SARS-CoV-2 detection in primary thyroid sarcoma: coincidence or interaction?

INTRODUCTION: Thyroid dysfunctions associated with SARS-CoV-2 are emerging in scientific literature. During the second COVID-19 epidemic spread, we evaluated a patient with the suspect of subacute thyroiditis. METHODS AND RESULTS: Specimen from fine-needle aspiration of a hypoechoic undefined area was analyzed for cytology and for SARS-CoV-2 detection. SARS-CoV-2 was retrieved by real-time polymerase chain reaction on the cytologic sample, which was then cultured on Vero E6 cells and demonstrated to be cytopathic. Whole-genome sequence was deposited. Histological exam diagnosed a rare case of primary thyroid sarcoma with diffuse and strong expression of mouse double minute 2 homolog (MDM2) oncoprotein. Ultrastructural examination confirmed, in several neoplastic cells, the presence of viral particles in cytoplasmic vacuoles. CONCLUSIONS: In our hypothesis, SARS-CoV-2 and sarcoma coexistence could represent a synergistic interplay, ultimately favoring both viral persistence and tumor proliferation: the overexpression of MDM2 in tumor cells might have generated a favorable immunological niche for SARS-CoV-2 localization and, in turn, SARS-CoV-2 could have favored tumor growth by inducing MDM2-mediated p53 downregulation. Functional studies are needed to confirm this suggestive pathway.

Rare autosomal trisomies: comparison of detection through cell-free DNA analysis and direct chromosome preparation of chorionic villus samples.

OBJECTIVE: Direct chromosome preparations of chorionic villus samples (CVS) and cell-free DNA (cfDNA) testing both involve analysis of the trophoblastic cell lineage. The aim of this study was to compare the spectrum of rare autosomal trisomies (RATs) detected by these two approaches and assess the available information on their clinical significance. METHODS: Data from 10 reports on genome-wide cfDNA testing were pooled to determine which chromosomes were most frequently involved in RAT-positive cases, and pregnancy outcome information was reviewed. CVS information was obtained from an updated database of 76 102 consecutive CVS analyses performed over a period of 18 years at TOMA laboratory, in which trophoblastic and mesenchymal layers were analyzed and amniotic fluid cell analysis was recommended for RAT-positive cases. Chromosomes involved and presence of confined placental mosaicism, true fetal mosaicism and uniparental disomy (UPD) for imprinted chromosomes were assessed. Also evaluated were the frequency and types of RATs in products of conception. RESULTS: RATs were present in 634 of 196 662 (0.32%) cfDNA samples and 237 of 57 539 (0.41%) CVS trophoblast samples (P < 0.01). The frequency of RATs varied over 8-fold between the cfDNA reports. Confirmation of abnormality through amniocentesis was more likely when RATs were ascertained through cfDNA (14 of 151; 9.3%) than through CVS trophoblasts (seven of 237; 3.0%) (P < 0.01). In cfDNA-ascertained cases, trisomies 15, 16 and 22, which are associated with fetal loss, were identified proportionately more often. Of 151 cases with RAT identified by cfDNA and outcome information available, 41.1% resulted in normal live birth; 27.2% in fetal loss; 7.3% had phenotypic abnormality detected through ultrasound or other follow-up evaluation; 2.0% had a clinically significant UPD; and 14.6% had fetal growth restriction or low birth weight. All autosomes were involved in trisomies in products of conception; the most common RATs detected were trisomies 16, 22 and 15 with a frequency of > 9% each. CONCLUSIONS: Although there are strong parallels between RATs ascertained through cfDNA analysis and direct chromosome preparation of CVS, caution is needed in applying conclusions from CVS analysis to cfDNA testing, and vice versa. RATs identified through genome-wide cfDNA tests have uncertain risks for fetal loss, growth restriction or fetal abnormality. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

Graves' orbitopathy, idiopathic orbital inflammatory pseudotumor and Epstein-Barr virus infection: a serological and molecular study.

