A 20 MHz Repetition Rate, Sub-Picosecond Ti-Sapphire Laser for Fiber Delivery in Nonlinear Microscopy of the Skin.

Nonlinear microscopy (NM) enables us to investigate the morphology or monitor the physiological processes of the skin through the use of ultrafast lasers. Fiber (or fiber-coupled) lasers are of great interest because they can easily be combined with a handheld, scanning nonlinear microscope. This latter feature greatly increases the utility of NM for pre-clinical applications and in vivo tissue imaging. Here, we present a fiber-coupled, sub-ps Ti-sapphire laser system being optimized for in vivo, stain-free, 3D imaging of skin alterations with a low thermal load of the skin. The laser is pumped by a low-cost, 2.1 W, 532 nm pump laser and delivers 0.5-1 ps, high-peak-power pulses at a ~20 MHz repetition rate. The spectral bandwidth of the laser is below 2 nm, which results in a low sensitivity for dispersion during fiber delivery. The reduction in the peak intensity due to the increased pulse duration is compensated by the lower repetition rate of our laser. In our proof-of-concept imaging experiments, a ~1.8 m long, commercial hollow-core photonic bandgap fiber was used for fiber delivery. Fresh and frozen skin biopsies of different skin alterations (e.g., adult hemangioma, basal cell cancer) and an unaffected control were used for high-quality, two-photon excitation fluorescence microscopy (2PEF) and second-harmonic generation (SHG) z-stack (3D) imaging.

A Cross-Sectional Study of the Dermatological Manifestations of Patients with Fabry Disease and the Assessment of Angiokeratomas with Multimodal Imaging.

Fabry disease (FD) is a multisystemic X-linked lysosomal storage disease that presents with angiokeratomas (AKs). Our objective was to investigate the clinical and morphologic features of AKs and to present two experimental techniques, multispectral imaging (MSI) and non-linear microscopy (NLM). A thorough dermatological examination was carried out in our 26 FD patients and dermoscopic images (n = 136) were evaluated for specific structures. MSI was used for the evaluation of AKs in seven patients. NLM was carried out to obtain histology samples of two AKs and two hemangiomas. Although AKs were the most common manifestation, the majority of patients presented an atypical distribution and appearance, which could cause a diagnostic challenge. Dermoscopy revealed lacunae (65%) and dotted vessels (56%) as the most common structures, with a whitish veil present in only 25%. Autofluorescence (405 nm) and diffuse reflectance (526 nm) images showed the underlying vasculature more prominently compared to dermoscopy. Using NLM, AKs and hemangiomas could be distinguished based on morphologic features. The clinical heterogeneity of FD can result in a diagnostic delay. Although AKs are often the first sign of FD, their presentation is diverse. A thorough dermatological examination and the evaluation of other cutaneous signs are essential for the early diagnosis of FD.

Scrofuloderma and granuloma annulare-like lesions: Challenges of diagnosing cutaneous tuberculosis in developed countries.

Tuberculosis remains a global health concern, as the increasing levels of urban poverty, higher number of immunodeficient patients and the development of drug resistance threaten the overall efforts made to induce a downward trend for the disease. Scrofuloderma, also known as tuberculosis cutis colliquativa is a subtype of cutaneous tuberculosis. Here we detail a case of a 70-year-old female patient presented with unilateral, left-sided, multiple palpable, painful, ulcerated and purulent cervical nodules, accompanied by persistent generalized erythematous popular granuloma annulare-like skin lesions on the upper extremities. Based on the result of the PCR assay, culture, imaging and histopathological findings, the diagnosis of scrofuloderma was established. To achieve prompt diagnosis and early treatment, it is crucial to include scrofuloderma in the differential diagnosis of ulcerated lesions in developed countries as well, and also be aware of the additional clinical symptoms, such as granuloma annulare-like lesions, possibly accompanying cutaneous tuberculosis.

