No evidence of Borrelia in cutaneous infiltrates of B-cell lymphomas with a highly sensitive, semi-nested real-time polymerase chain reaction targeting the 5S-23S intergenic spacer region.

BACKGROUND: The role of Borrelia in the development of skin lymphomas has been under discussion for decades. A similar association has been shown for Helicobacter pylori and gastric lymphomas (MALT type). Nevertheless, few molecular studies investigated Borrelia in skin lymphomas and the results are controversial. METHODS: We analysed 46 formalin-fixed, paraffin-embedded skin specimens of clincopathologically confirmed B-cell lymphomas (15 marginal zone lymphomas; 20 follicular lymphomas; three diffuse large B-cell lymphomas; eight secondary cutaneous infiltrates) taken from 36 patients from Northern Germany, an endemic area for Borrelia. Fifteen pseudolymphomatous lesions of cutaneous Borreliosis served as the control. Both groups were examined with a real-time (rt) PCR and a semi-nested PCR targeting the 5S-23S intergenic spacer region (IGS). A multiplex PCR was used to investigate B-cell clonality in all lymphomatous infiltrates (Biomed Primers). RESULTS: With both assays no Borrelia burgdorferi-specific DNA was identified in any of the B-cell lymphomas, while all 15 Borreliosis specimens gave a positive PCR result in the semi-nested PCR protocol, 12 were also positive in the rt PCR (P < 0.01). All B-cell lymphomas showed monoclonal IgH-Rearrangement. Analysis of cutaneous B-cell lymphomas from available studies including ours (n = 334) reveals an odds ratio <1. CONCLUSION: While some previous studies suggested an association between B. burgdorferi and the development of cutaneous B-cell lymphomas in endemic areas, we were unable to confirm this in our patients, despite a highly sensitive Borrelia PCR assay. Our results including meta-analysis of previous studies question the need for antibiotic therapy in patients with cutaneous B-cell lymphomas.

Species identification of dermatophytes in paraffin-embedded biopsies with a new polymerase chain reaction assay targeting the internal transcribed spacer 2 region and comparison with histopathological features.

BACKGROUND: Dermatophytosis is a very common skin infection with a broad clinical spectrum. Biopsies are often used to confirm the diagnosis, especially when the clinical presentation is unusual. Not uncommonly, organisms are hard to find even with periodic acid-Schiff stains. Polymerase chain reaction (PCR) for dermatophytes can be used in such cases. OBJECTIVES: To test a new PCR assay allowing species identification of dermatophytes on paraffin-embedded biopsies, and to reassess histopathological criteria for diagnosis of dermatophytosis. METHODS: In total, 121 biopsies of 92 patients with clinical suspicion of tinea were included. In 42 samples the clinical diagnosis had been confirmed histopathologically, and in 79 no fungal elements had been identified. PCRs targeting the internal transcribed spacer (ITS)2 region of dermatophytes were performed on the biopsies with subsequent sequencing. Sections were reassessed for the presence/absence of hyphae/spores, pattern and composition of infiltrate, and epidermal/follicular changes. Patient charts were reviewed for clinical data. RESULTS: The new ITS2 PCR assay detected 94% of the dermatophyte infections (compared with 79% identified by microscopy). Trichophyton rubrum was the dominant species (89%), and other species identified were Trichophyton verrucosum (2%), Microsporum canis (4%), Epidermophyton floccosum (2%) and Trichophyton interdigitale (4%). In particular, infections with T. interdigitale and manifestations with prominent spongiosis were not diagnosed histologically. Intracorneal neutrophils, which have been emphasized as a histopathological clue to dermatophytosis, were present in only 46% of PCR-positive samples. CONCLUSIONS: Molecular species identification of dermatophytes via ITS2 PCR can easily be implemented in a routine dermatopathology setting. It is fast and highly specific and improves the sensitivity of histopathological diagnosis of dermatophytosis.

Fast, sensitive and specific diagnosis of infections with Leishmania spp. in formalin-fixed, paraffin-embedded skin biopsies by cytochrome b polymerase chain reaction.

BACKGROUND: Northern spread of sandflies and Leishmania spp. has been observed in Europe. Diagnosis can be difficult owing to the various clinical manifestations. Species identification is important for patient management and therapy. Molecular diagnostics is increasingly used for pan-Leishmania detection but species identification remains challenging in formalin-fixed material. OBJECTIVES: To apply cytochrome b (cytb) polymerase chain reaction (PCR) and sequencing for identification of Leishmania species on formalin-fixed, paraffin-embedded (FFPE) skin biopsies; and to identify species-specific histological patterns. METHODS: Sixty-nine biopsies (48 patients) diagnosed with leishmaniasis based on the presence of amastigotes in the tissue (n = 41) or granulomatous infiltrates with positive pan-Leishmania real-time PCR (n = 28) were analysed with cytb PCR, sequencing and phylogenetic analysis. Histological sections were analysed; epidemiological data were collected. RESULTS: Cytb PCR identified Leishmania in all specimens: L. infantum (79%), L. major (8%), L. panamensis (4%), L. tropica (4%), L. killicki (2%) and L. aethiopica (2%). Of the detected species 95% were endemic to the country in which the infection was acquired. Amastigotes were found in 59%. Infiltrates were mainly tuberculoid granulomatous (65%), interstitial (15%) and sarcoidal (10%). Pseudolymphomatous features and pseudocarcinomatous hyperplasia were more common in L. major infections than in L. infantum (P < 0·01). CONCLUSIONS: Cytb PCR and sequencing is a fast, reliable and sensitive assay for identification of Leishmania spp. in FFPE biopsies. Leishmania infantum is the main cause of cutaneous leishmaniasis in Germany. Tuberculoid granulomas, other types of granulomas and pseudolymphomatous infiltrates may be encountered; the latter being indicative of infection with L. major.

