Methods for extraction and ex-vivo experimentation with the most complex human commensal, Demodex spp.

Demodex spp. mites are the most complex organisms of the human skin microbiome and were discovered more than 175 years ago, yet only little basic research is published about them. As they can be pathophysiologically relevant ectoparasites associated with rosacea, pityriasis folliculorum, and other inflammatory skin diseases, more research should be encouraged. Being a large microorganism or a tiny animal, there are no established basic methods to handle these mites. Here, we describe techniques enabling the extraction of Demodex mites from human skin, their analysis in different ex-vivo settings, the lysis of their exoskeleton, their preservation by freezing, and observation microscopically using specific fluorescent dyes or their inherent autofluorescence. These procedures should facilitate future Demodex research and fuel further the generation of knowledge. Furthermore it is intended to ultimatively enable the mite's cultivation in vitro and reveal its pathophysiological mechanisms.

In which rosacea patients should Demodex in the eyelashes be investigated?

AIM: The aim of this study was to investigate the relationship between the presence of Demodex on the face and within the eyelash follicles in patients with rosacea. SUBJECTS AND METHODS: This prospective cross-sectional study included 80 participants, 40 patients with rosacea and 40 individuals with no rosacea as controls. The presence of Demodex on the face was assessed by standard superficial skin biopsy. Sixteen eyelashes were epilated from each patient and control. RESULTS: The rate of Demodex infestation and severe infestation on the face in patients with rosacea was significantly higher than the control group. Demodex count within the eyelash follicle was significantly higher in patients with erythematotelangiectatic type rosacea than the control group. There was no increase in blepharitis in rosacea patients but when blepharitis was present, the rate of the presence of Demodex was higher in this group. There was a statistically significant relationship between the presence of Demodex within the eyelashes and itchy eyes in people without blepharitis. CONCLUSION: When at least one Demodex is found on the face in rosacea patients, the eyelashes should be examined for effective treatment of the mite. Itchy eyes may be an important sign of the presence of Demodex in people without blepharitis.

Erythematotelangiectatic rosacea may be associated with a subclinical stage of demodicosis: a case-control study.

BACKGROUND: Facial densities of Demodex mites have been observed to be greater in patients with demodicosis and papulopustular rosacea than in healthy control patients. In patients with erythematotelangiectatic rosacea (ETR), this density has been observed to be similar to or greater than that of healthy controls. Erythema and telangiectasia, characteristics of ETR, are often observed among patients with pityriasis folliculorum, a discreet demodicosis, suggesting a possible link between these conditions. OBJECTIVES: To compare the facial Demodex densities of patients with clinical ETR and patients with healthy skin, demodicosis, rosacea with papulopustules, and other facial dermatoses. METHODS: In this retrospective study, we recorded Demodex densities measured using two consecutive standardized skin surface biopsies (SSSB1 and SSSB2) in 23 patients with ETR, 20 healthy control patients, 590 patients with demodicosis, 254 with rosacea with papulopustules and 180 with other facial dermatoses. RESULTS: Patients with ETR had higher Demodex densities (D cm-2 ) than did the healthy controls (mean ± SEM; SSSB1: 15·7 ± 6·3 vs. 1·8 ± 1·1 D cm-2 , P = 0·042; SSSB2: 38·0 ± 13·7 vs. 5·1 ± 2·1 D cm-2 , P = 0·026) and patients with other dermatoses (SSSB1: 0·4 ± 0·1 D cm-2 , P = 0·004; SSSB2: 1·3 ± 0·3 D cm-2 , P = 0·004), but lower densities than patients with demodicosis (SSSB1: 82·7 ± 4·2 D cm-2 , P = 0·008; SSSB2: 172·2 ± 7·7 D cm-2 , P = 0·001) or rosacea with papulopustules (SSSB1: 86·6 ± 7·3 D cm-2 , P = 0·027; SSSB2: 197·0 ± 12·1 D cm-2 , P = 0·002). CONCLUSIONS: ETR may be associated with nonvisible Demodex proliferation, possibly corresponding to a subclinical stage of demodicosis. Dermatologists should be aware of this potential association and look for subclinical demodicosis in patients with ETR, so that topical acaricidal treatment can be offered if Demodex density is high.

