Background . The increased transmission of SARS-CoV-2 variants of concern (VOC) which originated in the United Kingdom (B.1.1.7/alpha), South Africa (B1.351/Beta), Brazil (P.1/Gamma), in the United States (B.1.427/429 or Epsilon) and in India (B.1.617.2/Delta) requires a vigorous public health response, including real time strain surveillance on a global scale. Although genome sequencing is the gold standard for identifying these VOCs, it is time consuming and expensive. Here, we describe a simple, rapid and high-throughput reverse-transcriptase PCR (RT-PCR) melting temperature (Tm) screening assay that identifies the first three major VOCs. Methods. RT-PCR primers and four sloppy molecular beacon (SMB) probes were designed to amplify and detect the SARS-CoV-2 N501Y (A23063T) and E484K (G23012A) mutations and their corresponding wild type sequences. After RT-PCR, the VOCs were identified by a characteristic Tm of each SMB. Assay optimization and testing was performed with RNA from SARS-CoV-2 USA WA1/2020 (WT), B.1.1.7 and B.1.351 variant strains. The assay was then validated using clinical samples. Results. The limit of detection (LOD) for both the WT and variants was 4 and 10 genomic copies/reaction for the 501 and 484 codon assays, respectively. The assay was 100% sensitive and 100% specific for identifying the N501Y and E484K mutations in cultured virus and in clinical samples as confirmed by Sanger sequencing. Conclusion. We have developed an RT-PCR melt screening test for the major VOCs which can be used to rapidly screen large numbers of patient samples providing an early warning for the emergence of these variants and a simple way to track their spread.

Background. The emergence of more transmissible SARS-CoV-2 variants in the United Kingdom (B.1.1.7), South Africa (B1.351) and Brazil (P.1) requires a vigorous public health response, including real time strain surveillance on a global scale. Although new SARS-CoV-2 variants can be most accurately identified by genomic sequencing, this approach is time consuming and expensive. A simple and more rapid screen for the key SARS-CoV-2 mutations that define variant strains is needed. We have developed a simple, rapid and high-throughput reverse-transcriptase PCR (RT-PCR) melting temperature assay that identifies the SARS-CoV-2 N501Y mutation, a key mutation which is present in all three known variant strains of concern. Methods. RT-PCR primers and two sloppy molecular beacon (SMB) probes were designed to amplify and detect the SARS-CoV-2 N501Y (A23063T) mutation. One SMB was designed with a probe region that was complementary to the wild type sequence (WT) and a second SMB was designed with a probe region that was complementary to the mutant (MT) sequence. Each SMB was labeled with a different fluorophore, and the assay was performed in a single test well. After RT-PCR, WT versus MT SARS-CoV-2 was identified by a characteristic shift in the melting temperature (Tm) of each SMB. Assay optimization and testing was performed with RNA from SARS-CoV-2 USA WA1/2020 (WT) and SARS-CoV-2 hCoV-19/England/204820464/2020 (a B.1.17 variant). The assay was then validated using clinical samples. Results. The limit of detection (LOD) of the assay for both the WT and the B1.1.7 variant was 4 genomic copies/reaction. The two SMBs produced Tm shifts that were 100% sensitive and 100% specific for identifying the N501Y mutation in cultured virus and in clinical samples as confirmed by Sanger sequencing. Conclusion. We have developed a rapid screening test for the SARS-CoV-2 N501Y mutation, which is a characteristic of all three SARS-CoV-2 stains of global concern. This assay can be used to rapidly screen large numbers of patient samples for these variants, providing an early warning for the emergence and spread of these strains of concern.

