Comparison of two techniques for measuring Demodex folliculorum and Demodex brevis in rosacea patients: standardized skin surface biopsy vs. direct microscopic examination

BackgroundDemodicosis is a parasitic skin disease caused by Demodex mites, and the determination of mite density per square centimeter is important to diagnose demodicosis. Standardized skin surface biopsy (SSSB) and direct microscopic examination (DME) are commonly used to determine Demodex mites density (Dd). However, no study has previously compared these two methods with respect to clinical types and distribution patterns of demodicosis.ObjectiveThe aim of this study was to compare the value of SSSB and DME findings in reference to the clinical types and distribution patterns of demodicosis.MethodsThe medical records of 35 patients diagnosed with demodicosis between December 2011 and June 2015 were retrospectively reviewed. Demodicosis was classified according to four clinical types (pityriasis folliculorum, rosacea type, acne type, and perioral type) and three distribution patterns (diffuse pattern, U-zone pattern, and T-zone pattern). Two samples, one for SSSB and one for DME, were obtained from a lesion of each patient.ResultsIn all patients, mean Dd and the proportion with a high Dd (>5D/cm2) by DME (14.5±3.3, 80.0%, respectively) were higher than by SSSB (5.5±1.3, 37.1%, respectively; p<0.01, p=0.02, respectively). In terms of clinical types, for rosacea type, mean Dd and proportion with a high Dd by DME (12.4±3.5, 84.6%, respectively) were significantly greater than those determined by SSSB (3.6±1.2, 23.1%; p=0.04, p=0.04, respectively). In terms of distribution pattern, for the diffuse pattern, mean Dd and the proportion with a high Dd by DME (17.5±3.7, 100%, respectively) were significantly higher than those determined by SSSB (6.0±2.7, 26.7%; p<0.01, p<0.01, respectively).ConclusionThe results of our study revealed that DME is a more sensitive method for detecting Demodex than SSSB, especially in patients with diffuse pattern and suspected rosacea type. Further research is needed to confirm this finding.

In daily dermatological practice, many dermatologists do not include demodicosis in their differential diagnoses, or the diagnosis of demodicosis is frequently masked by other skin diseases such as papulopustular or erythematotelangiectatic rosacea, seborrhoeic dermatitis, perioral dermatitis and contact dermatitis. There are two methods for measurement of the density of Demodex folliculorum (Dd): standardized skin surface biopsy (SSSB) and direct microscopic examination of fresh secretions from sebaceous glands (DME). No study has been reported in the literature comparing the diagnostic value of these two techniques. To compare the value of the two techniques, SSSB and DME, for the measurement of Dd in patients with suspected demodicosis. Mite density was investigated using SSSB and DME in 37 patients with facial skin lesions suggesting demodicosis. Two samples, one for SSSB and one for DME, were obtained from a cheek lesion of each patient. Twenty-three (62%) patients were diagnosed with demodicosis according to their clinical manifestations combined with a high Dd (Dd > 5 mites cm(-2)) with SSSB and/or DME. In all the patients, the mean Dd measured with SSSB was higher than that with DME (22.9 +/- 5.9 and 2.2 +/- 0.8, respectively; P = 0.001). Also, among the 23 patients with demodicosis, the mean Dd measured using SSSB was higher than the mean Dd with DME (36.5 +/- 8.3 and 3.4 +/- 1.2, respectively; P = 0.0001). We recommend the use of SSSB for the measurement of Dd as more patients with demodicosis can be diagnosed with this method compared with the DME method.