Genotyping of Borrelia from formalin-fixed paraffin-embedded skin biopsies of cutaneous borreliosis and tick bite reactions by assays targeting the intergenic spacer region, ospA and ospC genes.

BACKGROUND: Lyme borreliosis has a broad spectrum of clinical presentations involving the skin, joints and nervous system. The variable manifestations have been attributed to different Borrelia genospecies but genotyping required culture or fresh tissue. However, in dermatology practice, formalin-fixed paraffin-embedded biopsies are used for dermatopathological examination. Polymerase chain reaction (PCR) for Borrelia burgdorferi sensu latu has been established on such specimens, but studies attempting genotyping of subspecies or strains are lacking. OBJECTIVES: To adapt PCR assays for genotyping of Borrelia using paraffin-embedded biopsies, to identify Borrelia genospecies and to compare clinicopathological features of different genospecies. METHODS: Eighty-two paraffin-embedded biopsies from 68 patients, with erythema migrans, acrodermatitis chronica atrophicans, lymphocytoma cutis or tick bite reactions, were studied with assays targeting the intergenic spacer (IGS), ospA and ospC, followed by sequencing and phylogenetic analysis. Clinicopathological data were analysed comparing different Borrelia genospecies. RESULTS: Genotyping by IGS, ospA and ospC was successful in 85% of patients (91% B. afzelii, 7% B. garinii, 2% B. bavariensis). ospA serotyping identified type 2 (90%), type 3 (8%) and type 4 (2%). ospC-PCR was positive in 40% of the patients revealing 12 different groups, noninvasive forms being seen only in tick bite reactions and erythema migrans. No major clinicopathological differences could be identified between the genospecies, but neural inflammation and arthralgia were seen more often in lesions caused by invasive ospC strains. CONCLUSIONS: Genotyping of Borrelia can be easily implemented in a routine dermatopathology setting, especially as a fast method to confirm early cutaneous borreliosis. Genotyping could also enable earlier treatment of patients infected with invasive strains.

Benign mucinous metaplasia of the genital mucosa: histomorphological and immunohistochemical features and criteria for differentiation from extramammary Paget disease.

BACKGROUND: Benign mucinous metaplasia of the genitalia (BMM) is a rare condition typified by cells with foamy mucinous cytoplasm. Differential diagnoses include extramammary Paget disease (PD) and human papillomavirus (HPV)-induced vulval intraepithelial neoplasia (VIN) with mucinous differentiation. OBJECTIVES: To characterize histopathological and immunohistochemical features of BMM and to forge criteria for differentiation from PD and VIN with mucinous differentiation. METHODS: Eight biopsy specimens of BMM were stained with haematoxylin and eosin, periodic acid-Schiff and alcian blue, and for cytokeratin (CK) 7, CK10, CK14, CK20, carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA), S100, gross cystic disease fluid protein-15 (GCDFP-15), lysozyme and Ki67 and compared with PD. Polymerase chain reaction was performed in order to identify HPV-specific DNA. RESULTS: BMM showed mucin deposition in superficial epithelial layers ranging from numerous large goblet cells to subtle deposits. The epithelium often showed polygonal (squamoid) or cuboidal differentiation while columnar differentiation was an inconsistent feature. A band-like inflammatory infiltrate was consistently present. Metaplastic epithelium consistently expressed CK7, CEA and EMA either in the entire epithelium or in a superficial band, while CK14, CK10, GCDFP-15 and lysozyme were largely not expressed, and staining for CK20 and S100 was negative. Comparison with PD demonstrated similar staining characteristics, but in a scattered pattern of mucinous cells within preserved squamous epithelium and not in a band-like pattern as in BMM. Nuclear pleomorphism and Ki67-positive mucinous cells in superficial epithelial layers were seen only in PD; GCDFP-15 and/or lysozyme were expressed in the majority of cases of PD. No evidence of HPV-specific DNA was found in BMM. CONCLUSIONS: The spectrum of changes in BMM is distinctive, and BMM can be differentiated with surety from both PD and VIN with mucinous differentiation.

Pseudoclonality in cutaneous pseudolymphomas: a pitfall in interpretation of rearrangement studies.