Skin-homing T-cell responses associated with Demodex infestation and rosacea.

AIMS: Our aim was to investigate the skin-homing T-cell immune responses triggered in patients with Demodex infestation and/or rosacea. METHODS: Collected whole blood samples were divided into four groups: control subjects; nonrosacea patients with Demodex infestation (Demodex group); papulopustular rosacea (PPR) patients without Demodex infestation (Rosacea group); and PPR patients with Demodex infestation (Rosacea/Demodex group). Following ex vivo activation, skin-homing CLA+CD4+ T-cell subset levels were monitored by flow cytometry. RESULTS: When compared with control subjects, among skin-homing CD4+ T-cell subsets analysed, Demodex patients had higher TH 9 and Treg cell levels; Rosacea subjects displayed elevated TH 1 cell levels; and Rosacea/Demodex patients exhibited increased frequencies of TH 9 and TH 22 cells. In contrast to Rosacea subjects, Rosacea/Demodex group members displayed higher TH 2 cell levels; and when compared with Demodex groups, they had higher TH 1 and TH 2 but lower Treg cell levels. Demodex group members also exhibited higher Treg but lower TH 1 and TH 22 levels than Rosacea/Demodex group subjects. CONCLUSIONS: The skin-homing T-cell responses associated with Demodex infestation and rosacea formation seem to influence each other. The present as well as future studies could contribute to the development of effective treatment strategies for demodicosis and rosacea.

[THE RATIONALE FOR THE POSSIBLE ROLE OF DEMODEX FOLLICULORUM IN THE PATHOGENESIS OF ROSACEA].

The purpose of the study was to study the frequency of detection and population density of the mite in rosacea patients depending on the clinical form, the location of the morphological elements on the face and some parameters of the functional state of the skin. We observed 55 patients (38 women and 17 men) aged from 30 to 65 years old with disease duration from 6 months to 10 years. According to clinical forms, 35 had PPR and 20 - ETR (erythematous - telangiectatic rosacea). By location of the rash elements, the following types are distinguished: central, medial, asymmetric, lateral, and total. The mite was identified by a microscopic method. The functions of the skin barrier of the face skin: moisture, oiliness, dryness were determined using a bio-impedance analyzer (BIA). As shown by the results of the study of the above parameters in the observed patients, a high density of the mite population (> 5 per cm2) was noted in cases of total, medial and central type of their location. This indicator was in direct correlation with parameters such as skin fat content and moisture. 75% of patients in this group were diagnosed with PPR. In the group of patients with medium and low population density of mites (<5 per cm2), the lateral, asymmetric and central type of their location were dominated in 65% ​​of patients with ETNR. This group of patients showed low levels of skin fat and moisture, relatively high rates of dryness. Thus, the population density of the mites is dependent on the intensity of the anatomical location of the sebaceous glands of the face skin. An analysis of the results obtained in the study of the role of mite in the pathogenesis of rosacea led us to the conclusion: in rosacea patients, a correlation was found between the increase in the population density of mites, the type of element arrangement and the severity of dermatosis, which proves the role of the mite as one of the most frequent but not obligatory pathogenetic factors in the development of this dermatosis, especially its papulopustular form.

Demodex mites modulate sebocyte immune reaction: possible role in the pathogenesis of rosacea.