Introduction: Demodicidosis is a chronic skin disease, caused by two species of Demodex (D. folliculorum and D. brevis). Demodex infected individuals are mainly symptomless and may have pathogenic symptom only when mite density is high within the skin.Objectives: To compare the value of the Standardized skin surface biopsy (SSSB) and direct microscopic examination with KOH (DME) for assessing Demodex density.Methods: Fifty patients with demodicosis were determined Demodex by SSSB and DME. Comparision of the similarity of test results between the two methods was conducted by Cohen’s Kappa statistic.Results: The positive test rate of SSSB is 90.0%, of DME is 82.0%. The percentage of patients who tested positive for both methods was 76.0%, and the negative for both methods was 4.0%. Demodexdensity &gt; 5/cm2of SSSB was 47.9% higher than DME of 29.2%. There was quite similarity of Demodexdensity between SSSB and DME with coefficient kappa = 0.62. Conclusion: SSSB is more sensitive method for detecting Demodex than DME, particularly in patients with macular lesions, vasodilatation, U-shaped distribution, located on the nose and nasolabial sulcus.

Rosacea is an inflammatory disease with 50% of ocular involvement rate. Primary demodicosis is an eruption caused by Demodex mites, and there is no data about the rate of ocular involvement in primary demodicosis. In this cross-sectional study, it is aimed to reveal the frequency of Demodex blepharitis in rosacea and primary demodicosis patients. In total, 58 rosacea, 33 primary demodicosis patients, and 31 healthy volunteers were included in the study. Four samples were obtained from eyelashes with a forceps and from facial skin by standardized skin surface biopsy. A positive result is described as detecting at least one Demodex mite on an eyelash or at five mites in the face. The patients were also examined by an ophthalmologist in terms of ocular involvement. Both rosacea and primary demodicosis patients had significantly more complaints like burning and stinging in the eyes compared to the control patients (P=.001). Primary demodicosis and papulopustular rosacea patients had the highest numbers of eyelash mites, respectively, and significantly a higher rate of blepharitis than the control group. As a result, the Demodex count was significantly higher in the primary demodicosis and rosacea patients than the control group. We think that every Demodex-positive patients should be evaluated for also eyelash mites to prevent a possible chronic blepharitis.

Demodex mites are believed to contribute to the pathogenesis of both rosacea and demodicosis. While standardized skin surface biopsy (SSSB) remains the gold standard for detecting Demodex density, it is invasive and time-consuming. Ultraviolet (UV) dermoscopy, a non-invasive technique that visualizes fluorescent structures, may offer a rapid alternative for identifying Demodex proliferation. To investigate the relationship between Demodex folliculorum (D. folliculorum) and Demodex brevis (D.brevis) counts obtained by SSSB and fluorescent dots detected under UV dermoscopy, and to evaluate the diagnostic value of these findings in rosacea and demodicosis. Fifty-four patients with rosacea or demodicosis were evaluated using both UV dermoscopy and SSSB from the same facial region. Orange and bright blue fluorescent dots were recorded dermoscopically, and mite density and species were quantified via SSSB. Correlation analyses and ROC curve analysis were performed to assess diagnostic performance. A significant positive correlation was found between total Demodex count and bright blue fluorescent dots (r = 0.343, p = 0.013), but not with orange dots (r = -0.065, p = 0.649). Only D. folliculorum showed significant correlation with blue and total fluorescent dots. ROC analysis identified >11 bright blue dots as the optimal threshold for predicting Demodex positivity, with a sensitivity of 82.1% and specificity of 71.4% (AUC = 0.793, p = 0.003). Bright blue fluorescent dots observed under UV dermoscopy significantly correlate with Demodex density, particularly D. folliculorum. A threshold of >11 blue dots demonstrated good diagnostic accuracy and may serve as a practical, non-invasive alternative or adjunct to SSSB in clinical settings. Orange fluorescence was not associated with Demodex density and likely reflects unrelated sebaceous or microbial activity.

