Virus variant-specific clinical performance of a SARS-CoV-2 rapid antigen test with focus on Omicron variants of concern.

OBJECTIVES: Antigen rapid diagnostic tests (Ag-RDTs) play an important role in the diagnosis of SARS-CoV-2. They are easier, quicker, and less expensive than the 'reference standard' RT-PCR and therefore widely in use. Reliable clinical data with respect to Ag-RDT performance in SARS-CoV-2 Omicron variants of concern (VOCs) are limited. Consequently, the objective of this study was to determine the impact different VOCs-especially Omicron-have on the clinical performance of an Ag-RDT. METHODS: We compared the clinical performance of the Sofia SARS-CoV-2 Ag-RDT to RT-PCR in a real-world, single-centre study in a clinical point-of-care setting in patients admitted to a large hospital via the emergency department from 2 November 2020 to 4 September 2022. RESULTS: Among 38 434 Ag-RDT/RT-PCR tandems taken, 1528 yielded a SARS-CoV-2 positive RT-PCR test result, with a prevalence of 4.0% (95% CI, 3.8-4.2). Overall sensitivity of the Ag-RDT was 63.7% (95% CI, 61.3-66.1) and overall specificity was 99.6% (95% CI, 99.5-99.6). Ag-RDT sensitivity was dependent on viral load (VL), because the sensitivity increased to 93.2% (95% CI, 91.5-94.6) in samples with a VL > 106 SARS-CoV-2 copies/mL. Furthermore, the Ag-RDT was more sensitive in men, and older patients. Variant-dependent sensitivity assessment showed that the sensitivity was significantly lower in Omicron-VOC (64.1%; 95% CI, 60.5-67.6) compared with SARS-CoV-2 wild-type samples (70.0%; 95% CI, 59,8-78,6) (binomial test; p value < 0.001). Analysing the limits of detection showed a 27 times higher 95% limit of detection for the Omicron-VOC BA.5 compared with the SARS-CoV-2 wild-type. DISCUSSION: Ag-RDT sensitivity for detection of patients with lower VLs and with Omicron-VOC is reduced, limiting the effectiveness of Ag-RDTs. However, Ag-RDTs are still an unreplaceable tool for widely available, quick, and inexpensive point-of-care SARS-CoV-2 diagnostics.

Primary head and neck cancer cell cultures are susceptible to proliferation of Epstein-Barr virus infected lymphocytes.

BACKGROUND: New concepts for a more effective anti-cancer therapy are urgently needed. Experimental flaws represent a major counter player of this development and lead to inaccurate and unreproducible data as well as unsuccessful translation of research approaches into clinics. In a previous study we have created epithelial cell cultures from head and neck squamous cell carcinoma (HNSCC) tissue. METHODS: We characterize primary cell populations isolated from human papillomavirus positive HNSCC tissue for their marker expression by RT-qPCR, flow cytometry, and immunofluorescence staining. Their sensitivity to MDM2-inhibition was measured using cell viability assays. RESULTS: Primary HNSCC cell cultures showed the delayed formation of spheroids at higher passages. These spheroids mimicked the morphology and growth characteristics of other established HNSCC spheroid models. However, expression of epithelial and mesenchymal markers could not be detected in these cells despite the presence of the HNSCC stem cell marker aldehyde dehydrogenase 1 family member A1. Instead, strong expression of B- and T-lymphocytes markers was observed. Flow cytometry analysis revealed a heterogeneous mixture of CD3 + /CD25 + T-lymphocytes and CD19 + B-lymphocytes at a ratio of 4:1 at passage 5 and transformed lymphocytes at late passages (≥ passage 12) with CD45 + CD19 + CD20 + , of which around 10 to 20% were CD3 + CD25 + CD56 + . Interestingly, the whole population was FOXP3-positive indicative of regulatory B-cells (Bregs). Expression of transcripts specific for the Epstein-Barr-virus (EBV) was detected to increase in these spheroid cells along late passages, and this population was vulnerable to MDM2 inhibition. HPV + HNSCC cells but not EBV + lymphocytes were detected to engraft into immunodeficient mice. CONCLUSIONS: In this study we present a primary cell culture of EBV-infected tumor-infiltrating B-lymphocytes, which could be used to study the role of these cells in tumor biology in future research projects. Moreover, by describing the detailed characteristics of these cells, we aim to caution other researchers in the HNSCC field to test for EBV-infected lymphocyte contaminations in primary cell cultures ahead of further experiments. Especially researchers who are interested in TIL-based adopted immunotherapy should exclude these cells in their primary tumor models, e.g. by MDM2-inhibitor treatment. BI-12-derived xenograft tumors represent a suitable model for in vivo targeting studies.