OBJECTIVE: One of the hypotheses on the pathogenesis of autoimmune diseases, including Graves' disease (GD) and Graves' orbitopathy (GO), involves bacterial or viral infections. Recently, Epstein-Barr virus (EBV) has been proposed to play a role in the pathogenesis of idiopathic orbital inflammatory pseudotumor (IOIP) in Asians. The aim of the present study was to investigate the possible association of GO with EBV infection/exposure, as compared with IOIP, using serum and tissue samples, as well as primary cultures of orbital fibroblasts. METHODS: Thirty-one patients were studied, including four with IOIP, ten with GO, nine with GD without GO and eight control patients without IOIP, GD and GO. All patients with IOIP and GO underwent orbital decompression. Control patients underwent palpebral surgery. Fibroadipose orbital tissue samples were collected. Serum anti-EBV antibodies were measured in all patients. EBV-DNA was measured in blood samples, orbital tissue samples and primary cultures of orbital fibroblasts. RESULTS: Serum assays showed that the vast majority of patients have had a previous exposure to EBV, but no one had an acute infection. EBV-DNA was detected in ~40% of blood samples from GO, GD and control patients, but in none of the IOIP samples. EBV-DNA was not detected in any of the orbital tissue samples tested or in primary cultures of orbital fibroblasts. CONCLUSIONS: EBV infection does not seem to be associated with GD, GO and IOIP in Caucasians. Whether EBV is involved in IOIP in Asians or other populations remains to be confirmed.

Absence of xenotropic murine leukemia virus-related virus in Italian patients affected by chronic fatigue syndrome, fibromyalgia, or rheumatoid arthritis.

The xenotropic murine leukemia virus-related virus (XMRV) has been recently linked to chronic fatigue syndrome in a US cohort in whom the virus was demonstrated in 67% patients vs 3.7% healthy controls. Albeit this finding was not substantiated by subsequent reports and eventually considered a laboratory contamination, the matter is still the object of intense debate and scrutiny in various cohorts of patients. In this work we examined well-clinically characterized Italian patients affected by chronic fatigue syndrome, and also fibromyalgia and rheumatoid arthritis, two chronic illnesses of basically unknown etiology which show quite a few symptoms in common with chronic fatigue syndrome. Although we used recently updated procedures and controls, the XMRV was not found in 65 patients with chronic fatigue syndrome diagnosis, 55 with fibromyalgia, 25 with rheumatoid arthritis, nor in 25 healthy controls. These results add to the ever-growing number of surveys reporting the absence of XMRV in chronic fatigue syndrome patients and suggest that the virus is also absent in fibromyalgia and rheumatoid arthritis.

Giant condylomata (Buschke-Löwenstein tumours): our case load in surgical treatment and review of the current therapies.

BACKGROUND: Buschke-Löwenstein tumour (BLT) or giant condyloma is a verrucous infiltrating lesion, due to a sexually transmitted virus infection, human papilloma virus subtypes 6 and 11. Poor hygiene, promiscuity, chronic irritation and cellular immunocompromised states are often implicated in its genesis. Typical treatment of giant condyloma includes imiquimod cream, podophillin resin, cryotherapy, laser surgery, tangential shave excision with electrocautery. OBJECTIVE: The authors report their case load in the treatment of giant condyloma and the review of the modern therapies. METHODS AND MATERIALS: 27 consecutive patients (18 men, nine women) underwent surgery for giant condylomata of perianal region and externa genitalia at the Department of Plastic Surgery of the University of Palermo, from October 2006 to December 2009. All the patients had been treated before with conservative therapies without significant results. We performed the radical excision with split-thickness skin graft in all the patients. RESULTS: No significant complications have occurred in all the cases. The functional and aesthetic outcome were satisfying. No recurrence of disease were noticed in the follow-up. CONCLUSION: the radical excision with split-thickness skin graft appears to be a successful option of treatment for Buschke-Löwenstein tumours. Compared to other methods it does not necessitate several stages of treatment, moreover it has the advantage of a lower risk of recurrence, it allows a complete histologic examination, the healing process is rapid, the improvement of quality of patients's life is significant.

Detection of noncalcified pulmonary nodules on low-dose MDCT: comparison of the sensitivity of two CAD systems by using a double reference standard.

