Evaluation of the EasyNAT SARS-CoV-2 assay PCR test for the diagnosis of SARS-CoV-2 infection.

Reverse transcription polymerase chain reaction (RT-PCR) tests are commonly utilized in commercial settings but pose challenges due to labor-intensive procedures and extended response times during peak demand. In contrast, real-time fluorescence and isothermal amplification assays using Crossing Priming Amplification (CPA) offer faster genetic material analysis, eliminate subjectivity, and require less manipulation and personnel training. This study aimed to validate the EasyNAT SARS-CoV-2 Assay, a diagnostic kit based on CPA, using oral and nasopharyngeal samples. The EasyNAT kit was compared to the Xpert Xpress SARS-CoV-2 kit, evaluating 873 samples obtained during routine analysis at the Microbiology Laboratory of the Hospital Costa del Sol (Marbella, Spain). The overall sensitivity and specificity for the EasyNAT SARS-CoV-2 Assay were 79.1% (95%CI 74.5-83.7) and 99.5% (95%CI 98.7-100), respectively; with, validity index of 91.9%, positive predictive value of 98.9%, negative predictive value of 88.9%, positive likelihood ratio of 144.5, negative likelihood ratio of 0.21 and a total Youden Index of 0.79. Notably, sensitivity improved in fresh samples (91.4%), along with a high Youden Index (0.91). The EasyNAT SARS-CoV-2 Assay achieved a higher percentage of concordance in positive samples with Xpert Xpress SARS-CoV-2 when analyzing cycle threshold (Ct) intervals below 30 compared to intervals equal or greater than 30, and demons. In conclusion, the EasyNAT SARS-CoV-2 Assay demonstrated high sensitivity and agreement with Xpert Xpress SARS-CoV-2, particularly in fresh samples or when the signal was detected at Ct intervals below 30, indicating higher viral loads. This makes it suitable for rapid screening in various settings, including those with limited access to conventional molecular laboratory setting.

Human papillomavirus (HPV) detection in vaginal self-samples: evaluation of eNat® as an alternative suspension medium to ThinPrep®PreservCyt® for vaginal swabs.

Human Papillomavirus (HPV) testing on self-collected samples allows for improved coverage rates of cervical cancer (CC) screening programs. ThinPrep®PreservCyt® (HOLOGIC®, USA) medium is widely used for the suspension of cervical and vaginal self-samples. However, this medium is costly, toxic, and flammable, involving special handling procedures which make its use difficult in screening programs, particularly in low- and middle-income countries. This pilot study aimed to evaluate the analytical performance of eNat ® (Copan SpA), an alternative non-alcohol-based suspension medium, compared to ThinPrep®PreservCyt® (HOLOGIC®) for high-risk HPV (hrHPV) detection in vaginal self-collected swabs using three different real-time polymerase chain reaction (RT-PCR) HPV assays: Anyplex™II HPV28 (Seegene, Korea), Papilloplex® High Risk HPV (GeneFirst, UK), and HPV OncoPredict (Hiantis, Italy). 30 women, referred to colposcopy, were enrolled in this observational, prospective pilot study and asked to collect two vaginal self-taken samples, which were suspended in 5 mL of ThinPrep®PreservCyt® or eNat®. Nucleic acids were extracted from 200 µL using Microlab Nimbus platform (Seegene, Korea) and tested with the three different RT-PCR full-genotyping high-risk HPV assays. The HPV results of vaginal samples resuspended in the two different media were compared to those obtained from the reference clinician-collected cervical sample from the same woman. hrHPV detection in vaginal self-samples suspended in both media demonstrated a substantial agreement with cervical samples with the three assays under-investigation (0.667

Systemic Mastocytosis: Advances in Diagnosis and Current Management.