BACKGROUND: Rosacea is a common facial skin disorder mainly affecting middle-aged adults. Its aetiology is unknown and pathogenesis uncertain. Activation of the host innate immune response has been identified as an important factor. The Demodex mite population in the skin of rosacea patients is significantly higher than in patients with normal skin, suggesting that they may be of aetiological importance in this disorder. OBJECTIVES: To determine the potential of Demodex mites to interact with the host immune system. METHODS: Live Demodex mites were extracted from normal facial skin of control subjects and used in cell stimulation experiments with the immortalized SZ95 sebocyte line. Time- and mite-dose-dependent experiments were performed. Direct effects of Demodex and effects of the medium in which Demodex had been cultured were evaluated on the Toll-like receptor (TLR) signalling pathway on both a gene and protein expression level. RESULTS: Mites modulated TLR signalling events on both mRNA and protein levels in SZ95 sebocytes. An initial trend towards downmodulation of genes in this pathway was observed. A subsequent switch to positive gene upregulation was recorded after 48 h of coculture. Demodex secreted bioactive molecules that affected TLR2 receptor expression by sebocytes. High numbers of Demodex induced proinflammatory cytokine secretion, whereas lower numbers did not. CONCLUSIONS: Demodex mites have the capacity to modulate the TLR signalling pathway of an immortalized human sebocyte line. Mites have the capacity to secrete bioactive molecules that affect the immune reactivity of sebocytes. Increasing mite numbers influenced interleukin-8 secretion by these cells.

Role of Demodex mite infestation in rosacea: A systematic review and meta-analysis.

BACKGROUND: The reported prevalence and degrees of Demodex mite infestation in rosacea vary widely. OBJECTIVE: We sought to conduct an evidence-based meta-analysis of the prevalence and degrees of Demodex mite infestation in patients with rosacea. METHODS: Systematic literature review and meta-analysis were conducted. Odds ratios for prevalence of infestation and standardized mean difference (SMD) for Demodex density in patients with rosacea were pooled. Subgroup analysis for type of rosacea, control group, and sampling and examination methods were also performed. RESULTS: Twenty-three case-control studies included 1513 patients with rosacea. Compared with the control patients, patients with rosacea were more likely to be infested by Demodex mites [odds ratio, 9.039; 95% confidence interval (CI), 4.827-16.925] and had significantly higher Demodex density (SMD, 1.617; 95% CI, 1.090-2.145). Both erythematotelangiectatic rosacea (SMD, 2.686; 95% CI, 1.256-4.116) and papulopustular rosacea (SMD, 2.804; 95% CI, 1.464-4.145) had significantly higher Demodex density than did healthy control patients. LIMITATIONS: Interstudy variability was high, and a causal relationship could not be established by case-control studies. CONCLUSIONS: Patients with rosacea had significantly higher prevalence and degrees of Demodex mite infestation than did control patients. Demodex mites may play a role in both erythematotelangiectatic rosacea and papulopustular rosacea.

Two Consecutive Standardized Skin Surface Biopsies: An Improved Sampling Method to Evaluate Demodex Density as a Diagnostic Tool for Rosacea and Demodicosis.

Diagnosing papulopustular rosacea is not always straightforward; no specific diagnostic test is currently available. A high density of Demodex mites is consistently observed in this condition. This retrospective study assesses an improved method for evaluating Demodex density among 1,044 patients presenting to our dermatology practice. The skin was cleaned with ether and Demodex densities were measured in 2 consecutive standardized skin surface biopsies taken from the same site. Mean densities in patients with rosacea and demodicosis were much higher than those in healthy controls and patients with other facial dermatoses. The optimal cut-off values for the 2 biopsies were combined and the resultant criterion (presence of a first biopsy density < 5 Demodex/cm2 or a second biopsy density < 10 Demodex/cm2) enabled confirmation of a diagnosis of rosacea or demodicosis with a sensitivity of 98.7% and specificity of 95.5%, making this a valuable diagnostic tool for dermatologists in routine clinical practice.

Demodex mites in acne rosacea: reflectance confocal microscopic study.

