Skin Research and Technology 2014; 0: 1–5 Printed in Singapore All rights reserved doi: 10.1111/srt.12137
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Skin Research and Technology
Reflectance confocal microscopy vs. standardized skin surface biopsy for measuring the density of Demodex mites A. Turgut Erdemir, M. S. Gurel, A. E. Koku Aksu, F. Bilgin Karahalli, P. Incel, N. S. Kutlu Hayto glu and T. Falay Dermatology Department, Istanbul Training and Research Hospital, Istanbul, Turkey
(15.33 18.1) (P < 0.001). In the patients, RCM demonstrated the mean number of mites 40.90 20.9 and 4.11 6.4 in the controls per 10 mm2 area. The corresponding mean number of 2.63 0.77 mites was detected in the infested follicles per area of view compared to a mean of 0.77 0.98 mites in the infested follicles in the controls (P < 0.001). Conclusion: Reflectance confocal microscopy is a fast, direct and noninvasive method for Demodex-associated diseases and it is superior to SSSB for Demodex mite detection.
Background: Reflectance confocal microscopy (RCM) has been recently shown to be effective for measuring the Demodex mite density. Objectives: To compare and demonstrate the advantages and disadvantages of standardized skin surface biopsy (SSSB) and RCM for measuring the density of Demodex mites. Materials and methods: Forty-eight patients (30 female, 18 male) and 47 healthy controls (30 female, 17 male) were enrolled in the study. The patients diagnoses were pityriasis folliculorum (n = 40), papulopustulary rosecea (n = 7) and erythema-telengiectatic rosacea (n = 1). The area with the most intense erythema on the right cheek was selected for imaging with RCM (VivaScope 3000) and SSSB. Results: Forty-two patients demonstrated high Demodex density [(Dd) > 5 mites/cm2] with SSSB (85.7%). RCM identified demodicosis in 48 patients (100%). The mean Dd measured with RCM (409.8 209.2) was significantly higher than SSSB
Ó 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Accepted for publication 5 January 2014
of Demodex spp. have been identified on human skin: Demodex folliculorum and Demodex brevis (1). These mites live as parasites on the pilosebaceous unit and feed on the nutrients in the sebaceous glands. D. folliculorum is found in infundibular part of the hair follicles, while D. brevis lives in the sebaceous glands and in the deeper parts of the hair follicles. Demodex mites believed to become pathogenic when they penetrate into the dermis or with the increase in their number (2, 3). The prevalence of Demodex mites in middle-aged and elderly healthy skin is about 100%, although mite density is low. In 1930, Ayres identified the first two clinical infestation in humans. These were pityriasis folliculorum and rosacea-like demodicidosis. In later years, D. folliculorum has been reported to play a role in the pathogenesis of rosaceae, acne vulgaris, blepharitis, perioral dermatitis, pustular
folliculitis, papulo-pustular lesions of the scalp and pustular lesions in acquired immunodeficiency syndrome (2, 4–7). Different methods have been used for the determination of the Demodex mites. Standardized skin surface biopsy (SSSB) technique with cyanoacrylic adhesion is the most common method to measure the density of Demodex. Other sampling methods used in detection of the Demodex mites include direct microscopic examination of adhesive bands, skin scrapings and sebum extraction. Mite density per square centimetre (cm2) is important to determine pathogenicity (8, 9). The method of SSSB determined to be more sensitive and superior in the detection of Demodex mites compared with other methods (10). Reflectance confocal microscopy (RCM) provides noninvasive in vivo imaging of the epidermis and superficial dermis at cellular level resolution. RCM, which have been particularly
T
WO SPECIES
Key words: confocal microscopy – Demodex mites – demodicosis – standardized skin surface biopsy
1
Turgut Erdemir et al.
used in diagnosis of melanocytic lesions, demonstrated to be effective in detecting Demodex mites and for measuring the mite density (11–13). The aim of this study was to compare and demonstrate the advantages and disadvantages of SSSB and RCM and to compare the patients with demodiciosis-related disease with normal population.
