Bacteriology of hidradenitis suppurativa exacerbations and deep tissue cultures obtained during carbon dioxide laser treatment

Summary Background Previous studies have shown different bacteria in hidradenitis suppurativa (HS) lesions, but the literature regarding bacteria in acute exacerbation of HS is sparse. Objectives To determine the type of bacteria isolated from HS lesions during exacerbations of the disease. Methods Patients with HS with acute nodules or abscesses were examined and treated by carbon dioxide laser vaporization. Bacterial samples for aerobic and anaerobic cultures were taken from the skin surface (before surgery) and then from the deeper layers (during surgery) of the lesions. At each level two samples were taken, one with a punch biopsy and one by pressing a soft agar gel against the skin. The bacterial findings were typed and quantified. Results A total of 10 patients (eight women and two men), with a mean age of 37·2 years and a mean HS duration of 14·5 years, were included. All of them had an ongoing exacerbation (mean duration 5·6 days) of their HS, with one inflamed lesion that was treated by carbon dioxide laser vaporization. Coagulase‐negative staphylococci (CNS) were found in the deep layers in all 10 patients. Nine of the patients carried Corynebacterium spp. and two alpha‐haemolytic streptococci at various levels. Among the anaerobic microorganisms, Gram‐positive cocci were the most common bacteria. Conclusions As found in a previous study, CNS were the most common bacteria, but contrary to what we expected, Staphylococcus aureus was not found in any cultures from acute inflammatory nodules of HS exacerbations.     

Changes in HPV infection in patients with anogenital warts and their partners.

OBJECTIVES--To investigate the relationship between clinical findings and the detection of human papillomavirus (HPV) DNA in a range of anatomical sites in patients with and without anogenital warts. SUBJECTS--Men and women with a clinical diagnosis of anogenital warts, or a current partner with anogenital warts. SETTING--A department of genitourinary medicine in central London. METHODS--The anogenital areas of the patients were thoroughly examined using a colposcope before and after application of acetic acid. Different types of specimens were taken from a variety of anatomical sites. Superficial skin sampling was performed by the application of slides covered with "Superglue" (SG) to clinically normal and abnormal areas of anogenital skin. The presence of human cells in the SG samples was confirmed by detection of the beta-globin gene using the polymerase chain reaction (PCR). HPV DNA was extracted from the specimens and amplified by using consensus primers with the PCR. HPV types 6, 11, 16, 18, 31 and 33 were identified by Southern blotting followed by hybridisation. RESULTS--In women, HPV DNA was detected in 83% of wart biopsies, 29% of cervical biopsies, 36% of cervical scrapes, 25% of urethral loop specimens, 37% of vaginal washes and 33% of rectal swab specimens. In men, HPV DNA was detected in 67% of wart biopsies, 37% of urethral loop specimens and 12% of rectal swab specimens. Of the SG samples containing the beta-globin gene, 49% from women and 50% from men contained HPV DNA. HPV DNA was not detected in buccal scrapes and serum samples from women or men. Of all specimens with detectable HPV DNA, there was evidence of a single HPV type in 41%, multiple types in 48% and undetermined types in 11%. Samples taken from different sites of a patient tended to have HPV types in common. Sexual partners, however, did not consistently have HPV types in common. CONCLUSIONS--HPV DNA was distributed widely in the anogenital area, in warts, acetowhite areas and clinically normal skin. The SG technique was well tolerated by patients and produced results consistent with other findings. Sampling from a single site of the genitalia on one occasion may significantly underestimate the infection rate with HPV. Multifocal infection of the anogenital area with HPV should be taken into consideration when interpreting epidemiological studies and management strategies.     

Bacteraemia in patients with hidradenitis suppurativa undergoing carbon dioxide laser surgery: detection and quantification of bacteria by lysis-filtration

