Pooled SARS-CoV-2 antigen tests in asymptomatic children and their caregivers: Screening for SARS-CoV-2 in a pediatric emergency department.

Background: Universal admission screening for SARS-CoV-2 in children and their caregivers (CG) is critical to prevent hospital outbreaks. We evaluated pooled SARS-CoV-2 antigen tests (AG) to identify infectious individuals while waiting for polymerase chain reaction (PCR) test results.

Methods: This single-center study was performed from November 5, 2020 to March 1, 2021.

Time-Dependent Apparent Increase in dd-cfDNA Percentage in Clinically Stable Patients Between One and Five Years Following Kidney Transplantation.

Background: Donor-derived cell-free DNA (dd-cfDNA) is reportedly a valuable tool for graft surveillance following kidney transplantation (KTx). Possible changes in dd-cfDNA(%) reference values over time have not been evaluated. For long-term monitoring after KTx, changes in host cfDNA might represent a biasing factor in dd-cfDNA(%) determinations.

The Impact of on Tacrolimus Pharmacokinetics and Outcome in Clinical Practice at a Single Kidney Transplant Center.

Although there is evidence that the variant should be considered in tacrolimus dosing in renal transplantation, its impact beyond tacrolimus dose requirements remains controversial. In a cohort of 121 kidney transplant recipients, we analyzed the , , and alleles and the variants , , and for their impact on exposure and dose requirement. Relevant clinical outcome measures such as acute rejection within the first year after transplantation, delayed graft function, and renal function at discharge (estimated glomerular filtration rate) were evaluated.

Absolute quantification of donor-derived cell-free DNA as a marker of rejection and graft injury in kidney transplantation: Results from a prospective observational study.

Donor-derived cell-free DNA (dd-cfDNA) is a noninvasive biomarker for comprehensive monitoring of allograft injury and rejection in kidney transplantation (KTx). dd-cfDNA quantification of copies/mL plasma (dd-cfDNA[cp/mL]) was compared to dd-cfDNA fraction (dd-cfDNA[%]) at prespecified visits in 189 patients over 1 year post KTx. In patients (N = 15, n = 22 samples) with biopsy-proven rejection (BPR), median dd-cfDNA(cp/mL) was 3.

Multicenter Evaluation of a New Electrochemiluminescence Immunoassay for Everolimus Concentrations in Whole Blood.

Background: The precise monitoring of everolimus, an immunosuppressant drug, is vital for transplant recipients due to its narrow therapeutic range. This study evaluated the analytical performance of a new electrochemiluminescence immunoassay (ECLIA) for everolimus concentrations in whole blood.

Methods: Accuracy, imprecision, and sensitivity studies for the Roche Elecsys everolimus ECLIA were performed at 5 European laboratories.

Therapeutic Drug Monitoring of Everolimus: Comparability of Concentrations Determined by 2 Immunoassays and a Liquid Chromatography Tandem Mass Spectrometry Method.

Background: Therapeutic drug monitoring is recommended to guide therapy with the immunosuppressant everolimus (EVL) in solid organ transplantation to prevent rejections and to limit toxicity. For therapeutic drug monitoring, predose EVL concentrations are measured in whole blood mainly by liquid chromatography tandem mass spectrometry (LC-MS/MS). In addition, 2 immunoassays [Quantitative Microsphere System (QMS) EVL and Elecsys EVL] are commercially available.

Simultaneous determination of mycophenolate and its metabolite mycophenolate-7-o-glucuronide with an isocratic HPLC-UV-based method in human plasma and stability evaluation.

Objectives: Mycophenolic acid (MPA) is an immunosuppressive agent which is commonly used in a fixed dose regime in solid organ transplantation. For clinical trials and therapeutic drug monitoring measuring plasma concentrations is necessary. Also, stability issues have to be addressed.

Performance of a phosphoflow assay to determine phosphorylation of S6 ribosomal protein as a pharmacodynamic read out for mTOR inhibition.

Objectives: The S6 ribosomal protein (S6RP) is phosphorylated by the mammalian target of rapamycin (mTOR). The objective of this study was to assess the analytical suitability of a commercial kit-based phosphoflow cytometry protocol using whole blood (WBS) to measure the level of phosphorylated S6RP (p-S6RP) in T-cell subsets to study the pharmacodynamic effects of mTOR inhibitors (mTORi).

Design And Methods: A kit was used for fixation and permeabilization of mitogen-stimulated cells, and p-S6RP was assessed separately in CD3+CD4+ and CD3+CD8+ cells by employing an anti-phospho-Ser235/236 antibody.

Analytical evaluation of a real-time PCR-based DNA demethylation assay to assess the frequency of naturally occurring regulatory T cells in peripheral blood.