Performance of the Sofia SARS-CoV-2 rapid antigen test as frontline test in a university hospital, Germany.

The rapid and reliable detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of high importance for individual patient care and hospital infection prevention. We aimed to evaluate the performance of the Sofia SARS-CoV-2 antigen rapid diagnostic test (Ag-RDT) in comparison to real-time reverse-transcription polymerase chain reaction (RT-PCR). We conducted a prospective, monocentric cross-sectional study in an emergency department of a German university hospital from November 2020 to March 2021. We tested all samples using both Sofia SARS-CoV-2 Ag-RDT and real-time RT-PCR. A total of 7877 patients were included. Overall sensitivity of the Ag-RDT was 62.9% and specificity was 99.4%. Sensitivity varied across study months, whereas specificity remained high. Sensitivity increased to 94.2% in samples with a cycle threshold (Ct)-value ≤25. The Sofia Ag-RDT proved to be a rapid tool to detect samples with high viral loads (Ct-value ≤25) and might thus help to identify infectious patients.

Argatroban for anticoagulation of a blood salvage system - an ex-vivo study.

BACKGROUND: Blood salvage systems help to minimize intraoperative transfusion of allogenic blood. So far no data is available on the use of argatroban for anticoagulation of such systems. We conducted an ex-vivo trial to evaluate the effectiveness of three different argatroban doses as compared to heparin and to assess potential residual anticoagulant in the red cell concentrates. METHODS: With ethical approval and individual informed consent, blood of 23 patients with contraindications for use of blood salvage systems during surgery was processed by the Continuous-Auto-Transfusion-System (C.A.T.S. ® Cell Saver System, Fresenius Kabi, Bad Homburg, Germany) using 5,50 or 250 mg of argatroban or 25.000 U of heparin in 1000 ml saline for anticoagulation of the system. Emergency and high-quality washing modes were applied in random order. Patency of the system and residual amount of anticoagulants in the re-transfusion bag were measured. The collected blood was not re-infused, but only used for analysis of hematocrit, heparin and argatroban concentrations. RESULTS: Patency of the system was provided by all anticoagulants except for 3/8 cases with 5 mg of argatroban. Residual anticoagulant was found in 2/10 (20 %) heparin samples in two different patients (1 emergency and 1 high-quality washing) and in all argatroban samples. High quality washing eliminated 89-95 % and emergency washing 60-90 % of the initial argatroban concentration. Residual argatroban concentrations ranged from 55 ng ml(-1) to 6810 ng ml(-1), with initial argatroban concentrations of 5 and 250 mg, respectively. CONCLUSION: The C.A.T.S. does not reliably remove heparin and should therefore not be used in HIT patients. Anticoagulation with 50 and 250 mg argatroban, maintains the systems patency and is significantly removed during washing. In this ex-vivo study a concentration of 50 µg ml(-1) argatroban provided the best ratio of system patency and residual argatroban concentration. Additional dose-finding studies with different blood salvage systems are needed to evaluate the optimal argatroban concentration.

Seeding a human tendon matrix with bone marrow aspirates compared to previously isolated hBMSCs--an in vitro study.

Injuries of tendons and ligaments give rise to significant morbidity. Tissue engineering offers promising treatment concepts such as seeding a scaffold with human bone marrow stem cells (hBMSCs) to create high-quality tendon replacement tissue. HBMSCs are usually isolated and cultured prior to seeding. Studies evaluating if previous isolation is superior to seeding with bone marrow aspirates have not been published yet. The aim of this study was to compare these two seeding methods in terms of cell viability, proliferation and differentiation. HBMSCs were harvested from the iliac crest during routine trauma surgery. In group A the scaffold (human achilles tendons) was seeded with bone marrow aspirates, while in group B hBMSCs were isolated, harvested and then seeded. Constructs were stimulated in perfusion bioreactors according to established protocols. Mean cell proliferation was significantly increased (p< 0.05) on tendons seeded with bone marrow aspirates. Cell viability, the concentration of alkaline phosphatase in the perfused media and the synthesis of procollagen - III - polypeptide (PIIIP) were not significantly different when comparing the two groups. The synthesis of procollagen-I-polypeptide (PIP) was significantly increased on tendons seeded with previously isolated hBMSCs p < 0.05). The results indicate that seeding a human tendon matrix scaffold with bone marrow aspirates may be equal to seeding with previously isolated hBMSCs. This new seeding method could facilitate and speed up the tissue engineering process.

Oncofetal fibronectin mRNA is highly abundant in the blood of patients with papillary thyroid carcinoma and correlates with high-serum thyroid-stimulating hormone levels.

