Detection of low-level HCV variants in DAA treated patients: comparison amongst three different NGS data analysis protocols

Background: Notwithstanding the efforts of direct-acting antivirals (DAAs) for the treatment of chronically infected hepatitis C virus (HCV) patients, concerns exist regarding the emergence of resistance-associated substitutions (RAS) related to therapy failure. Sanger sequencing is still the reference technique used for the detection of RAS and it detects viral variants present up to 15%, meaning that minority variants are undetectable, using this technique. To date, many studies are focused on the analysis of the impact of HCV low variants using next-generation sequencing (NGS) techniques, but the importance of these minority variants is still debated, and importantly, a common data analysis method is still not defined. Methods: Serum samples from four patients failing DAAs therapy were collected at baseline and failure, and amplification of NS3, NS5A and NS5B genes was performed on each sample. The genes amplified were sequenced using Sanger and NGS Illumina sequencing and the data generated were analyzed with different approaches. Three different NGS data analysis methods, two homemade in silico pipeline and one commercially available certified user-friendly software, were used to detect low-level variants. Results: The NGS approach allowed to infer also very-low level virus variants. Moreover, data processing allowed to generate high accuracy data which results in reduction in the error rates for each single sequence polymorphism. The results improved the detection of low-level viral variants in the HCV quasispecies of the analyzed patients, and in one patient a low-level RAS related to treatment failure was identified. Importantly, the results obtained from only two out of the three data analysis strategies were in complete agreement in terms of both detection and frequency of RAS. Conclusions: These results highlight the need to find a robust NGS data analysis method to standardize NGS results for a better comprehension of the clinical role of low-level HCV variants. Based on the extreme importance of data analysis approaches for wet-data interpretation, a detailed description of the used pipelines and further standardization of the in silico analysis could allow increasing diagnostic laboratory networking to unleash true potentials of NGS

Clinical-Genetic Features Influencing Disability in Spastic Paraplegia Type 4: A Cross-sectional Study by the Italian DAISY Network

Background and objectives: Hereditary spastic paraplegias (HSPs) are a group of inherited rare neurologic disorders characterized by length-dependent degeneration of the corticospinal tracts and dorsal columns, whose prominent clinical feature is represented by spastic gait. Spastic paraplegia type 4 (SPG4, SPAST-HSP) is the most common form. We present both clinical and molecular findings of a large cohort of patients, with the aim of (1) defining the clinical spectrum of SPAST-HSP in Italy; (2) describing their molecular features; and (3) assessing genotype-phenotype correlations to identify features associated with worse disability. Methods: A cross-sectional retrospective study with molecular and clinical data collected in an anonymized database was performed. Results: A total of 723 Italian patients with SPAST-HSP (58% men) from 316 families, with a median age at onset of 35 years, were included. Penetrance was 97.8%, with men showing higher Spastic Paraplegia Rating Scale (SPRS) scores (19.67 ± 12.58 vs 16.15 ± 12.61, p = 0.009). In 26.6% of patients with SPAST-HSP, we observed a complicated phenotype, mainly including intellectual disability (8%), polyneuropathy (6.7%), and cognitive decline (6.5%). Late-onset cases seemed to progress more rapidly, and patients with a longer disease course displayed a more severe neurologic disability, with higher SPATAX (3.61 ± 1.46 vs 2.71 ± 1.20, p < 0.001) and SPRS scores (22.63 ± 11.81 vs 12.40 ± 8.83, p < 0.001). Overall, 186 different variants in the SPAST gene were recorded, of which 48 were novel. Patients with SPAST-HSP harboring missense variants displayed intellectual disability (14.5% vs 4.4%, p < 0.001) more frequently, whereas patients with truncating variants presented more commonly cognitive decline (9.7% vs 2.6%, p = 0.001), cerebral atrophy (11.2% vs 3.4%, p = 0.003), lower limb spasticity (61.5% vs 44.5%), urinary symptoms (50.0% vs 31.3%, p < 0.001), and sensorimotor polyneuropathy (11.1% vs 1.1%, p < 0.001). Increasing disease duration (DD) and abnormal motor evoked potentials (MEPs) were also associated with increased likelihood of worse disability (SPATAX score>3). Discussion: The SPAST-HSP phenotypic spectrum in Italian patients confirms a predominantly pure form of HSP with mild-to-moderate disability in 75% of cases, and slight prevalence of men, who appeared more severely affected. Early-onset cases with intellectual disability were more frequent among patients carrying missense SPAST variants, whereas patients with truncating variants showed a more complicated disease. Both longer DD and altered MEPs are associated with worse disability

Dati archeologici e fenomeni vulcanici nell’area nolana. Nota preliminare

Questa breve nota riguardante l’area nord orientale della provincia di Napoli vuole portare un contributo alla soluzione dei problemi connessi ai fenomeni sismici e vulcanici in età antica con la presentazione preliminare di una serie di osservazioni effettuate in una zona di interesse archeologico, quale l’area nolana, nota dalle fonti e dalla tradizione ma assai poco conosciuta attraverso i risultati dello scavo. L’attività di tutela della Soprintendenza Archeologica delle Province di Napol..

