Total and subgenomic RNA viral load in patients infected with SARS-CoV-2 Alpha, Delta, and Omicron variants.

BACKGROUND: SARS-CoV-2 genomic and subgenomic RNA levels are frequently used as a correlate of infectiousness. The impact of host factors and SARS-CoV-2 lineage on RNA viral load is unclear. METHODS: Total nucleocapsid (N) and subgenomic N (sgN) RNA levels were measured by RT-qPCR in specimens from 3,204 individuals hospitalized with COVID-19 at 21 hospitals. RT-qPCR cycle threshold (Ct) values were used to estimate RNA viral load. The impact of time of sampling, SARS-CoV-2 variant, age, comorbidities, vaccination, and immune status on N and sgN Ct values were evaluated using multiple linear regression. RESULTS: Ct values at presentation for N (mean ±standard deviation) were 24.14±4.53 for non-variants of concern, 25.15±4.33 for Alpha, 25.31±4.50 for Delta, and 26.26±4.42 for Omicron. N and sgN RNA levels varied with time since symptom onset and infecting variant but not with age, comorbidity, immune status, or vaccination. When normalized to total N RNA, sgN levels were similar across all variants. CONCLUSIONS: RNA viral loads were similar among hospitalized adults, irrespective of infecting variant and known risk factors for severe COVID-19. Total N and subgenomic RNA N viral loads were highly correlated, suggesting that subgenomic RNA measurements adds little information for the purposes of estimating infectivity.

Incidence of COVID-19 reinfection among Midwestern healthcare employees.

Given the overwhelming worldwide rate of infection and the disappointing pace of vaccination, addressing reinfection is critical. Understanding reinfection, including longevity after natural infection, will allow us to better know the prospect of herd immunity, which hinges on the assumption that natural infection generates sufficient, protective immunity. The primary objective of this observational cohort study is to establish the incidence of reinfection of COVID-19 among healthcare employees who experienced a prior COVID-19 infection over a 10-month period. Of 2,625 participants who experienced at least one COVID-19 infection during the 10-month study period, 156 (5.94%) experienced reinfection and 540 (20.57%) experienced recurrence after prior infection. Median days were 126.50 (105.50-171.00) to reinfection and 31.50 (10.00-72.00) to recurrence. Incidence rate of COVID-19 reinfection was 0.35 cases per 1,000 person-days, with participants working in COVID-clinical and clinical units experiencing 3.77 and 3.57 times, respectively, greater risk of reinfection relative to those working in non-clinical units. Incidence rate of COVID-19 recurrence was 1.47 cases per 1,000 person-days. This study supports the consensus that COVID-19 reinfection, defined as subsequent infection ≥ 90 days after prior infection, is rare, even among a sample of healthcare workers with frequent exposure.

SARS-CoV-2 Genomic Surveillance Reveals Little Spread From a Large University Campus to the Surrounding Community.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has had high incidence rates at institutions of higher education (IHE) in the United States, but the transmission dynamics in these settings are poorly understood. It remains unclear to what extent IHE-associated outbreaks have contributed to transmission in nearby communities. METHODS: We implemented high-density prospective genomic surveillance to investigate these dynamics at the University of Michigan and the surrounding community during the Fall 2020 semester (August 16-November 24). We sequenced complete severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from 1659 individuals, including 468 students, representing 20% of cases in students and 25% of total cases in Washtenaw County over the study interval. RESULTS: Phylogenetic analysis identified >200 introductions into the student population, most of which were not related to other student cases. There were 2 prolonged student transmission clusters, of 115 and 73 individuals, that spanned multiple on-campus residences. Remarkably, <5% of nonstudent genomes were descended from student clusters, and viral descendants of student cases were rare during a subsequent wave of infections in the community. CONCLUSIONS: The largest outbreaks among students at the University of Michigan did not significantly contribute to the rise in community cases in Fall 2020. These results provide valuable insights into SARS-CoV-2 transmission dynamics at the regional level.

Severe Acute Respiratory Syndrome Coronavirus 2 Total and Subgenomic RNA Viral Load in Hospitalized Patients.

BACKGROUND: Previous studies demonstrated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA can be detected for weeks after infection. The significance of this finding is unclear and, in most patients, does not represent active infection. Detection of subgenomic RNA has been proposed to represent productive infection and may be a useful marker for monitoring infectivity. METHODS: We used quantitative reverse-transcription polymerase chain reaction (RT-qPCR) to quantify total and subgenomic nucleocapsid (sgN) and envelope (sgE) transcripts in 185 SARS-CoV-2-positive nasopharyngeal swab samples collected on hospital admission and to relate to symptom duration. RESULTS: We find that all transcripts decline at the same rate; however, sgE becomes undetectable before other transcripts. The median duration of symptoms to a negative test is 14 days for sgE and 25 days for sgN. There is a linear decline in subgenomic compared to total RNA, suggesting that subgenomic transcript copy number is dependent on copy number of total transcripts. The mean difference between total and sgN is 16-fold and the mean difference between total and sgE is 137-fold. This relationship is constant over duration of symptoms, allowing prediction of subgenomic copy number from total copy number. CONCLUSIONS: Subgenomic RNA may be no more useful in determining infectivity than a copy number threshold determined for total RNA.

Neutrophil calprotectin identifies severe pulmonary disease in COVID-19.