Mastocytosis is a rare hematologic disorder characterized by abnormal proliferation and accumulation of neoplastic mast cells in various body sites. Isolated skin involvement is termed cutaneous mastocytosis (CM) and the term systemic mastocytosis (SM) refers to multi-organ involvement, most commonly of the bone marrow, skin, liver, and spleen. A subset of patients with SM have an associated clonal hematologic neoplasm which is most commonly myelodysplastic syndrome, chronic myelomonocytic leukemia, or acute myelogenous leukemia and this entity is termed SM with associated hematological neoplasm (AHN). Bone marrow involvement is present in all patients regardless of the subtype of SM. The genetic hallmark of SM is a somatic gain-of-function point mutation within the KIT gene. Other molecular aberrations that have been reported include somatic mutations in TET2, SRSF2, ASXL1, CBL, RUNX1, and RAS and these are common in SM-AHN. The clinical presentation of SM can range from indolent to advanced depending on extent of mast cell burden and genetic profile. In the case of indolent SM, the goal of treatment is to control mediator release-related effects as well as to reduce mast cell burden. In the case of SM-AHN, therapy is primarily that of the AHN and allogeneic hematopoietic stem cell transplantation is the preferred therapy in suitable candidates.

Tumor Mutational Burden Guides Therapy in a Treatment Refractory POLE-Mutant Uterine Carcinosarcoma.

Gynecologic carcinosarcomas, previously known as malignant mixed Müllerian tumors, are uncommon malignancies that demonstrate an aggressive biology and lack a standard therapeutic approach. Molecular analyses have revealed recurrent alterations in chromatin remodeling genes, but clinical support for therapeutic significance is lacking. We prospectively identified a patient with refractory uterine carcinosarcoma whose tumor was subject to molecular profiling at diagnosis and again at radiographic progression. Initial molecular testing did not assess tumor mutational burden, DNA polymerase ɛ (POLE), or microsatellite status. After the failure of several lines of chemotherapy, comprehensive genomic profiling of a repeat biopsy identified two missense mutations of the exonuclease domain of POLE (P286R and T323A). Tumor mutational burden was elevated (169 mutations per DNA megabase), consistent with an ultramutator phenotype. As seen in previously reported POLE-endometrioid cases, our patient harbored alterations in PIK3CA, ARID1A, and PTEN and was microsatellite stable, with appreciable tumor-infiltrating lymphocytes. She achieved an ongoing durable response with pembrolizumab. This is the first report of programmed cell death protein 1 response in uterine carcinosarcoma. KEY POINTS: Uterine carcinosarcoma is an uncommon and aggressive histologic variant of endometrial carcinoma with a poor prognosis.Inactivating DNA polymerase ɛ (POLE) mutations have been associated with high tumor mutational burden (TMB) and response to immune checkpoint inhibition.To the authors' knowledge, this is the first report of response to immune checkpoint inhibitor therapy in a patient with uterine carcinosarcoma.This case further supports expanding genomic profiling to include assessment of tumor mutational burden across tumor types, given the potential for immune checkpoint inhibitor therapy in TMB-high tumors.

Parallel walk test: its correlation with balance and motor functions in people with chronic stroke.

OBJECTIVES: To investigate (1) the intrarater, interrater, and test-retest reliability of the times and scores generated in the parallel walk test (PWT); (2) their correlations with impairments and activity limitations of individuals with stroke; and (3) the cutoff times that best discriminate individuals with stroke from healthy elderly subjects. DESIGN: Cross sectional study. SETTING: University-based rehabilitation center. PARTICIPANTS: Participants (N=72) comprised individuals with stroke (n=37) and healthy individuals (n=35). INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: The PWT was administered along with the Fugl-Meyer Motor Assessment of the Lower Extremities (FMA-LE), handheld dynamometer measurements of ankle dorsiflexor and plantarflexor muscle strength, the 5-Times-Sit-to-Stand Test, the Berg Balance Scale (BBS), a limits of stability (LOS) test, the 10-m walk test (10-MWT), and the timed Up and Go (TUG) test. RESULTS: PWT times and scores showed good to excellent intrarater, interrater, and test-retest reliability in individuals with stroke. PWT times using paths of 3 different widths significantly correlated with FMA-LE scores, 5-Times-Sit-to-Stand Test times, BBS scores, some LOS test results, 10-MWT gait speed, and TUG test times. PWT times of 6.30 to 7.48 seconds, depending on the path width, were shown reliably to discriminate individuals with stroke from healthy individuals. CONCLUSION: The PWT is a reliable, easy-to-administer clinical tool for assessing dynamic walking balance in individuals with chronic stroke.

Strong expression of chemokine receptor CCR9 in diffuse large B-cell lymphoma and follicular lymphoma strongly correlates with gastrointestinal involvement.