BACKGROUND: Pseudoclonality is a well-known problem in the interpretation of rearrangement studies of lymph node biopsies. Recently, pseudoclonality has been demonstrated in skin lesions of borreliosis. Studies on pseudoclonality in cutaneous pseudolymphomas are lacking but pseudoclones may pose a risk for overinterpretation of such lesions as cutaneous lymphoma. OBJECTIVES: To determine the frequency of pseudoclonality in cutaneous pseudolymphomas identified by clinicopathological correlation and follow up. METHODS: Study of 30 lesions of pseudolymphomatous cutaneous infiltrates (including insect bite reactions, borrelial pseudolymphomas and pseudolymphomatous drug eruptions) by histopathology, immunophenotyping, T-cell receptor gamma rearrangement and IgH rearrangement. RESULTS: Seven infiltrates were B-cell pseudoclonal; four were T-cell pseudoclonal. Moreover, B-cell clonality was identified in four cases. Immunophenotyping demonstrated that B-cell pseudoclonality and B-cell clonality occurred when infiltrates were moderately dense and included only a minority of B lymphocytes. T-cell pseudoclonality also occurred mostly in moderately dense infiltrates. CONCLUSIONS: B-cell and T-cell pseudoclones are not uncommonly encountered in moderately dense pseudolymphomatous infiltrates (23% and 13%, respectively). B-cell clonality is seen occasionally in pseudolymphomatous infiltrates (13%), especially when they are sparse in B lymphocytes. Therefore, rearrangement studies cannot be interpreted without correlation with morphological patterns and immunophenotyping of infiltrates and they need to be confirmed by duplicate or triplicate tests in order to prevent overinterpretation.

Erythema migrans: a reassessment of diagnostic criteria for early cutaneous manifestations of borreliosis with particular emphasis on clonality investigations.

BACKGROUND: Controversy exists about the relationship of borrelia infection with B-cell lymphomas because B-cell clonality has been identified in infiltrates that contained borrelia-specific DNA. Systematic clinicopathological, immunophenotypical and molecular pathological studies of early borreliosis are lacking. OBJECTIVES: (i) To clarify whether clonal B-cell populations are present already in early borreliosis of the skin (erythema migrans); (ii) to re-evaluate clinicopathological, immunophenotypical and molecular pathological criteria for diagnosis of erythema migrans. METHODS: Study of 34 patients with erythema migrans confirmed by polymerase chain reaction (PCR). Infiltrates were analysed by histopathological and immunohistochemical methods and multiplex PCR for clonal IgH rearrangements. RESULTS: Erythema migrans shows a broad spectrum of changes including sparse infiltrates of T lymphocytes, dense interstitial granulomatous infiltrates (CD68+), and pseudolymphomatous patterns with germinal centre formation. There were accompanying epidermal changes in 59% of patients, and plasma cells were an inconsistent finding. B cells were few when infiltrates were sparse, but increased disproportionately when infiltrates were dense. IgH rearrangement studies revealed one pseudo-oligoclonal, three pseudoclonal and three clonal infiltrates. Pseudoclonality was encountered when infiltrates contained only few B lymphocytes. CONCLUSIONS: Infiltrates in erythema migrans are dominated by T cells followed by CD68+ histiocytes and B lymphocytes. Plasma cells are an inconsistent finding. Pseudoclonality of IgH rearrangement is a result of infiltrates being sparse in B lymphocytes and represents a pitfall in molecular pathological diagnosis that can only be avoided by duplicate or triplicate tests. Incidental B-cell clonality may be encountered in patients with unequivocal erythema migrans and should not be interpreted as a malignant lymphomatous process induced by borrelia.

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.

Heterogeneity and subunit composition of the haemoglobins of five tilapiine species (Teleostei, Cichlidae) of the genera Oreochromis and Sarotherodon.

Haemoglobins of five tilapiine species of the genera Oreochromis and Sarotherodon were investigated. By gel filtration chromatography a molecular weight of 67-69 kDa was determined for the tetrameric molecules which remained stable between pH 5.0 and pH 9.1. When subjected to sodium dodecyl sulphate-Ureapolyacrylamide gel electrophoresis (PAGE), haemoglobins of all species each were split into monomers of three different molecular weights ranging between 16.3 kDA and 17.6 kDa. Subsequently, isoelectric focusing separated haemolysates into about 23 differently charged tetrameric haemoglobins that were arranged in species-specific patterns. This diversity was shown to result from the occurrence of different types of globin chains. By acidic urea PAGE a total of seven major alpha-globins and five major beta-globins were detected and species-characteristic chain variants were identified. To determine the globin chain composition of particular haemoglobin tetramers, 26 bands were isolated by isoelectric focusing and analysed by acidic urea PAGE. Tetramers consisted of doublets of identical alpha- and identical beta-chains (alpha 2 beta 2, symmetric tetramers), or combinations of three (alpha 2 beta beta *; alpha alpha * beta 2) or four (alpha alpha * beta beta *) distinct chains (asymmetric tetramers). Finally, globin chains of Oreochromis niloticus were subjected to partial N-terminal amino acid sequencing. Differences in the composition of the three major beta-chains could be shown, whereas the alpha-chains were N-terminally blocked.