Highly-Sensitive Lineage Discrimination of SARS-CoV-2 Variants through Allele-Specific Probe Polymerase Chain Reaction

Introduction: Tools to detect SARS-Coronavirus-2 variants of concern and track the ongoing evolution of the virus are necessary to support ongoing public health efforts and the design and evaluation of novel COVID-19 therapeutics and vaccines. Although next-generation sequencing (NGS) has been adopted as the gold standard method for discriminating SARS-CoV-2 lineages, alternative methods may be required when processing samples with low viral loads or low RNA quality. Methods: An allele-specific probe polymerase chain reaction (ASP-PCR) targeting lineage-specific single nucleotide polymorphisms (SNPs) was developed and used to screen 1,082 samples from two clinical trials in the United Kingdom and Brazil. Probit regression models were developed to compare ASP-PCR performance against 1,771 NGS results for the same cohorts. Results: Individual SNPs were shown to readily identify specific variants of concern. ASP-PCR was shown to discriminate SARS-CoV-2 lineages with a higher likelihood than NGS over a wide range of viral loads. Comparative advantage for ASP-PCR over NGS was most pronounced in samples with Ct values between 26-30 and in samples that showed evidence of degradation. Results for samples screened by ASP-PCR and NGS showed 99% concordant results. Discussion: ASP-PCR is well-suited to augment but not replace NGS. The method can differentiate SARS-COV-2 lineages with high accuracy and would be best deployed to screen samples with lower viral loads or that may suffer from degradation. Future work should investigate further destabilization from primer:target base mismatch through altered oligonucleotide chemistry or chemical additives.

Elastin-specific MRI of extracellular matrix-remodelling following hepatic radiofrequency-ablation in a VX2 liver tumor model

Hepatic radiofrequency ablation (RFA) induces a drastic alteration of the biomechanical environment in the peritumoral liver tissue. The resulting increase in matrix stiffness has been shown to significantly influence carcinogenesis and cancer progression after focal RF ablation. To investigate the potential of an elastin-specific MR agent (ESMA) for the assessment of extracellular matrix (ECM) remodeling in the periablational rim following RFA in a VX2 rabbit liver tumor-model, twelve New-Zealand-White-rabbits were implanted in the left liver lobe with VX2 tumor chunks from donor animals. RFA of tumors was performed using a perfused RF needle-applicator with a mean tip temperature of 70 °C. Animals were randomized into four groups for MR imaging and scanned at four different time points following RFA (week 0 [baseline], week 1, week 2 and week 3 after RFA), followed by sacrifice and histopathological analysis. ESMA-enhanced MR imaging was used to assess ECM remodeling. Gadobutrol was used as a third-space control agent. Molecular MR imaging using an elastin-specific probe demonstrated a progressive increase in contrast-to-noise ratio (CNR) (week 3: ESMA: 28.1 ± 6.0; gadobutrol: 3.5 ± 2.0), enabling non-invasive imaging of the peritumoral zone with high spatial-resolution, and accurate assessment of elastin deposition in the periablational rim. In vivo CNR correlated with ex vivo histomorphometry (ElasticaVanGiesson-stain, y = 1.2x − 1.8, R2 = 0.89, p < 0.05) and gadolinium concentrations at inductively coupled mass spectroscopy (ICP-MS, y = 0.04x + 1.2, R2 = 0.95, p < 0.05). Laser-ICP-MS confirmed colocalization of elastin-specific probe with elastic fibers. Following thermal ablation, molecular imaging using an elastin-specific MR probe is feasible and provides a quantifiable biomarker for the assessment of the ablation-induced remodeling of the ECM in the periablational rim.

Feasibility study of a 3D-printed personalized immobilization device integrating a patient-specific probe holder for prostate radiotherapy combined with adjuvant MR-guided transperineal ultrasound hyperthermia

The authors have requested that this preprint be removed from Research Square.

Hyperpolarized 15N-labeled, deuterated tris(2-pyridylmethyl)amine as an MRI sensor of freely available Zn2+

Dynamic nuclear polarization (DNP) coupled with 15N magnetic resonance imaging (MRI) provides an opportunity to image quantitative levels of biologically important metal ions such as Zn2+, Mg2+ or Ca2+ using appropriately designed 15N enriched probes. For example, a Zn-specific probe could prove particularly valuable for imaging the tissue distribution of freely available Zn2+ ions, an important known metal ion biomarker in the pancreas, in prostate cancer, and in several neurodegenerative diseases. In the present study, we prepare the cell-permeable, 15N-enriched, d6-deuterated version of the well-known Zn2+ chelator, tris(2-pyridylmethyl)amine (TPA) and demonstrate that the polarized ligand had favorable T1 and linewidth characteristics for 15N MRI. Examples of how polarized TPA can be used to quantify freely available Zn2+ in homogenized human prostate tissue and intact cells are presented.