Diagnosis of Lyme Borreliosis With a Novel, Seminested Real-Time Polymerase Chain Reaction Targeting the 5S-23S Intergenic Spacer Region: Clinical Features, Histopathology, and Immunophenotype in 44 Patients.

ABSTRACT: Lyme borreliosis (LB) is the most common tick-borne infection in Europe and North America. Polymerase chain reaction (PCR) is an important tool to confirm the diagnosis, but not always successful, especially when organisms are sparse. We developed a novel, seminested real-time PCR assay [target: 5S-23S intergenic spacer region (IGS)] and compared it with 3 well-established conventional PCR assays (IGS/OspA/real-time IGS) on 596 formalin-fixed, paraffin-embedded routine skin biopsies. The seminested real-time assay identified 46 cases of borreliosis while 25, 27, and 38 were identified by the 3 other assays, respectively (P 0.01, P 0.02, and P 0.42; significance P < 0.05). Clinicopathologic and immunophenotypic analysis of PCR-positive cases revealed 38 erythema migrans (EM), 6 Borrelia lymphocytomas, and 2 acrodermatitis chronica atrophicans (ACA). In the 44 PCR-confirmed cases, plasma cells were present in only a third of EM cases. By contrast, CD123-positive plasmacytoid dendritic cells were common (74%) and therefore are unlikely to be helpful in the differential diagnosis between EM and tumid lupus erythematosus. A loss of CD34 in a third of all LB specimens limits its diagnostic value in the differential diagnosis with morphea. Interstitial macrophages were common in cutaneous LB (42/43) forming interstitial granulomas in a third of all cases, and 3/38 EM, 3/6 Borrelia lymphocytomas, and 1/2 ACA were only identified by the new seminested real-time assay, suggesting that it is especially helpful in confirming the diagnosis of Borrelia lymphocytoma.

A Prognostic Gene Signature Expressed in Primary Cutaneous Melanoma: Synergism With Conventional Staging.

BACKGROUND: Current clinico-pathological American Joint Committee on Cancer (AJCC) staging of primary cutaneous melanoma is limited in its ability to determine clinical outcome, and complementary biomarkers are not available for routine prognostic assessment. We therefore adapted a gene signature, previously identified in fresh-frozen (FF) melanomas and adjacent stroma, to formalin-fixed paraffin-embedded (FFPE) melanomas. The aim was to develop a gene expression profiling (GEP) score to define patient survival probability at the time of first diagnosis. METHODS: Expression of 11 FF melanoma signature genes was quantified by reverse transcription polymerase chain reaction in an FFPE melanoma training cohort (n = 125), corresponding to the combined FF melanoma training and validation cohorts. The resulting GEP score was validated technically and clinically in an independent FFPE melanoma cohort (n = 211). All statistical tests were two-sided. RESULTS: We identified a prognostic eight-gene signature in the tumor area (tumor and adjacent tissue) of AJCC stage I-III melanomas. A signature-based GEP score correlated with melanoma-specific survival (MSS; Kaplan-Meier analysis: P < .0001) was independent of tumor stage (multivariable regression analysis: P = .0032) and stroma content (<5%-90%) and complemented conventional AJCC staging (receiver operating characteristic curve analysis: area under the curve = 0.91). In the clinical validation cohort, the GEP score remained statistically significant (P = .0131) in a multivariable analysis accounting for conventional staging. The GEP score was technically robust (reproducibility: 93%; n = 84) and clinically useful, as a binary as well as a continuous score, in predicting stage-specific patient MSS. CONCLUSIONS: The GEP score is a clinically significant prognostic tool, contributes additional information regarding the MSS of melanoma patients, and complements conventional staging.

