Original Article

Comparative study of two diagnostic methods of in patients with and

Anzhela Kravchenko

Department of Dermatovenereology and Cosmetology, Russian Medical Academy of Continuous Professional Education, the Ministry of Healthcare of Russian Federation, Moscow, Russia

Email: [email protected]

Abstract

Background: Demodicosis is a disease, caused by parasitisation of the opportunistic parasites from the group – . This article presents a comparative study of two methods (light microscopy of scrapings and confocal laser scanning in vivo microscopy) for identification of Demodex mites on the facial skin in acne and rosacea patients. The use of confocal laser scanning in vivo microscopy in dermatology today is considered as one of the most promising methods. Methods: A total of 90 subjects were included in the study, comprising 30 patients with acne and rosacea complicated by demodicosis, 30 patients with acne and rosacea not complicated by demodicosis, and 30 healthy volunteers. All patients were examined by scraping of the skin, eyebrow and/or epilation and confocal laser scanning in vivo microscopy. Results: The specificity of light microscopy of skin scrapings was 65.5%, while the specificity of confocal laser scanning in vivo microscopy for the diagnosis of demodicosis was 68.8%. Conclusion: The study showed advantages of confocal laser scanning in vivo microscopy compared to the traditional method of investigation.

Keywords: acne, confocal laser scanning in vivo microscopy, Demodex mites, light microscopy, rosacea

Background nymphs, eggs and adult form counts. The criterion of mites’ activity is the detection of more than five Demodicosis is a disease caused by parasitisation adults, larvae or eggs per one cm2. Diagnosis of of the opportunistic parasites from the acariasis demodicosis is established by detection group – Demodex mites. Mites, with size of 0.2-0.5 of one on 2-4 eyelashes. The activity of mites- mm, live in the ducts of the sebaceous and borne invasion can be assessed by differences in Meibomian glands, in the orifices of the hair the number of mites per cm2.4 follicles, and the excretory ducts of the sebaceous glands of and .1,2 Demodex The classic method of detecting Demodex mites is mites are part of the microflora of the skin light microscopy of the skin scraping specimens, and commonly asymptomatic. However, they can using a disposable scarifier and tweezers. The be identified in acute inflammatory dermatoses; material is taken from an area which has the largest such as acne, rosacea, seborrheic dermatitis, accumulation of sebaceous glands on the face, perioral dermatitis, or as independent disease.3 eyebrows, and eyelashes. The magnification of the microscope is 10 and 40 times. Diagnosis The diagnosis is confirmed by identifying the comprises clinical presentations (papulopustules, presence of Demodex mites. The most common comedones, erythema of the face, telangiectasias) method used to identify Demodex mites is a and positive confirmation by laboratory compilation of an acarogram consisted of larvae, investigation (quantitative load of more than five adult mites, larvae or eggs or both per one cm2).5

J Gen Proced Dermatol Venereol Indones. 2018;2(3):98-104. 98 It is essential to repeat the examination in cases of method is the ability to detect, quantify and high clinical suspicion with that negative laboratory determine the Demodex mites on the facial skin of result. Specimens must be taken from large areas patients with rosacea and acne by counting mites of the rash, since random selection of the study site and follicles per unit area, and by measuring the can cause negative laboratory analysis and may size of mites. T. Kojima et al. demonstrated the use not be able to prove the absence of mites-borne of confocal laser scanning microscopy for the invasion.6 diagnosis of eye demodicosis.15 The authors managed to find mites in the bulb terminal of the Modern laser-based confocal microscopes operate eyelashes, inflammatory infiltrates around the the optical system of the microscope more meibomian glands and conjunctiva. Thus, accurately, reduce the glare of the images, and according to the scientific literature, confocal laser improve the focusing of the light beam.7 The work scanning microscopy is a non-invasive and rapid of a confocal microscope is based primarily on the method for detecting Demodex mites.16 ability of various structures of the skin to refract laser radiation, thus obtaining images of the layers Given the relevance of this topic, we conducted a of the epidermis and dermis8 and assessing the diagnostic survey of healthy volunteers and condition of the skin as well as fibres of the dermis.9 patients with acne, rosacea, complicated and non- Confocal laser scanning in vivo microscopy is a complicated demodicosis using light microscopy of new method for studying the structure of the skin in the facial skin scrapings and confocal laser the form of pictures with grayscale hue. Confocal scanning in vivo microscopy. The study aimed to laser scanning microscopy allows you to determine evaluate the sensitivity and specificity of the the thickness of the skin and visualizes different methods of light microscopy of facial skin scrapings layers of the skin. Thus, the method provides compared to confocal laser scanning in vivo additional information on the composition and microscopy for diagnosing "Demodicosis" in structure of the skin.10 In ophthalmology, it is patients with acne and rosacea. possible to visualize changes in Meibomian glands in the form of enlargement or obstruction, the Methods presence of inflammatory infiltrates, as well as to detect Demodex mites.11 The scanning method of This study was conducted in 2014-2017. Thirty laser confocal microscopy can be compared with a patients with acne and rosacea complicated by histological examination of the skin, with an demodicosis (group I), 30 patients with acne and advantage that this method is performed non- rosacea, not complicated by demodicosis (group II) invasively.8 According to various data, the and 30 healthy volunteers (group III) were included sensitivity of this method to identify demodicosis is in the study. Sixteen patients with acne and 83-91%, and the specificity of this method is 95- rosacea had Demodex mites on their facial skin, 99%.12-14 but their presence was confirmed only by one of the investigation methods. Despite the fact that these The use of confocal laser scanning in vivo patients were tested, they did not enter the study. microscopy in dermatology today is considered as Thus, the initial number of patients analysed in the one of the most promising methods to identify course of work was 106 people. The grouping of demodicosis, despite the fact that it has a number patients included in the study is presented in Figure of disadvantages, which are 1. 1. Obtaining relatively superficial images with penetrating ability up to 200 μm, which The diagnosis of acne and rosacea was limits the possibility of studying deeper established at the primary clinic based on the layers of the skin. clinical manifestation of the diseases. The study 2. A relative high cost equipment and included participants with age over 18 years old, operational. after signing an informed consent to participate in 3. Inaccessible for large number of the study. The range of the participants’ age was dermatologists.11 18 to 79 years (mean age 30.0 ± 11.9). Subjects comprised 39 males and 51 females. There were In comparison with the conventional light 40 patients with acne and 20 patients with rosacea, microscope, the advantages of the method are divided into group I and group II. The high-contrast images with high resolution, its three- characteristics of patients are presented in Table 1. dimensional reconstruction, and digital processing of the data obtained.9 One of the advantages of the

