Magnetic hydrogel particles improve nanopore sequencing of SARS-CoV-2 and other respiratory viruses.

Presented here is a magnetic hydrogel particle enabled workflow for capturing and concentrating SARS-CoV-2 from diagnostic remnant swab samples that significantly improves sequencing results using the Oxford Nanopore Technologies MinION sequencing platform. Our approach utilizes a novel affinity-based magnetic hydrogel particle, circumventing low input sample volumes and allowing for both rapid manual and automated high throughput workflows that are compatible with Nanopore sequencing. This approach enhances standard RNA extraction protocols, providing up to 40 × improvements in viral mapped reads, and improves sequencing coverage by 20-80% from lower titer diagnostic remnant samples. Furthermore, we demonstrate that this approach works for contrived influenza virus and respiratory syncytial virus samples, suggesting that it can be used to identify and improve sequencing results of multiple viruses in VTM samples. These methods can be performed manually or on a KingFisher automation platform.

Hydrogel particles improve detection of SARS-CoV-2 RNA from multiple sample types.

Here we present a rapid and versatile method for capturing and concentrating SARS-CoV-2 from contrived transport medium and saliva samples using affinity-capture magnetic hydrogel particles. We demonstrate that the method concentrates virus from 1 mL samples prior to RNA extraction, substantially improving detection of virus using real-time RT-PCR across a range of viral titers (100-1,000,000 viral copies/mL) and enabling detection of virus using the 2019 nCoV CDC EUA Kit down to 100 viral copies/mL. This method is compatible with commercially available nucleic acid extraction kits (i.e., from Qiagen) and a simple heat and detergent method that extracts viral RNA directly off the particle, allowing a sample processing time of 10 min. We furthermore tested our method in transport medium diagnostic remnant samples that previously had been tested for SARS-CoV-2, showing that our method not only correctly identified all positive samples but also substantially improved detection of the virus in low viral load samples. The average improvement in cycle threshold value across all viral titers tested was 3.1. Finally, we illustrate that our method could potentially be used to enable pooled testing, as we observed considerable improvement in the detection of SARS-CoV-2 RNA from sample volumes of up to 10 mL.

"Rush" skin biopsy specimens in a tertiary medical center: diagnostic yield and clinical utility.

BACKGROUND: Skin biopsy specimens are submitted for "rush" or "stat" processing, thereby indicating a sense of urgency about the clinical situation. OBJECTIVE: Our purpose was to determine the patient population who underwent biopsy on a "rush" basis and assess the way in which interpretation of the skin biopsy specimen influences clinical management. METHODS: A retrospective study was performed on the "rush" biopsy specimens sequentially requested during 1 year. Histologic findings, the clinical situation, and the effect of the biopsy result on clinical management were determined. RESULTS: A total of 90 adult patients, many critically ill, were identified. The majority of patients were admitted to hematology-oncology services (58%). The other 42% were admitted to a variety of clinical services. The chief clinical concerns in hematology-oncology patients were graft-versus-host disease and cutaneous infection. Only 5.5% of biopsy specimens taken to exclude graft-versus-host disease were used in immediate clinical decision-making compared with 45% of biopsy specimens for oncology patients with suspected infection and 42% for all other services. CONCLUSION: Many urgent skin biopsy specimens were not used for immediate clinical decision-making. The usefulness of skin biopsy varies with the clinical situation, and some situations are more prone to yield equivocal histologic data. Knowledge of these situations may reduce the number of unhelpful biopsy specimens. Therapy based on clinical findings had often been initiated before receiving the biopsy results, but biopsy findings are often helpful as confirmatory data. Diagnostic findings were not necessary for a biopsy specimen to provide useful data and nonspecific findings interpreted in light of clinical findings were also useful.

Prognostic factors for cutaneous squamous cell and basal cell carcinoma. Determinants of risk of recurrence, metastasis, and development of subsequent skin cancers.

Squamous cell carcinoma and basal cell carcinoma are the most common cancers in humans. This article discusses general prognostic factors for these nonmelanocytic skin cancers. Anatomic and histologic considerations for both carcinomas are also presented.

The prognostic significance of basic fibroblast growth factor in cutaneous malignant melanoma.