BACKGROUND/OBJECTIVES: Demodex mite density is emphasised in the aetiopathogenesis of acne rosacea. Reflectance confocal microscopy (RCM) has been shown to be a good method for determining demodex mite density. The objective was to determine demodex mite density using RCM in acne rosacea patients and compare them with controls. METHODS: In all, 30 papulopustular rosacea (PPR) and 30 erythematotelangiectatic rosacea (ETR) totally 60 acne rosacea patients and 40 controls, were enrolled in the study. The right cheek was selected for imaging and RCM was used for scanning. Ten images of 1000 × 1000 µm (total 10 mm2 ) area were scanned from adjacent areas. The numbers of follicles, infested follicles and mites were counted. The mean numbers of mites per follicle and infested follicles were calculated and compared in the patients and control groups. RESULTS: The mean number of mites was 44.30 ± 23.22 in PPR, 14.57 ± 15.86 in ETR and 3.55 ± 6.48 in the control group (P < 0.001). The mean number of mites per follicle was 1.77 ± 0.90 in PPR, 0.57 ± 0.63 in ETR and 0.13 ± 0.23 in the control group (P < 0.001). The cut-off for the mean number of mites for determining mite infestation was 0.17 and above. CONCLUSIONS: Demodex mite density was markedly increased in both ETR and PPR patients. It is believed that the presence of demodex mites plays an important role in rosacea aetiopathogenesis. Demodex mite treatment may reduce the severity of the disease and slow its progressive nature.

Histopathological Analysis of 226 Patients With Rosacea According to Rosacea Subtype and Severity.

BACKGROUND: The histopathological features of rosacea have not been described in detail. OBJECTIVE: To evaluate the histopathological features of rosacea according to clinical characteristics such as subtype and severity. METHODS: We retrospectively analyzed histopathological findings in 226 patients with rosacea, which included 52 patients with the erythematotelangiectatic rosacea (ETR) and 174 patients with the papulopustular rosacea (PPR) subtype. The frequency of each histopathological finding was compared between subtypes. Histopathological features were also compared according to the severity, through subgroup analysis within each subtype group. RESULTS: Perivascular and perifollicular lymphohistiocytic infiltration were common dermal findings in both subtype groups, but the intensity of dermal inflammatory infiltration was higher in PPR than in ETR. Follicular spongiosis and exocytosis of inflammatory cells into hair follicles were noted in both subtypes; but these findings were significantly more common in the PPR subtype. Vascular changes (telangiectasia and proliferation) and solar elastosis were common histopathological findings in both subtypes, with no difference in frequency between subtype groups. Demodex mites were identified in about 40% of patients, and the frequency of demodex mites did not differ between subtype groups. The intensity of perifollicular inflammation and the presence of follicular inflammatory reactions were dependent on the severity of rosacea in both subtype groups. CONCLUSIONS: The intensity of inflammatory reactions, especially perifollicular infiltration, was higher in PPR patients than in ETR patients. Rosacea has a spectrum of histopathological features that are related to clinical progression between rosacea subtypes.

Major pathophysiological correlations of rosacea: a complete clinical appraisal.

BACKGROUND: Rosacea is a characteristic cutaneous disorder with a diverse clinical manifestations ranging from facial vascular hyper-reactivity to sebaceous gland hyperplasia. Many theories on pathophysiology of rosacea were proposed over the past decade, however the pathogenicity is poorly understood. AIM: To review the evidence on different pathophysiological correlations of rosacea. METHODS: A literature search was conducted for studies published between 1990 to March 2014. The inclusion criteria was pathophysiology, randomized controlled trials, controlled trials on rosacea. RESULTS: Out of 5141 articles, 14 high quality studies met all the selection criteria. Of 14 articles, 5 are randomized control trials (RCTs), 2 are controlled trial, 3 comparative trials, 2 observational trials, 1 prospective and 1 diagnostic trial. The studies were categorized into two groups: the trigger factors and sub-types & symptoms. Of 7 high quality studies, 4 provided strong evidence that immune responses causing disease triggered by external/internal factors such as sunlight, food and chemical agents, 3 trials provided significant evidence of microorganisms as causative agents. The remaining trials did not provide significant evidences on pathophysiology. CONCLUSION: Vasculature, chronic inflammatory responses, environmental triggers, food and chemicals ingested and microorganisms either alone or in combination are responsible for rosacea. Many promising drugs are under various phases of clinical trials and interestingly, probiotics could also possibly be used as one of the treatment option.

Additional evidence that rosacea pathogenesis may involve demodex: new information from the topical efficacy of ivermectin and praziquantel.