BACKGROUND: Hidradenitis suppurativa (HS) is a cicatrising and persistent disease of apocrine gland-bearing areas in adults. The severity of this condition varies from a few suppurating lesions to widespread, disabling disease. The aetiology is obscure, but suggested contributory factors include a genetic predisposition, comedones occluding the pilosebaceous apparatus, bacterial infections, and hormonal factors. Treatment consists mainly of surgery, while medical therapies serve principally as adjunct therapy. OBJECTIVES: The aim of the study was to determine the number and type of bacteria circulating in the bloodstream in patients with HS undergoing surgical treatment with a carbon dioxide laser stripping-secondary intention technique. METHODS: Twenty-one patients (20 females and 1 male, mean age 36, range 20-55 years) were included in the study. One blood sample (8.3 ml) was taken before surgery, one during the operation and the last one 10 min after surgery. Five healthy persons (all females, mean age 36, range 23-48 years) not undergoing any operation were used as the controls. The blood was cultured by a lysis-filtration technique which had been shown to be very sensitive. Since the filter catches the microorganisms and colonies are formed during culturing, the number of bacteria in the samples is easily determined. RESULTS: In 6 patients, all samples were negative, which indicates that the method of surgery itself caused no spread of bacteria from the lesions. Bacterial growth in the first blood sample was found in 9 patients, from the second sample in 10 and from the third one in 6. In 1 patient, bacteria were detected in three samples. At least 12 bacterial species were identified. The dominating bacteria were coagulase-negative staphylococci of which most were subtyped as Staphylococcus warneri. Among the anaerobic microorganisms, Propionibacterium acnes and P.granulosum were the most frequently isolated bacteria. The bacterial findings in the blood samples accord well with the results from a previous study in which cultures were taken from the deep parts of the HS lesions. In the 5 controls, no microbial growth was detected. CONCLUSION: The carbon dioxide laser stripping technique caused no additional spread of bacteria into the bloodstream. The evaluation of cultures containing microorganisms from normal skin flora is controversial. Since the bacteria encountered in this study are in close agreement with the findings in cultures from the deeper parts of HS lesions they seem to be relevant. The growth of bacteria in the first blood sample taken before surgery may indicate that some of these patients have bacteria continuously circulating in their blood.     

Improved method for rhesus monkey or rat skin preparation and cryosectioning for topical or transdermal skin distribution studies

Abstract A method for preparing skin biopsies for cryosectioning was developed to accurately obtain samples from specific areas of the dermis, while minimizing contamination with epidermal tissue. Routine preparation of 6 mm punch biopsies from freshly excised, full-thickness skin produced contraction and folding of the edges of the biopsy prior to mounting for snap-freezing and cryosectioning. Sample orientation was ruined, and cryosections were heterogeneous with respect to dermal structures and/or to dermal and epidermal layers. Biopsy artifacts were prevented by prefreezing skin over dry ice prior to taking biopsies. The biopsies were held frozen on dry ice until they were mounted on cryostat pegs with flattened, frozen OCT surfaces; then they were snap-frozen in chilled OCT in an isopentane bath cooled with liquid nitrogen. The method for determining skin level homogeneity of cryosections consisted of taking 10 μm cryosections for histology between sections sampled for drug level analysis. The histological sections were fixed in 5% acetic acid in methanol and stained with hematoxylin and eosin to define the skin layers and structures associated with each sample for analysis. Histological sections from prefrozen skin had fewer processing artifacts, and dermal cryosections free of epidermal contamination were dramatically increased compared to the routine procedure.     

Immunofluorescent and FISH analysis of skin biopsies

Cutaneous biopsies are traditionally studied for the expression of cellular markers by immunoenzymatic techniques. However, immunofluorescent analysis is a valuable, and largely overlooked, ancillary technique that can resolve questions arising from conventional immunostaining, since it allows pairs of antigens to be simultaneously visualized. Furthermore, a novel technique, based on a combination of immunoperoxidase and immunofluorescent staining, allows three markers to be demonstrated together. Fluorescent microscopy also allows skin biopsies from lymphoma cases to be analyzed for chromosomal abnormalities by the fluorescent in situ hybridization (FISH) technique, which is now applicable to routine biopsy samples. In this review, we describe the technical aspects of immunofluorescent and FISH analysis of routine cutaneous biopsy samples.     