Basic fibroblast growth factor (bFGF) is a growth factor and an angiogenesis factor which may play a role in the evolution of cutaneous malignant melanoma (CMM). In this study, we evaluated the distribution of bFGF in CMM using immunochemical methods and correlated the pattern of bFGF expression with the clinical course. Formalin-fixed, paraffin-embedded sections of 46 CMMs were immunostained with a high-affinity purified antibody raised against human bFGF. CMM were categorized into lesions that exhibited subsequent recurrence (local, regional and/or systemic) or recurrence-free lesions. The minimum follow-up time was 5 years. Expression of bFGF within the tumors and in peritumoral and intratumoral blood vessels was similar in the two groups. Comparable results were attained when 8 recurring vs 8 non-recurring CMM, selected from the above tumors, were matched for age, gender, anatomic site and tumor thickness. These results suggest that the biologic behavior of CMM may not be predicted by immunoreactivity to bFGF in CMM cells or in the local tumor vasculature.

Differentiation-specific alternative splicing of bovine papillomavirus late mRNAs.

Activation of the late promoter (PL) of bovine papillomavirus type 1 (BPV-1) is dependent on the differentiation state of keratinocytes and occurs in the upper layers of the bovine fibropapilloma. In this study, we show by in situ hybridization that a differentiation-specific pattern of BPV-1 late RNA splicing is also seen in the fibropapilloma. RNAs containing the 7385/3605 and 3764/5609 splice junctions were confined to the granular cell layer. In contrast, RNAs containing the 7385/3225 splice junction were present in both the granular and spinous layers. The switch in splice site usage in the granular cell layer limits the expression of the mRNA encoding the major capsid protein to these most terminally differentiated cells. Thus, BPV-1 late mRNA expression is regulated at both transcriptional and posttranscriptional levels.

Proliferative activity in metastatic malignant melanoma: comparison of pre-treatment and post-treatment metastases.

In this study, the proliferative activity of malignant melanoma metastases was assessed before and after isolated limb perfusion chemotherapy by quantitating AgNORs, mitoses and PCNA activity. No significant difference in either AgNOR count, mitotic activity or PCNA index was observed. We conclude that AgNOR count, mitotic activity and PCNA index were not significantly effected by isolated limb perfusion chemotherapy.

The human immunodeficiency virus type 1 Rev protein and the Rev-responsive element counteract the effect of an inhibitory 5' splice site in a 3' untranslated region.

A 5' splice site located in a 3' untranslated region (3'UTR) has been shown previously to inhibit gene expression. Natural examples of inhibitory 5' splice sites have been identified in the late 3'UTRs of papillomaviruses and are thought to inhibit viral late gene expression at early stages of the viral life cycle. In this study, we demonstrate that the interaction of the human immunodeficiency virus type 1 Rev protein with the Rev-responsive element (RRE) overcomes the inhibitory effects of a 5' splice site located within a 3'UTR. This was studied by using both a bovine papillomavirus type 1 L1 cDNA expression vector and a chloramphenicol acetyltransferase expression vector containing a 5' splice site in the 3'UTR. In both systems, coexpression of Rev enhanced cytoplasmic expression from vectors containing the RRE even when the RRE and the inhibitory 5' splice site were separated by up to 1,000 nucleotides. In addition, multiple copies of a 5' splice site in a 3'UTR were shown to act synergistically, and this effect could also be moderated by the interaction of Rev and the RRE. These studies provide additional evidence that at least one mechanism of Rev action is through interactions with the splicing machinery. We have previously shown that base pairing between the U1 small nuclear RNA and a 3'UTR 5' splice site is required for inhibition of gene expression. However, experiments by J. Kjems and P. A. Sharp (J. Virol. 67:4769-4776, 1993) have suggested that Rev acts on spliceosome assembly at a stage after binding of the U1 small nuclear ribonucleoprotein to the 5' splice site. This finding suggests that binding of additional small nuclear ribonucleoproteins, as well as other splicing factors, may be necessary for the inhibitory action of a 3'UTR 5' splice site. These data also suggest that expression of the papillomavirus late genes in terminally differentiated keratinocytes can be regulated by a viral or cellular Rev-like activity.

Cutaneous pathology in Wegener's granulomatosis. A clinicopathologic study of 75 biopsies in 46 patients.

