Abstract Application of sunscreen is a widely used mechanism for protecting skin from the harmful effects of UV light. However, protection can only be achieved through effective application, and areas that are routinely missed are likely at increased risk of UV damage. Here we sought to determine if specific areas of the face are missed during routine sunscreen application, and whether provision of public health information is sufficient to improve coverage. To investigate this, 57 participants were imaged with a UV sensitive camera before and after sunscreen application: first visit; minimal pre-instruction, second visit; provided with a public health information statement. Images were scored using a custom automated image analysis process designed to identify areas of high UV reflectance, i.e. missed during sunscreen application, and analysed for 5% significance. Analyses revealed eyelid and periorbital regions to be disproportionately missed during routine sunscreen application (median 14% missed in eyelid region vs 7% in rest of face, p<0.01). Provision of health information caused a significant improvement in coverage to eyelid areas in general however, the medial canthal area was still frequently missed. These data reveal that a public health announcement-type intervention could be effective at improving coverage of high risk areas of the face, however high risk areas are likely to remain unprotected therefore other mechanisms of sun protection should be widely promoted such as UV blocking sunglasses.

Citation: Pratt H, Hassanin K, Troughton LD, Czanner G, Zheng Y, McCormick AG, et al. (2017) UV imaging reveals facial areas that are prone to skin cancer are disproportionately missed during sunscreen application. PLoS ONE 12(10): e0185297. https://doi.org/10.1371/journal.pone.0185297 Editor: Andrzej T. Slominski, University of Alabama at Birmingham, UNITED STATES Received: June 1, 2017; Accepted: September 8, 2017; Published: October 2, 2017 Copyright: © 2017 Pratt et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: Data are available from the University of Liverpool Ethics Committee for researchers who meet the criteria for access to confidential data. Requests for access can be made to the University of Liverpool Research Governance Officer via ethics@liv.ac.uk or Faculty of Health and Life Sciences, Cedar House, Ashton Street, Liverpool, L69 3GE, Tel 0151 793 4358. Funding: This work was supported by bench fees raised from a University of Liverpool Masters in Research Course. Competing interests: The authors have declared that no competing interests exist.

Introduction Despite increasing sun awareness and sun protection usage, between 70–90 percent of basal cell carcinomas (BCCs) develop in sun-exposed head and neck regions, and 5 to 10 percent of all skin cancers occur on the eyelids alone[1]. Specifically within England, 33610 eyelid BCCs were recorded in the 11 years between 2000 and 2010[1]. Within the eyelid area, the medial canthus, a region where the medial corner of the upper and lower eyelids meet, has been shown to be not only a particularly common site for BCC, but is also associated with poor prognosis [2,3,4,5]. It has been postulated that the high prevalence of non-melanoma skin cancer on the eyelids is due to the skin being the thinnest on the body and hence specifically vulnerable to damage from prolonged ultraviolet (UV) light exposure, a well-established risk factor for BCCs and for squamous cell carcinomas [6,7,8,9,10]. Therefore, the importance of adequately protecting this vulnerable area is clear, and the use of sunscreen formulations has been widely promoted. Use of sunscreens for sun protection requires two conditions to be met: i) adequate quantities of the substance to be applied with appropriate frequency of reapplication, and ii) effective coverage of all sun exposed areas. Importantly, it has been demonstrated that even when the frequency of application and quantity applied are appropriate, the application technique in terms of coverage, is often inadequate [11,12,13,14]. Studies investigating sunscreen application to the face with emphasis on identifying commonly missed areas have very rarely been performed, however, one study in 1994 suggested inferior application to the medial canthal area in a study of 50 participants [15]. In the 23 years since this publication, through numerous public health information streams, awareness of the risks associated with UV exposure has increased dramatically. However, the positive health benefits of UV exposure in terms of supporting the complex sensory functions of the skin, including effects on brain, neuroendocrine, and immune function, as well as the deleterious effects of vitamin D insufficiency have been widely demonstrated leading to guidelines designed to balance the benefits of UV exposure against the potential DNA damage [16,17,18,19,20,21,22,23]. Therefore an updated investigation into application habits is warranted. Recent data suggest that sunscreens are increasingly becoming the method of choice for sun protection meaning that it is more important than ever that sunscreen is applied effectively [24,25,26]. To date, the majority of sunscreen application publications use surrogates in place of real sun creams to determine coverage. Often these surrogates are of different texture or visibility which may influence application [27,28]. Recent improvements in the availability of UV sensitive cameras have opened up the possibility of investigating sunscreen application directly using actual formulations available to the public and obtaining superior image quality compared with the use of a Wood’s lamp [14] or by imaging fluorescent creams [29]. UV photographic imaging has thus been demonstrated as being useful as a method not only to assess sunscreen application but also to assess skin damage and drive behavioral change in sun bed users [30,31,32]. In this study, we have adopted the UV imaging approach to determine if skin cancer prone facial regions are ineffectively covered, and if an information based intervention could be used to improve sunscreen application.