An intensified dosing schedule of subcutaneous methotrexate in patients with moderate to severe plaque-type psoriasis (METOP): a 52 week, multicentre, randomised, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: Methotrexate is one of the most commonly used systemic drugs for the treatment of moderate to severe psoriasis; however, high-quality evidence for its use is sparse and limited to use of oral dosing. We aimed to assess the effect of an intensified dosing schedule of subcutaneous methotrexate in patients with moderate to severe plaque-type psoriasis. METHODS: We did this prospective, multicentre, randomised, double-blind, placebo-controlled, phase 3 trial (METOP) at 16 sites in Germany, France, the Netherlands, and the UK. Eligible patients were aged 18 years or older, had a diagnosis of chronic plaque psoriasis for at least 6 months before baseline, had currently moderate to severe disease, and were methotrexate treatment-naive. Participants were randomly assigned (3:1), via a computer-generated random number sequence integrated into an electronic data capture system, to receive either methotrexate at a starting dose of 17·5 mg/week or placebo for the first 16 weeks, followed by methotrexate treatment of all patients up to 52 weeks (methotrexate-methotrexate vs placebo-methotrexate groups). Dose escalation to 22·5 mg/week was allowed after 8 weeks of methotrexate treatment if patients had not achieved at least a 50% reduction in baseline Psoriasis Area and Severity Index score (PASI), with corresponding volume increases in placebo injections. Treatment was combined with folic acid 5 mg/week. Group allocation was concealed from participants and investigators from the time of randomisation until an interim database lock at week 16, and was open label from week 16 onwards, with no masking of participants or investigators. The primary efficacy endpoint was a 75% reduction in PASI score (PASI 75) from baseline to week 16. We did analysis by modified intention to treat, with non-responder imputation. This study is registered with EudraCT, number 2012-002716-10. FINDINGS: Between Feb 22, 2013, and May 13, 2015, we randomly assigned 120 patients to receive methotrexate (n=91) or placebo (n=29). At week 16, a PASI 75 response was achieved in 37 (41%) patients in the methotrexate group compared with three (10%) patients in the placebo group (relative risk 3·93, 95% CI 1·31-11·81; p=0·0026). Subcutaneous methotrexate was generally well tolerated; no patients died or had serious infections, malignancies, or major adverse cardiovascular events. Serious adverse events were recorded in three (3%) patients who received methotrexate for the full 52 week treatment period. INTERPRETATION: Our findings show a favourable 52 week risk-benefit profile of subcutaneous methotrexate in patients with psoriasis. The route of administration and the intensified dosing schedule should be considered when methotrexate is used in this patient group. FUNDING: Medac.

Histopathology and immunophenotype of acrodermatitis chronica atrophicans correlated with ospA and ospC genotypes of Borrelia species.

BACKGROUND: Chronic cutaneous borreliosis (acrodermatitis chronica atrophicans, ACA) is a relatively rare manifestation of borreliosis attributed mainly to Borrelia afzelii. Chronic borreliosis has been associated with ospA and ospC genotypes. Literature on molecular investigations of Borrelia in lesions of ACA is scant. METHODS: Histopathological and immmunohistochemical features in 22 biopsies of ACA (16 patients) were examined. Paraffin-embedded biopsies were analyzed with polymerase chain reaction (PCR) assays targeting ospA and ospC genes, sequencing and phylogenetic analysis. RESULTS: Genotyping of ospA identified B. afzelii, serotype 2, in 12 of 16 patients. ospC-PCR was positive in seven patients revealing genotypes Af5 (n = 4), Af2 (n = 2) and Af6 (n = 1). Histopathologically, interstitial granulomatous infiltrates (CD68 positive) were common, combined with thickened collagen bundles and band-like infiltrates of CD4 positive T lymphocytes. Plasma cells were sparse/absent in 9 of 22 specimens even on staining with CD138. On CD34-staining, interstitial fibroblasts were often reduced akin to the situation in morphea. CONCLUSIONS: With assays targeting ospA and ospC genes we confirmed from paraffin-embedded biopsies that B. afzelii, serotype 2, osp C groups Af5, Af2 and Af6 is the main cause of ACA. Specimens commonly showed a combination of band-like T-cell-rich infiltrates with interstitial granulomatous features, a pattern previously under-recognized in ACA. This finding was particularly typical for lesions infected with ospC genotype Af5.

Evidence that a neutrophil-keratinocyte crosstalk is an early target of IL-17A inhibition in psoriasis.