PURPOSE: This study compared the sensitivity of two commercial computer-aided detection (CAD) systems in identifying noncalcified pulmonary nodules on low-dose multidetector computed tomography (MDCT) scans by using a double reference standard. MATERIALS AND METHODS: Three chest low-dose MDCT scans of patients who had undergone lung cancer screening were retrospectively analysed using two distinct commercial CAD systems: LungCAD VC10A, Siemens Medical Solutions (CAD1) and LungVCAR, GE Healthcare (CAD2). The exact location of each finding suggested by each system was recorded by an independent reader according to spatial coordinates (x, y, z). Two panels of experienced thoracic radiologists from two different institutions independently established two reference standards (RS1, RS2) by identifying the true positive findings with spatial coordinates without using CAD. Sensitivity of the two CAD systems, defined by lesionlevel analysis, was tested and sensitivities compared. RESULTS: RS1 identified 34 noncalcified pulmonary nodules, whereas RS2 identified 54. The total number of findings detected by the two CAD systems was 684. CAD1 correctly identified 13/34 nodules (sensitivity 38%) for RS1 and 17/54 (sensitivity 30%) for RS2, whereas CAD2 correctly identified 11/34 nodules (sensitivity 35%) for RS1 and 13/54 (sensitivity 23%) for RS2. Comparison between the two CAD systems did not show a statistically significant difference in terms of sensitivity (p<0.05) for both RS1 (p=0.42) and RS2 (p=0.33). CONCLUSIONS: The two commercial CAD systems had similar sensitivity in detecting noncalcified pulmonary nodules on low-dose MDCT of the chest.

Anti-SARS-CoV-2 antibody levels and kinetics of vaccine response: potential role for unresolved inflammation following recovery from SARS-CoV-2 infection.

The immune response after SARS-CoV-2 vaccine administration appears to be characterized by high inter-individual variation, even in SARS-CoV-2 positive subjects, who could have experienced different post-infection, unresolved conditions. We monitored anti-SARS-CoV-2 IgG levels and kinetics along with circulating biomarkers in a cohort of 175 healthcare workers during early immunization with COVID-19 mRNA-LNP BNT162b2 vaccine, to identify the associated factors. Subjects with a previous SARS-CoV-2 infection were characterized by higher BMI and CRP levels and lower neutrophil count with respect to naïve subjects. Baseline IgG levels resulted associated with CRP independently on BMI and inflammatory diseases. Among 137 subjects undergoing vaccination and monitored after the first and the second dose, three kinetic patterns were identified. The pattern showing a rapid growth was characterized by higher IgG levels at baseline and higher CRP and MCHC levels than negative subjects. Subjects previously exposed to SARS-CoV-2 showed higher levels of CRP, suggesting persistence of unresolved inflammation. These levels are the main determinant of IgG levels at baseline and characterized subjects belonging to the best performing, post-vaccine antibody kinetic pattern.

Nasal nitric oxide and nitric oxide synthase expression in primary ciliary dyskinesia.

No study has evaluated the correlation between different expression of nitric oxide synthase (NOS) isoforms in nasal epithelial cells and nasal NO (nNO) level in primary ciliary dyskinesia (PCD). Gene expression of endothelial (NOS3) and inducible NOS (NOS2) and their correlation with nNO level, ciliary function and morphology were studied in patients with PCD or secondary ciliary dyskinesia (SCD). NOS3 gene polymorphisms were studied in blood leukocytes. A total of 212 subjects were studied (48 with PCD, 161 with SCD and three normal subjects). nNO level correlated with mean ciliary beat frequency (p = 0.044; r = 0.174). The lower the nNO level the higher was the percentage of immotile cilia (p<0.001; r = -0.375). A significant positive correlation between NOS2 gene expression and nNO levels was demonstrated in all children (p = 0.001; r = 0.428), and this correlation was confirmed in patients with PCD (p = 0.019; r = 0.484). NOS2 gene expression was lower in PCD than in SCD (p = 0.04). The NOS3 isoform correlated with missing central microtubules (p = 0.048; r = 0.447). nNO levels were higher in PCD subjects with the NOS3 thymidine 894 mutation, and this was associated with a higher ciliary beat frequency (p = 0.045). These results demonstrate a relationship between nNO level, NOS mRNA expression and ciliary beat frequency.

