Journal of General - Procedural Dermatology & Venereology Journal of General - Procedural Dermatology & Venereology Indonesia
Indonesia
Volume 2
Number 3 Vol. 2, No. 3 (June 2018) Article 3
6-30-2018
Comparative study of two diagnostic methods of demodicosis in Comparative study of two diagnostic methods of demodicosis in patients with acne and rosacea
patients with acne and rosacea
Anzhela Kravchenko
Department of Dermatovenereology and Cosmetology, Russian Medical Academy of Continuous Professional Education, the Ministry of Healthcare of Russian Federation, Moscow, Russia
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Recommended Citation Recommended Citation
Kravchenko, Anzhela (2018) "Comparative study of two diagnostic methods of demodicosis in patients with acne and rosacea," Journal of General - Procedural Dermatology & Venereology Indonesia: Vol. 2: No.
3, Article 3.
DOI: 10.19100/jdvi.v2i3.62
Available at: https://scholarhub.ui.ac.id/jdvi/vol2/iss3/3
This Article is brought to you for free and open access by UI Scholars Hub. It has been accepted for inclusion in Journal of General - Procedural Dermatology & Venereology Indonesia by an authorized editor of UI Scholars Hub.
Original Article
Comparative study of two diagnostic methods of demodicosis in patients with acne and rosacea
Anzhela Kravchenko
Department of Dermatovenereology and Cosmetology, Russian Medical Academy of Continuous Professional Education, the Ministry of Healthcare of Russian Federation, Moscow, Russia
Email: angkravchenko@gmail.com
Abstract
Background: Demodicosis is a disease, caused by parasitisation of the opportunistic parasites from the acariasis group – Demodex mites. This article presents a comparative study of two methods (light microscopy of skin 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 eyelash 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
Demodicosis is a disease caused by parasitisation of the opportunistic parasites from the acariasis group – Demodex mites. Mites, with size of 0.2-0.5 mm, live in the ducts of the sebaceous and Meibomian glands, in the orifices of the hair follicles, and the excretory ducts of the sebaceous glands of humans and mammals.1,2 Demodex mites are part of the microflora of the human skin and commonly asymptomatic. However, they can be identified in acute inflammatory dermatoses;
such as acne, rosacea, seborrheic dermatitis, perioral dermatitis, or as independent disease.3 The diagnosis is confirmed by identifying the presence of Demodex mites. The most common method used to identify Demodex mites is a compilation of an acarogram consisted of larvae,
nymphs, eggs and adult form counts. The criterion of mites’ activity is the detection of more than five adults, larvae or eggs per one cm2. Diagnosis of eyelashes demodicosis is established by detection of one mite on 2-4 eyelashes. The activity of mites- borne invasion can be assessed by differences in the number of mites per cm2.4
The classic method of detecting Demodex mites is light microscopy of the skin scraping specimens, using a disposable scarifier and tweezers. The material is taken from an area which has the largest accumulation of sebaceous glands on the face, eyebrows, and eyelashes. The magnification of the microscope is 10 and 40 times. Diagnosis comprises clinical presentations (papulopustules, comedones, erythema of the face, telangiectasias) and positive confirmation by laboratory investigation (quantitative load of more than five adult mites, larvae or eggs or both per one cm2).5
It is essential to repeat the examination in cases of high clinical suspicion with that negative laboratory result. Specimens must be taken from large areas of the rash, since random selection of the study site can cause negative laboratory analysis and may not be able to prove the absence of mites-borne invasion.6
Modern laser-based confocal microscopes operate the optical system of the microscope more accurately, reduce the glare of the images, and improve the focusing of the light beam.7 The work of a confocal microscope is based primarily on the ability of various structures of the skin to refract laser radiation, thus obtaining images of the layers of the epidermis and dermis8 and assessing the condition of the skin as well as fibres of the dermis.9 Confocal laser scanning in vivo microscopy is a new method for studying the structure of the skin in the form of pictures with grayscale hue. Confocal laser scanning microscopy allows you to determine the thickness of the skin and visualizes different layers of the skin. Thus, the method provides additional information on the composition and structure of the skin.10 In ophthalmology, it is possible to visualize changes in Meibomian glands in the form of enlargement or obstruction, the presence of inflammatory infiltrates, as well as to detect Demodex mites.11 The scanning method of laser confocal microscopy can be compared with a histological examination of the skin, with an advantage that this method is performed non- invasively.8 According to various data, the sensitivity of this method to identify demodicosis is 83-91%, and the specificity of this method is 95- 99%.12-14
The use of confocal laser scanning in vivo microscopy in dermatology today is considered as one of the most promising methods to identify demodicosis, despite the fact that it has a number of disadvantages, which are
1. Obtaining relatively superficial images with penetrating ability up to 200 μm, which limits the possibility of studying deeper layers of the skin.