Background COVID-19 is a multi-system infection with emerging evidence-based antiviral and anti-inflammatory therapies to improve disease prognosis. However, a subset of patients with COVID-19 signs and symptoms have repeatedly negative RT-PCR tests, leading to treatment hesitancy. We used comparative serology early in the COVID-19 pandemic when background seroprevalence was low to estimate the likelihood of COVID-19 infection among RT-PCR negative patients with clinical signs and/or symptoms compatible with COVID-19. Methods Between April and October 2020, we conducted serologic testing of patients with (i) signs and symptoms of COVID-19 who were repeatedly negative by RT-PCR (‘Probables’; N = 20), (ii) signs and symptoms of COVID-19 but with a potential alternative diagnosis (‘Suspects’; N = 15), (iii) no signs and symptoms of COVID-19 (‘Non-suspects’; N = 43), (iv) RT-PCR confirmed COVID-19 patients (N = 40), and (v) pre-pandemic samples (N = 55). Results Probables had similar seropositivity and levels of IgG and IgM antibodies as propensity-score matched RT-PCR confirmed COVID-19 patients (60.0% vs 80.0% for IgG, p-value = 0.13; 50.0% vs 72.5% for IgM, p-value = 0.10), but multi-fold higher seropositivity rates than Suspects and matched Non-suspects (60.0% vs 13.3% and 11.6% for IgG; 50.0% vs 0% and 4.7% for IgM respectively; p-values < 0.01). However, Probables were half as likely to receive COVID-19 treatment than the RT-PCR confirmed COVID-19 patients with similar disease severity. Conclusions Findings from this study indicate a high likelihood of acute COVID-19 among RT-PCR negative with typical signs/symptoms, but a common omission of COVID-19 therapies among these patients. Clinically diagnosed COVID-19, independent of RT-PCR positivity, thus has a potential vital role in guiding treatment decisions.

Saliva has been a COVID-19 diagnostic specimen of interest due to its simple collection, scalability, and yield. Yet COVID-19 testing and estimates of the infectious period remain largely based on nasopharyngeal and nasal swabs. We sought to evaluate whether saliva testing captured prolonged presence of SARS-CoV-2 and potential infectiousness later in the disease course. We conducted an observational study of symptomatic COVID-19 patients at University Hospital in Newark, NJ. Paired saliva and nasal specimens from 96 patients were analyzed, including longitudinal analysis of paired observations from 28 of these patients who had multiple time-points. Saliva detected significantly more cases of COVID-19 beyond 5 days (86.1% [99/115] saliva vs 48.7% [56/115] nasal, p-value < 0.001), 9 days (79.4% [50/63] saliva vs 36.5% [23/63] nasal, p-value < 0.001) and 14 days (71.4% [20/28] saliva vs 32.1% [9/28] nasal, p-value = 0.010) of symptoms. Additionally, saliva yielded lower cycle thresholds across all time periods, indicative of higher viral loads in saliva. In the longitudinal analysis, a log-rank analysis indicated that the survival curve for saliva was significantly different from the curve for nasal swabs (p<0.001) with a median survival time for saliva of 18 days compared to 13 days for nasal swabs. We additionally performed saliva viral cultures among a similar COVID-19 patient cohort and noted patients with positive saliva viral cultures between 7 to 28 days of symptoms. Findings from this study suggest that SARS-CoV-2 RNA persists longer and in higher abundance in saliva compared to nasal swabs, with potential of prolonged propagating virus. Testing saliva may thus increase yield for detecting potentially infectious virus even beyond the first five days of symptomatic COVID-19.

Plasmacytoid dendritic cells (pDC) are innate immune cells and potent producers of interferon alpha (IFN-α). Sars-CoV-2, an RNA virus that causes Coronavirus Disease 2019 (COVID-19), has taken the lives of more than 400,000 people in the United States. Reports indicate that COVID-19 patients have reduced plasma IFN-α, suggesting a potential use of IFN-α as a disease therapeutic. However, investigations on pDC function and phenotype in COVID-19 patients are needed. We isolated peripheral blood mononuclear cells from fifty hospitalized COVID-19 patients. PBMC were stimulated with HSV-1 or Influenza A virus and IFN-α production and phenotype were assessed by flow cytometry. After stimulation, there were fewer IFN-α+ pDC from COVID-19 patients compared to controls. To reduce inflammation, COVID-19 patients may be treated with dexamethasone, a corticosteroid that has negative effects on pDC function. Although there was more impairment of pDC numbers and function in dexamethasone-treated subjects, the pDC dysregulation was also seen prior to dexamethasone treatment. Phenotypically, we identified reduced expression of pDC markers BDCA2 and CD123, and an upregulation of co-stimulatory markers on pDC from COVID-19 patients. We also observed an increased proportion of Ki67+ pDCs, which indicates increased turnover of pDCs during moderate to severe COVID-19 disease. In summary, pDC from COVID-19 patients produce significantly less IFN-α, express costimulatory ligands used to stimulate adaptive immunity, and may be undergoing rapid turnover. Overall, these changes may compromise the antiviral and adaptive immune response or represent pDC exhaustion during SARS-CoV-2 infection.