Materials and methods This was a prospective, controlled clinical trial comparing and demonstrating the advantages and disadvantages of SSSB and RCM for measuring the density of Demodex mites. The study was carried out between 2012 August and 2013 August in Dermatology Department of Istanbul Training and Research Hospital. The study was granted ethical approval by the Ethics Committee of Istanbul Training and Research Hospital, adhered to the Declaration of Helsinki guidelines. Informed consent document was obtained from the patients. Forty-eight patients (30 female, 18 male) and 47 healthy controls (30 female, 17 male) were enrolled in the study. The study was conducted on patients considered to be infested with Demodex clinically (erythema, burning sensation, dryness, numerous follicular scales, few superficial vesiculopustules) and who have not received any topical or local treatment for 1 month were included in the study. Facial skin lesions compatible with demodicosis clinically were pityriasis folliculorum (n = 40), papulopustulary rosecea (n = 7) and erythema-telengiectatic rosacea (n = 1). The area with the most intense erythema on the right cheek was selected for imaging with VivaScope 3000 (VivaScope® 3000, Lucid Inc., Rochester, NY, USA). Ten images of 1 mm2 were taken from a 1 cm2 area. Then SSSB was applied on the same area. First, the area was cleaned with alcohol, then cyanoacrylate glue (super instant glue, Scotch; 3M, St. Paul, MN, USA) was dropped on a marked 1 cm2 area on a glass slide. The adhesive bearing surface of the glass slide was applied for 1 min on the selected area. The specimen is examined by light microscopy at 940 and 9100 magnifications. The number of the Demodex mites was counted with RCM and SSSB. The number of mites detected with RCM was multiplied with
2
10 for synchronizing the imaged fields. And the results were compared with each other. Also 47 age- and gender-matched healthy controls were included in the study. Their right cheek was imaged with VivaScope 3000 and ten images of 1 mm2 (10 mm2) were taken from a 1 cm2 area. The number of follicles, the number of mites per follicle, the number of infested follicles and the number of mites per infested follicles were counted. The results are compared to the patient group.
Statistics All analyses were performed using SPSS software (version 15.0 for Windows; SPSS, Inc., Chicago, IL, USA). Descriptive statistics of the data: for categorical variables, numbers and percentages, for numeric variables, the mean, standard deviation and median were given. Student’s t-test was used in the comparison of the groups when the variables distributed normally and Mann– Whitney U-test was employed when the variables do not distributed normally. Chi-squared (v2) analysis was used to compare differences between groups of categorical variables. The Wilcoxon test was used to analyse the difference of numerical variables in the dependent groups when the variables were not distributed normally. For all statistical tests, P ≤ 0.05 was considered statistically significant.
Results Forty-eight patients (30 female, 18 male) and 47 control group (30 female, 17 male) were participated in the study. All patients were Caucasian. The mean age of the patient group was 40.8 11.7 while the mean age of the control group was 40.3 15.7. There was no statistically significant difference between age and gender in the patient and control group. The diagnosis of the patient group was as follows: pityriasis folliculorum (83.3%), papulopustulary rosecea (14.6%) and erythema-telengiectatic rosacea (2.1%). Demodex mites were displayed as elongated structures with their tales superficially located in the follicle (Fig. 1). When RCM is positioned completely perpendicular to the follicle, Demodex mites were viewed as clusters of bright, round structures approximately 4–9 lm sized detected by RCM (Fig. 2). The best images were taken at the level of 10–90 lm. The maximum
Reflectance confocal microscopy use in demodicosis
Fig. 3. Demodex folliculorum under a light microscope. (original magnification 940). Fig. 1. Demodex mites appear as lengthy cone-shaped body within a hair follicle imaged with reflectance confocal microscopy.
Fig. 2. Demodex mites appear as clusters of bright, roundish structures when reflectance confocal microscopy images taken perpendicularly.
number of mite per follicle was 11. Demodex mites were detected as transparent and spindle shaped with SSSB (Fig. 3). There was no statistically significant difference between gender and the mean number of the Demodex mites for both methods. RCM identified high number of mites in all of the patients (48 patients, 100%) and SSSB identified demodicosis in 42 of 48 patients (85.7%). The mean Demodex density (Dd) measured with RCM (409.8 209.2) was higher than SSSB (15.33 18.1) and the difference was statistically significant (P < 0.001).