Additional evidence that Demodex folliculorum may contribute to the pathogenesis of papulopustular rosacea are new studies of two topical antiparasitic agents. Ivermectin and praziquantel have recently been shown to be effective in decreasing the severity of papulopustular rosacea. These two agents significantly differ in molecular structure, but yield similar antiparasitic mechanisms of action. Higher numbers of Demodex mites are found in the skin of patients with rosacea than in people with normal skin. If Demodex play a role in pathogenesis, then hypersensitivity to the mites, their flora, or their products could explain the observed efficacy of antidemodectic therapy.

[IMPACT OF OPISTHORCHIS INVASION ON THE COURSE OF A SKIN PROCESS IN PATIENTS WITH ROSACEA].

The authors give the data-and results of their investigations of 47 patents with rosacea concurrent with chronic opisthorchiasis (Group 1) and 40 patients with rosacea without opisthorhiasis (Group 2): It is found that the severer clinical manifestations of rosacea are, the lower the quality of life is in patients. The patients with rosacea concurrent with chronic opisthorchiasis were found to have severe clinical forms. Rosacea diagnostic rating scores was 1.1-fold higher than in the patients without helminthiasis, suggesting a severer course. Quality-of-life dissatisfaction in patients with rosacea concurrent with chronic opisthorchiasis was significantly higher than in those with rosacea only.

Microbiota of Demodex mites from rosacea patients and controls.

Proliferation of Demodex mites is associated with rosacea. Furthermore, Demodex-associated bacteria were suggested to play a role in the pathogenesis of rosacea. We decided to analyze Demodex microbiota. Mites were collected by standardized skin surface biopsies from patients with erythematotelangiectatic, papulopustular rosacea or from control subjects. The microbiota from each mite was characterized by 16S rRNA clone library approach. The 16S rRNA clone library consisted of 367 clones obtained from 73 extracts originating from 5 samples per study group (ETR, PPR or healthy subjects). A total of 86 species were identified with 36 as Demodex-specific microbiota. In the papulopustular group, proportions of Proteobacteria and Firmicutes increased whereas proportion of Actinobacteria decreased. Here, we report preliminary results on the microbiota of Demodex mites based on a molecular approach showing an unexpected diversity. Differences according to the host status need to be confirmed but open new perspectives for diagnostic of rosacea.

Which factors influence Demodex mite density in standardized superficial skin biopsy in patients with rosacea? A prospective cross-sectional analysis.

Rosacea is a chronic cutaneous disease that manifests with facial erythema, telangiectasia, papules and pustules on the central face. Although the pathogenesis is not well established, rosacea appears to have a close relationship with Demodex mites. The aim of the study was to elucidate the factors influencing Demodex mite density by standardized superficial skin biopsy (SSSB) in patients with rosacea. This prospective, cross-sectional study included 200 patients with rosacea. Clinical characteristics of the patients were recorded and SSSB was used to measure Demodex density (Dd). If Dd was < 5 D/cm2 in the first SSSB, SSSB was repeated 4 more times to avoid false negative results. Of 200 patients, 152 (76%) were females and 48 (24%) males with a mean age of 43.47 ± 11.87 years. Ninety-nine patients (49.5%) had erythematotelangiectatic (ETR) and 101 patients (50.5%) had papulopustular (PPR) subtype of rosacea. Among 200 patients, the ratio of cumulative positive results of the consecutive SSSBs were as follows: 1st SSSB = 125 (62.5%), 2nd SSSB = 155 (77.5%), 3rd SSSB = 170 (85%), 4th SSSB = 173 (86.5%) and 5th SSSB = 174 (87%). The ratio of detecting Demodex infestation in the first SSSB was significantly lower in patients with PPR (55/101, 54.5%) than in patients with ETR (70/99, 70.7%). Median total Demodex mite density and D. folliculorum density were significantly higher in the ETR group than in the PPR group. There was a statistically significant relationship between density of Demodex tails in dermoscopy and positive/negative results of Demodex infestation in SSSB. As a conclusion, Demodex mite density by SSSB was influenced by various factors such as subtypes of rosacea, types of Demodex species, and dermoscopic findings.

Emerging therapies in rosacea.