Severe cases of coronavirus disease 2019 (COVID-19) are regularly complicated by respiratory failure. Although it has been suggested that elevated levels of blood neutrophils associate with worsening oxygenation in COVID-19, it is unknown whether neutrophils are drivers of the thrombo-inflammatory storm or simple bystanders. To better understand the potential role of neutrophils in COVID-19, we measured levels of the neutrophil activation marker S100A8/A9 (calprotectin) in hospitalized patients and determined its relationship to severity of illness and respiratory status. Patients with COVID-19 (n = 172) had markedly elevated levels of calprotectin in their blood. Calprotectin tracked with other acute phase reactants including C-reactive protein, ferritin, lactate dehydrogenase, and absolute neutrophil count, but was superior in identifying patients requiring mechanical ventilation. In longitudinal samples, calprotectin rose as oxygenation worsened. When tested on day 1 or 2 of hospitalization (n = 94 patients), calprotectin levels were significantly higher in patients who progressed to severe COVID-19 requiring mechanical ventilation (8039 ± 7031 ng/ml, n = 32) as compared to those who remained free of intubation (3365 ± 3146, P < 0.0001). In summary, serum calprotectin levels track closely with current and future COVID-19 severity, implicating neutrophils as potential perpetuators of inflammation and respiratory compromise in COVID-19.

Temporal dynamics of SARS-CoV-2 mutation accumulation within and across infected hosts.

Analysis of SARS-CoV-2 genetic diversity within infected hosts can provide insight into the generation and spread of new viral variants and may enable high resolution inference of transmission chains. However, little is known about temporal aspects of SARS-CoV-2 intrahost diversity and the extent to which shared diversity reflects convergent evolution as opposed to transmission linkage. Here we use high depth of coverage sequencing to identify within-host genetic variants in 325 specimens from hospitalized COVID-19 patients and infected employees at a single medical center. We validated our variant calling by sequencing defined RNA mixtures and identified viral load as a critical factor in variant identification. By leveraging clinical metadata, we found that intrahost diversity is low and does not vary by time from symptom onset. This suggests that variants will only rarely rise to appreciable frequency prior to transmission. Although there was generally little shared variation across the sequenced cohort, we identified intrahost variants shared across individuals who were unlikely to be related by transmission. These variants did not precede a rise in frequency in global consensus genomes, suggesting that intrahost variants may have limited utility for predicting future lineages. These results provide important context for sequence-based inference in SARS-CoV-2 evolution and epidemiology.

Disparities in Seroprevalence of SARS-CoV-2 Immunoglobulin Antibodies in a Large Midwestern Health Care System.

OBJECTIVES: Increased exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a result of having an essential job is compounded by factors such as age, race, and ethnicity. We used a cross-sectional study design to describe disparities in the seroprevalence of SARS-CoV-2 immunoglobulin G (IgG) test results by demographic characteristics and clinical roles among a cohort of health care workers employed by the largest Midwestern health care system in the United States. METHODS: We collected 16 233 SARS-CoV-2 IgG serum samples from June 8 through July 10, 2020, from a convenience sample of Illinois- and Wisconsin-based adult health care workers. The research team, in collaboration with ACL Laboratories, used a SARS-CoV-2 IgG assay to detect the presence of SARS-CoV-2 IgG antibodies. Study data included SARS-CoV-2 IgG assay results and demographic characteristics of workers (age, sex, race, ethnicity, clinical role, zip code). We generated crude and adjusted odds ratios (ORs) to describe disparities in seroprevalence distribution among demographic and social factors. RESULTS: Of 16 233 IgG serum samples tested, 622 (3.8%) test results were positive for SARS-CoV-2. We found significant disparities in SARS-CoV-2 positivity by age, race, ethnicity, and clinical role. Participants aged 32-82 had lower adjusted ORs (aORs) of positive IgG than participants aged 18-31 (aOR range, 0.54-0.66). Odds of positivity were higher among Black (aOR = 3.86), Asian (aOR = 1.42), and mixed-race (aOR = 1.99) workers than among White workers; among Hispanic workers (aOR = 1.80) than among non-Hispanic workers; and among coronavirus disease 2019 (COVID-19) clinical workers (aOR = 1.86) than among nonclinical workers. CONCLUSIONS: Public health efforts should focus on increasing COVID-19 safety messaging, testing, vaccination, and other prevention efforts for people who are young, non-White, Hispanic, and working in COVID-19-clinical units.

Neutrophil extracellular traps in COVID-19.

In severe cases of coronavirus disease 2019 (COVID-19), viral pneumonia progresses to respiratory failure. Neutrophil extracellular traps (NETs) are extracellular webs of chromatin, microbicidal proteins, and oxidant enzymes that are released by neutrophils to contain infections. However, when not properly regulated, NETs have the potential to propagate inflammation and microvascular thrombosis - including in the lungs of patients with acute respiratory distress syndrome. We now report that sera from patients with COVID-19 have elevated levels of cell-free DNA, myeloperoxidase-DNA (MPO-DNA), and citrullinated histone H3 (Cit-H3); the latter 2 are specific markers of NETs. Highlighting the potential clinical relevance of these findings, cell-free DNA strongly correlated with acute-phase reactants, including C-reactive protein, D-dimer, and lactate dehydrogenase, as well as absolute neutrophil count. MPO-DNA associated with both cell-free DNA and absolute neutrophil count, while Cit-H3 correlated with platelet levels. Importantly, both cell-free DNA and MPO-DNA were higher in hospitalized patients receiving mechanical ventilation as compared with hospitalized patients breathing room air. Finally, sera from individuals with COVID-19 triggered NET release from control neutrophils in vitro. Future studies should investigate the predictive power of circulating NETs in longitudinal cohorts and determine the extent to which NETs may be novel therapeutic targets in severe COVID-19.