ABSTRACT
Background. SARS-CoV-2 variants of concern (VOC) have challenged real-time reverse transcriptase polymerase chain reaction (RT-PCR) methods for the diagnosis of COVID-19. Methods. The CDC 2019-Novel Coronavirus real-time RT-PCR panel was modified to create a single-plex extraction-free proxy RT-PCR assay, VOCFast™. This assay uses the nucleocapsid N1 as well as novel primer/probe pairs to target VOC mutations in the Orf1a and spike (S) genes. For analytical validation of VOCFast, synthetic controls for the Wuhan, alpha/B.1.1.7, beta/B.1.351, and gamma/P.1 strains were tested at various concentrations. Clinical validation was performed using patient anterior nares swab and saliva specimens collected in the Denver, CO area between Nov 2020 and Feb 2021 or in March 2021. Orthogonal next-generation sequencing (NGS) was also performed. Results. Similar N1 quantification cycle (Cq) values corresponding to viral load were observed for all strains, suggesting that VOC mutations do not affect performance of the N1 primer/probe. Orf1a-mut and S1-mut primer/probes generated a stable high Cq value for the Wuhan strain. Conversely, Orf1a-mut Cq values were inversely correlated with viral load for all VOC. The S1-mut Cq was inversely correlated with viral load of the alpha strain, but did not reliably amplify beta/gamma VOC. The limit of detection was 8 copies/uL. The first set of COVID-19 patient specimens revealed no amplification using Orf1amut whereas 53% of specimens collected in Mar 2021 demonstrated amplification by Orf-1a. Orthogonal testing by the SARS-CoV-2 NGS Assay and COVID-DX software demonstrated that 12/12 alpha strains, 2/2 beta/gamma strains, and 33/33 Wuhan strains were correctly identified by VOCFast. Conclusion. The combination of the N1, Orf1a-mut, and S1-mut primers/probes in VOCFast can distinguish the Wuhan, alpha, and beta/gamma strains and it consistent with NGS results. Testing of clinical samples revealed that VOC emerged in Denver, CO in March 2021. Future work to discriminate beta, gamma, and emerging VOC is ongoing. In summary, VOCFast is an extraction-free RT-PCR assay for nasal swab and saliva specimens that can identify VOC with a turnaround time suitable for clinical testing.
ABSTRACT
Statistics show that 76.74% (4 688) of 6 109 patients with chronic wounds are over 50 years of age; the proportion of patients with underlying diseases in all age groups above 50 years ranges from 78.25% to 100.00%. Among the underlying diseases of chronic wound patients, the top four diseases are diabetes mellitus, cardiovascular and cerebrovascular diseases, hypertension, and respiratory diseases. The above underlying diseases and ages of patients are the susceptibility factors of coronavirus disease 2019 released by National Health Commission of China. It is an unavoidable fact that the patients with chronic wounds have to go to the hospital for treatment prescribed by the physician. At the same time, we found that quite a few patients preferred going far afield in choosing hospital for treatment due to various reasons. During the prevention and control of coronavirus epidemic, this " go far afield" style of seeking medical treatment may increase the exposure risk during travelling. Accordingly, we convened 36 wound care clinics in different regions in Shanghai to implement the " Five Measures" encouraging the patients with chronic wounds to seek medical treatment nearby. The principle of this operation is that when seeking medical treatment, patients with chronic wounds should try their best to reduce the travel distance as much as possible to minimize the exposure risk during the epidemic period, which will in turn support the campaign of epidemic prevention and control.