SARS-CoV-2 Variants Associated with Vaccine Breakthrough in the Delaware Valley through Summer 2021.

The severe acute respiratory coronavirus-2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. Evidence suggests that the virus is evolving to allow efficient spread through the human population, including vaccinated individuals. Here, we report a study of viral variants from surveillance of the Delaware Valley, including the city of Philadelphia, and variants infecting vaccinated subjects. We sequenced and analyzed complete viral genomes from 2621 surveillance samples from March 2020 to September 2021 and compared them to genome sequences from 159 vaccine breakthroughs. In the early spring of 2020, all detected variants were of the B.1 and closely related lineages. A mixture of lineages followed, notably including B.1.243 followed by B.1.1.7 (alpha), with other lineages present at lower levels. Later isolations were dominated by B.1.617.2 (delta) and other delta lineages; delta was the exclusive variant present by the last time sampled. To investigate whether any variants appeared preferentially in vaccine breakthroughs, we devised a model based on Bayesian autoregressive moving average logistic multinomial regression to allow rigorous comparison. This revealed that B.1.617.2 (delta) showed 3-fold enrichment in vaccine breakthrough cases (odds ratio of 3; 95% credible interval 0.89-11). Viral point substitutions could also be associated with vaccine breakthroughs, notably the N501Y substitution found in the alpha, beta and gamma variants (odds ratio 2.04; 95% credible interval of1.25-3.18). This study thus overviews viral evolution and vaccine breakthroughs in the Delaware Valley and introduces a rigorous statistical approach to interrogating enrichment of breakthrough variants against a changing background. IMPORTANCE SARS-CoV-2 vaccination is highly effective at reducing viral infection, hospitalization and death. However, vaccine breakthrough infections have been widely observed, raising the question of whether particular viral variants or viral mutations are associated with breakthrough. Here, we report analysis of 2621 surveillance isolates from people diagnosed with COVID-19 in the Delaware Valley in southeastern Pennsylvania, allowing rigorous comparison to 159 vaccine breakthrough case specimens. Our best estimate is a 3-fold enrichment for some lineages of delta among breakthroughs, and enrichment of a notable spike substitution, N501Y. We introduce statistical methods that should be widely useful for evaluating vaccine breakthroughs and other viral phenotypes.

Anti-nucleocapsid antibody levels and pulmonary comorbid conditions are linked to post-COVID-19 syndrome.

BACKGROUNDProlonged symptoms after SARS-CoV-2 infection are well documented. However, which factors influence development of long-term symptoms, how symptoms vary across ethnic groups, and whether long-term symptoms correlate with biomarkers are points that remain elusive.METHODSAdult SARS-CoV-2 reverse transcription PCR-positive (RT-PCR-positive) patients were recruited at Stanford from March 2020 to February 2021. Study participants were seen for in-person visits at diagnosis and every 1-3 months for up to 1 year after diagnosis; they completed symptom surveys and underwent blood draws and nasal swab collections at each visit.RESULTSOur cohort (n = 617) ranged from asymptomatic to critical COVID-19 infections. In total, 40% of participants reported at least 1 symptom associated with COVID-19 six months after diagnosis. Median time from diagnosis to first resolution of all symptoms was 44 days; median time from diagnosis to sustained symptom resolution with no recurring symptoms for 1 month or longer was 214 days. Anti-nucleocapsid IgG level in the first week after positive RT-PCR test and history of lung disease were associated with time to sustained symptom resolution. COVID-19 disease severity, ethnicity, age, sex, and remdesivir use did not affect time to sustained symptom resolution.CONCLUSIONWe found that all disease severities had a similar risk of developing post-COVID-19 syndrome in an ethnically diverse population. Comorbid lung disease and lower levels of initial IgG response to SARS-CoV-2 nucleocapsid antigen were associated with longer symptom duration.TRIAL REGISTRATIONClinicalTrials.gov, NCT04373148.FUNDINGNIH UL1TR003142 CTSA grant, NIH U54CA260517 grant, NIEHS R21 ES03304901, Sean N Parker Center for Allergy and Asthma Research at Stanford University, Chan Zuckerberg Biohub, Chan Zuckerberg Initiative, Sunshine Foundation, Crown Foundation, and Parker Foundation.

