Evaluation of Swab-Seq as a scalable, sensitive assay for community surveillance of SARS-CoV-2 infection.

The ongoing SARS-CoV-2 pandemic and subsequent demand for viral testing has led to issues in scaling diagnostic lab efforts and in securing basic supplies for collection and processing of samples. This has motivated efforts by the scientific community to establish improved protocols that are more scalable, less resource intensive, and less expensive. One such developmental effort has resulted in an assay called "Swab-Seq", so named because it was originally developed to work with dry nasal swab samples. The existing gold standard test consists of RNA extracted from a nasopharyngeal (NP) swab that is subjected to quantitative reverse transcription polymerase chain reaction (qRT-PCR). Swab-Seq adapts this method to a next-generation sequencing readout. By pairing this modification with extraction-free sampling techniques, Swab-Seq achieves high scalability, low cost per sample, and a reasonable turnaround time. We evaluated the effectiveness of this assay in a community surveillance setting by testing samples collected from both symptomatic and asymptomatic individuals using the traditional NP swab. In addition, we evaluated extraction-free sampling techniques (both saliva and saline mouth gargle samples). We found the assay to be as clinically sensitive as the qRT-PCR assay, adaptable to multiple sample types, and able to easily accommodate hundreds of samples at a time. We thus provide independent validation of Swab-Seq and extend its utility regarding sample type and sample stability. Assays of this type greatly expand the possibility of routine, noninvasive, repeated testing of asymptomatic individuals suitable for current and potential future needs.

Age-related arterial immune cell infiltration in mice is attenuated by caloric restriction or voluntary exercise.

Age-related arterial inflammation is associated with dysfunction of the arteries and increased risk for cardiovascular disease. To determine if aging increases arterial immune cell infiltration as well as the populations of immune cells principally involved, we tested the hypothesis that large elastic and resistance arteries in old mice would exhibit increased immune cell infiltration compared to young controls. Additionally, we hypothesized that vasoprotective lifestyle interventions such as lifelong caloric restriction or 8weeks of voluntary wheel running would attenuate age-related arterial immune cell infiltration. The aorta and mesenteric vasculature with surrounding perivascular adipose was excised from young normal chow (YNC, 4-6months, n=10), old normal chow (ONC, 28-29months, n=11), old caloric restricted (OCR, 28-29months, n=9), and old voluntary running (OVR, 28-29months, n=5) mice and digested to a single cell suspension. The cells were then labeled with antibodies against CD45 (total leukocytes), CD3 (pan T cells), CD4 (T helper cells), CD8 (cytotoxic T cells), CD19 (B cells), CD11b, and F4/80 (macrophages) and analyzed by flow cytometry. Total leukocytes, T cells (both CD4+ and CD8+ subsets), B cells, and macrophages in both aorta and mesentery were all 5- to 6-fold greater in ONC compared to YNC. Age-related increases in T cell (both CD4+ and CD8+), B cell, and macrophage infiltration in aorta were abolished in OCR mice. OVR mice exhibited 50% lower aortic T cell and normalized macrophage infiltration. B cell infiltration was not affected by VR. Age-related mesenteric CD8+ T cell and macrophage infiltration was normalized in OCR and OVR mice compared to young mice, whereas B cell infiltration was normalized by CR but not VR. Splenic CD4+ T cells from ONC mice exhibited a 3-fold increase in gene expression for the T helper (Th) 1 transcription factor, Tbet, and a 4-fold increase in FoxP3, a T regulatory cell transcription factor, compared to YNC. Splenic B cells and mesenteric macrophages from old mice exhibited decreased proinflammatory cytokine gene expression regardless of treatment group. These results demonstrate that aging is associated with infiltration of immune cells around both the large-elastic and resistance arteries and that the vasoprotective lifestyle interventions, CR and VR, can ameliorate age-related arterial immune cell infiltration.