance of the mean differences in scores.

POEM
A Wilcoxon signed-rank test determined that there was a statistically significant decrease in the POEM 3 total scores between the pre-garment intervention and the with-garment intervention phases (Mdn decrease= 1.67) z=-2.79, p=0.005, r=0.518. There was also a significant decrease in POEM 3 scores between the pregarment intervention and the post-garment intervention phases (Mdn decrease=2.33), z=-3.385, p<0.001, r=0.63.

EASI
The total EASI 2 score was assessed using a Wilcoxon signedrank test. Note that Participant 31's post-garment intervention phase EASI 2 scores were removed because it was identified as a significant outlier as determined by an assessment of box-plot.
The tests that showed a statistically significant decrease between the pre-garment intervention and the with-garment intervention phases (Mdn decrease=2.82 ) z=-3.712, p<0.001, r=0.70. Also, an overall statistically significant decrease in EASI 2 scores between the pre-garment intervention and post-garment intervention phases (Mdn decrease=1.15 ) z=-2.14, p=0.032, r=0.41 was demonstrated.

DSA
Finally, a Wilcoxon signed-rank test determined that there was a similar statistically significant decrease in DSA 4 scores between the pre-garment intervention and the with-garment intervention phases (Mdn decrease=1.33) z=-3.464, p<0.001, r=0.64. There was also a significant decrease in POEM 3 scores between the pregarment intervention and the post-garment intervention phases (Mdn decrease=1.17), z=-2.47, p=0.014, r=0.46.

Assessment of Safety and Tolerability
Safety and tolerability of garments were measured through the AD severity scores. There were no significant increases in mean scores, physical signs or subjective symptoms between the pre-garment IP and with-garment IP or post-garment IP for all measures of AD Severity (redness, dryness, pruritus, swelling, pain, bleeding, weeping, cracking, flaking, roughness, papulation, oozing and crust, excoriation, lichenification, or sleep loss). Twenty

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(3) (Table 3), requiring oral antibiotics, and one (1) experienced urticaria whilst patting an animal. One (1) participant experienced a serious adverse event requiring hospitalization to treat a lower respiratory tract infection with antibiotics. None of these events was considered by the principal investigator to be related to the garments. Throughout the entirety of the trial all participants tolerated the Martha Gardner Wool Mix™ laundry detergent.

SCORAD
A Wilcoxon signed-rank test determined that there was a statistically significant decrease in all SCORAD1 total scores between the following intervention phases as seen in table 3

Discussion
Overall, the intervention of SMWBG exhibited improvement in physical signs and subjective symptoms as demonstrated by improvement in EASI 2 , SCORAD 1 , POEM 3 and DSA 4 scores.
Additionally, we identified positive aspects of wearing wool including; no garment-related reported adverse events, reusability of garments and practicality, and a sense of ownership of treatment for children and young adults being reported by patients.

Physical Signs of Improvement
The physical signs of improvement were evident in the decreased EASI 2 , SCORAD 1 extent and POEM 3 scores. This is in keeping with the findings of [16]. were an unexpected outcome. This improvement may be explained by a decrease in the auto eczematization phenomenon i.e., by improving significant foci of skin inflammation, reactivity of the skin in general can be seen to improve [17]. This theory needs to be further investigated to support this hypothesis.
The POEM 3 scores were measures of the participants' perception of their AD severity as opposed to investigator reviews in the EASI 2 and SCORAD 1  participants also had short courses of oral antibiotic therapy for unrelated diseases during the trial which may have affected the course of their AD. This study was a single-arm trial and therefore has generalisability limitations. This study is part of a series of studies investigating the efficacy of SMWBLG in AD and future research is being performed to further explore this relationship.

Future Studies
From our initial investigations, it seems that there is an improvement in AD symptoms related to use of SMWBG. Further investigations into the complex relationship between SMW and AD severity remain to be performed. Future studies measuring transepidermal water loss (TEWL) in participants wearing SMW garments should be explored to assess the effect of hygral regulation and fatigue on AD. A proposed mechanism for wearing a superfine woolen keratin layer leading to a decrease in inflammatory signalling and degeneration of filaggrin (known to promote the construction of the cornified cell envelope) needs to be explored [18][19]. Double blinded designed studies would be appropriate to undertake. Studies comparing different fiber sources such as cotton, silk and polyester and other man-made fabrics measuring TEWL, AD severity scoring and thermal regulation in participants with AD may further clarify the therapeutic benefit and mechanism of action of SMWBG.

Conclusion
The intervention of SMWBG demonstrated improvement in the physical signs of AD in our cohort. This study supports the proposal that SMWBG provides a valuable adjunct therapy in the management of AD. There were no adverse garment-related effects, no reports of allergic or contact dermatitis with use and most importantly, no reports of worsening of AD symptoms. Patients reported the added benefits of comfort, practicality and reusability.
Patients also reported a sense of ownership of treatment in children and young adults. Further investigation into the use of SMWBG is required to demonstrate the mechanisms behind the improvement of SMW in patients with AD.