The gastrointestinal (GI) tract is the most common site of extranodal B-cell lymphomas. However, it is unclear how neoplastic lymphoid cells preferentially home there. We hypothesize that expression of the GI-homing chemokine receptor CCR9 may account for the dissemination of B-cell lymphomas to the GI tract. To test our hypothesis, we compared the expression of CCR9 using immunohistochemistry on GI versus nodal diffuse large B-cell lymphoma and follicular lymphoma. We found that 27 (66%) of 41, 12 (29%) of 41, and 2 (5%) of 41 of GI lymphoma cases demonstrated 3+, 2+, and 1+ CCR9 staining, respectively. In contrast, 2 (5%) of 39, 5 (13%) of 39, 8 (20.5%) of 39, and 24 (61.5%) of 39 nodal-restricted lymphoma cases demonstrated 3+, 2+, 1+, and 0+ CCR9 staining (P < .0001). This was observed for both diffuse large B-cell lymphoma (P < .001) and follicular lymphoma (P < .001). We also compared the expression of CCR9 on nodal B-cell lymphomas with involvement of the GI tract with those restricted to the lymph node. We found that 10 (62%) of 16, 3 (19%) of 16, and 3 (19%) of 16 nodal lymphomas with GI involvement showed 3+, 2+, and 1+ CCR9 staining, respectively. In contrast, 2 (5%) of 39, 5 (13%) of 39, 8 (20.5%) of 39, and 24 (61.5%) of 39 nodal lymphomas without GI involvement demonstrated 3+, 2+, 1+, and 0+ CCR9 staining, respectively (P < .001). Our finding that CCR9 expression is elevated in the nodal lymphomas of patients with GI involvement suggests the potential clinical utility of chemokine receptor status, as assessed by immunohistochemistry, to potentially predict GI dissemination and progression to higher stage in patients who initially present with limited nodal-restricted disease.

Primary central nervous system histiocytic sarcoma presenting as a postradiation sarcoma: case report and literature review.

Histiocytic sarcoma (HS) is a rare neoplasm that occurs most commonly in the intestinal tract, skin, soft tissue, and lymph node. The incidence of primary central nervous system (CNS) HS is even rarer, with a total of 6 cases reported in the literature. An etiologic link has not been identified for CNS HS, and the current case of primary CNS HS is unique in that an etiologic link to prior radiation therapy is identified, associated with complex cytogenetic abnormalities in the tumor. Although radiation-associated sarcomas can present as any number of different pathologic entities, this is the first reported case of a radiation-associated CNS HS. The pathologic and immunophenotypic characteristics of this case, with a nearly obscuring heavy inflammatory infiltrate and expression of monocytic/histiocytic markers (CD163, CD68, CD4, fascin), are characteristic of CNS HS. A discussion of the differential diagnosis and review of relevant literature are presented.

Poorly differentiated adenocarcinoma arising from a cervical bronchial cyst.

Bronchogenic cysts with malignant change are rarely reported. We describe a case of poorly differentiated adenocarcinoma arising from a cervical bronchial cyst in a patient presenting with a thyroid mass, cervical lymphadenopathy, and initial biopsy suggestive of papillary thyroid carcinoma. The clinical presentation, intraoperative findings, radiographic images, and pathology slides are presented. To our knowledge, this is the first report of a poorly differentiated adenocarcinoma arising from a bronchial cyst in the cervical region.

ERK MAP kinase-activated Arf6 trafficking directs coxsackievirus type B3 into an unproductive compartment during virus host-cell entry.

Clathrin- and caveolae-mediated endocytosis have been implicated in the productive entry of many viruses into host cells. ADP-ribosylation factor 6 (Arf6)-dependent endocytosis is another endocytosis pathway that traffics from the cell surface and it is the only Arf that traffics at the plasma membrane. However, little is known about Arf6-dependent trafficking during virus entry. This study showed that coxsackievirus type B3 (CVB3) associated with decay-accelerating factor in non-polarized HeLa cells can be redirected into non-productive compartments by Arf6-dependent internalization, thus restricting infection. Overexpression of wild-type (WT) and constitutively active (CA) Arf6 in HeLa cells resulted in a 2.3- and 3.6-fold decrease in infection, respectively. A dominant-negative inhibitor of Arf6 recovered restriction of infection by WT-Arf6 and CA-Arf6. RNA interference of endogenous Arf6 resulted in a 3.3-fold increase in CVB3 titre in HeLa cells. It was shown that coxsackie-adenovirus receptor (CAR) ligation by virus or CAR-specific antibody could activate extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase family and lead to Arf6-mediated viral restriction. In the absence of ERK activation, CVB3 internalization into early endosomes was inhibited and subsequent infection was reduced, but Arf6-mediated restriction was also abolished. In conclusion, receptor-mediated signalling enhances CVB3 entry whilst also activating non-productive pathways of virus entry; thus, virus infection is an equilibrium of productive and non-productive pathways of entry.