BACKGROUND: Papillary thyroid carcinoma (PTC) cells express oncofetal fibronectin (onfFN) mRNA, which may be useful to detect circulating tumor cells. The objective of this study was to determine the fraction of PTC patients having onfFN mRNA in their peripheral blood and to determine if onfFN mRNA levels are correlated with the status of the disease or with thyroid-stimulating hormone (TSH) serum concentrations. METHODS: This study included 95 PTC patients, who were previously treated by thyroidectomy and radioactive iodine administration. Patients were examined by cervical sonography, whole-body (131)I scintigraphy, thyroglobulin measurement, and onfFN mRNA quantification both when they were being treated with L-thyroxine (L-T4) and after L-T4 withdrawal. The mean value for onfFN mRNA in blood from 25 healthy subjects was used as control for the onfFN mRNA assay. RESULTS: After L-T4 withdrawal, serum TSH levels rose and onfFN mRNA was found in the peripheral blood of 33 of 64 (52%) disease-free patients, 15 of 23 (65%) patients with local residual disease, and 6 of 8 (75%) patients with known local or distant metastases. Continuous administration of L-T4 repressed serum TSH. In this state none of 17 (0%) disease-free patients and 1 of 4 (25%) patients with local residual disease had an elevated onfFN mRNA level, and 2 of 4 (50%) patients with metastasis had positive tests for serum onfFN mRNA. CONCLUSIONS: onfFN transcripts are highly abundant in the peripheral blood of patients with PTC. L-T4 withdrawal, which produced elevated serum TSH concentrations in these athyroidic patients, markedly increased the fraction with positive tests for serum onfFN mRNA at all stages of the disease. Analyzing onfFN mRNA in the absence of a TSH stimulus allows a much better discrimination of different states of PTC disease and, based on current concepts of the significance of circulating mRNA, may be a useful tool to detect circulating thyroid cancer cells.

Gilbert's syndrome and hyperbilirubinemia in protease inhibitor therapy--an extended haplotype of genetic variants increases risk in indinavir treatment.

BACKGROUND/AIMS: Gilbert's syndrome is a frequent genetic conjugation abnormality associated with adverse drug effects. Genetic UDP glucuronosyltransferase (UGT)1A gene variants can influence gene transcription, inducibility and glucuronidation activity. Protease inhibitors used in human immunodeficiency virus (HIV) infection and chronic viral hepatitis can inhibit UGTs. Indinavir (IDV) can lead to hyperbilirubinemia in Gilbert's syndrome (UGT1A1*28), which does not explain interindividual severity differences and may thus involve additional UGT1A variants. METHODS: One hundred and twenty-five HIV patients receiving IDV and 427 healthy blood donors were genotyped for the presence of UGT1A1*28, UGT1A3 -66T/C, UGT1A7 -57T/G, UGT1A7(N129K/R131K) using Taqman 5' nuclease assays. RESULTS: Hyperbilirubinemia was observed in 42%. UGT1A1*28 frequencies did not differ between HIV patients and controls but were significantly higher in hyperbilirubinemic patients. The frequency of homozygous carriers of the 4 UGT1A marker haplotype increased with hyperbilirubinemia affecting all patients with bilirubin levels >85 micromol/l. CONCLUSIONS: In IDV treatment the risk of severe hyperbilirubinemia is associated with genetic variants of the UGT1A3 and UGT1A7 genes in addition to Gilbert's syndrome (UGT1A1*28). This haplotype is a useful predictor of protease inhibitor-induced side effects.

Rapid allelic discrimination by TaqMan PCR for the detection of the Gilbert's syndrome marker UGT1A1*28.

Gilbert's syndrome causes mild, unconjugated hyperbilirubinemia and is present in approximately 10% of the Caucasian population. The basis of the disorder is a 70% reduction in bilirubin glucuronidation catalyzed by the UDP-glucuronosyltransferase 1A1 (UGT1A1), which, in Caucasians, is the result of a homozygous TA insertion into the promoter region of the UGT1A1 gene (UGT1A1*28). Homozygous carriers of UGT1A1*28 as well as those with additional UGT1A variants can suffer from severe irinotecan toxicity or jaundice during treatment with the protease inhibitor atazanavir. UGT1A1*28 genotyping identifies patients at risk for drug toxicity and can increase drug safety by dose individualization. Rapid and facile UGT1A1*28 genotyping is therefore of great clinical importance. Two hundred ninety-one patients with suspected Gilbert's syndrome were genotyped using the TaqMan 5'nuclease assay with minor groove binder-non fluorescent quench probes; results were confirmed by direct sequencing. Ninety-six patients (33%) were homozygous for UGT1A1*28, which was verified by direct sequencing of a different PCR product showing 100% concordance with the TaqMan PCR results. We describe a novel UGT1A1*28 genotyping method that employs allelic discrimination by TaqMan PCR. This assay provides a rapid, high-throughput, and cost-effective method for Gilbert's syndrome genotyping, which is of value for pretreatment screening of potential irinotecan toxicity. The method utilizes a technological platform that is widely used in clinical practice and could therefore be easily adapted for routine clinical applications.