Note sulla topografia di Puteoli

La prima colonia romana in Pozzuoli sorse sulla collina oggi conosciuta come Rione Terra, già sede dell’insediamento greco di Dicearchia. La limitata area di due ettari, occupata al momenta della deduzione della colonia, ben si accorda con il numero di 300 coloni tramandatoci da Livio. Malgrado tutti i limiti che si presentano nell’indagare le strutture di una città antica quando su di essa insiste un aggregate moderno, se il passaggio dall’antico al nuovo è stato graduale e senza periodi di ..

Pareti dipinte. Dallo scavo alla valorizzazione

Proceedings of the 14th AIPMA's International Conference, helf in Naples and Herculaneum in 2019 (September 9-13

Assessment of bone marrow infiltration diagnosed by flow cytometry in canine large B cell lymphoma: Prognostic significance and proposal of a cut-off value

The aims of this study were to assess the prognostic significance of bone marrow (BM) infiltration in canine large B cell lymphoma (LBCL) and to establish cut-off values for designating the BM as infiltrated by lymphoid blasts. The degree of BM infiltration by large CD21 positive cells in dogs with LBCL was assessed by flow cytometry (FC) and related to time to progression (UP) and lymphoma-specific survival (LSS). Forty-six dogs were prospectively enrolled, staged and treated with a dose-intense chemotherapeutic protocol. BM infiltration was directly correlated with peripheral blood infiltration (P = 0.001), high lactate dehydrogenase activity (P = 0.0024) and substage b disease (P < 0.001). In the univariate analysis, there was a significant association between BM infiltration diagnosed by FC and both UP (P = 0.001) and LSS (P < 0.001). Substage was the only factor associated with TTP in the multivariate analysis (P = 0.002), whereas substage (P < 0.001) and anaemia (P = 0.008) were associated with LSS. A cut-off of 3% BM infiltration had the strongest prognostic value, since it discriminated between dogs with a poorer prognosis (median TTP 69 days; median LSS 155 days) and dogs with a better prognosis (median UP 149 days; median LSS 322 days). BM analysis is an essential step in the staging of LBCL. The presence of BM infiltration by FC at diagnosis is a negative prognostic indicator in canine LBCL. (C) 2013 Elsevier Ltd. All rights reserved

Assessment of bone marrow infiltration diagnosed by flow cytometry in canine large B cell lymphoma: prognostic significance and proposal of a cut-off value

The aims of this study were to assess the prognostic significance of bone marrow (BM) infiltration in canine large B cell lymphoma (LBCL) and to establish cut-off values for designating the BM as infiltrated by lymphoid blasts. The degree of BM infiltration by large CD21 positive cells in dogs with LBCL was assessed by flow cytometry (FC) and related to time to progression (UP) and lymphoma-specific survival (LSS). Forty-six dogs were prospectively enrolled, staged and treated with a dose-intense chemotherapeutic protocol. BM infiltration was directly correlated with peripheral blood infiltration (P = 0.001), high lactate dehydrogenase activity (P = 0.0024) and substage b disease (P < 0.001). In the univariate analysis, there was a significant association between BM infiltration diagnosed by FC and both UP (P = 0.001) and LSS (P < 0.001). Substage was the only factor associated with TTP in the multivariate analysis (P = 0.002), whereas substage (P < 0.001) and anaemia (P = 0.008) were associated with LSS. A cut-off of 3% BM infiltration had the strongest prognostic value, since it discriminated between dogs with a poorer prognosis (median TTP 69 days; median LSS 155 days) and dogs with a better prognosis (median UP 149 days; median LSS 322 days). BM analysis is an essential step in the staging of LBCL. The presence of BM infiltration by FC at diagnosis is a negative prognostic indicator in canine LBCL. (C) 2013 Elsevier Ltd. All rights reserved

Utility of breast ultrasonography in the diagnostic work-up of precocious puberty and proposal of a prognostic index for identifying girls with rapidly progressive central precocious puberty.

To determine the utility of breast ultrasono- graphy in the diagnostic work-up of precocious puberty and to create a prognostic index for early differentiation between non/slowly progressive or transient forms of precocious puberty and rapidly progressive central precocious puberty.We recruited consecutively 60 girls with precocious pubertal development. In all the girls we evaluated Tanner stage, basal and gonadotropin-releasing hormone (GnRH)-stimulated follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels, estradiol (E2) levels, and bone age, and performed pelvis and breast ultrasound examinations. Logistic regression models were fitted to identify possible diagnostic factors for rapidly progressive central precocious puberty and non/slowly progressive or transient forms.Ultrasound breast volume>or=0.85 cm3 was associated with rapidly progressive central precocious puberty (P=0.01). Uterine volume>or=5 cm3, LH peak>or=7 IU/L, presence of an endometrial echo, E2 levels>or=50 pmol/L and bone age>2 SD above expected were significantly associated with rapidly progressive central precocious puberty. A multivariate model including uterine volume, E2 level, bone age, presence of an endometrial echo and ultrasound breast volume revealed a strong ability to classify rapidly progressive forms. From this multivariate analysis a prognostic index for rapidly progressive central precocious puberty was defined.Ultrasound imaging allows better definition of the breast and the maturation stage than does use of Tanner's stages. Ultrasound breast volume>or=0.85 cm3 is an independent predicting factor of rapidly progressive central precocious puberty. A prognostic index that was created from a multivariate model including uterine volume, E2 level, presence of an endometrial echo, bone age and ultrasonographically determined breast volume, may help in the early differentiation between rapidly progressive central precocious puberty and non/slowly progressive or transient forms