Hypoxia divergently regulates production of reactive oxygen species in human pulmonary and coronary artery smooth muscle cells.

Acute hypoxia causes pulmonary vasoconstriction and coronary vasodilation. The divergent effects of hypoxia on pulmonary and coronary vascular smooth muscle cells suggest that the mechanisms involved in oxygen sensing and downstream effectors are different in these two types of cells. Since production of reactive oxygen species (ROS) is regulated by oxygen tension, ROS have been hypothesized to be a signaling mechanism in hypoxia-induced pulmonary vasoconstriction and vascular remodeling. Furthermore, an increased ROS production is also implicated in arteriosclerosis. In this study, we determined and compared the effects of hypoxia on ROS levels in human pulmonary arterial smooth muscle cells (PASMC) and coronary arterial smooth muscle cells (CASMC). Our results indicated that acute exposure to hypoxia (Po(2) = 25-30 mmHg for 5-10 min) significantly and rapidly decreased ROS levels in both PASMC and CASMC. However, chronic exposure to hypoxia (Po(2) = 30 mmHg for 48 h) markedly increased ROS levels in PASMC, but decreased ROS production in CASMC. Furthermore, chronic treatment with endothelin-1, a potent vasoconstrictor and mitogen, caused a significant increase in ROS production in both PASMC and CASMC. The inhibitory effect of acute hypoxia on ROS production in PASMC was also accelerated in cells chronically treated with endothelin-1. While the decreased ROS in PASMC and CASMC after acute exposure to hypoxia may reflect the lower level of oxygen substrate available for ROS production, the increased ROS production in PASMC during chronic hypoxia may reflect a pathophysiological response unique to the pulmonary vasculature that contributes to the development of pulmonary vascular remodeling in patients with hypoxia-associated pulmonary hypertension.

Impact of cytosine 5-halogens on the interaction of DNA with restriction endonucleases and methyltransferase.

Growing evidence from both prokaryotes and eukaryotes indicates that pyrimidine 5-methyl groups can have profound biological consequences that are mediated by the affinity of DNA-protein interactions. The presence of the 5-methyl group could potentially create a steric block preventing the binding of some proteins whereas the affinity of many other proteins is substantially increased by pyrimidine methylation. In this paper, we have constructed a series of oligonucleotides containing cytosine and a series of 5-substituted cytosine analogues including all halogens. This set of oligonucleotides has been used to probe the relationship between the size of the substituent and its capacity to modulate cleavage by the methylation-sensitive restriction endonucleases MspI and HpaII. Additionally, we have examined the impact of the halogen substitution on the corresponding bacterial methyltransferase (M.HpaII). We observed that MspI cleavage is only subtly affected by substituted cytosine analogues at the inner position of the CCGG recognition site. In contrast, HpaII cleaves cytosine-containing oligonucleotides completely whereas 5-fluorocytosine-containing oligonucleotides are cleaved at a reduced rate. The presence of the larger halogens Cl, Br, or I as well as a methyl group completely prevents cleavage by HpaII. These data suggest that the steric wall is encountered by HpaII slightly beyond the fluorine substituent, at about 2.65 A from the pyrimidine C5-position. It is known that 5-fluorocytosine in an oligonucleotide can form a covalent irreversible suicide complex with either prokaryotic or eukaryotic methyltransferases. Kinetic data reported here suggest that the 5-fluorocytosine-containing oligonucleotide can also inhibit M.HpaII by formation of a reversible, noncovalent complex. Our results indicate that although a 5-Cl substituent has electronic properties similar to 5-F, 5-chlorocytosine duplexes neither form a complex with M.HpaII nor inhibit enzymatic methylation. Emerging data suggest that halogenation of cytosine can occur in DNA in vivo from inflammation-mediated reactive molecules. The results reported here suggest that the inadvertent halogenation of cytosine residues in DNA could alter the affinity of sequence-specific DNA-binding proteins.