Neutrophil degranulation and severely impaired extracellular trap formation at the basis of susceptibility to infections of hemodialysis patients.

BACKGROUND: Chronic kidney disease patients are at increased risk of mortality with cardiovascular diseases and infections as the two leading causes of death for end-stage kidney disease treated with hemodialysis (HD). Mortality from bacterial infections in HD patients is estimated to be 100-1000 times higher than in the healthy population. METHODS: We comprehensively characterized highly pure circulating neutrophils from HD and healthy donors. RESULTS: Protein levels and transcriptome of HD patients' neutrophils indicated massive neutrophil degranulation with a dramatic reduction in reactive oxygen species (ROS) production during an oxidative burst and defective oxidative cellular signaling. Moreover, HD neutrophils exhibit severely impaired ability to generate extracellular NET formation (NETosis) in NADPH oxidase-dependent or independent pathways, reflecting their loss of capacity to kill extracellular bacteria. Ectopic hydrogen peroxidase (H2O2) or recombinant human SOD-1 (rSOD-1) partly restores and improves the extent of HD dysfunctional neutrophil NET formation. CONCLUSIONS: Our report is one of the first singular examples of severe and chronic impairment of NET formation leading to substantial clinical susceptibility to bacteremia that most likely results from the metabolic and environmental milieu typical to HD patients and not by common human genetic deficiencies. In this manner, aberrant gene expression and differential exocytosis of distinct granule populations could reflect the chronic defect in neutrophil functionality and their diminished ability to induce NETosis. Therefore, our findings suggest that targeting NETosis in HD patients may reduce infections, minimize their severity, and decrease the mortality rate from infections in this patient population.

High throughput SARS-CoV-2 variant analysis using molecular barcodes coupled with Next Generation Sequencing

The identification of SARS-CoV-2 variants across the globe and their implications on the outspread of the pandemic, infection potential and resistance to vaccination, requires modification of the current diagnostic methods to map out viral mutations rapidly and reliably. Here, we demonstrate that integrating DNA barcoding technology, sample pooling and Next Generation Sequencing (NGS) provide an applicable solution for large-population viral screening combined with specific variant analysis. Our solution allows high throughput testing by barcoding each sample, followed by pooling of test samples using a multi-step procedure. First, patient-specific barcodes are added to the primers used in a one-step RT-PCR reaction, amplifying three different viral genes and one human housekeeping gene (as internal control). Then, samples are pooled, purified and finally, the generated sequences are read using an Illumina NGS system to identify the positive samples with a sensitivity of 82.5% and a specificity of 97.3%. Using this solution, we were able to identify six known and one unknown SARS-CoV-2 variants in a screen of 960 samples out of which 258 (27%) were positive for the virus. Thus, our diagnostic solution integrates the benefits of large population and epidemiological screening together with sensitive and specific identification of positive samples including variant analysis at a single nucleotide resolution.