The response of psoriasis to antibodies targeting the interleukin (IL)-23/IL-17A pathway suggests a prominent role of T-helper type-17 (Th17) cells in this disease. We examined the clinical and immunological response patterns of 100 subjects with moderate-to-severe psoriasis receiving 3 different intravenous dosing regimens of the anti-IL-17A antibody secukinumab (1 × 3 mg/kg or 1 × 10 mg/kg on Day 1, or 3 × 10 mg/kg on Days 1, 15 and 29) or placebo in a phase 2 trial. Baseline biopsies revealed typical features of active psoriasis, including epidermal accumulation of neutrophils and formation of microabscesses in >60% of cases. Neutrophils were the numerically largest fraction of infiltrating cells containing IL-17 and may store the cytokine preformed, as IL-17A mRNA was not detectable in neutrophils isolated from active plaques. Significant clinical responses to secukinumab were observed 2 weeks after a single infusion, associated with extensive clearance of cutaneous neutrophils parallel to the normalization of keratinocyte abnormalities and reduction of IL-17-inducible neutrophil chemoattractants (e.g. CXCL1, CXCL8); effects on numbers of T cells and CD11c-positive dendritic cells were more delayed. Histological and immunological improvements were generally dose dependent and not observed in the placebo group. In the lowest-dose group, a recurrence of neutrophils was seen in some subjects at Week 12; these subjects relapsed faster than those without microabscesses. Our findings are indicative of a neutrophil-keratinocyte axis in psoriasis that may involve neutrophil-derived IL-17 and is an early target of IL-17A-directed therapies such as secukinumab.

Increased detection of cutaneous leishmaniasis in Norway by use of polymerase chain reaction.

Cutaneous leishmaniasis (CL) is a parasitic infection and occurs in tropical and subtropical regions worldwide and in the region of the Mediterranean Sea. The diagnosis is based on the clinical appearance and biopsy findings that may be supplemented with polymerase chain reaction (PCR). In this study 20 cases were selected if (i) a histopathological diagnosis of granulomatous dermatitis was made, (ii) CL was taken into consideration or (iii) the diagnosis was CL. PCR analysis with primers specific for leishmania was performed on archived histological specimens and was positive in 6 of the 20 cases. In two cases both the clinical and histopathological diagnosis concurred with CL. In the remaining four cases a clinical diagnosis other than CL was made. In two of these cases the histopathology showed granulomatous dermatitis, and detection of parasites led to consideration of CL. In the last two cases leishmaniasis was not taken into consideration by clinicians or pathologists. Our study shows that CL may occur more often than anticipated in Norway, but clinicians do not consider the diagnosis as often as they should. Pathologists may also fail to diagnose or suggest CL especially when parasites are not visualized in the histopathological specimen.

BRAF mutations in cutaneous melanoma: no correlation with histological prognostic factors or overall survival / Mutações BRAF em melanomas cutâneos: nenhuma correlação com fatores prognósticos e sobrevida global

INTRODUCTION: Molecular biology techniques allow identification of molecular markers such as BRAF and c-Kit gene mutations in melanomas. Studies on genetic alterations in melanomas of South-American patients are sparse. OBJECTIVES: To identify the incidence of BRAF and c-Kit gene mutations in primary cutaneous melanomas in Brazilian patients and to evaluate pathogenetic and prognostic implications of these mutations correlating them with clinical and histopathological data. MATERIAL AND METHODS: Ninety-six surgical specimens of primary cutaneous melanoma and 15 corresponding metastasis were analyzed using TaqMan Real-Time polymerase chain reaction (PCR) assays. RESULTS: In comparison with the medical literature, a relatively low frequency of BRAF mutation in primary (39 percent) and metastatic (40 percent) melanomas and complete absence of c-Kit gene mutations were demonstrated. BRAF mutations arose at an early stage during melanoma progression and were not involved in the transition of thin (< 1 mm) to thick (> 1 mm) melanomas. BRAF mutations are related to patients' younger age and to the pattern of sun exposure, although there was no correlation with any histological prognostic factor or overall survival. CONCLUSION: The identification of both BRAF and c-Kit mutation is not a suitable prognostic indicator in the Brazilian population. Moreover, the relatively low frequency of BRAF mutations brings into question if it actually plays a key role in melanoma pathogenesis.