Comparison of immunofluorescence microscopy, immunoblotting and enzyme-linked immunosorbent assay methods in the laboratory diagnosis of bullous pemphigoid

This prospective study investigated patients with a clinical diagnosis of bullous pemphigoid (BP) who presented to a tertiary dermatology referral centre in Singapore. All patients had blood samples and skin biopsies taken for histology, immunofluorescence (IF) and immunoblot analysis prior to initiation of treatment. We analysed 23 new cases of BP during the 1-year study period. Seventeen of 22 biopsy specimens showed subepidermal blister formation, and 12 of the 17 (71%) had a predominance of eosinophils (>50%) in the blister cavity. The dermal inflammatory infiltrate of 22 biopsy specimens was predominantly lymphocytic in nine (41%) and eosinophilic in eight (36%). The histological picture was highly suggestive of BP in 15 of 22 patients (68%), suggestive in two (9%) and poorly suggestive in five (23%). Twenty-one of 23 (91%) patients had linear deposits of IgG and C3 along the dermo-epidermal junction. Serum indirect IF was positive in 22 of 23 (96%) patients, all showing antibody binding to the roof of the induced blister on salt-split skin. All of the 23 serum samples demonstrated positive immunoblot reactivity to BP180 and/or BP230 from epidermal extracts of normal human skin. Immunoblot reactivity with BP180 and BP230 was 78% (n=18) and 52% (n=12), respectively. The BP180 NC16A antibody could be detected in 22 of 23 (96%) sera using the enzyme-linked immunosorbent assay (ELISA) technique. The sensitivity of traditional diagnostic techniques, i.e. direct IF (91%) and indirect IF (96%), was comparable with that of the newer techniques, i.e. immunoblot analysis (100%) and ELISA (96%). ELISA in combination with routine indirect IF may be a useful diagnostic tool in patients with suspected BP who refuse a skin biopsy but consent to give a serum sample.     

K16 expression in uninvolved psoriatic skin: a possible marker of pre-clinical psoriasis

BACKGROUND: K16, a type I keratin, is upregulated in hyperproliferative states including psoriasis. It has been used as a marker of psoriasis and its expression is upregulated in relapsing psoriasis and downregulating in resolving. We evaluated non-lesional psoriatic skin for K16 expression. METHODS: Sixty-seven non-lesional and lesional skin samples from patients with psoriasis and normal skin from 19 non-psoriatic patients were studied by immunohistochemistry on frozen sections with K16. RESULTS: Seventeen of 19 normal skin samples showed staining of basal cells in the deeper part of the rete ridges. Sixty-two non-lesional psoriatic skin samples showed intense basal staining of K16. Of the remaining five non-lesional samples, diffuse intense suprabasal staining in one, pan-epidermal staining in two, and no staining was seen in two samples. Suprabasal (37), diffuse (14), sandwich (12), and basal (3) pattern staining were seen in psoriatic skin. One psoriatic skin sample did not show any expression. CONCLUSION: Our results demonstrate that K16 expression is also observed in non-lesional psoriatic skin and may serve as a marker of preclinical psoriasis.     

High prevalence of cutaneous human papillomavirus DNA on the top of skin tumors but not in "Stripped" biopsies from the same tumors

Genomes of human papillomaviruses (HPV) are common in biopsies from non-melanoma skin cancers but are also found on healthy skin and it is possible that HPV positivity in tumor biopsies by PCR may merely reflect contamination of the lesion surface. To investigate this issue, 229 immunocompetent patients were tested for HPV DNA in swab samples collected on top of skin tumors and in biopsies of the same tumors, obtained after stripping with tape to remove superficial layers. HPV DNA was detected on top of 69% (159 of 229) of the lesions, and in 12% (28 of 229) of the stripped biopsies (p<0.001). The difference was seen for all four types of tumors studied. Seborrheic keratosis had 79% (34 of 43) HPV positivity on top of lesions versus 19% (eight of 43) in biopsies; actinic keratosis had 83% (38 of 46) HPV positivity on top versus 11% (five of 46) in biopsies; basal cell carcinoma had 63% (69 of 109) on top versus 8% (nine of 109) in biopsies and squamous cell carcinoma had 58% (18 of 31) on top versus 19% (six of 31) in biopsies. HPV DNA is common in superficial layers of lesions, but is not necessarily present throughout tumors.     

Ultraviolet-B-induced apoptosis and cytokine release in xeroderma pigmentosum keratinocytes