Influence of fibrin glue on proliferation and differentiation of human bone marrow stromal cells seeded on a biologic 3-dimensional matrix.

For tissue engineering of bone, a carrier matrix and efficient cell seeding are desirable. This study analysed the effect of fibrin glue on bone marrow stromal cells (BMSC) adhesion, proliferation (MTS-Test), differentiation (alkaline phosphatase (AP), osteocalcin (OC), ELISA) and compared the results with cells seeded within culture media on a decellularized, xenogenic bone matrix. There was no significant difference regarding cell adhesion. Proliferation after one week was significantly increased without fibrin glue. AP was increased in both groups when compared with porous scaffolds without cells. OC secretion was increased under both seeding conditions. Microscopic investigation of the cells with fibrin-glue showed less cell-cell contacts. This study reveals that cell seeding with medium demonstrates similar adherence rates compared with fibrin glue. Fibrin glue significantly decreases cell proliferation. Cell differentiation with respect to ALP and OC is not affected. Further studies are required to assess the long term and in vivo effects of both methods with respect to BMSC viability and differentiation. Fibrin sealants seem not necessary to achieve cell adherence when using a porous bone matrix.

Hepatic expression of A disintegrin and metalloproteinase (ADAM) and ADAMs with thrombospondin motives (ADAM-TS) enzymes in patients with chronic liver diseases.

BACKGROUND/AIMS: ADAMs (A Disintegrin And Metalloprotease) are multifunctional, membrane-bound and soluble cell surface glycoproteins with numerous functions in cell physiology. We assessed the expression of ADAMs in fibrotic liver disease of different aetiologies and clarified whether the expression of ADAMs is related to histological staging of fibrosis. In addition, the expression of ADAMs was determined in different cell types of liver. METHODS: Seventy-one biopsy samples from patients with chronic liver diseases were analyzed for mRNA expression of ADAM-8, -9, -12, -28, -TS1, -TS2, matrix metalloprotease (MMP)-2, -9 and tissue inhibitor of metalloproteinases-1 and -2 by quantitative real-time RT-PCR. RESULTS: The ADAM expression in liver injury is independent of aetiology. A strong correlation between ADAM -9, -28, -TS1 versus MMP-2 and SMA was identified. Activated hepatic stellate cells (HSC) showed increased mRNA expression of ADAM-8, -9, -12, -28, -TS2 compared to quiescent HSC. Significant differences between histological stages of fibrosis were found for ADAM-28, MMP-2 and MMP-9. CONCLUSIONS: The results suggest that ADAMs are differentially expressed in the liver. We assume that ADAM-9, -TS1 and -TS2 play a crucial role in extracellular matrix remodeling during fibrotic processes in the liver.

Cyclic strain induces FosB and initiates osteogenic differentiation of mesenchymal cells.

Mechanical loading is crucial for bone remodeling and osteoblast differentiation. FosB belongs to the AP-1 family of transcription factors, a group of proteins known to regulate osteoblast differentiation and bone formation. In mice, FosB is rapidly induced by mechanical stress at the transcriptional level. The aim of this study was to determine the effect of different mechanical stretch patterns on FosB gene expression and on osteogenic differentiation of human osteoblast precursor cells. Human bone-marrow-derived mesenchymal precursor cells were grown in flexible silicone dishes and stimulated by a daily application of three rounds of 2 h of cyclic stretch of either 2% or 8% elongation at 1 Hz on 3 consecutive days using a special motor-driven apparatus. By real-time PCR, we quantified FosB mRNA and the expression of genes involved in osteoblast differentiation such as Runx2 and collagen 1 to determine the osteogenic effect of mechanical stretch. Stretching induced FosB transcription and the expression of osteoblast markers in partly committed human mesenchymal precursor cells in a stretch- and time-dependent manner. We conclude that cyclic stretch-induced FosB expression and the upregulation of osteoblast genes plays a role in osteogenic differentiation of human mesenchymal precursor cells.

Gilbert's disease and atazanavir: from phenotype to UDP-glucuronosyltransferase haplotype.