In the patients with Demodex-related disease, RCM demonstrated the mean number of mites 41.0 20.9 in the patients and 4.11 6.4 in the controls per 10 mm2 area. A mean number of 2.63 0.77 mites were detected in the infested follicles per area of view, compared to a mean of 0.77 0.98 mites in the infested follicles in the controls (P < 0.001). Mean numbers per follicle for the 10 mm2 area were also counted and divided into seven groups. The number of follicles without Demodex mites was lower in the patient than control group (P < 0.001). In the patient group, the number of mites usually ranged from 1 to 3 mites, while in the controls
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Skin Research and Technology
Reflectance confocal microscopy vs. standardized skin surface biopsy for measuring the density of Demodex mites A. Turgut Erdemir, M. S. Gurel, A. E. Koku Aksu, F. Bilgin Karahalli, P. Incel, N. S. Kutlu Hayto glu and T. Falay Dermatology Department, Istanbul Training and Research Hospital, Istanbul, Turkey
(15.33 18.1) (P < 0.001). In the patients, RCM demonstrated the mean number of mites 40.90 20.9 and 4.11 6.4 in the controls per 10 mm2 area. The corresponding mean number of 2.63 0.77 mites was detected in the infested follicles per area of view compared to a mean of 0.77 0.98 mites in the infested follicles in the controls (P < 0.001). Conclusion: Reflectance confocal microscopy is a fast, direct and noninvasive method for Demodex-associated diseases and it is superior to SSSB for Demodex mite detection.
Background: Reflectance confocal microscopy (RCM) has been recently shown to be effective for measuring the Demodex mite density. Objectives: To compare and demonstrate the advantages and disadvantages of standardized skin surface biopsy (SSSB) and RCM for measuring the density of Demodex mites. Materials and methods: Forty-eight patients (30 female, 18 male) and 47 healthy controls (30 female, 17 male) were enrolled in the study. The patients diagnoses were pityriasis folliculorum (n = 40), papulopustulary rosecea (n = 7) and erythema-telengiectatic rosacea (n = 1). The area with the most intense erythema on the right cheek was selected for imaging with RCM (VivaScope 3000) and SSSB. Results: Forty-two patients demonstrated high Demodex density [(Dd) > 5 mites/cm2] with SSSB (85.7%). RCM identified demodicosis in 48 patients (100%). The mean Dd measured with RCM (409.8 209.2) was significantly higher than SSSB
Ó 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Accepted for publication 5 January 2014
of Demodex spp. have been identified on human skin: Demodex folliculorum and Demodex brevis (1). These mites live as parasites on the pilosebaceous unit and feed on the nutrients in the sebaceous glands. D. folliculorum is found in infundibular part of the hair follicles, while D. brevis lives in the sebaceous glands and in the deeper parts of the hair follicles. Demodex mites believed to become pathogenic when they penetrate into the dermis or with the increase in their number (2, 3). The prevalence of Demodex mites in middle-aged and elderly healthy skin is about 100%, although mite density is low. In 1930, Ayres identified the first two clinical infestation in humans. These were pityriasis folliculorum and rosacea-like demodicidosis. In later years, D. folliculorum has been reported to play a role in the pathogenesis of rosaceae, acne vulgaris, blepharitis, perioral dermatitis, pustular
folliculitis, papulo-pustular lesions of the scalp and pustular lesions in acquired immunodeficiency syndrome (2, 4–7). Different methods have been used for the determination of the Demodex mites. Standardized skin surface biopsy (SSSB) technique with cyanoacrylic adhesion is the most common method to measure the density of Demodex. Other sampling methods used in detection of the Demodex mites include direct microscopic examination of adhesive bands, skin scrapings and sebum extraction. Mite density per square centimetre (cm2) is important to determine pathogenicity (8, 9). The method of SSSB determined to be more sensitive and superior in the detection of Demodex mites compared with other methods (10). Reflectance confocal microscopy (RCM) provides noninvasive in vivo imaging of the epidermis and superficial dermis at cellular level resolution. RCM, which have been particularly
T
WO SPECIES
Key words: confocal microscopy – Demodex mites – demodicosis – standardized skin surface biopsy
1
Turgut Erdemir et al.
used in diagnosis of melanocytic lesions, demonstrated to be effective in detecting Demodex mites and for measuring the mite density (11–13). The aim of this study was to compare and demonstrate the advantages and disadvantages of SSSB and RCM and to compare the patients with demodiciosis-related disease with normal population.