Rosacea is a common skin disorder with multiple symptoms. The emergence of research that furthers understanding of pathophysiological mechanisms has created new targets for disease treatment. Specifically, there is a need for new treatments that address the various erythematic symptoms associated with rosacea. Systemic and topical therapies have both yielded positive results in treating rosacea with various medications. Subantimicrobial-dose doxycycline is one such promising treatment. Development of novel products in the near future should help achieve more satisfactory outcomes for patients.

Rosacea: current state of epidemiology.

Case definitions are critical in epidemiologic research. However, modern disease indicators must now consider complex data from gene-based research along with traditional clinical parameters. Rosacea is a skin disorder with multiple signs and symptoms. In individuals, these features may be multiple or one may predominate. While studies on the epidemiology of rosacea have previously been sparse, there has been a recent increase in research activity. A broader body of epidemiological information that includes a greater variety of countries beyond Northern Europe and general population-based demographics is needed. As there are operational issues in current case definitions of rosacea subtypes--rationalization and standardization--universal consistent applications in future research is also imperative. Further improvement in disease definition combining new research information along with clinical pragmatism should increase the accuracy of rosacea case ascertainment and facilitate further epidemiological research.

Quantification of Demodex folliculorum by PCR in rosacea and its relationship to skin innate immune activation.

The aim of this study is to quantify D. folliculorum colonisation in rosacea subtypes and age-matched controls and to determine the relationship between D. folliculorum load, rosacea subtype and skin innate immune system activation markers. We set up a multicentre, cross-sectional, prospective study in which 98 adults were included: 50 with facial rosacea, including 18 with erythematotelangiectatic rosacea (ETR), and 32 with papulopustular rosacea (PPR) and 48 age- and sex-matched healthy volunteers. Non-invasive facial samples were taken to quantify D. folliculorum infestation by quantitative PCR and evaluate inflammatory and immune markers. Analysis of the skin samples show that D. folliculorum was detected more frequently in rosacea patients than age-matched controls (96% vs 74%, P < 0.01). D. folliculorum density was 5.7 times higher in rosacea patients than in healthy volunteers. Skin sample analysis showed a higher expression of genes encoding pro-inflammatory cytokines (Il-8, Il-1b, TNF-a) and inflammasome-related genes (NALP-3 and CASP-1) in rosacea, especially PPR. Overexpression of LL-37 and VEGF, as well as CD45RO, MPO and CD163, was observed, indicating broad immune system activation in patients with rosacea. In conclusion, D. folliculorum density is highly increased in patients with rosacea, irrespective of rosacea subtype. There appears to be an inverse relationship between D. folliculorum density and inflammation markers in the skin of rosacea patients, with clear differences between rosacea subtypes.

Noninvasive in vivo detection and quantification of Demodex mites by confocal laser scanning microscopy.

BACKGROUND: In many Demodex-associated skin diseases Demodex mites are present in abundance and seem to be at least partially pathogenic. So far all diagnostic approaches such as scraping or standardized superficial skin biopsy are (semi-)invasive and may cause discomfort to the patient. OBJECTIVES: To see whether confocal laser scanning microscopy (CLSM) - a noninvasive method for the visualization of superficial skin layers - is able to detect and quantify D. folliculorum in facial skin of patients with rosacea. METHODS: Twenty-five patients (34-72 years of age) with facial rosacea and 25 age- and sex-matched normal controls were examined by CLSM. Mosaics of 8 × 8 mm and 5 × 5 mm were created by scanning horizontal layers of lesional skin and quantification of mites per follicle and per area as well as follicles per area was performed. RESULTS: In all patients D. folliculorum could be detected by CLSM and presented as roundish or lengthy cone-shaped structures. CLSM allowed the quantification of Demodex mites and revealed significant differences (P < 0·0001): the mean number of mites was 165·4 per 8 × 8 mm area and 94·2 per 5 × 5 mm area in the patients compared with 34·7 and 22·4, respectively, in the controls. The corresponding mean number of mites per follicle was 0·7 and 0·8, respectively, in the patients and 0·1 and 0·2, respectively, in the controls. CONCLUSIONS: With the help of CLSM it is possible to detect, image and quantify Demodex mites noninvasively in facial skin of patients with rosacea.