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

IntroductionTools to detect SARS-Coronavirus-2 variants of concern and track the ongoing evolution of the virus are necessary to support 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. MethodsAn 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. ResultsIndividual 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. DiscussionASP-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.

Mononeuritis multiplex: an unexpectedly common feature of severe COVID-19

The prolonged mechanical ventilation required by patients with severe COVID-19 is expected to result in significant Intensive Care Unit - Acquired Weakness (ICUAW) in many of the survivors. However, in our post-COVID-19 follow up clinic we have found that, as well as the anticipated global weakness related to loss of muscle mass, a significant proportion of these patients also have disabling focal neurological deficits relating to an axonal mononeuritis multiplex. Amongst the 69 patients with severe COVID-19 that have been discharged from the intensive care units in our hospital, we have seen 11 individuals (16%) with such neuropathies. In many instances, the multi-focal nature of the weakness in these patients was initially unrecognised as symptoms were wrongly assumed to simply relate to "critical illness neuropathy". While mononeuropathy is well recognised as an occasional complication of intensive care, our experience suggests that such deficits are common and frequently disabling in patients recovering from COVID-19.