Considerations for the Safe Operation of Schools During the Coronavirus Pandemic.

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, providing safe in-person schooling has been a dynamic process balancing evolving community disease burden, scientific information, and local regulatory requirements with the mandate for education. Considerations include the health risks of SARS-CoV-2 infection and its post-acute sequelae, the impact of remote learning or periods of quarantine on education and well-being of children, and the contribution of schools to viral circulation in the community. The risk for infections that may occur within schools is related to the incidence of SARS-CoV-2 infections within the local community. Thus, persistent suppression of viral circulation in the community through effective public health measures including vaccination is critical to in-person schooling. Evidence suggests that the likelihood of transmission of SARS-CoV-2 within schools can be minimized if mitigation strategies are rationally combined. This article reviews evidence-based approaches and practices for the continual operation of in-person schooling.

Detection of SARS-CoV-2 RNA using RT-LAMP and molecular beacons.

BACKGROUND: Rapid spread of SARS-CoV-2 has led to a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse transcription-polymerase chain reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but instrument costs are high and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse transcription and loop-mediated isothermal amplification (RT-LAMP) provides an alternative that uses orthogonal and often less expensive reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however, a common artifact is nonspecific DNA amplification, which complicates detection. RESULTS: Here we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. To optimize beacons for RT-LAMP, multiple locked nucleic acid monomers were incorporated to elevate melting temperatures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in "single pot" reactions, including incorporation of a human RNA LAMP-BEAC assay to confirm sample integrity. Comparison of LAMP-BEAC and RT-qPCR on clinical saliva samples showed good concordance between assays. To facilitate implementation, we developed custom polymerases for LAMP-BEAC and inexpensive purification procedures, which also facilitates increasing sensitivity by increasing reaction volumes. CONCLUSIONS: LAMP-BEAC thus provides an affordable and simple SARS-CoV-2 RNA assay suitable for population screening; implementation of the assay has allowed robust screening of thousands of saliva samples per week.

Signatures of COVID-19 Severity and Immune Response in the Respiratory Tract Microbiome.

Viral infection of the respiratory tract can be associated with propagating effects on the airway microbiome, and microbiome dysbiosis may influence viral disease. Here, we investigated the respiratory tract microbiome in coronavirus disease 2019 (COVID-19) and its relationship to disease severity, systemic immunologic features, and outcomes. We examined 507 oropharyngeal, nasopharyngeal, and endotracheal samples from 83 hospitalized COVID-19 patients as well as non-COVID patients and healthy controls. Bacterial communities were interrogated using 16S rRNA gene sequencing, and the commensal DNA viruses Anelloviridae and Redondoviridae were quantified by qPCR. We found that COVID-19 patients had upper respiratory microbiome dysbiosis and greater change over time than critically ill patients without COVID-19. Oropharyngeal microbiome diversity at the first time point correlated inversely with disease severity during hospitalization. Microbiome composition was also associated with systemic immune parameters in blood, as measured by lymphocyte/neutrophil ratios and immune profiling of peripheral blood mononuclear cells. Intubated patients showed patient-specific lung microbiome communities that were frequently highly dynamic, with prominence of Staphylococcus. Anelloviridae and Redondoviridae showed more frequent colonization and higher titers in severe disease. Machine learning analysis demonstrated that integrated features of the microbiome at early sampling points had high power to discriminate ultimate level of COVID-19 severity. Thus, the respiratory tract microbiome and commensal viruses are disturbed in COVID-19 and correlate with systemic immune parameters, and early microbiome features discriminate disease severity. Future studies should address clinical consequences of airway dysbiosis in COVID-19, its possible use as biomarkers, and the role of bacterial and viral taxa identified here in COVID-19 pathogenesis. IMPORTANCE COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of the respiratory tract, results in highly variable outcomes ranging from minimal illness to death, but the reasons for this are not well understood. We investigated the respiratory tract bacterial microbiome and small commensal DNA viruses in hospitalized COVID-19 patients and found that each was markedly abnormal compared to that in healthy people and differed from that in critically ill patients without COVID-19. Early airway samples tracked with the level of COVID-19 illness reached during hospitalization, and the airway microbiome also correlated with immune parameters in blood. These findings raise questions about the mechanisms linking SARS-CoV-2 infection and other microbial inhabitants of the airway, including whether the microbiome might regulate severity of COVID-19 disease and/or whether early microbiome features might serve as biomarkers to discriminate disease severity.