2. A relative high cost equipment and operational.
3. Inaccessible for large number of dermatologists.11
In comparison with the conventional light microscope, the advantages of the method are high-contrast images with high resolution, its three- dimensional reconstruction, and digital processing of the data obtained.9 One of the advantages of the
method is the ability to detect, quantify and determine the Demodex mites on the facial skin of patients with rosacea and acne by counting mites and follicles per unit area, and by measuring the size of mites. T. Kojima et al. demonstrated the use of confocal laser scanning microscopy for the diagnosis of eye demodicosis.15 The authors managed to find mites in the bulb terminal of the eyelashes, inflammatory infiltrates around the meibomian glands and conjunctiva. Thus, according to the scientific literature, confocal laser scanning microscopy is a non-invasive and rapid method for detecting Demodex mites.16
Given the relevance of this topic, we conducted a diagnostic survey of healthy volunteers and patients with acne, rosacea, complicated and non- complicated demodicosis using light microscopy of the facial skin scrapings and confocal laser scanning in vivo microscopy. The study aimed to evaluate the sensitivity and specificity of the methods of light microscopy of facial skin scrapings compared to confocal laser scanning in vivo microscopy for diagnosing "Demodicosis" in patients with acne and rosacea.
Methods
This study was conducted in 2014-2017. Thirty patients with acne and rosacea complicated by demodicosis (group I), 30 patients with acne and rosacea, not complicated by demodicosis (group II) and 30 healthy volunteers (group III) were included in the study. Sixteen patients with acne and rosacea had Demodex mites on their facial skin, but their presence was confirmed only by one of the investigation methods. Despite the fact that these patients were tested, they did not enter the study.
Thus, the initial number of patients analysed in the course of work was 106 people. The grouping of patients included in the study is presented in Figure 1.
The diagnosis of acne and rosacea was established at the primary clinic based on the clinical manifestation of the diseases. The study included participants with age over 18 years old, after signing an informed consent to participate in the study. The range of the participants’ age was 18 to 79 years (mean age 30.0 ± 11.9). Subjects comprised 39 males and 51 females. There were 40 patients with acne and 20 patients with rosacea, divided into group I and group II. The characteristics of patients are presented in Table 1.
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)
30 (100%) 12
(40%)
18 (60%)
Diagnosis Acne Rosacea Acne Rosacea
8 (26.7%)
4 (13.35%)
10 (3.3%)
8 (26.7%)
Mean age (years) 23±5.5 44±10.4 29±6.4 51±17.6
Group II - absence of Demodex mites
30 (100%)
Gender Male (n) Female (n)
12 (40%)
18 (60%)
Diagnosis Acne Rosacea Acne Rosacea
9 (30%)
3 (10%)
13 (43.3%)
5 (16.7%)
Mean age (years) 24±2.2 41±6.3 33±2.9 50±5.4
Group III– healthy volunteers
30 (100%)
Gender Male (n) Female (n)
15 (50%)
15 (50%)
Mean age (years) 26±1.4 31±3.1
n = number of patients
The presence of Demodex mites was determined by performing scraping of the skin of the face, squeezing the contents of the sebaceous glands, and epilation of the eyelashes and eyebrows. At a given location, an area of one cm2 was selected and marked by a pencil. The material was placed on a watch glass with a drop of 10% potassium hydroxide solution and covered with a slide for 10- 15 minutes. Then, the material was examined with a light microscope to determine the presence of Demodex mites and their quantitative load per unit area (one cm2). Patients with a quantitative load of more than five adult mites, larvae and/or eggs per
one cm2, and clinical manifestation of the diseases are accounted for Group I.