Correlation of systemic involvement and presence of pathological skin calcification assessed by ex vivo nonlinear microscopy in Pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE (OMIM 264800)) is an autosomal recessive connective tissue disorder mainly caused by mutations in the ABCC6 gene. PXE results in ectopic calcification primarily in the skin, eye and blood vessels that can lead to blindness, peripheral arterial disease and stroke. Previous studies found correlation between macroscopic skin involvement and severe ophthalmological and cardiovascular complications. This study aimed to investigate correlation between skin calcification and systemic involvement in PXE. Ex vivo nonlinear microscopy (NLM) imaging was performed on formalin fixed, deparaffinized, unstained skin sections to assess the extent of skin calcification. The area affected by calcification (CA) in the dermis and density of calcification (CD) was calculated. From CA and CD, calcification score (CS) was determined. The number of affected typical and nontypical skin sites were counted. Phenodex + scores were determined. The relationship between the ophthalmological, cerebro- and cardiovascular and other systemic complications and CA, CD and CS, respectively, and skin involvement were analyzed. Regression models were built for adjustment to age and sex. We found significant correlation of CA with the number of affected typical skin sites (r = 0.48), the Phenodex + score (r = 0.435), extent of vessel involvement (V-score) (r = 0.434) and disease duration (r = 0.48). CD correlated significantly with V-score (r = 0.539). CA was significantly higher in patients with more severe eye (p = 0.04) and vascular (p = 0.005) complications. We found significantly higher CD in patients with higher V-score (p = 0.018), and with internal carotid artery hypoplasia (p = 0.045). Significant correlation was found between higher CA and the presence of macula atrophy (ß = - 0.44, p = 0.032) and acneiform skin changes (ß = 0.40, p = 0.047). Based on our results, the assessment of skin calcification pattern with nonlinear microscopy in PXE may be useful for clinicians to identify PXE patients who develop severe systemic complications.

Emergency Use and Efficacy of an Asynchronous Teledermatology System as a Novel Tool for Early Diagnosis of Skin Cancer during the First Wave of COVID-19 Pandemic.

Background: After the outbreak of the corona virus disease-19 (COVID-19) pandemic, teledermatology was implemented in the Hungarian public healthcare system for the first time. Our objective was to assess aggregated diagnostic agreements and to determine the effectiveness of an asynchronous teledermatology system for skin cancer screening. Methods: This retrospective single-center study included cases submitted for teledermatology consultation during the first wave of the COVID-19 pandemic. Follow-up of the patients was performed to collect the results of any subsequent personal examination. Results: 749 patients with 779 lesions were involved. 15 malignant melanomas (9.9%), 78 basal cell carcinomas (51.3%), 21 squamous cell carcinomas (13.8%), 7 other malignancies (4.6%) and 31 actinic keratoses (20.4%) were confirmed. 87 malignancies were diagnosed in the high-urgency group (42.2%), 49 malignancies in the moderate-urgency group (21.6%) and 16 malignancies in the low-urgency group (4.6%) (p < 0.0001). Agreement of malignancies was substantial for primary (86.3%; κ = 0.647) and aggregated diagnoses (85.3%; κ = 0.644). Agreement of total lesions was also substantial for primary (81.2%; κ = 0.769) and aggregated diagnoses (87.9%; κ = 0.754). Conclusions: Our findings showed that asynchronous teledermatology using a mobile phone application served as an accurate skin cancer screening system during the first wave of the COVID-19 pandemic.

Quantitative Multispectral Imaging Differentiates Melanoma from Seborrheic Keratosis.

Melanoma is a melanocytic tumor that is responsible for the most skin cancer-related deaths. By contrast, seborrheic keratosis (SK) is a very common benign lesion with a clinical picture that may resemble melanoma. We used a multispectral imaging device to distinguish these two entities, with the use of autofluorescence imaging with 405 nm and diffuse reflectance imaging with 525 and 660 narrow-band LED illumination. We analyzed intensity descriptors of the acquired images. These included ratios of intensity values of different channels, standard deviation and minimum/maximum values of intensity of the lesions. The pattern of the lesions was also assessed with the use of particle analysis. We found significantly higher intensity values in SKs compared with melanoma, especially with the use of the autofluorescence channel. Moreover, we found a significantly higher number of particles with high fluorescence in SKs. We created a parameter, the SK index, using these values to differentiate melanoma from SK with a sensitivity of 91.9% and specificity of 57.0%. In conclusion, this imaging technique is potentially applicable to distinguish melanoma from SK based on the analysis of various quantitative parameters. For this application, multispectral imaging could be used as a screening tool by general physicians and non-experts in the everyday practice.

Autofluorescence Imaging of the Skin Is an Objective Non-Invasive Technique for Diagnosing Pseudoxanthoma Elasticum.

Pseudoxanthoma elasticum (PXE) is a rare multisystemic autosomal recessive connective tissue disease. In most cases, skin manifestations of PXE are the first to develop, followed later by severe ocular and cardiovascular complications. In our present study, in addition to dermoscopy, we introduced novel techniques, autofluorescence (AF) and diffuse reflectance (DR) imaging for the assessment of affected skin sites of five PXE patients. PXE-affected skin areas in most skin sites showed a previously observed pattern upon dermoscopic examination. With the novel imaging, PXE-affected skin lesions displayed high AF intensity. During our measurements, significantly higher mean, minimum and maximum AF intensity values were found in areas of PXE-affected skin when compared to uninvolved skin. Conversely, images acquired with the use of 660 and 940 nm illumination showed no mentionable difference. Our results demonstrate that AF imaging may be used in the in vivo diagnostics and quantification of the severity of the skin lesions of PXE patients. In addition, it is a safe, fast and cost-effective diagnostic method. AF imaging may be also used to objectively monitor the efficacy of the possible novel therapeutic approaches of PXE in the future.