We have assessed the ability of xeroderma pigmentosum and normal keratinocytes grown out from skin biopsies to undergo apoptosis after irradiation with ultraviolet B. Keratinocytes have been studied from xeroderma pigmentosum complementation groups A (three biopsies), C (three biopsies), D (one biopsy), xeroderma pigmentosum variant (two biopsies), and Cockayne syndrome (one biopsy). The three xeroderma pigmentosum group A and the xeroderma pigmentosum group D samples were at least six times more sensitive than normal cells to ultraviolet B-induced apoptosis. The xeroderma pigmentosum variant samples showed intermediate susceptibility. Xeroderma pigmentosum group C samples proved heterogeneous: one showed high sensitivity to apoptosis, whereas two showed near normal susceptibility. The Cockayne syndrome sample showed the high susceptibility of xeroderma pigmentosum groups A and D only at a higher fluence. These results suggest that the relationships between repair deficiency, apoptosis, and susceptibility to skin cancer are not straightforward. Ultraviolet B-induced skin cancer is also thought to be due in part to ultraviolet B-induced impairment of immune responses. The release of the inflammatory cytokines interleukin-6 and tumor necrosis factor-alpha from cultured xeroderma pigmentosum keratinocytes tended to occur at lower fluences than in normals, but was less extensive, and was more readily inhibited at higher fluences of ultraviolet B.     

Diagnostic impact and sensitivity of skin biopsies in Sneddon's syndrome. A report of 15 cases

BACKGROUND: Sneddon's syndrome is defined as a combination of idiopathic livedo racemosa generalisata and symptoms of cerebrovascular defect. The disease usually starts with vascular symptoms in the epidermis, with neurological deficits becoming evident later. For this reason, histological examination of skin biopsies and determination of arteriolar occlusion is of particular importance for reliable categorization and early diagnosis. To date, these methods have been considered to be too insensitive. OBJECTIVES: To evaluate the sensitivity of skin biopsies in Sneddon's syndrome. METHODS: We took a total of five deep punch biopsies (4 mm) from different areas of the livedo (three from white and two from red areas) in 15 patients. Present knowledge of the pathogenic relationships and the particular anatomical features of the skin were taken into account. RESULTS: The method had a sensitivity of 27% with one biopsy, 53% with two biopsies and 80% with three biopsies taken from white areas in all cases. CONCLUSIONS: Skin biopsies using the method presented achieved a high sensitivity, suggesting that the diagnosis in clinically suspected cases could be confirmed in the majority of cases with this technique.     

The aniline blue fluorescence staining of eosinophilic granulocytes

An aniline blue fluorescence staining technique specific for eosinophils was compared to haematoxylin and eosin to determine the percentages of eosinophils in the peripheral blood of patients with atopic or with allergic contact dermatitis. The aniline blue technique proved to be excellent for demonstrating eosinophilic granulocytes. Although the eosinophilic granulocytes were reasonably demonstrable in thin layers stained with haematoxylin and eosin, the aniline blue staining seems to be better in cases of doubt. Using the same techniques the presence of eosinophil granulocytes was studied in 5 punch biopsies taken from positive patch tests (after 48 h) and 5 biopsies from positive skin reactions after and intradermal injection with an atopic allergen (after 20--30 min). In all punch biopsies taken after an intradermal injection with an atopic allergen, eosinophilic granulocytes, often numerous, were seen in the vessels of the dermis. In punch biopsies taken from positive patch tests the eosinophilic granulocytes were seen scattered through the dermis in variable numbers.     

Mast cells in sun-exposed and non-sun-exposed skin. An autopsy study

One hundred ten skin biopsies were taken from 55 consecutive autopsies to evaluate the number and location of mast cells in the dermis. For each autopsy, one biopsy was taken from the V of the neck (sun-exposed area) and the other from the upper thigh (non-sun-exposed area). Normal-appearing skin was biopsied. There were 28 men and 27 women ranging in age from 16 to 94 years. Fifty-three patients were Caucasian and two were Negro. Mast cells were counted in 10 random high-power fields in the papillary dermis only. The average number of mast cells per high-power field (X 400) in sun-exposed skin for both men and women was 8.19 (65/mm2 or 13,000/mm3) with one standard deviation of 4.08, while that of non-sun-exposed skin was 7.52 (60/mm2 or 11,900/mm3) with one standard deviation of 3.62. The difference between the number of mast cells in sun-exposed and non-sun-exposed skin was not statistically significant. In addition, no statistically significant differences were observed for the average number of mast cells per high-power field in regard to sex or the presence of malignancies.     