Prenatal BACs-on-Beads™ : a new technology for rapid detection of aneuploidies and microdeletions in prenatal diagnosis.

OBJECTIVE: Molecular cytogenetic techniques on uncultured prenatal samples are the sole tests applied in some countries in cases with advanced maternal age (AMA) or increased risk after prenatal screening. Moreover, there is a trend to perform invasive prenatal diagnosis (PD) during the first trimester before ultrasound manifestations, so new rapid and reliable assays are necessary to investigate microdeletions not detectable with the conventional karyotype. We report the validation study of the prenatal bacterial artificial chromosomes-on-Beads™ (BoBs™ ; CE-IVD), a bead-based multiplex assay detecting chromosomes 13, 18, 21, X/Y aneuploidies and nine microdeletion regions having an overall detection rate of 1/1700. METHOD: We retrospectively studied 408 selected samples and prospectively tested 212 consecutive samples ascertained for conventional karyotyping. RESULTS: We did not find false-positive results. Triploidies were not detected. Maternal cell contamination of male samples up to 90% was unmasked inspecting gonosome profiles. Mosaic conditions at 20 to 30% were revealed. Failures were due to low amount of DNA. CONCLUSION: Prenatal BoBs™ is a robust technology for the investigation of fetuses with normal karyotype with or without sonographic abnormalities. Running in parallel with the karyotype analysis, it can be proposed instead of rapid FISH or QF-PCR providing rapid results on common aneuploidies and additional information regarding the microdeletion syndromes.

Burden of an educational program on end of life management in a Internal Medicine ward: a real life report.

BACKGROUND AND AIM: Appropriate end of life (EOL) management in Internal Medicine wards is challanging. The aim of this study was to analyze the burden of an educational program on EOL management in a Internal Medicine ward. Materials and methods: We retrospectively analysed characteristics and management of patients consecutively died in an italian Internal Medicine ward along one year. We compared demographic, co-morbidity, pharmacological treatment in the last 48-hours of life and procedures during hospital stay in patients died six months before and after an educational program on palliative cares and EOL management addressed to a team of physicians and nurses. RESULTS: Study population was composed by 354 patients (190 females), with mean age ± DS 83.5 ± 10.6 years, one half admitted after the educational program. Eighty-four percent of deaths was exepected in the last 48 hours before exitus. Demographic characteristics and causes of hospitalization were not different before and after educational program. After the educational program the sharing of palliative care program with patient, relatives and/or caregivers (97.7% vs 85.8%, p=0.0001) and written order to withdrawal vital parameters relevation (39.5% vs 22%, p=0.0005) significantly increased, while difference in pharmacological classes prescribed in the last 48 hours of life was not find. Blood (54.8% vs 67.2%, p=0.0219) and arterial gas analysis (28.8% vs 39.5%, p=0.0435) samples in the last 48 hours of life were significantly reduced. Radiological and/or endoscopic examinations, red cells or platelets transfusion were reduced and palliative therapy was increased, despite difference between the two periods was not statistically significant. CONCLUSION: Educational program in Internal Medicine wards aimed to improve skills could contribute to make EOL management more appropriate and patient-oriented and it should be strongly encour-aged.

Reproducibility of a local cooling test to assess microvascular function in human skin.