Materials and methods Ethical approval The study was approved by University of Liverpool Ethics Review Board with approval number 201606181. Written consent was obtained from all participants prior to both phases of the trial. The individuals who’s images have been used in this manuscript have given written informed consent (as outlined in PLOS consent form) to publish their case details here. Study design Sample sizes were determined based on preliminary data where 4 participants were analysed from two identical non-intervention visits and one intervention visit (SD = 8). Power analyses were performed based on 95% power and 5% type I error rate; to detect an increase of at least 5% after intervention requires 57 participants assuming paired t-test. 57 people (27 male and 30 female) were recruited in October to December through poster advertising and an email cascade to all staff and students of the Institute of Ageing and Chronic Disease, University of Liverpool. There were no exclusion criteria based on demographics, ethnicity or other personal criteria, however, excluded from the study were volunteers who self-identified as having allergies to sun lotion. Volunteers were required to fill in a pre-questionnaire before participating in the study (S1 Fig), requiring them to confirm whether they had used any form of sun protection previously, and whether they had any known allergies. Participants were also requested to self-identify their skin-type based on provided Fitzpatrick scale diagram. Participants were then allowed to self-select from either SPF50 spray or SPF50 cream sunscreen formulations (both Nivea, Birmingham, UK) and instructed to apply sunscreen in their usual manner. No instructions were provided regarding volume of solution to use. UV images were acquired before and after sunscreen application. The same participants were invited to return for a second visit two weeks later, where they received an information sheet stating “Skin cancers most commonly occur on sun exposed areas of the body. Most skin cancers occur in the face with 10% of all skin cancers occurring on the sensitive eyelid area which is thinner and therefore more sensitive to sunlight. Using sunblock reduces the risk of getting skin cancer.” They were then given the same sunscreen formulation as used initially and imaged before and after application. Participants were not shown the images from their first visit prior to sunscreen application. Following the second phase of the study, participants were requested to complete a second questionnaire asking about their experience and to identify behavioural trends within the population (S2 Fig). Image acquisition Participants were sat in front of a plain white background and images captured using a tripod mounted DSLR (Canon EOS Rebel XTi 400D) with 60mm EF-S macro lens (both; Canon, Surrey, UK). The camera was modified to be sensitive only to UV light by replacing the internal hot mirror with a UV band pass filter (Lifepixel, Mukilteo, WA, USA) and were photgraphed with the use of an electronic flash (Vivitar Auto Thyristor Model 285 with fresnel lens removed, Vivitar, Edison, NJ, USA). Camera settings (F 2.8, ISO 1800, shutter speed 1.2s,), lighting and distances from the camera were kept constant throughout. The original images were greyscale ten million pixels images in JPG format. Image analysis Manual segmentation was performed using image J (NIH, Bethesda, MA) by an observer manually drawing around areas deemed to be not covered using the freehand selection tool. Due to observers reporting difficulty in identifying glare/reflection and resultant intraobserver variability, this approach was deemed ineffective. Therefore, an automated image analysis method was developed to objectively detect, segment and quantify the areas of the face within the UV images that were not covered by sunscreen. To counteract differences in skin tones and reflection, contrast limited adaptive histogram equalisation was first applied to the images using openCV (http://opencv.org/)[33]. This divided the image into small blocks and each of these blocks were then histogram equalized using the following algorithm: Let f be the source image and m x n matrix from the small block The pixel intensity values in a greyscale image vary from 0–255 The probability of an image having intensity x is given by: Next, the dlib package (http://dlib.net/) was used to detect facial landmarks within the image. These landmarks were used as markers for determining the facial region and theletterbox region surrounding the eyes of the participant. The landmark located at the inner eye was used to define the medial canthus. The face was segmented from the original image and the relative letterbox points collated for later use. Gaussian blur (openCV code library) was then applied to the normalised image to smooth the image and reduce unwanted noise [34]. The Gaussian equation for a 2D image is defined as: Where μ is the mean pixel value and θ represents the variance. The image was then mapped to Hue Saturation Value and thresholding performed to produce a binary segmented mask of the image [35]. Results for applying the substance were determined from this binary mask and reported as a percentage of the pixels in each image/letter box region. A binary, yes/no classification was used for the medial canthal region, where images were scored as not covered if the segmentation detected any missed skin within the defined region. Statistical approaches Data were tested for normality with Shapiro Wilk test. Mann-Whitney tests were performed to compare coverage between eyelid regions and non eyelid regions relative to gender, skin type, and sun cream versus sun spray. Spearman’s correlation was used to assess correlation between percentages of eyelid regions missed with percentage of rest of face. Wilcoxon tests were used to assess the improvement in coverage. Chi square test was used to assess change in medial canthal region coverage. Differences were deemed statistically significant where p<0.05.