Intraepidermal nerve fiber expression of calcitonin gene-related peptide, vasoactive intestinal peptide and substance P in psoriasis

In order to evaluate more fully the role of neuropeptides in the pathogenesis of psoriasis, skin biopsies were obtained from 36 patients with psoriasis to identify substance P (SP), vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP). Lesional and nonlesional skin was examined from these biopsies and the results compared with those from biopsies taken from patients with a variety of other inflammatory dermatoses, including lichen planus, lichen simplex chronicus, spongiotic dermatitis, and seborrheic dermatitis. Also studied was a series of nine biopsies taken from patients with no known skin disorders. We found an increase in the number of SP-positive nerve fibers within the epidermis in biopsies from lesional skin of psoriasis patients (8.4 nerves per 3-mm biopsy) compared with nonlesional psoriatic skin (2.6 nerves per 3-mm biopsy) and normal skin (2.0 nerves per 3 mm biopsy). Other inflammatory disorders also demonstrated fewer SP-positive nerves than lesional psoriatic skin; lichen planus (0 nerves per 3 mm biopsy) and lichen simplex chronicus (1.3 nerves per 3 mm biopsy). The difference in SP-positive nerve expression between lesional psoriatic skin and the group comprising nonlesional skin, normal skin, lichen planus, and lichen simplex chronicus attained statistical significance ( P < 0.013). SP-positive intraepidermal nerve fibers in lesional psoriatic specimens were fewer than in spongiotic dermatitis (17.4 nerves per 3 mm biopsy). There was no significant difference in numbers of VIP- or CGRP-immunopositive intraepidermal nerve fibers between psoriatic skin and the group comprising all other material tested. However, in five patients with psoriasis, there was a marked increase in the expression of intraepidermal CGRP (up to 10.7 nerves per 3-mm biopsy) and VIP (up to 8.3 nerves per 3-mm biopsy) which was not observed in control groups. These findings suggest that neuropeptides SP, CGRP, and VIP play a role in the pathogenesis of psoriasis. Received: 3 March 1997     

Is increased 5 alpha-reductase activity a primary phenomenon in androgen-dependent skin disorders?

Testosterone metabolism was investigated in fractions of human skin, enriched in epidermis, dermis, sebaceous glands, and sweat glands, by histologic sectioning of skin punch biopsies, and the results were compared with two culturable skin cells, i.e., keratinocytes and fibroblasts. Since sebocytes could not be brought in culture, metabolism was also investigated in the hamster flank model. In the epidermal tissue of the skin biopsies the predominant metabolite was androstenedione, formed by the enzyme 17 beta-hydroxysteroid dehydrogenase. The same was true for cultured hair follicle keratinocytes. In the deeper skin layers the formation of androstenedione was markedly reduced, whereas the formation of 5 alpha-reduced metabolites was highly increased, with a maximum in the skin fractions containing large sebaceous glands. Cultured shoulder skin fibroblasts showed a markedly different testosterone metabolism compared with the sectioned skin biopsies, suggesting that dermal fibroblasts play a less important role in the overall skin testosterone metabolism. The present approach, allowing the comparison of testosterone metabolism in different substructures of the same skin biopsy provides new evidence that the high 5 alpha-reductase activity in the specific skin fractions must be mainly ascribed to the sebaceous glands. These results render a previous hypothesis, stating that the elevated level of 5 alpha-reductase and subsequent formation of dihydrotestosterone in androgenetic alopecia and acne (usually accompanied by seborrhea) could therefore simply be the consequence of sebaceous gland enlargement, much stronger. This hypothesis is further evaluated by quantitative correlation of sebaceous gland size with enzyme activity in the hamster flank model.     

Pretreatment of photoaged forearm skin with topical tretinoin accelerates healing of full-thickness wounds

Pretreatment of skin with all-trans retinoic acid (tretinoin) has been shown to enhance wound healing. Previous studies have mainly used animal models to demonstrate this effect. We wanted to determine whether pretreatment could promote wound healing in severely photoaged dorsal forearm skin. Four elderly men with severely actinically damaged forearms were treated daily for 16 weeks. One arm was treated with 0.05-0.1% tretinoin cream (Retin A®, Ortho), and the other with Purpose® cream (Ortho) as a vehicle control. Four-millimetre punch biopsies were taken from both dorsal forearms prior to treatment. After 16 weeks, full-thickness 2-mm punch biopsies were taken from both sides. Serial photographs were taken, and healing of the wounds quantitatively assessed by image analysis. On the 11th day, the wounds were excised using a 4-mm biopsy punch. Biopsies were processed for light microscopy. After 16 weeks, the tretinoin-treated Forearms showed moderate erythema and scaling. Polarized tight photographs revealed multiple, red, vascularized foci and/or a diffuse network of small vessels. The histological effects were typical for tretinoin, i.e. compaction of the stratum corneum, epidermal acanthosis with correction of atypia, an increase in small vessels, and increased cellularity in the upper dermis. Purpose® cream had no effect, either clinically or historically. On the tretinoin-treated side, the wound areas were 35-37% smaller on days 1 and 4, and 47-50% smaller on days 6, 8, 11, compared with the controls. Clinically and histologically. reepithelialization occurred more rapidly. Thus tretinoin dramatically accelerated wound healing in photodamaged skin.     