While no cutaneous lesion is specific for Wegener's granulomatosis (WG), several histopathologic entities, including leukocytoclastic vasculitis and necrotizing granulomatous inflammation, are characteristic. This report details the histopathologic features of 75 cutaneous biopsies from 46 patients with WG. Biopsies were subdivided into histologic groups that included leukocytoclastic vasculitis (31%), granulomatous inflammation (GI) (19%), nonspecific ulceration (4%), superficial dermal and epidermal necrosis without inflammation (2.7%), erythema nodosum (2.7%), granuloma annulare (1%), chronic inflammation (31%), and acute inflammatory lesions without vasculitis (9%). No convincing example of granulomatous vasculitis was observed. The histopathologic subgroups were correlated with clinical features, and the results were compared with those from a control group of 82 WG patients with no skin involvement. We found that the histopathologic subgroups of leukocytoclastic vasculitis and granulomatous inflammation correlated with different clinical courses. Patients with leukocytoclastic vasculitis developed WG at an earlier age (median age, 30 years) than did the control group (median age, 45 years). Leukocytoclastic vasculitis developed shortly after onset of WG (median, 15 months vs. 35 months for patients with nonspecific chronic inflammation). All lesions occurred during active disease. Active disease with leukocytoclastic vasculitis was associated with a mean erythrocyte sedimentation rate twice that of active disease in the same patient when leukocytoclastic vasculitis was absent. The patients with leukocytoclastic vasculitis had more rapidly progressive and widespread WG than patients with granulomatous skin lesions or patients without skin lesions. A marked excess of joint and musculoskeletal symptoms and renal disease was seen in patients with leukocytoclastic vasculitis. Patients with granulomatous inflammation also developed WG at an early age (median age, 30 years) when compared with the control group. Cutaneous granulomatous lesions also developed shortly after presentation (median, 12 months). Only 64% of granulomatous biopsies were from patients with active disease. These patients frequently had neither renal nor pulmonary manifestations of WG, and their disease progressed at a slower rate than that of the patients with leukocytoclastic vasculitis. These findings suggest that the cutaneous lesions characteristic of WG may correlate with the activity, distribution, and course of the disease.

Granuloma annulare in patients with malignant lymphoma: clinicopathologic study of thirteen new cases.

BACKGROUND: Reports of necrobiotic granulomas or granuloma annulare in patients with malignant lymphoma are rare. OBJECTIVE: Our intent was to determine any unique clinical or histopathologic features in patients with granuloma annulare and lymphoma. METHODS: We reviewed the medical records and biopsy material from 13 patients with granuloma annulare and lymphoma. RESULTS: Three patients had Hodgkin's disease and 10 had non-Hodgkin's lymphoma. The granuloma annulare lesions showed typical histopathologic features. However, the clinical pattern was frequently atypical, with painful lesions in unusual locations including the palms and soles. Three patients displayed granulomatous inflammation in noncutaneous sites, either within the malignant lymphoma or in uninvolved tissues, and all three had atypical clinical presentations of granuloma annulare. CONCLUSION: Granuloma annulare with atypical clinical presentations may be associated with an underlying hematopoietic malignancy and may be part of a generalized granulomatous reaction to malignant lymphoma.

Differentiation-specific expression from the bovine papillomavirus type 1 P2443 and late promoters.

The papillomavirus life cycle is tightly linked with keratinocyte differentiation in squamous epithelia. Vegetative viral DNA replication begins in the spinous layer, while synthesis of capsid proteins and virus maturation is restricted to the most differentiated or granular layer of the epithelium. In this study, in situ hybridization of bovine fibropapillomas was used to demonstrate that the activity of two promoters of bovine papillomavirus type 1 (BPV-1) is regulated in a differentiation-specific manner. In situ hybridization with a late promoter (PL)-specific oligonucleotide probe suggested that PL is dramatically upregulated in the granular layer of the fibropapilloma. Northern (RNA) blot analysis of RNA from BPV-1-infected fibropapillomas indicated that the three major BPV-1 late-region mRNAs were transcribed from PL. These RNAs include the previously described L1 (major capsid) mRNA as well as two larger mRNAs. The two larger mRNAs were characterized and shown to contain the L2 (minor capsid protein) open reading frame as well as the L1 open reading frame. In contrast to PL, the P2443 promoter was maximally active in basal keratinocytes and the fibroma. The major mRNA transcribed from P2443 is the putative E5 oncoprotein mRNA which is spliced between nucleotides 2505 and 3225. No signal was detected above the basal layer with use of a probe specific for this mRNA. The E5 oncoprotein has previously been localized by immunoperoxidase staining to the granular cell layer as well as the basal cell layer of the fibropapilloma (S. Burnett, N. Jareborg, and D. DiMaio, Proc. Natl. Acad. Sci. USA 89:5665-5669, 1992). These data suggest that E5 proteins in the basal cell and granular cell layers are not translated from the same mRNA.

Carcinosarcoma of the adrenal cortex presenting with mineralocorticoid excess.

An adrenal carcinosarcoma is reported in a 79-year-old woman presenting with clinical signs of hyperaldosteronism. The tumor weighed 199 g and consisted of areas typical of adrenal carcinoma and areas of sarcoma. The sarcomatous component of the tumor showed osteogenic and chondroid differentiation. Vimentin stained both the carcinomatous and sarcomatous regions. Four months after resection, the patient developed metastases. This is the third reported case of adrenal carcinosarcoma and the only case in which hyperaldosteronism or bony differentiation was observed.