Visualization of Keratin with Diffuse Reflectance and Autofluorescence Imaging and Nonlinear Optical Microscopy in a Rare Keratinopathic Ichthyosis.

Keratins are one of the main fluorophores of the skin. Keratinization disorders can lead to alterations in the optical properties of the skin. We set out to investigate a rare form of keratinopathic ichthyosis caused by KRT1 mutation with two different optical imaging methods. We used a newly developed light emitting diode (LED) based device to analyze autofluorescence signal at 405 nm excitation and diffuse reflectance at 526 nm in vivo. Mean autofluorescence intensity of the hyperkeratotic palmar skin was markedly higher in comparison to the healthy control (162.35 vs. 51.14). To further assess the skin status, we examined samples from affected skin areas ex vivo by nonlinear optical microscopy. Two-photon excited fluorescence and second-harmonic generation can visualize epidermal keratin and dermal collagen, respectively. We were able to visualize the structure of the epidermis and other skin changes caused by abnormal keratin formation. Taken together, we were able to show that such imaging modalities are useful for the diagnosis and follow-up of keratinopathic diseases.

Dermatologic aspects of bed bug epidemic: an atlas of differential diagnosis.

Bed bug infestation rate has gone through an unforeseen increase in the past decades worldwide. Their resurgence is a consequence of numerous factors, including growing population density, increased international travel and the spread of insecticide resistance. Bed bug infestation is often revealed by skin symptoms appearing after their bite in sensitive patients. Medical professionals encountering patients with bed bug bites have responsibility for recognizing the condition and for instructing patients about the necessary measures for eradication. Setting the correct diagnosis, however, is not unequivocal as several skin diseases with autoimmune, immune-mediated aetiology or other arthropod stings and bites may present with similar symptoms. In this review we provide a differential diagnostic guide and an atlas of clinical pictures assigned to the diagnoses. We highlight those dermatological findings where the possibility of bed bug bite arises and identify key elements that help in the differentiation so as to avoid unnecessary diagnostic tests and force early start of extermination.

Efficiency of long-term high-dose intravenous ascorbic acid therapy in locally advanced basal cell carcinoma - a pilot study.

INTRODUCTION: The anti-cancer properties of high-dose intravenous ascorbic acid have been demonstrated in various malignancies. In our recent study, we tested topically applied ascorbic acid to treat basal cell carcinoma (BCC), and achieved a good clinical response. AIM: Based on these results, we decided to examine the efficacy and tolerability of high-dose intravenous ascorbic acid (IVA) for locally advanced BCC. MATERIAL AND METHODS: In this pilot study, patients diagnosed with locally advanced BCC who were not amenable to radiation, surgical or local therapy (no other treatment option was available at the time) received intravenous ascorbic acid (1-1.8 g/kg), in an outpatient setting, 1-3 times per week for a mean duration of 42 ±23.6 weeks. This therapy was generally well tolerated. RESULTS: Among 4 patients who had a total of 165 (mean: 41 ±51, range: 1-114) skin lesions, 3 patients achieved stable disease and one had progressive disease. There was substantial variability in individual tumor response to therapy. With the aid of two-photon microscopy and second harmonic generation imaging techniques, alterations in collagen structure were observed between tumor nests during IVA therapy. CONCLUSIONS: Our results suggest that IVA is well tolerated in a small group of patients with extensive BCCs. However, in the era of smoothened (Smo) receptor inhibitors, it may only be considered as an adjuvant therapy in treatment-resistant cases.

Nonlinear optical microscopy is a novel tool for the analysis of cutaneous alterations in pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE, OMIM 264800) is a rare autosomal recessive disorder with ectopic mineralization and fragmentation of elastin fibers. It is caused by mutations of the ABCC6 gene that leads to decreased serum levels of inorganic pyrophosphate (PPi) anti-mineralization factor. The occurrence of severe complications among PXE patients highlights the importance of early diagnosis so that prompt multidisciplinary care can be provided to patients. We aimed to examine dermal connective tissue with nonlinear optical (NLO) techniques, as collagen emits second-harmonic generation (SHG) signal, while elastin can be excited by two-photon excitation fluorescence (TPF). We performed molecular genetic analysis, ophthalmological and cardiovascular assessment, plasma PPi measurement, conventional histopathological examination, and ex vivo SHG and TPF imaging in five patients with PXE and five age- and gender-matched healthy controls. Pathological mutations including one new variant were found in the ABCC6 gene in all PXE patients and their plasma PPi level was significantly lower compared with controls. Degradation and mineralization of elastin fibers and extensive calcium deposition in the mid-dermis was visualized and quantified together with the alterations of the collagen structure in PXE. Our data suggests that NLO provides high-resolution imaging of the specific histopathological features of PXE-affected skin. In vivo NLO may be a promising tool in the assessment of PXE, promoting early diagnosis and follow-up.