Funding sources: None. Conflicts of interest: None declared. Madam, We thank Lacey et al.1 for their interesting and very welcome thoughts concerning our article on the usefulness of confocal laser scanning microscopy (CLSM) in the quantification of Demodex mites, and for their excellent pictures of Demodex mites, seen under the light microscope after extraction using the standardized skin surface biopsy (SSSB) method. As a centre specialized in acne and rosacea we are also using SSSB routinely as the standard method for Demodex quantification and have gathered ample experience within the past decades. We agree that mainly Demodex folliculorum is detected with SSSB and especially with CLSM (with a penetration depth limited to about 200–300 μm), as D. folliculorum is usually located at the upper part of the infundibulum of the hair follicle, while Demodex brevis is usually seen at the lower portion close to the sebaceous glands. Thus D. brevis may be more frequently identified by SSSB, as well as the different life stages. This may be important in understanding the role of Demodex mites in human skin, but in our experience the main question in daily practice is: ‘Is the number of Demodex mites normal or are there too many?’ Our data suggest that CLSM offers a noninvasive and quick method to answer this question in vivo.2

Successful treatment of ivermectin refractory demodicosis with isotretinoin and permethrin cream

Rosacea is a chronicskin condition that typicallyaffects the face and it results in redness and inflammation. The main risk factors of this disease are Demodex folliculorum, living in the pilosebaceous units. To evaluatethe efficacy and safty of permethrin 2.5% in combination withtea tree oil (TTO) topical gel versusplacebo on Demodex density (Dd) and clinical manifestationusingstandard skin surface biopsy (SSSB) in rosacea patients. In this double-blind, randomized clinical trial, 47 papulopustular rosacea patients were enrolled, with 35 patients finishing the 12weeks of treatment. Each patient used permethrin 2.5% with TTO on one side of the face and a placebo on the other, twice daily for 12weeks. SSSB, photography and clinical rosacea scores according to National Rosacea Society, as well as adverse drug reaction (ADRs) were reported at the baseline, 2nd, 5th, 8th, and 12th weeks. A total of 47 patients were enrolled with papulopustular rosacea, and 35 patients finished the study. The effects of permethrin 2.5% with TTO gel on mite density were significant at week 5, 8, 12 (P value=.001). Clinical features and global assessments showed papules, pustules and nontransient erythema had improvement in drug group after 12weeks (P values <.05). The improvement of burning and stinging and dry appearance was greater than the placebo gel (P value <.05). Itching in placebo group was significantly more than other group (P value=.002). Administration of permethrin 2.5% with TTO gel demonstrated good efficacy and safety in rosacea. This topical gel inhibited the inflammatory effects of rosacea and reduced Demodex mite.

Efficacy and safety of permethrin 5% topical gel vs. placebo for rosacea: a double-blind randomized controlled clinical trial.

Reflectance confocal microscopy (RCM) has been recently shown to be effective for measuring the Demodex mite density. To compare and demonstrate the advantages and disadvantages of standardized skin surface biopsy (SSSB) and RCM for measuring the density of Demodex mites. 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. Forty-two patients demonstrated high Demodex density [(Dd)>5mites/cm(2) ] 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 (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 10mm(2) 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). Reflectance confocal microscopy is a fast, direct and noninvasive method for Demodex-associated diseases and it is superior to SSSB for Demodex mite detection.

Microbiota of Demodex mites from rosacea patients and controls

Context: Cetuximab is an epidermal growth factor receptor inhibitor. It is frequently used in the treatment of solid tumors. However, it has a high potential to cause acne-like rash. Demodex mites, which are known to increase in number in immunosuppressive circumstances, are closely related to the acneiform lesions.Objective: The aim of this study is to evaluate the presence of demodex mites in acne-like rash that appears under the treatment of Cetuximab.Methods: We reviewed the medical records of patients who applied to our clinic with cetuximab induced papulopustular rashes between November 2014 and March 2016. Demodex sampling was performed by standardized skin surface biopsy (SSSB) in a total of 11 patients (eight males and three females). Infestation was defined as at least 5 living parasites/cm2 of skin.Results: Upon the SSSB examination in 10 out of the 11 patients, no demodex mites were detected. Demodex mites were found in only one of the patients. This patient, in whom two dead Demodex folliculorums were found through facial sampling, was also regarded as negative since his demodex density was under the threshold limit value.Conclusion: In this study, it has been concluded that acne-like rash that develops under the treatment of cetuximab is not related to the presence of demodex mites. Papulopustular eruptions that develop under cetuximab treatment should not be directly correlated with the presence of demodex; first SSSB and demodex presence should be evaluated.