Punch biopsy of the skin: effect of tissue fixation with CO2-freezing. An experimental study

The effect of tissue fixation with CO₂-freezing on the size and quality of punch biopsies was studied post mortem. Four millimeter punch biopsies were taken from the abdominal skin with a motor drill operating at 250 RPM, before and after freezing with CO₂ in acetone. The dry defatted weight was determined from 32 biopsies in each series, and light microscopy was performed on 16 biopsies in each series after setting up a double blind system. The dry weight of the whole biopsy was higher (p<0.05) after CO₂-freezing, but the weight of the epidermis/dermis alone was the same. The conical shape of the punch biopsies was due to retraction occurring during and after biopsy and not to loss of tissue. In the CO₂-frozen series, there were more (p<0.001) biopsies cylindrical in shape, more (p<0.001) biopsies including fascia and muscle, fewer (p<0.001) biopsies with trauma, more (p<0.01) biopsies with subcutaneous tissue represented, in a larger amount (p<0.001), and more (p<0.001) biopsies optimal for microscopy. After tissue fixation, the quality of the biopsies for microscopy was much better with regard to tissues represented and tissue archilecture. Further efforts to develop an easy in vivo technique for tissue fixation are indicated.     

The expression of congenital ichthyosiform erythroderma in second trimester fetuses of the same family: morphologic and biochemical studies

The first born offspring of first-cousin parents was affected with a keratinization disorder thought to be nonbullous congenital ichthyosiform erythroderma (CIE). In each of three subsequent pregnancies, the parents elected to have prenatal diagnosis based on evaluation of fetal skin biopsies. The epidermis of fetus 1 was identical to normal 21-wk estimated gestation age (EGA) fetal epidermis, but because keratinization begins normally around 24 wk EGA, the procedure was repeated 4 wk later. A thin epidermis with a few layers of stratum corneum indicated a normal fetus and a healthy infant was born at term. Skin biopsy samples from fetus 2 gave conflicting results; the epidermis of one sample appeared normal but the second had 5-15 layers of incompletely keratinized cells superficial to basal and intermediate layers. The hair canals of both samples were hyperkeratotic. Pelleted amniotic fluid cells contained aggregates of incompletely keratinized epidermal cells and concentric rings of keratinized cells. The fetus was thought to be affected and the pregnancy terminated. Regional variation in epidermal thickness and keratinization was noted upon gross examination of the fetus and by histology of the skin. Marked hyperkeratinization of follicles was evident in all regions. No abnormal keratins were expressed in the affected epidermis but epidermal lipids analyzed from two body regions had a lower triglyceride content and a higher content of free sterols compared with age-matched, normal fetal epidermis. Immunolabeling for markers of differentiation revealed variable stages of epidermal differentiation according to region. Four structurally identical biopsy samples were obtained from a third fetus. The epidermis appeared normal for age and hair canals were keratinized to various extents. The pregnancy was continued and at 33 wk a male infant was born with a severe ichthyosis of the face and scalp and fine, white scaling on the body. The epidermis of both the severely and mildly affected regions of the newborn had a thick, compact stratum corneum and other features of CIE. Scars from all four fetal biopsies were identified on the trunk, in areas which appeared less affected clinically. This study reports, for the first time, the criteria for prenatal diagnosis of CIE and the variable expression of this disorder in the midtrimester fetus. More importantly, it demonstrates the risks and pitfalls of this in utero diagnosis based on epidermal morphology.     