OBJECTIVE: In the present study we aimed to assess the reproducibility of skin microvascular reactivity while fast cooling locally with a custom-designed laser-Doppler flowmetry (LDF) probe. METHODS: Twenty-two healthy volunteers underwent local 15 degrees C cooling on the forearm during 5 (protocol 1, n=12) or 30 min (protocol 2, n=10). Skin blood flow was concomitantly assessed using LDF. Measurements were repeated after 30 min (protocol 1) or 7 days (protocols 1 and 2). Data were expressed as cutaneous vascular conductance (CVC) and percentage of baseline (%BL). Within subject coefficients of variation (CV) and intra-class correlation coefficients (ICC) were calculated. RESULTS: Immediate reproducibility of the 5-min cooling was very good, either expressed as CVC or %BL (CV were 8% and 18%; ICC were 0.85 and 0.78, respectively). However, the 30-min cooling was the most reproducible at 1 week, either as CVC or %BL (CV were 26% and 23%; ICC were 0.86 and 0.75, respectively). Local cooling was well tolerated by all volunteers. CONCLUSIONS: We propose in the present work a reproducible 30-min LDF cooling test. Such a tool could be of great interest to assess microvascular reactivity to local cooling in diseases such as Raynaud's syndrome, and to further evaluate drugs for such diseases.

Immunobiology of the Torque teno viruses and other anelloviruses.

Many features of the Torque teno virus and the other anelloviruses (AVs) that have been identified after this virus was discovered in 1997 remain elusive. The immunobiology of the AVs is no exception. However, evidence is progressively accumulating that at least some AVs have an interesting interplay with cells and soluble factors known to contribute to the homeostasis of innate and adaptive immunity. Evidence is also accumulating that this interplay can have a significant impact on how effectively an infected host can deal with superimposed infectious and non-infectious noxae. This review article discusses the scanty information available on these aspects and highlights the ones that would be more urgent to precisely understand in order to get an adequate assessment of how important for human health these extremely ubiquitous and pervasive viruses really are.

Assessment of prevalence and load of torquetenovirus viraemia in a large cohort of healthy blood donors.

OBJECTIVES: Torquetenovirus (TTV) is an emerging marker of functional immune competence with the potential to predict transplant-related adverse events. A large-scale epidemiological study was performed to understand how basal values vary in healthy individuals according to age and gender. METHODS: We tested plasma from 1017 healthy blood donors aged 18-69 years. The presence and load of TTV were determined by a real-time PCR assay. A sub-cohort of 384 donors was tested for anti-cytomegalovirus IgG antibodies, and 100 participants were also tested for TTV viraemia on a paired whole blood sample. RESULTS: The overall prevalence of TTV was 65% (657/1017) with a mean (±SD) growth of 5 ± 4% every 10 years of age increase, but stably higher in males (465/690, 67%) than in females (192/327, 59%). Mean (±SD) TTV load was 2.3 ± 0.7 Log copies/mL with no sex difference. TTV viraemia showed modest increases along 10-year age intervals (mean ± SD: 0.3 ± 0.1). TTV viraemia in donors sampled 2 years later remained stable (mean ± SD: 2.3 ± 0.8 versus 2.2 ± 0.7 Log copies between samples). Twenty-six per cent (9/34) of blood donors with TTV-negative plasma scored positive when whole blood was tested, and the donors with positive plasma showed a mean (±SD) 1.4 ± 0.5 Log increase in copy numbers when whole blood was tested. CONCLUSIONS: This study establishes the mean value of TTV viraemia in plasma in healthy blood donors and suggests that ageing causes only minimal increases in TTV viraemia.

Effectiveness of nanofiltration in removing small non-enveloped viruses from three different plasma-derived products.

The objective of this study was to assess the ability of nanofiltration of albumin solution, prothrombin complex (PTC) and factor IX (FIX) to remove two small, non-enveloped DNA viruses, parvovirus B19 (B19V) and torque teno virus (TTV). Virus removal was investigated with down-scale experiments performed with sequential steps of 35-nm and 15-nm nanofiltrations of products spiked with virus DNA-positive sera. Viral loads were determined by real-time PCRs. The 15-nm nanofiltration removed more than 4.0 B19V log from all the products, TTV was reduced of more than 3.0 log from albumin solution and FIX by 35-nm and 15-nm nanofiltrations, respectively, being viral DNA undetectable after these treatments. Traces of TTV were still found in PTC after the 15-nm nanofiltration. In conclusion, nanofiltration can be efficacious in removing small naked viruses but, since viruses with similar features can differently respond to the treatment, a careful monitoring of large-scale nanofiltration should be performed.

Future management of viral diseases: role of new technologies and new approaches in microbial interactions.