Skin parameters were assessed in patients in vivo using a VivaScope® 1500 confocal laser scanning in vivo microscope (Lucid Inc., Rochester, NY, USA), in real time using the VivaScan VS 007.11.12 software package. The maximum output power of the laser was 21.7 mW. The penetrating ability of the microscope is 200-300 μm, which is up to the papillary dermis.
Patients with acne and rosacea
(16)
Demodex mites were identified by 1 investigation method
Healthy volunteers (30)
Patients with acne and rosacea
(30)
Demodex mites were not identified
by 2 investigation methods
Total of 106 patients
Patients with acne and rosacea
(30)
Demodex mites were identified by 2
investigation methods
The patient was asked to not moving during specimen collection. A microscope object glass was glued to facial skin with acrylic glue in order to limit the displacement of the tissues. The affected surface of the skin was located in the centre of the object glass. A drop of immersion oil was placed between the skin surface and the microscope sensor. Chromdamol STS® oil or water-based ultrasound gel was used as an immersion medium since their refractive index (1.50) is the closest to the refractive index of the stratum corneum (1.55).
Study of pathomorphological characteristics of the facial skin was performed in three locations (both cheeks and forehead), with the two modes of the microscope, which are VivaBlock and VivaStack.
The VivaBlock mode was used to analyse the horizontal image of the skin at a predetermined depth. In this mode, up to 256 consecutive pictures
can be taken. When using VivaStack, a series of images was obtained from the stratum corneum of the epidermis to the papillary layer of the dermis.
Using VivaCam mode, a dermatoscopic image of the site was obtained. All layers of the epidermis to the papillary dermis were scanned. The cut-off distance from one shot to the next was three μm.
The size of the hair follicles and excretory ducts of the sebaceous glands were assessed. The researcher performed a quantitative evaluation of the Demodex mites in the hair follicles and excretory ducts of the sebaceous glands, the depth of the mites, as well as the number of excretory ducts of the sebaceous glands and hair follicles per unit area. One unit area was a plot of 5 × 5 mm, (a total area of 25 mm2). Demodex mites are visualized by rounded or oval formations with a hyper contour around the peripheral orifices (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, the number of mites in the excretory duct of the sebaceous gland or hair follicle, the number of hair follicles and excretory ducts of sebaceous glands per unit area, the size of the mites, the gland ducts of the sebaceous glands, as well as the orifices of hair follicle were counted. The ImageJ software version 1.34 was used to measure all the given value.
To assess the validity of the confocal laser scanning in vivo microscopy method, the study was conducted in all subjects and in participants who were not included in the study (n=106). A
comparative study was performed on the effectiveness of confocal laser scanning in vivo microscopy and scraping methods followed by microscopic examination. The data obtained are presented in Table 2.
The sensitivity and specificity were calculated using standard formulas with positive, false positive, negative, and false negative results calculations.17 Statistical data analysis was carried out with SPSS Statistic 21.0 software. The relationship of categorical indicators was established with Fisher's exact test. The researcher used one-way analysis of variance with
a paired comparison to assess the significance of differences between the follicle sizes. Mann- Whitney criteria and Kruskal-Wallis tests for multiple comparisons were used to assess the
significance of differences in the absence of normal distribution. Differences were considered significant at p < 0.05.
Table 2. Comparative analysis of research methods for the presence of Demodex mites (n=106; 100%)
Method
Identification of Demodex mites (>5 mites per 1cm2)
Patients diagnosed with acne and rosacea
Healthy volunteers
Total
Light microscopy of skin scrapings
+ 28.3% 2.8% 31.1%
- 37.7% 31.2% 68.9%
Method
Identification of Demodex mites (>5 mites per 1cm2)
Patients diagnosed with acne and rosacea
Healthy volunteers
Total Confocal laser scanning in
vivo microscopy
+ 37.7% 5.7% 43.4%
- 28.3% 28.3% 56.6%
Results
The quantitative distribution of occurring cases diagnosed by light microscope and confocal laser scanning in vivo microscope is shown in Table 3.