Smoothened receptor inhibitor vismodegib for the treatment of basal cell carcinoma: a retrospective analysis of efficacy and side effects.

Background: Smoothened receptor inhibitor vismodegib is indicated to treat locally advanced basal cell carcinoma (laBCC) and lesions in nevoid basal cell carcinoma syndrome (NBCCS).Methods: We treated 11 patients - including four NBCCS and seven laBCC patients - with vismodegib at our department.Results: Complete remission was achieved in three cases, without relapse after discontinuation. Two of the aforementioned patients had NBCCS, in their cases further treatment might be needed. Two patients showed improvement, but later passed away due to unrelated conditions. Two patients with laBCC initially showed remission, then the treatment was suspended due to side effects. After re-administration of the drug, loss of efficacy was observed. We did not observe therapy resistance in our NBCCS group. The rest of the patients showed good response to therapy, but have not reached full remission yet. The main side effects of vismodegib were muscle cramps, dysgeusia, nausea and alopecia. The frequency of adverse events did not show significant differences between the patient groups.Conclusions: Our results show that vismodegib therapy is effective in the treatment of BCC; however, side effects are often severe. Since the suspension of treatment can lead to therapy resistance, the management of side effects is of great importance.

Intralesional therapy for the treatment of keratoacanthoma.

Keratoacanthoma (KA) is a common epidermal tumor that originates from the hair follicle of the skin. It is generally considered as a benign neoplasm, but in rare cases, it can also transform into squamous cell carcinoma. Although surgical excision with a safety margin is considered to be the gold standard treatment for most subtypes of KA, several other treatment options are also available. Intralesional therapy is one of these options, which could be cosmetically and functionally a better alternative to surgical removal, while it provides similar outcomes. It is more effective than topical treatments, yet fewer side effects may be seen than in systemic treatments. Based on the literature, the most commonly used intralesional agent is methotrexate, followed by 5-fluorouracil and interferon alpha. Regardless of the advantages, which make intralesional therapy a desirable treatment alternative, guidelines for the intralesional treatment of KA are not yet established. A histopathological confirmation before the start of treatment is still recommended to prevent any possible misdiagnosis of KA for SCC. In our present study, we set out to review the current state of the art of the intralesional treatment of KA.

Association Between Serum Lipid Profile and Obstructive Respiratory Events During REM and Non-REM Sleep.

PURPOSE: Obstructive sleep apnoea (OSA) represents a risk for dyslipidaemia. Obstructive respiratory events during rapid eye movement (REM) sleep are more strongly related to the development of hypertension and diabetes than in non-REM. However, the relationship between sleep phases and serum lipid profile is unclear. We aimed to analyse the relationship between obstructive respiratory events in REM and non-REM sleep as well as serum lipid profile. METHODS: Polysomnography was performed in 94 adult subjects who did not take any lipid-modifying medications. Fasting venous blood sample was taken the following morning for total cholesterol, triglyceride, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol, lipoprotein(a), apoprotein A1 (ApoA1) and for apoprotein B (ApoB) measurements. Lipid profiles were correlated with apnoea-hypopnoea index (AHI) during REM (AHIREM) and non-REM (AHINREM) stages in all subjects. In addition, lipid profiles were compared between REM-dependent OSA patients (AHIREM ≥ 5/h, but AHINREM < 5/h) and control subjects (both AHIREM and AHINREM < 5/h). RESULTS: AHIREM correlated only with triglyceride concentrations (p = 0.04, Spearman's rho, ρ = 0.21). In contrast, there was a significant association between AHINREM and triglyceride (p = 0.02, ρ = 0.23), ApoB (p = 0.03, ρ = 0.21), HDL-C (p < 0.01, ρ = - 0.32) as well as ApoA1 levels (p = 0.04, ρ = - 0.21). However, these correlations were not present after adjustment for BMI (all p > 0.05). There was no difference in the lipid profile of REM-dependent OSA subjects and healthy controls (p > 0.05). CONCLUSIONS: Altered serum lipid profile is equally associated with a disturbed REM and non-REM sleep in OSA. Obesity must be considered as a strong covariate when interpreting lipid data in sleep apnoea.