Discussion In this study, we have demonstrated that although overall sunscreen application to the face regularly achieves high levels of coverage, problem areas still exist in the eyelid regions, particularly the medial canthus. However, we demonstrate that provision of a short information sheet is sufficient to drive a small but statistically significantly improved coverage to eyelids regions, which was particularly effective in those individuals that initially performed poorly. These data highlight the need for greater public awareness of eyelid cancer risk and suggest that simple pubic information intervention could be effective. However, our data also demonstrate that, despite improved awareness, the medial canthal areas were still frequently ineffectively covered and therefore use of alternate strategies for protection, such as UV blocking sunglasses, should be promoted wherever possible. This will have the dual effect of protecting cancer at risk areas and also protecting eyes from UV damage, thereby reducing incidence of corneal damage, macular degeneration and cataract formation [44,45]. The participants in our study were drawn from University students and staff members and therefore there is a selection biased toward well-educated participants. Moreover, our participants reflected the local University population and were skewed toward lighter skin types. This has two important implications for the generalizability of our findings. First; one would predict that our cohort would have a base line cancer-risk awareness levels and as such are likely to be vigilant when applying sun protection [43,46]. This may suggest that the disproportionately poor coverage of the eyelid region could be even more striking in the general population. It should be noted, that in our post questionnaire just under half of the respondents did not cite a specific reason for missing the eyelids so, despite a perceived general awareness, their application was still relatively poor. Second; our cohort may respond more to our information based intervention strategy than a broader cross section of the public and therefore we may be over estimating the magnitude of the improvement. However, information based intervention is a standard and proven efficacious approach in wide variety of contexts in diverse groups of patients/participants and as such we do not believe this to be a major limitation here [47,48,49]. When considering the data within this study it is important to consider the behaviour modifying effect wearing sun protection has. If sunscreen application is only performed when preparing for an extended period in the sun, ineffective application will lead to a false sense of security in terms of perceived protection. Our data strongly indicate that in routine application, the eyelid region and particularly the medial canthal areas are relatively poorly protected hence at increased risk. The belief that the whole face is protected may repeatedly increase UV exposure to vulnerable areas that have been missed as people spend longer exposed to the sun. An important additional consideration is that any public health message must weigh the benefits of reduced UV induced DNA damage against the positive health benefits of UV-B induced vitamin D production. Specifically, insufficient levels of vitamin D have been shown to occur in 50% of the UK adult population with seasonal variations showing severe deficiencies during winter and spring [50,51]. Low vitamin D levels are associated with increased risk of certain cancer subtypes, and other health risks including bone disease, muscle weakness and diabetes mellitus [22,23,52,53,54]. Dietary vitamin D supplementation can reduce some of these health risks [22,53], however, it has also been demonstrated that relatively short UV exposure is sufficient to produce the daily recommended serum vitamin D levels [55]. This is reflected in National Institute for Clinical Excellence (NICE) guidelines which recommend short periods of non-protected exposure to ensure adequate vitamin D production. The findings presented here do not countermand these recommendations, but rather that emphasis be made that, when protection is required, extra attention be paid that sunscreens are applied in a way that does not repeatedly leave the same areas unprotected each time. Taken together, our findings strongly suggest that a public information campaign is warranted to stress the importance of eye protection from the sun. The ongoing problem of irritation caused by the sun cream/spray needs to be addressed with an appropriate education system put in place to educate the public in newer tear-free formulations [56], whilst the importance of seeking alternate protection mechanisms should be further emphasized.

Supporting information S1 Fig. Pre study questionnaire. https://doi.org/10.1371/journal.pone.0185297.s001 (PDF) S2 Fig. Post study questionnaire. Note that this was presented to participants via an online link. https://doi.org/10.1371/journal.pone.0185297.s002 (PDF) S3 Fig. Comparison between manual and automated segmentation. A. Percentage of cropped eyelid region not covered by sunscreen as determined by manual segmentation. Blue dots represent maximal values without attempting to account for flash reflection/glare. Red dots represent manual segmentation where the observer has attempted to adjust for glare. B. Comparison of automated scoring output (green dots) and manual scoring (red dots) for percentage cover. C. Comparison between mean manual score vs automated score. Dotted line represents exact agreement between scores. D. Differences between manual and automated segmentation scores plotted as divergence from automatic score. For all graphs, data are from 19 images, in A, B, C each vertical column is a single image sorted by self-assessed skin types. https://doi.org/10.1371/journal.pone.0185297.s003 (TIF) S4 Fig. Montage of eyelid region images. Eyelid regions images from all participants showing images taken before sunscreen application (left panels), after sunscreen application in visit 1 (central panels) and after sunscreen application in visit 2(right panels). https://doi.org/10.1371/journal.pone.0185297.s004 (TIF)

Acknowledgments The authors would like to thank all participants for their involvement in this study and allowing the use of their images. HP developed the image analysis platforms and analysed images, KH recruited participants and acquired images, LDT performed manual analysis of the images, GZ performed statistical analyses and contributed to study design, YZ aided in design of image analysis platform, AM conceived the study, drafted the manuscript and contributed to study design, KJH directed the study, recruited participants, drafted the manuscript and contributed to study design. The authors have declared that no competing interests exist.