Demodex mites are highly found in the skin of patients with rosacea. The diagnosis of Demodex can be made by standardized skin surface biopsy. Dermoscopy is a tool used in the noninvasive diagnosis of various dermatological diseases. To determine whether dermoscopic features of demodicosis are associated with the result of standardized skin surface biopsy in patients with rosacea and to compare dermoscopic features of rosacea in Demodex-positive and negative samples and Demodex type. A total of 30 patients (7 male, 23 female) were included in the study. Dermoscopic examination was performed on both the clinically most severely affected areas and adjacent healthy skin. The skin surface biopsy sample was taken from the same place from where the dermoscopic image was taken. A total of 83 (lesion n = 60, non-lesion n = 23) areas were evaluated. Demodex was detected in 60.2% (n = 50) of the samples. Half of these samples revealed only Demodex folliculorum, and the remaining half revealed D folliculorum and Demodex brevis. Of the Demodex-positive samples, 88% had Demodex tails (P =0.001) and 68% Demodex follicular openings (P = 0.002) on dermoscopy. In D folliculorum+D brevis-positive samples, the rate of scale and pustule was higher than D folliculorum-positive samples (P = 0.017 and P = 0032, respectively). The sensitivity and specificity of Demodex tail are higher than Demodex follicular opening and scale and pustule detection with dermoscopy and may indicate the coexistence of both D folliculorum and D brevis.

A standardized skin surface biopsy was performed in 34 patients suffering from skin diseases with high Demodex folliculorum density (Dd) > 5D/cm2 before, during and after topical treatment. The patients were randomized into six comparable groups to study six topical treatments: metronidazole 2%, permethrin 1%, sublimed sulphur 10%, lindane 1%, crotamiton 10% and benzyl benzoate (BB) 10%. Their acaricidal activity was measured according to three criteria: (i) for each treatment, decrease of Dd to under the normal threshold (< or = 5 D/cm2); (ii) for each treatment, a significant decrease in Dd; and (iii) comparison of the relative difference in Dd between treatments. These three criteria converged to establish the acaricidal activity of BB on D. folliculorum; the efficacy of crotamiton was demonstrated by the second criterion. An important irritating effect was observed with BB and sulphur.

Standardized skin surface biopsy (SSSB) is considered to be the gold standard technique to evaluate the density of Demodex mites for the diagnosis of demodicidosis. Potassium hydroxide (KOH) preparation of skin scrapings is a much simpler procedure that can be used to detect pathogens in the superficial skin. To evaluate the reliability of potassium hydroxide preparation of skin scrapings as compared to the standard skin biopsy technique with regard to capacity to detect Demodex mites, time consumed and technician satisfaction. One hundred outpatients presenting with facial erythema of uncertain cause were enrolled. Standardized skin surface biopsy and potassium hydroxide preparation of skin scrapings were undertaken in adjacent areas on the patients' right cheek. Patients with normal facial skin were excluded from the study. The accuracy of Demodex mite detection by potassium hydroxide preparation of skin-scrapings when compared to the standard procedure is 82%. The sensitivity, specificity, positive and negative predictive values of this method are 75%, 84.2%, 60% and 91.43%, respectively. There was no statistically significant difference between the standard and skin scraping techniques (P = 0.238) with regard to mite detection. Mean preparation time while using the skin scraping technique was 6 times less than that of the standard technique. For interpretation also, skin scraping technique (3.6 min) consumed much less time than the biopsy technique (9.8 min). Moreover, experienced technicians were more satisfied with skin scraping. Potassium hydroxide preparation of skin scrapings is an effective, time saving and practical technique to detect Demodex mites with accuracy comparable to the standard biopsy method.