INTRODUÇÃO: Técnicas de biologia molecular permitem a identificação de marcadores moleculares como mutações dos genes BRAF e c-Kit em melanomas. Estudos de alterações genéticas em pacientes sul-americanos são escassos. OBJETIVOS: Identificar a incidência de mutações dos genes BRAF e c-Kit em melanomas cutâneos primários em uma série de pacientes brasileiros e avaliar as implicações patogenéticas e prognósticas dessas mutações, correlacionando-as com dados clínicos e histopatológicos. MATERIAL E MÉTODOS: Noventa e seis espécimes cirúrgicos de melanomas cutâneos e 15 metástases correspondentes foram analisados por meio da técnica TaqMan Real-Time polymerase chain reaction (PCR). RESULTADOS: Uma frequência relativamente baixa de mutações BRAF em melanomas cutâneos primarios (39 por cento) e metastáticos (40 por cento) em comparação com os dados da literatura e ausência de mutações c-Kit foi demonstrado. Mutações BRAF surgiram em um estágio inicial da progressão do melanoma e não foram envolvidas na transição de melanomas finos (< 1 mm) para grossos (> 1 mm). Essas mutações estavam presentes em pacientes mais jovens e se correlacionaram com o padrão de exposição solar dos pacientes estudados, mas não houve correlação com nenhum fator prognóstico histológico ou sobrevida global dos mesmos. CONCLUSÃO: A identificação de ambas as mutações (BRAF e c-Kit) não servem como indicadores de prognóstico na população brasileira. Além disso, a baixa frequência de mutações BRAF encontrada neste estudo nos faz questionar se essa mutação realmente tem papel-chave na patogênese do melanoma.

Correlation of histopathological patterns in cutaneous melanoma with BRAF mutations / Correlação de padrões histopatológicos de melanomas cutâneos com mutações BRAF

INTRODUCTION: Mutations on BRAF gene located on chromosome 7q are the most frequently found in cutaneous melanomas (60 percent-80 percent). The only study correlating histopathological patterns of cutaneous melanomas with the presence of BRAF mutations was undertaken by Viros et al. in 2008. The authors observed that morphological features of melanomas are associated with BRAF mutations. OBJECTIVES: To correlate histopathological patterns in cutaneous melanoma with the presence of BRAF mutations in order to corroborate the results of the study performed by Viros et al. METHODS: Paraffin embedded surgical specimens of 20 primary cutaneous melanomas with BRAF mutation and 20 specimens without BRAF mutation were evaluated independently by two dermatologists that carried out a blind experiment. The features analyzed were nesting, circumscription, presence of isolated melanocytes in the lesion, size and shape of neoplastic cells, and tumor cell pigmentation. RESULTS: "Nesting" was the most prevalent variable for the determination of melanomas with BRAF mutations according to both observers (r = 0.46; p = 0.04). CONCLUSION: As far as mutational status is concerned, it was not possible to confirm any predictive value for histopathological patterns such as circumscription, presence of isolated melanocytes in the lesion and cytological features. Difficulties in the interpretation of some histological criteria were demonstrated by the variation in the observers' conclusions. It is difficult to state if genetic alterations such as BRAF mutations may serve as biomarkers for melanoma classification.

INTRODUÇÃO: Mutações do gene BRAF localizado no cromossomo 7q são as mais frequentemente encontradas em melanomas cutâneos (60 por cento-80 por cento). O único estudo que correlacionou padrões histopatológicos de melanomas cutâneos com a presença de mutações BRAF foi realizado por Viros et al., em 2008, que observaram que características morfológicas de melanomas estavam associadas a mutações BRAF. OBJETIVOS: Correlacionar padrões histopatológicos de melanomas cutâneos com a presença de mutações BRAF, a fim de confirmar os achados de Viros et al. MÉTODOS: Espécimes em parafina de 20 casos de melanomas cutâneos primários com mutações BRAF e 20 casos sem mutações foram avaliados independentemente por dois dermatologistas sem o conhecimento da presença ou não das mutações. Os padrões analisados foram formação de "ninhos", circunscrição, presença de melanócitos isolados na lesão, tamanho e forma das células neoplásicas e pigmentação das células tumorais. RESULTADOS: A formação de "ninhos" foi a variável com o maior poder de determinação para melanomas com mutações BRAF para ambos os observadores (r = 0,46; p = 0,04). CONCLUSÃO: Não foi possível confirmar nenhum valor preditivo em relação ao status mutacional de um melanoma para os padrões histológicos circunscrição e presença de melanócitos isolados na lesão, bem como para características citológicas. Dificuldades na interpretação de alguns critérios histológicos foram demonstradas pela variação da concordância entre os observadores. É difícil afirmar se alterações genéticas como as mutações BRAF podem servir como biomarcadores para a classificação de melanomas.