Gilbert's disease leads to intermittent non-hemolytic hyperbilirubinemia by a reduction of hepatic bilirubin glucuronidation associated with the presence of the UDP-glucuronosyltransferase (UGT) 1A1*28 polymorphism. It is considered benign because it does not result in hepatocellular damage. However, pharmacogenetic analyses have linked UGT1A1*28 to drug toxicity and cancer predisposition. The protease inhibitor atazanavir (ATV) is an inhibitor of hepatic UGT activity leading to hyperbilirubinemia in individual patients. Whether this is linked specifically to UGT1A1*28 or to more complex variants influencing glucuronidation is unclear. One hundred and six ATV-treated patients were characterized and genotyped for UGT1A1*28, the UGT1A3 (-66C) and UGT1A7 (-57G) promoter variants, and UGT1A7(129K/131K). ATV treatment increased median bilirubin levels from 10 to 41 micromol/L (P = .001) with hyperbilirubinemia exceeding 43 micromol/L in 37%. Hyperbilirubinemia over 43 micromol/L was significantly associated not only with UGT1A1*28 but also with UGT1A3-66C, UGT1A7-57G, and UGT1A7(129K/131K), although these variants do not naturally occur in linkage dysequilibrium in blood donors. Homozygous combinations of UGT1A1*28 with the other variants increased from 7.4% (normal bilirubin to 42 micromol/L) to 41% to 46.1% (43 to >85 micromol/L), and 100% (>85 micromol/L). All six patients with hyperbilirubinemia greater than 85 micromol/L were homozygous for all four variants identifying a haplotype inherited on a single allele. In conclusion, the genetic variant associated with Gilbert's disease is identified as part of a haplotype of four UGT1A variants spanning three genes at the UGT1A gene locus. This haplotype predisposes to hyperbilirubinemia in ATV treatment and may have an additional role as a pharmacogenomic risk factor for drug therapy.

Evaluation and quality assessment of glucose concentration measurement in blood by point-of-care testing devices.

BACKGROUND: In this study the analytical performance of eight glucose point-of-care testing (POCT) devices was evaluated. For this purpose, POCT measurement of glucose in heparinized blood collected from patients was paralleled by determination of the glucose concentration in the respective plasma by an analyzer (Hitachi 917) in the central laboratory, providing traceable results. METHODS: Trueness of POCT measurements was studied by comparing the plasma POCT values (mean of five measurements) with the results from the traceable measurement procedure (TMP). RESULTS: The percentage of POCT results within +/-6% of the TMP mean value ranged from 24% to 50%, depending on the POCT device. Within the reference interval of plasma glucose (4.4-6.0 mmol/L), up to 67% of the POCT values were lower than 4.4 mmol/L, leading to a false diagnosis of hypoglycemia. In the hypoglycemic range (<4.4 mmol/L) up to 29% of the POCT analyses were false normoglycemic. CONCLUSIONS: In conclusion, this study shows an insufficient trueness of glucose measurements by POCT devices in the normo- and hypoglycemic range. To improve quality assessment, sample splitting and simultaneous measurement of blood glucose concentration every 4 weeks by POCT devices and of plasma glucose concentration by a reliable TMP is recommended.

Expression and coordinated regulation of matrix metalloproteinases in chronic hepatitis C and hepatitis C virus-induced liver cirrhosis.

Matrix metalloproteinases (MMPs) are central to tissue remodelling; however, little is known about the temporal pattern and differential regulation of hepatic MMP expression in the course of chronic human liver disease. Using quantitative reverse transcription-PCR ELISA assays, we studied hepatic mRNA expression of MMP-1, -2, -3, -7, -9, -10, -11, -13 and -14 in patients with chronic hepatitis C and hepatitis C virus-induced end-stage liver cirrhosis and controls. Results were compared with histology, hepatic expression of tissue inhibitor of metalloproteinases (TIMP)-1, -2 and -3, procollagen types I and IV, laminin, and with circulating protein levels of hyaluronate, TIMP-1 and -2 and MMP proenzymes, as measured by ELISA. The impact of the MMP-3(-1171) promoter polymorphism on hepatic MMP-3 expression was analysed. Hepatic mRNA expression data identified differentially regulated groups of MMPs during the course of chronic hepatitis C, showing either steadily increasing mRNA expression with disease progression (MMP-1, -2, -7 and -14) or transiently elevated expression (MMP-9, -11 and -13). The first group closely correlated to the parameters of fibrogenesis. Hepatic MMP-3 expression was unrelated to disease stage, but was determined by the MMP-3(-1171) promoter polymorphism. In conclusion, MMP expression during the course of chronic hepatitis C appears to be a closely regulated process, with different clusters of coordinately regulated MMP genes being identified.