Histology‐guided protein digestion/extraction from formalin‐fixed and paraffin‐embedded pressure ulcer biopsies

Herein we present a simple, reproducible and versatile approach for in situ protein digestion and identification on formalin‐fixed and paraffin‐embedded (FFPE) tissues. This adaptation is based on the use of an enzyme delivery platform (hydrogel discs) that can be positioned on the surface of a tissue section. By simultaneous deposition of multiple hydrogels over select regions of interest within the same tissue section, multiple peptide extracts can be obtained from discrete histological areas. After enzymatic digestion, the hydrogel extracts are submitted for LC‐MS/MS analysis followed by database inquiry for protein identification. Further, imaging mass spectrometry (IMS) is used to reveal the spatial distribution of the identified peptides within a serial tissue section. Optimization was achieved using cutaneous tissue from surgically excised pressure ulcers that were subdivided into two prime regions of interest: the wound bed and the adjacent dermal area. The robust display of tryptic peptides within these spectral analyses of histologically defined tissue regions suggests that LC‐MS/MS in combination with IMS can serve as useful exploratory tools.     

Unusual histopathological features of cutaneous leishmaniasis identified by polymerase chain reaction specific for Leishmania on paraffin-embedded skin biopsies

BACKGROUND: Cutaneous leishmaniasis (CL) is rare in Northern Europe and may be overlooked because colleagues have little experience with it. OBJECTIVES: To identify manifestations of CL that may escape diagnosis. METHODS: Correlation of clinical diagnosis and histopathological findings in 28 biopsy specimens taken from 19 patients with CL confirmed by polymerase chain reaction (PCR) specific for Leishmania. RESULTS: In only one patient was the clinical diagnosis CL; other diagnoses included: malignant epithelial neoplasms (5), follicular cyst (2), atypical mycobacteriosis (1), sarcoidosis (2) and lymphoma (1). Lesions were single (15) or few (4) nodules predominantly situated on the extremities or face (16). Histopathological findings were diagnostic of CL in only 10 cases. In nine cases Leishmania was not identified microscopically; histopathological diagnoses were: granulomatous dermatitis (6), lupoid rosacea (1), foreign body granuloma (1) and granuloma annulare (1). Unaltered epidermis (9), nodular infiltrates (5), numerous multinucleated histiocytes (3), palisaded granulomas with fibrinoid centres (2), sarcoidal granulomas (4) and elastophagocytosis (1) misled the histopathologists in these cases. CONCLUSIONS: CL seems often to be misdiagnosed clinically in countries where it is not endemic. Histopathologically, CL may be misinterpreted as sarcoidosis, foreign body granuloma, lupoid rosacea and granuloma annulare, especially when Leishmania is not seen microscopically. We suggest that in Northern Europe, PCR for Leishmania-specific DNA should be performed routinely in any granulomatous dermatitis presenting as a single or few nodules on the extremities or face, even when a diagnosis of CL was not considered by the referring clinician.     

Molecular diagnosis of skin infections using paraffin‐embedded tissue – review and interdisciplinary consensus

Nucleic acid amplification techniques (NATs), such as PCR, are highly sensitive and specific methods that have become valuable supplements to culture and serology in the diagnosis of infectious disorders. However, especially when using formalin‐fixed and paraffin‐embedded tissue, these techniques are associated with both false‐negative and false‐positive results, a pitfall that is frequently misjudged. Representatives of the German Society of Hygiene and Microbiology (DGHM) and the German Society of Dermatology (DDG) therefore set out to develop a consensus – in the form of a review article – on the appropriate indications for NATs using paraffin‐embedded tissue, its contraindications, and the key points to be considered in the pre‐ and post‐analytical phase. Given that fresh, naive tissue is preferably to be used in the workup of a suspected infection, PCR analysis on paraffin sections represents an exception. The latter may be considered if an infection is suspected at a later point in time and fresh tissue has not been preserved or can no longer be obtained. Potential indications include confirmation of histologically suspected infections with Leishmania spp., Bartonella spp., Rickettsia spp., or in case of ecthyma contagiosum. Infections with, for example, mycobacteria or RNA viruses, on the other hand, are not considered useful indications for NATs using paraffin sections. In order to avoid misinterpretation of test results, it is essential that laboratory reports on NATs using paraffin‐embedded tissue contain information on the indication/diagnostic circumstances, the required and chosen pre‐analytical steps, the limitations of the method, and on diagnostic alternatives.     

Detection of Borrelia burgdorferi in skin biopsies from patients with morphea by polymerase chain reaction

Aim We looked for the evidence of Borrelia infection in patients with morphea by serologic means and by polymerase chain reaction (PCR) analysis of skin biopsy samples. Background The possible relationship between Lyme borreliosis and morphea has been suggested by certain clinical, immunological and microbiological findings, but many authors were not be able to demonstrate Borrelia burgdorferi infection in patients with morphea and cast doubts on an etiological role for B. burgdorferi in this skin lesion. Patients and methods Ten patients with morphea, 9 females (range: 8–65 years) and one 44-year-old man were examined. Serological tests for Lyme borreliosis were performed by immunofluorescence assay and flagellin enzyme-linked immunosorbent assay. Skin biopsy specimens were taken from the periphery of morphea lesions for histological examination and PCR. Results Antibodies to B. burgdorferi were detected in 3 patients and B. burgdorferi DNA was demonstrated in 5 patients. Conclusions The amplification of DNA with PCR analysis seems to open new prospects for the detection of Borrelia genome in tissues, in the present study we were able to demonstrate the presence of B. burgdorferi DNA in patients with morphea, even in seronegative patients. These data confirm that PCR is an interesting tool in skin lesion diagnosis and support the hypothesis of an etiological association between B. burgdorferi infection and some cases of morphea.     