Refining criteria for diagnosis of cutaneous infections caused by herpes viruses through correlation of morphology with molecular pathology.

BACKGROUND: Infections of the skin by herpes viruses do not always present themselves in typical fashion. Early diagnosis, however, is crucial for appropriate treatment. Polymerase chain reaction (PCR) allows diagnosis and differential diagnosis of herpes virus infections, but the method is not yet available in large parts of the world, where diagnosis is made based on morphology alone. AIM: To refine criteria for the diagnosis of herpes virus infections of the skin by way of correlation of clinical and histopathologic findings with results of PCR studies. METHODS: We studied 75 clinically diagnosed patients of "zoster," "varicella," and "herpes simplex", to correlate clinical and histopathological findings with results of PCR studies on paraffin embedded biopsy specimens. RESULTS: Clinical suspicion of infection by herpes viruses was confirmed by histopathology in 37% of the cases and by PCR studies in 65% of the cases. Zoster was frequently misdiagnosed as infection with herpes simplex viruses (30%). When diagnostic signs of herpes virus infection were encountered histopathologically, PCR confirmed the diagnosis in 94%. By way of correlation with results of PCR studies, initial lesions of herpes virus infections could be identified to have a distinctive histopathological pattern. Herpetic folliculitis appeared to be a rather common finding in zoster, it occurring in 28% of the cases. CONCLUSION: We conclude that correlation of clinical and histopathological features with results of PCR studies on one and the same paraffin embedded specimen permits identification of characteristic morphologic patterns and helps to refine criteria for diagnosis both clinically and histopathologically.

Phylogenetic patterns in populations of Chilean species of the genus Orestias (Teleostei: Cyprinodontidae): results of mitochondrial DNA analysis.

Patterns of molecular genetic differentiation among taxa of the "agassii species complex" (Parenti, 1984) were analysed based on partial mtDNA control region sequences. Special attention has been paid to Chilean populations of Orestias agassii and species from isolated lakes of northern Chile, e.g., O. agassii, Orestias chungarensis, Orestias parinacotensis, Orestias laucaensis, and Orestias ascotanensis. Orestias tschudii, Orestias luteus, and Orestias ispi were analysed comparatively. Our findings support the utility of mtDNA control region sequences for phylogenetic studies within the "agassii species complex" and confirmed the monophyly of this particular lineage, excluding O. luteus. However, the monophyly of further morphologically defined lineages within the "agassii complex" appears doubtful. No support was found for the utility of these data sets for inferring phylogenetic relationships between more distantly related taxa originating from Lake Titicaca.

Phylogeographic patterns in populations of the black-chinned tilapia complex (Teleostei, Cichlidae) from coastal areas in West Africa: support for the refuge zone theory.

Within the tilapiines, a major African cichlid lineage, quite limited genetic informations are available on phylogenetic interrelationships and phylogeographical patterns at both macro- and micro-evolutionary scales. The present study examines the genetic diversity of the black-chinned tilapia complex from coastal areas in West Africa (Senegal to Congo-Brazzaville) based on mtDNA control region sequences. Phylogenetic inferences provide support for the monophyly of both taxa involved. It is suggested that Sarotherodon melanotheron and Sarotherodon nigripinnis have diverged probably since the early Pleistocene. The occurrence of the main intraspecific lineages could be dated back to about 500,000-900,000 years. Our data suggest that West African Pleistocene refuge zones may have strongly influenced the degree and level of genetic differentiation among populations and thus the observed phylogeographic patterns. The spatial distribution of mtDNA lineages and the centres of intrapopulational genetic diversity clearly correspond to proposed lowland refuge zones and core areas of Central Africa. Moreover, this study genetically supports the existence of Sarotherodon melanotheron leonensis for the first time and further confirms the validity of the most recently introduced subspecies, e.g., Sarotherodon nigripinnis dolloi.