Femtomolar SARS-CoV-2 Antigen Detection Using the Microbubbling Digital Assay with Smartphone Readout Enables Antigen Burden Quantitation and Tracking.

BACKGROUND: High-sensitivity severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen assays are desirable to mitigate false negative results. Limited data are available to quantify and track SARS-CoV-2 antigen burden in respiratory samples from different populations. METHODS: We developed the Microbubbling SARS-CoV-2 Antigen Assay (MSAA) with smartphone readout, with a limit of detection of 0.5 pg/mL (10.6 fmol/L) nucleocapsid antigen or 4000 copies/mL inactivated SARS-CoV-2 virus in nasopharyngeal (NP) swabs. We developed a computer vision and machine learning-based automatic microbubble image classifier to accurately identify positives and negatives and quantified and tracked antigen dynamics in intensive care unit coronavirus disease 2019 (COVID-19) inpatients and immunocompromised COVID-19 patients. RESULTS: Compared to qualitative reverse transcription-polymerase chain reaction methods, the MSAA demonstrated a positive percentage agreement of 97% (95% CI 92%-99%) and a negative percentage agreement of 97% (95% CI 94%-100%) in a clinical validation study with 372 residual clinical NP swabs. In immunocompetent individuals, the antigen positivity rate in swabs decreased as days-after-symptom-onset increased, despite persistent nucleic acid positivity. Antigen was detected for longer and variable periods of time in immunocompromised patients with hematologic malignancies. Total microbubble volume, a quantitative marker of antigen burden, correlated inversely with cycle threshold values and days-after-symptom-onset. Viral sequence variations were detected in patients with long duration of high antigen burden. CONCLUSIONS: The MSAA enables sensitive and specific detection of acute infections and quantification and tracking of antigen burden and may serve as a screening method in longitudinal studies to identify patients who are likely experiencing active rounds of ongoing replication and warrant close viral sequence monitoring.

SARS-CoV-2 Genomic Variation in Space and Time in Hospitalized Patients in Philadelphia.

The severe acute respiratory coronavirus 2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. The epidemic accelerated in Philadelphia, PA, in the spring of 2020, with the city experiencing a first peak of infections on 15 April, followed by a decline through midsummer. Here, we investigate spread of the epidemic in the first wave in Philadelphia using full-genome sequencing of 52 SARS-CoV-2 samples obtained from 27 hospitalized patients collected between 30 March and 17 July 2020. Sequences most commonly resembled lineages circulating at earlier times in New York, suggesting transmission primarily from this location, though a minority of Philadelphia genomes matched sequences from other sites, suggesting additional introductions. Multiple genomes showed even closer matches to other Philadelphia isolates, suggestive of ongoing transmission within Philadelphia. We found that all of our isolates contained the D614G substitution in the viral spike and belong to lineages variously designated B.1, Nextstrain clade 20A or 20C, and GISAID clade G or GH. There were no viral sequence polymorphisms detectably associated with disease outcome. For some patients, genome sequences were determined longitudinally or concurrently from multiple body sites. In both cases, some comparisons showed reproducible polymorphisms, suggesting initial seeding with multiple variants and/or accumulation of polymorphisms after infection. These results thus provide data on the sources of SARS-CoV-2 infection in Philadelphia and begin to explore the dynamics within hospitalized patients.IMPORTANCE Understanding how SARS-CoV-2 spreads globally and within infected individuals is critical to the development of mitigation strategies. We found that most lineages in Philadelphia had resembled sequences from New York, suggesting infection primarily but not exclusively from this location. Many genomes had even nearer neighbors within Philadelphia, indicating local spread. Multiple genome sequences were available for some subjects and in a subset of cases could be shown to differ between time points and body sites within an individual, indicating heterogeneous viral populations within individuals and raising questions on the mechanisms responsible. There was no evidence that different lineages were associated with different outcomes in patients, emphasizing the importance of individual-specific vulnerability.