Objectives: Regulatory T cells (Tregs) which may indicate operational tolerance provide a promising biomarker for individualization of immunosuppression. Naturally thymus-derived Tregs (nTregs) represent the major suppressive phenotype and can be identified by their demethylation status in the Tregs Specific Demethylated Region (TSDR) of the Forkhead-Box-P3 (FOXP3) gene using quantitative PCR (qPCR).

Design And Methods: The analytical performance of a TSDR demethylation qPCR assay was assessed in whole blood of healthy individuals (HI) and kidney transplant recipients (KTR).

Editorial: Immune monitoring in solid organ transplantation.

Solid organ transplantation is inevitably associated with the activation of the immune system of the graft recipient. An advanced knowledge of the immunological mechanisms leading to acute and chronic rejection, the advent of powerful immunosuppressive drugs, and refined surgical techniques have made solid organ transplantation a standard therapy to replace irretrievable loss of vital functions. The immune system is a complex network involving immune cells, cytokines, chemokines, antibodies, and the complement system.

Donor-Derived Cell-Free DNA Is a Novel Universal Biomarker for Allograft Rejection in Solid Organ Transplantation.

Background: In solid organ transplantation, sensitive real-time biomarkers to assess the graft health are desirable to enable early intervention, for example, to avoid full-blown rejections. During rejection, high amounts of graft-derived cell-free DNA (GcfDNA) are shed into the blood stream. The quantification of this GcfDNA in allotransplantation is considered to fulfill this need, because it can be measured with great precision and at reasonable cost.

T-Cell Surface Antigens and sCD30 as Biomarkers of the Risk of Rejection in Solid Organ Transplantation.

T-cell activation is a characteristic of organ rejection. T cells, located in the draining lymph nodes of the transplant recipient, are faced with non-self-molecules presented by antigen presenting cells and become activated. Activated T cells are characterized by up-regulated surface antigens, such as costimulatory molecules, adhesion molecules, chemokine receptors, and major histocompatibility complex class II molecules.

Lymphocyte surface molecules as immune activation biomarkers.

Immunosuppression is mandatory after solid organ transplantation between HLA mismatched individuals. It is a lifelong therapy that needs to be closely monitored to avoid under- and over-immunosuppression. For many drugs, pharmacokinetic monitoring has been proven to be beneficial.

Association between adverse effects under azathioprine therapy and inosine triphosphate pyrophosphatase activity in patients with chronic inflammatory bowel disease.

Background: Inosine triphosphate pyrophosphatase (ITPA) catalyzes the pyrophosphohydrolysis of inosine triphosphate to inosine monophosphate. Recently, single-nucleotide polymorphisms in the ITPA gene, associated with decreased enzyme activity, have been reported. Some clinical studies have demonstrated that the 94C>A mutation is linked to flu-like symptoms, rash, and pancreatitis during azathioprine (AZA) therapy and to early AZA discontinuation.

Simultaneous quantification and genotyping of hepatitis C virus RNA by a two-step real-time PCR assay on the lightcycler instrument.

Background: Clinically, both viral load and genotypes have been found to be major predictors of antiviral therapy outcome regarding chronic hepatitis C and they are, under normal circumstances, performed as separate assays.

Design And Methods: In order to improve the diagnostic strategy and subsequently reduce the reagent costs we have developed and established the simultaneous quantification and genotyping of hepatitis C virus RNA by a two-step real-time PCR on the LightCycler Instrument (Roche Diagnostics).

Results: The quantification assay was calibrated against WHO Standard 96/790.

Investigation of the crossreactivity of mycophenolic acid glucuronide metabolites and of mycophenolate mofetil in the Cedia MPA assay.

The immunosuppressant mycophenolic acid (MPA) used for solid organ transplantation is predominantly metabolized to a pharmacologically inactive phenolic glucuronide (MPAG) and, to a lesser extent, to the pharmacologically active acyl glucuronide (AcMPAG). The recently introduced CEDIA Mycophenolic Acid Assay from Microgenics has been reported to overestimate MPA in clinical samples and crossreactivity with AcMPAG has been suspected. A detailed investigation of the crossreactivity of AcMPAG and the prodrug mycophenolate mofetil (MMF) in the CEDIA assay is presented using pure substances.

Two-step real-time PCR quantification of all subtypes of human immunodeficiency virus type 1 by an in-house method using locked nucleic acid-based probes.

Current HIV-1 viral-load assays are too expensive and time-consuming for small sample quantity or resource-limited setting. In addition, some commercial assays have shown shortcomings in quantifying rare genotypes. We developed an internally controlled, two-step, reverse transcription-initiated real-time PCR protocol on the LightCycler instrument achieving a favourable detection limit with an extended quantification range, detecting all HIV-1 subtypes suitable for laboratories with low sample throughput.