J Gen Proced Dermatol Venereol Indones. 2018;2(3):98-104. 99 Total of 106 patients

Patients with acne Patients with acne Patients with acne Healthy volunteers and rosacea and rosacea and rosacea (30) (30) (30) (16)

Demodex mites Demodex mites Demodex mites were identified by 2 were not identified were identified by 1 investigation by 2 investigation investigation method methods methods

Figure 1. Grouping of patients included in the study

Table 1. Characteristics of patients in three groups

Group I - presence of Demodex mites (5 mites per one cm2) Total (n) Gender Male (n) Female (n) 12 18 30 (40%) (60%) (100%) Diagnosis Acne Rosacea Acne Rosacea 8 4 10 8 (26.7%) (13.35%) (3.3%) (26.7%) Mean age (years) 23±5.5 44±10.4 29±6.4 51±17.6 Group II - absence of Demodex mites Gender Male (n) Female (n) 12 18 30 (40%) (60%) (100%) Diagnosis Acne Rosacea Acne Rosacea 9 3 13 5 (30%) (10%) (43.3%) (16.7%) Mean age (years) 24±2.2 41±6.3 33±2.9 50±5.4 Group III– healthy volunteers Gender Male (n) Female (n) 15 15 30 (50%) (50%) (100%) Mean age (years) 26±1.4 31±3.1 n = number of patients

The presence of Demodex mites was determined one cm2, and clinical manifestation of the diseases by performing scraping of the skin of the face, are accounted for Group I. squeezing the contents of the sebaceous glands, and epilation of the eyelashes and eyebrows. At a Skin parameters were assessed in patients in vivo given location, an area of one cm2 was selected using a VivaScope® 1500 confocal laser scanning and marked by a pencil. The material was placed in vivo microscope (Lucid Inc., Rochester, NY, on a watch glass with a drop of 10% potassium USA), in real time using the VivaScan VS hydroxide solution and covered with a slide for 10- 007.11.12 software package. The maximum output 15 minutes. Then, the material was examined with power of the laser was 21.7 mW. The penetrating a light microscope to determine the presence of ability of the microscope is 200-300 μm, which is Demodex mites and their quantitative load per unit up to the papillary dermis. area (one cm2). Patients with a quantitative load of more than five adult mites, larvae and/or eggs per

J Gen Proced Dermatol Venereol Indones. 2018;2(3):98-104. 100 The patient was asked to not moving during can be taken. When using VivaStack, a series of specimen collection. A microscope object glass images was obtained from the stratum corneum of was glued to facial skin with acrylic glue in order to the epidermis to the papillary layer of the dermis. limit the displacement of the tissues. The affected Using VivaCam mode, a dermatoscopic image of surface of the skin was located in the centre of the the site was obtained. All layers of the epidermis to object glass. A drop of immersion oil was placed the papillary dermis were scanned. The cut-off between the skin surface and the microscope distance from one shot to the next was three μm. sensor. Chromdamol STS® oil or water-based The size of the hair follicles and excretory ducts of ultrasound gel was used as an immersion medium the sebaceous glands were assessed. The since their refractive index (1.50) is the closest to researcher performed a quantitative evaluation of the refractive index of the stratum corneum (1.55). the Demodex mites in the hair follicles and excretory ducts of the sebaceous glands, the depth Study of pathomorphological characteristics of the of the mites, as well as the number of excretory facial skin was performed in three locations (both ducts of the sebaceous glands and hair follicles per cheeks and forehead), with the two modes of the unit area. One unit area was a plot of 5 × 5 mm, (a microscope, which are VivaBlock and VivaStack. total area of 25 mm2). Demodex mites are The VivaBlock mode was used to analyse the visualized by rounded or oval formations with a horizontal image of the skin at a predetermined hyper contour around the peripheral orifices depth. In this mode, up to 256 consecutive pictures (Figure 2).