No detection of HTLV-I DNA in punch skin biopsies from patients with cutaneous T-cell lymphoma by the polymerase chain reaction.

In this study we have tried to take advantage of the high genetic homology between conserved regions of human T-cell lymphotropic virus (HTLV) types I and II, hoping that a possible retrovirus in patients with cutaneous T-cell lymphoma (CTCL) would have regions with homology to types I/II. DNA was extracted from punch skin biopsies from 21 patients and subjected to the polymerase chain reaction (PCR), using primer sets designed to match conserved regions in the HTLV-I/II genome. The PCR products were subjected to agarose gel electrophoresis with subsequent Southern blotting and hybridization to an HTLV-I probe. No bands of exogenous origin were seen on the agarose gel or by hybridization. If a retrovirus is present in the skin in CTCL patients, it is either not related to HTLV-I/II, present at a copy number below the PCR detection limit, or has been cleared from the skin before the clinical symptoms appear.     

Polymerase chain reaction for diagnosis of cutaneous leishmaniasis in histologically positive,suspicious and negative skin biopsies

BACKGROUND: Cutaneous leishmaniasis (CL) is a major problem in many tropical and subtropical countries. The clinical diagnosis should be confirmed by identification of the parasite in biopsy or smear or by tissue culture. The sensitivity of direct microscopy is not high, and tissue culture is not uniformly available and successful. Polymerase chain reaction (PCR) is a sensitive test for the detection of low amounts of DNA in tissues. OBJECTIVE: We applied PCR on paraffin-embedded skin biopsies to assess the validity of this method in the diagnosis of cutaneous leishmaniasis. METHODS: DNA extraction from paraffin blocks was performed by the heat method, and PCR was carried out using the primers for the Leishmania-specific 120-base-pair fragment of kinetoplast DNA minicircles. Paraffin blocks of gelatin-embedded formalin-fixed Leishmania tropica, taken from culture, served as positive controls. Negative controls were the skin biopsies of patients whose clinicopathologic diagnoses were not cutaneous leishmaniasis. RESULTS: PCR showed the parasite in all 33 cases whose skin biopsies had shown the Leishmania parasite by light microscopy. PCR results were also positive in 24 cases out of 29 where microscopic examination of skin biopsies had failed to detect the amastigote but their clinical diagnosis was CL. The sensitivity of PCR in the diagnosis of CL was 92%. None of the nonleishmaniasis cases showed positive results (specificity 100%). PCR results were positive in 52 out of 54 cases whose skin biopsies showed granulomatous inflammation. Evaluation of the histopathologic findings showed that the presence of vaguely formed immature granuloma was directly and the detection of mature well-formed granuloma was inversely correlated with the detection of the parasite in biopsies (p < 0.01). CONCLUSION: PCR on paraffin-embedded tissue is a highly sensitive and specific test for the diagnosis of CL, and detection of granulomatus inflammation is a highly reliable histopathologic finding in suspected cases.     

C3d immunohistochemistry on formalin‐fixed tissue is a valuable tool in the diagnosis of bullous pemphigoid of the skin

Background: Direct immunofluorescence (DIF) testing is an important procedure in the diagnosis of autoimmune bullous dermatoses. We investigated the expression of C3d in formalin‐fixed, paraffin‐embedded tissue of autoimmune bullous dermatoses. Methods: The immunohistochemical expression of C3d in bullous pemphigoid (BP) (n = 32), pemphigoid gestationis (PG) (n = 3), pemphigus (n = 14), dermatitis herpetiformis Duhring (DHD) (n = 10), linear immunoglobulin A (IgA) dermatosis (n = 4), mixed forms of BP and linear IgA dermatosis (n = 2), and 44 controls was analyzed on formalin‐fixed tissue. Results: Thirty‐one of 32 cases (97%) of BP and 3 out of 3 cases (100%) of PG showed a linear positivity of C3d along the basement membrane. Only 3 out of 14 (21%) cases of pemphigus showed an intraepidermal intercellular expression of C3d. The two mixed forms of linear IgA dermatosis and BP showed a linear positivity of C3d along the basement membrane. All cases of DHD, linear IgA dermatosis and all of the controls were negative for C3d. Conclusions: C3d immunohistochemistry is a valuable tool in the diagnosis of BP and PG of the skin with a sensitivity of at least 97%. Mixed forms of linear IgA dermatosis, and BP, DHD and linear IgA dermatosis can only be identified by DIF. A positive result may prompt serologic confirmation of BP without further need for DIF. Pfaltz K, Mertz K, Rose C, Scheidegger P, Pfaltz M, Kempf W. C3d immunohistochemistry on formalin‐fixed tissue is a valuable tool in the diagnosis of bullous pemphigoid of the skin.     