Materials and methods This was a prospective, controlled clinical trial comparing and demonstrating the advantages and disadvantages of SSSB and RCM for measuring the density of Demodex mites. The study was carried out between 2012 August and 2013 August in Dermatology Department of Istanbul Training and Research Hospital. The study was granted ethical approval by the Ethics Committee of Istanbul Training and Research Hospital, adhered to the Declaration of Helsinki guidelines. Informed consent document was obtained from the patients. Forty-eight patients (30 female, 18 male) and 47 healthy controls (30 female, 17 male) were enrolled in the study. The study was conducted on patients considered to be infested with Demodex clinically (erythema, burning sensation, dryness, numerous follicular scales, few superficial vesiculopustules) and who have not received any topical or local treatment for 1 month were included in the study. Facial skin lesions compatible with demodicosis clinically were pityriasis folliculorum (n = 40), papulopustulary rosecea (n = 7) and erythema-telengiectatic rosacea (n = 1). The area with the most intense erythema on the right cheek was selected for imaging with VivaScope 3000 (VivaScope® 3000, Lucid Inc., Rochester, NY, USA). Ten images of 1 mm2 were taken from a 1 cm2 area. Then SSSB was applied on the same area. First, the area was cleaned with alcohol, then cyanoacrylate glue (super instant glue, Scotch; 3M, St. Paul, MN, USA) was dropped on a marked 1 cm2 area on a glass slide. The adhesive bearing surface of the glass slide was applied for 1 min on the selected area. The specimen is examined by light microscopy at 940 and 9100 magnifications. The number of the Demodex mites was counted with RCM and SSSB. The number of mites detected with RCM was multiplied with
2
10 for synchronizing the imaged fields. And the results were compared with each other. Also 47 age- and gender-matched healthy controls were included in the study. Their right cheek was imaged with VivaScope 3000 and ten images of 1 mm2 (10 mm2) were taken from a 1 cm2 area. The number of follicles, the number of mites per follicle, the number of infested follicles and the number of mites per infested follicles were counted. The results are compared to the patient group.
Statistics All analyses were performed using SPSS software (version 15.0 for Windows; SPSS, Inc., Chicago, IL, USA). Descriptive statistics of the data: for categorical variables, numbers and percentages, for numeric variables, the mean, standard deviation and median were given. Student’s t-test was used in the comparison of the groups when the variables distributed normally and Mann– Whitney U-test was employed when the variables do not distributed normally. Chi-squared (v2) analysis was used to compare differences between groups of categorical variables. The Wilcoxon test was used to analyse the difference of numerical variables in the dependent groups when the variables were not distributed normally. For all statistical tests, P ≤ 0.05 was considered statistically significant.
Results Forty-eight patients (30 female, 18 male) and 47 control group (30 female, 17 male) were participated in the study. All patients were Caucasian. The mean age of the patient group was 40.8 11.7 while the mean age of the control group was 40.3 15.7. There was no statistically significant difference between age and gender in the patient and control group. The diagnosis of the patient group was as follows: pityriasis folliculorum (83.3%), papulopustulary rosecea (14.6%) and erythema-telengiectatic rosacea (2.1%). Demodex mites were displayed as elongated structures with their tales superficially located in the follicle (Fig. 1). When RCM is positioned completely perpendicular to the follicle, Demodex mites were viewed as clusters of bright, round structures approximately 4–9 lm sized detected by RCM (Fig. 2). The best images were taken at the level of 10–90 lm. The maximum
Reflectance confocal microscopy use in demodicosis
Fig. 3. Demodex folliculorum under a light microscope. (original magnification 940). Fig. 1. Demodex mites appear as lengthy cone-shaped body within a hair follicle imaged with reflectance confocal microscopy.
Fig. 2. Demodex mites appear as clusters of bright, roundish structures when reflectance confocal microscopy images taken perpendicularly.
number of mite per follicle was 11. Demodex mites were detected as transparent and spindle shaped with SSSB (Fig. 3). There was no statistically significant difference between gender and the mean number of the Demodex mites for both methods. RCM identified high number of mites in all of the patients (48 patients, 100%) and SSSB identified demodicosis in 42 of 48 patients (85.7%). The mean Demodex density (Dd) measured with RCM (409.8 209.2) was higher than SSSB (15.33 18.1) and the difference was statistically significant (P < 0.001).
In the patients with Demodex-related disease, RCM demonstrated the mean number of mites 41.0 20.9 in the patients and 4.11 6.4 in the controls per 10 mm2 area. A mean number of 2.63 0.77 mites were detected in the infested follicles per area of view, compared to a mean of 0.77 0.98 mites in the infested follicles in the controls (P < 0.001). Mean numbers per follicle for the 10 mm2 area were also counted and divided into seven groups. The number of follicles without Demodex mites was lower in the patient than control group (P < 0.001). In the patient group, the number of mites usually ranged from 1 to 3 mites, while in the controls