Prenatal exclusion of harlequin ichthyosis; potential pitfalls in the timing of the fetal skin biopsy

BACKGROUND: Harlequin ichthyosis (HI) is a severe and usually fatal congenital skin disorder with autosomal recessive inheritance. Several cases of HI prenatal diagnosis have been performed using fetal skin biopsy, mainly at around 23 weeks estimated gestational age (EGA), and reported in the literature. However, prenatal testing must be done earlier than 21 weeks EGA in several countries including Japan where the present HI families live, because termination is legally allowed only until 22 weeks EGA. OBJECTIVES: We report the successful prenatal exclusion of HI in two fetuses from two independent families and discuss the technical difficulties and potential pitfalls in the prenatal exclusion of HI at early gestation stages. METHODS: Fetal skin biopsy specimens and amniotic fluid samples at 19 and 20 weeks EGA from two fetuses at risk of HI were examined by light and electron microscopy. RESULTS: For the prenatal diagnosis in case 1, the fetal skin biopsy samples were obtained at 20 weeks EGA and showed normal keratinization in the hair canals; no abnormalities were observed in the keratinized cells. In case 2, the interfollicular epidermis and the hair follicles in the samples obtained at 19 weeks EGA had not differentiated enough to show proper keratinization. However, lamellar granules were normally formed in the inner root sheath cells of the late bulbous hair pegs. From these ultrastructural findings, the case 1 fetus was diagnosed as unaffected with HI, and the case 2 fetus was diagnosed as unlikely to be affected. Subsequently, both were born as healthy, unaffected babies. CONCLUSIONS: The timing of biopsies at 19 weeks EGA is not ideal for fetal skin biopsy because the samples are not always sufficiently differentiated for the prenatal diagnosis of HI. However, morphological observations of lamellar granules gives us important additional information useful for HI prenatal diagnosis.     

Atomic force microscopy for biomechanical and structural analysis of human dermis: A complementary tool for medical diagnosis and therapy monitoring

Skin mechanical properties are usually measured considering the entire skin thickness and very little is known about the mechanical behaviour of individual skin layers. We propose atomic force microscopy (AFM) as a tool to quantify nanoscale changes in the biomechanical properties and ultrastructure of human papillary dermis exposed to different mechanical and physical stimuli. Samples from 3 human skin biopsies were studied: one stretched by obesity, one subjected to a high level of sun exposure and normal skin as control. Slices of the papillary dermis layer were harvested at controlled depths from each skin biopsy and 25 μm² areas of each slice were imaged and D‐periodicity of collagen fibres measured by AFM, together with their stiffness. Standard histological analysis was also carried out to correlate biochemical properties and their distribution with stiffness and topography. We obtained similar stiffness values between the sample affected by obesity and the control sample at any depth level into the dermis, while the sun‐exposed sample presented a significantly lower stiffness. Additionally, all samples presented an increase in the stiffness at higher depths into the papillary dermis layer. Collagen fibres close to the epidermis of sample affected either by obesity and sun exposure—the former even more than the latter—are thicker and present a larger D‐period than those in the control sample. Our results open the possibility to use structural and mechanical analysis based on AFM as a complementary tool for medical diagnosis and therapy monitoring.     

Immunohistochemical detection of Treponema pallidum in skin samples with clinical and histopathological correlations and Warthin-Starry staining critical analysis

BackgroundSyphilis in its different phases may be a difficult diagnosis in clinical and histopathological grounds.ObjectivesThe present study objectives were to evaluate the detection and tissue distribution of Treponema pallidum in skin lesions of syphilis.MethodsA blinded diagnostic accuracy study was performed with immunohistochemistry and Warthin-Starry silver staining in skin samples from patients with syphilis and other diseases. Patients attended two tertiary hospitals between 2000 and 2019. Prevalence ratios (PR) and 95% confidence intervals (95% CI) were calculated for the association between immunohistochemistry positivity and clinical-histopathological variables.ResultsThirty-eight patients with syphilis and their 40 biopsy specimens were included in the study. Thirty-six skin samples were used as non-syphilis controls. The Warthin-Starry technique was unable to accurately demonstrate bacteria in all samples. Immunohistochemistry showed spirochetes only in skin samples from patients with syphilis (24/40) with 60% sensitivity (95% CI 44.8‒75.2). Specificity was 100% and accuracy, 78.9% (95% CI 69.8‒88.1). Most cases had spirochetes in both dermis and epidermis and there was a high bacterial load.Study limitationsCorrelation between immunohistochemistry and clinical or histopathological characteristics was observed but was limited statistically due to the small sample size.ConclusionsSpirochetes were promptly seen in an immunohistochemistry protocol, which can contribute to the diagnosis of syphilis in skin biopsy samples. On the other hand, the Warthin-Starry technique showed to be of no practical value.