Figure 2. Images obtained with the confocal laser scanning microscope. Hair follicles and excretory ducts of the sebaceous glands with presence (left, arrow) and absence (right) of Demodex mites

Using confocal laser scanning in vivo microscopy, comparative study was performed on the the number of mites in the excretory duct of the effectiveness of confocal laser scanning in vivo or hair follicle, the number of hair microscopy and scraping methods followed by follicles and excretory ducts of sebaceous glands microscopic examination. The data obtained are per unit area, the size of the mites, the gland ducts presented in Table 2. of the sebaceous glands, as well as the orifices of hair follicle were counted. The ImageJ software The sensitivity and specificity were calculated version 1.34 was used to measure all the given using standard formulas with positive, false value. positive, negative, and false negative results calculations.17 Statistical data analysis was carried To assess the validity of the confocal laser out with SPSS Statistic 21.0 software. The scanning in vivo microscopy method, the study was relationship of categorical indicators was conducted in all subjects and in participants who established with Fisher's exact test. The were not included in the study (n=106). A researcher used one-way analysis of variance with

J Gen Proced Dermatol Venereol Indones. 2018;2(3):98-104. 101 a paired comparison to assess the significance of significance of differences in the absence of normal differences between the follicle sizes. Mann- distribution. Differences were considered Whitney criteria and Kruskal-Wallis tests for significant at p < 0.05. multiple comparisons were used to assess the

Table 2. Comparative analysis of research methods for the presence of Demodex mites (n=106; 100%)

Identification of Patients diagnosed Healthy Total Method Demodex mites with acne and rosacea volunteers (>5 mites per 1cm2) Light microscopy of skin + 28.3% 2.8% 31.1% scrapings - 37.7% 31.2% 68.9% Identification of Patients diagnosed Healthy Total Method Demodex mites with acne and rosacea volunteers (>5 mites per 1cm2) Confocal laser scanning in + 37.7% 5.7% 43.4% vivo microscopy - 28.3% 28.3% 56.6%

Results microscopic diagnosis of facial skin scraping. A confocal laser scanning in vivo microscope makes The quantitative distribution of occurring cases it possible to visualize mites located in deeper diagnosed by light microscope and confocal laser layers of the skin that are not accessible for scanning in vivo microscope is shown in Table 3. scarification. This method has high potency in The Demodex mites were detected in 30 patients diagnostic capabilities, combining a set of different with acne and rosacea (28.3%) by light microscope diagnostic methods in dermatology (scraping, and 40 patients (37.7%) by confocal laser scanning dermatoscopy, histological examination). The in vivo microscopy. Using confocal laser scanning absence of traumatization of the epithelium and the in vivo microscopy, it was also possible to identify painfulness of the procedure are additional Demodex mites in healthy volunteers (n=6, 5.7%). advantages of this method. Using light microscopy, only three healthy volunteers were identified with Demodex mites with The higher specificity of confocal laser scanning in the number of 5 mites per one cm2 area (n=3, vivo microscopy (68.8%) predisposes to more 2.8%), while the remaining 33 participants were frequent production of false negative results, which negative (31.2%). The sensitivity of the laboratory were detected in six healthy volunteers (5.7%), method using a light microscope for diagnosing which again proves the high accuracy of the Demodex mites is 90.9%, with a specificity of method and a higher probability of detection of 65.5%. The sensitivity of the instrumental Demodex mites even in healthy volunteers, diagnostic method using confocal laser scanning in compared to light microscopy of skin scrapings. At vivo microscopy is 87%, with a specificity of 68.8%. the same time, Demodex mites were identified in only three healthy volunteers (2.8%) using the light The obtained data also proved confocal laser microscopy method for scrapings. scanning in vivo microscopy`s superiority over light

Table 3. Quantitative distribution of occurring cases diagnosed by light microscopy of skin scrapings and on a confocal laser scanning in vivo microscope (n=106; 100%)

Positive False positive False negative Negative Methods results results results results (%) (%) (%) (%) Light microscopy of skin 28.3 37.7 2.8 31.2 scrapings Confocal laser scanning in 37.7 28.3 5.7 28.3 vivo microscopy

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