The Demodex mites were detected in 30 patients with acne and rosacea (28.3%) by light microscope and 40 patients (37.7%) by confocal laser scanning in vivo microscopy. Using confocal laser scanning in vivo microscopy, it was also possible to identify Demodex mites in healthy volunteers (n=6, 5.7%).
Using light microscopy, only three healthy volunteers were identified with Demodex mites with the number of 5 mites per one cm2 area (n=3, 2.8%), while the remaining 33 participants were negative (31.2%). The sensitivity of the laboratory method using a light microscope for diagnosing Demodex mites is 90.9%, with a specificity of 65.5%. The sensitivity of the instrumental diagnostic method using confocal laser scanning in vivo microscopy is 87%, with a specificity of 68.8%.
The obtained data also proved confocal laser scanning in vivo microscopy`s superiority over light
microscopic diagnosis of facial skin scraping. A confocal laser scanning in vivo microscope makes it possible to visualize mites located in deeper layers of the skin that are not accessible for scarification. This method has high potency in diagnostic capabilities, combining a set of different diagnostic methods in dermatology (scraping, dermatoscopy, histological examination). The absence of traumatization of the epithelium and the painfulness of the procedure are additional advantages of this method.
The higher specificity of confocal laser scanning in vivo microscopy (68.8%) predisposes to more frequent production of false negative results, which were detected in six healthy volunteers (5.7%), which again proves the high accuracy of the method and a higher probability of detection of Demodex mites even in healthy volunteers, compared to light microscopy of skin scrapings. At the same time, Demodex mites were identified in only three healthy volunteers (2.8%) using the light microscopy method for scrapings.
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%)
Methods
Positive results
(%)
False positive results
(%)
False negative results
(%)
Negative results
(%) Light microscopy of skin
scrapings 28.3 37.7 2.8 31.2
Confocal laser scanning in
vivo microscopy 37.7 28.3 5.7 28.3
Discussion
When comparing methods for detecting Demodex mites, it can be seen that both methods have their advantages and disadvantages. Attention is drawn to the fact that confocal laser scanning in vivo microscopy has an order of magnitude, more advantages compared to the method of light microscopy of skin scrapings. Comparing the results obtained by the method of light microscopy of scrapings and confocal laser scanning in vivo microscopy in acne and rosacea patients, as well as healthy volunteers, it was found that Demodex mites were more frequently detected by confocal laser scanning in vivo microscopy, whereas scraping in these patients produced a negative result. The data obtained demonstrated not only a highly informative content of the confocal laser scanning in vivo microscopy method, but also its superiority over microscopic diagnostics. A positive result in group III, the healthy volunteers, confirms the hypotheses of some authors that mites can be saprophytes.
The high sensitivity of both methods of detection of mites suggests a high probability of obtaining a positive result of the study when Demodex mites are suspected in patients with acne and rosacea. A more significant sensitivity of light microscopy of skin scrapings is in question since the results of detection of Demodex mites in both methods of study in patients with acne and rosacea diverge.
The confocal laser scanning in vivo microscopy data refute the data obtained during light microscopy of skin scrapings. For example, mites were not detected by light microscope in patients with clinical symptoms while confocal laser scanning in vivo microscopy detected mites in sample from patients with acne and rosacea.
Despite the lower sensitivity of confocal laser scanning in vivo microscopy in the diagnosis of demodicosis, the method showed more accurate results compared to light microscopy, revealing the presence of Demodex mites in patients who were considered negative by light microscopy method.
The new method of confocal laser scanning in vivo microscopy is comparable in term of effectiveness to the conventional standard method of light microscopy of skin scrapings and is recommended for examining patients with acne and rosacea to detect Demodex mites for the diagnosis of
"Demodicosis."
Conclusion
As a result of the study of patients with acne and rosacea as well as healthy volunteers, highly
informative content and specificity of confocal laser scanning in vivo microscopy in the diagnosis of demodicosis were established. The obtained data also proved confocal laser scanning in vivo microscopy’s superiority over light microscopic diagnosis of facial skin scraping. Therefore, confocal laser scanning microscopy showed advantages compared to the traditional methods.
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