Detection of Chlamydia trachomatis by ligase chain reaction compared with polymerase chain reaction and cell culture in urogenital specimens.

OBJECTIVE--The aim of this study was to evaluate the newly developed ligase chain reaction (LCR) assay for the detection of Chlamydia trachomatis in urogenital specimens using cell culture and Amplicor PCR for comparison. SUBJECTS--Two hundred and eighty patients attending hospital or urban STD clinics (high-risk population, 62 men and 84 women) and obstetric/gynaecology clinics (low-risk population, 134 women) in Bordeaux, France. METHODS--Specimens from men were tested with LCR on urethral swabs and urine, with Amplicor or urine, with cell culture on urethral swabs. Specimens from women were tested with LCR, Amplicor and cell culture on endocervical swabs and with LCR on urine. When the three methods generated different results, the LCR and Amplicor tests were repeated on the remaining samples. Samples with discordant LCR and Amplicor results and a negative culture were further analysed by major outer membrane protein gene omp1-PCR. RESULTS--After analysis of discrepant results, the overall prevalence was 7.5% (21/280) calculated on the basis of an expanded "gold standard" defined as culture positive or LCR plus Amplicor positive or omp1-PCR positive for discrepant results between LCR and Amplicor tests. Of the 21, 20 were detected by LCR, 17 by Amplicor and culture. The specificity of LCR and Amplicor was 99.6%. CONCLUSION--The LCR Chlamydia trachomatis test is a highly sensitive nonculture technique and a good alternative test for the detection of chlamydial infections.     

A case of zoster sine herpete presenting with thoracic radicular pain diagnosed by polymerase chain reaction in skin exudate

Varicella zoster virus (VZV) can cause radicular pain in the absence of skin lesions; such cases are referred to as zoster sine herpete (ZSH) and are usually diagnosed by using serological assays or polymerase chain reaction (PCR). An effort is underway to detect VZV DNA in novel specimens rather than conventional samples (e.g., blood or cerebrospinal fluid) for PCR. There are two reports that PCR analysis in the exudate of the auricular skin can be a useful diagnostic tool for the diagnosis of ZSH in patients presenting with cranial nerve paralysis without herpetic eruptions. Here, we report a case of ZSH diagnosed by using PCR analysis of skin exudates in a patient who developed thoracic radicular pain. This is believed to be the first case of ZSH diagnosed using PCR analysis of skin exudate in a patient in whom the cranial nerve was not involved.     

Detection of herpesvirus DNA in peripheral blood mononuclear cells and skin lesions of patients with pemphigus by polymerase chain reaction

Pemphigus is an autoimmune disease where both endogenous (genetic) and exogenous (environmental) factors play a part. Viral infections, in particular herpesvirus infections, have been identified as a possible triggering factor for pemphigus. In this study, using the polymerase chain reaction, we studied peripheral blood mononuclear cells (PBMC) and skin biopsies from patients with pemphigus, and in some of these were able to demonstrate the presence of DNA sequences of herpes simplex virus 1/2 (50% in PBMC and 71% in skin biopsies), Epstein-Barr virus (15% in PBMC and 5% in skin biopsies) and human herpesvirus 6 (20% in PBMC only). However, the inability to detect herpesvirus DNA consistently in these cases suggests that viral infection may only be an occasional factor triggering the outbreak or exacerbation of the disease. The possible role of interferons and interleukins in the pathogenesis of virus-induced pemphigus is discussed.     

Morphoea is neither associated with features of Borrelia burgdorferi infection, nor is this agent detectable in lesional skin by polymerase chain reaction

BACKGROUND: The aetiology of morphoea is still unknown. Borrelia burgdorferi as a causative agent of morphoea has been discussed since 1985, but the relationship remains uncertain. OBJECTIVES: We aimed to find evidence for infection with B. burgdorferi by combined evaluation of different clinical and laboratory data in a group of 54 patients with morphoea. METHODS: In each patient, an evaluation of the case history was performed with regard to infection with B. burgdorferi, using a standardized questionnaire. Questions focused on previous tick bites and skin changes suspicious for erythema migrans (EM). The case history data of 52 patients were compared with those of 104 matched control subjects and of 25 patients with acrodermatitis chronica atrophicans (ACA). Serological examinations were performed in 53 patients with morphoea. Furthermore, lesional skin was examined for borrelial DNA in 33 patients, using nested polymerase chain reaction (PCR) for the ospA and the borrelial rRNA gene. RESULTS: Results of the questionnaire showed no differences between patients with morphoea and matched controls. In contrast, patients with ACA showed a much higher prevalence of tick bites and skin changes suspicious for EM as compared with patients with morphoea. Serological examination was positive in only one patient with morphoea alone and in two additional patients with coexistent ACA. No borrelial DNA was detected by PCR in lesional skin of 33 patients with morphoea. CONCLUSIONS: No evidence was found for B. burgdorferi infection in patients with morphoea.