BACKGROUND: New technologies allow rapid detecting and counting of virus genomes in clinical specimens, defining susceptibility to specific antivirals, pinpointing molecular sequences correlated to virulence traits, and identifying viral and host factors driving resolution or chronicity of infections. As a result, during the past three decades the diagnostic virology laboratory has become crucial for patient care and an integral component of the multifarious armamentarium for patient management. This change in paradigm has caused obsolescence of methods once considered the reference standard of infectious disease diagnosis that were used to detect whole or specific components of virions in the specimen. OBJECTIVES: This review provides an overview of standard and novel technologies applied to molecular diagnosis of viral infections and illustrates some crucial points for correcting interpretation of the laboratory data. SOURCES: Peer-reviewed literature of topics pertinent to this review. CONTENT AND IMPLICATIONS: New technologies are reinventing the way virologic diagnoses are made, with a conversion to new, simpler-to-use platforms. Although indicated for the same purpose, not all methods are equal and can yield different results. Further, tests identifying multiple analytes at once can detect microorganisms present or activated as a result of pathologic processes triggered by other pathogens or noninfectious causes. Thus, new directions will have to be taken in the way in which the diagnoses of viral diseases are performed. This represents a breakthrough in the clinical virology laboratory.

Chromosome 11 segmental paternal isodisomy in amniocytes from two fetuses with omphalocoele: new highlights on phenotype-genotype correlations in Beckwith-Wiedemann syndrome.

BACKGROUND: The phenotypic variability in Beckwith-Wiedemann syndrome (BWS) reflects the genetic heterogeneity of the mechanism which by default leads to the deregulation of genes located at 11p15.5. Genotype-phenotype correlation studies have demonstrated an association between omphalocoele and CDKN1C/p57 mutations or hypermethylation. Paternal uniparental disomy 11 (pUPD11) has been described only in the mosaic condition with both uniparental and biparental cell lines, and no association with omphalocoele has been pointed out. METHODS: Two cases are presented here, in which a paternal segmental UPD11 was detected by molecular investigation of amniotic fluid cell cultures after the presence of apparently isolated omphalocoele was revealed in the fetuses by ultrasound scan. Further studies were performed on additional autoptic feto-placental tissues to characterise the distribution of the uniparental cell line and to unmask any biparental lineage in order to document in more detail the as yet unreported association between omphalocoele and pUPD11. RESULTS: Results on the UPD distribution profile showed that the abdominal organs have a predominant uniparental constitution. This condition could mimic the effect of CDKN1C/p57 inactivation, causing the omphalocoele. CONCLUSION: New genotype-phenotype correlations emerge from the investigated cases, suggesting that molecular analysis be extended to all cases with fetal omphalocoele in order to establish the incidence of pUPD11 in complete BWS and in monosymptomatic/mild forms.

Nature of packs used in propellant modeling.

In recent years we have constructed closely packed spheres using the Lubachevsky-Stillinger algorithm to generate morphological models of heterogeneous solid propellants. Improvements to the algorithm now allow us to create large polydisperse packs on a laptop computer, and to create monodisperse packs with packing fractions greater than 70% which display significant crystal order. The use of these models in the physical context motivates efforts to examine in some detail the nature of the packs, including certain statistical properties. We compare packing fractions for binary packs with long-known experimental data. Also, we discuss the near-neighbor number and the radial distribution function (RDF) for monodisperse packs and make comparisons with experimental data. We also briefly discuss the RDF for bidisperse packs. We also consider bounded monodisperse packs, and pay particular attention to the near-wall structure where we identify significant order.

Phytochemical and antioxidant analysis of eight Hypericum taxa from Central Italy.

Eight taxa of the Hypericum spp. growing in Central Italy (Appennino Umbro-Marchigiano) were analyzed by HPLC-DAD for constituents quantitation, for antioxidant and free radical scavenging activities. H. perforatum subsp. veronense was the richest in phenolic compounds and hyperforin was detected for the first time in H. hircinum subsp. majus. Significant values of antioxidant activity were found in the investigated Hypericum taxa.