Background: Repeated coronavirus disease 2019 (COVID-19) molecular testing can lead to positive test results after negative results and to multiple positive results over time. The association between positive test results and infectious virus is important to quantify.Methods: A 2-month cohort of retrospective data and consecutively collected specimens from patients with COVID-19 or patients under investigation were used to understand the correlation between prolonged viral RNA positive test results, cycle threshold (Ct) values and growth of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in cell culture. Whole-genome sequencing was used to confirm virus genotype in patients with prolonged viral RNA detection. Droplet digital polymerase chain reaction was used to assess the rate of false-negative COVID-19 diagnostic test results.Results: In 2 months, 29 686 specimens were tested and 2194 patients underwent repeated testing. Virus recovery in cell culture was noted in specimens with a mean Ct value of 18.8 (3.4) for SARS-CoV-2 target genes. Prolonged viral RNA shedding was associated with positive virus growth in culture in specimens collected up to 21 days after the first positive result but mostly in individuals symptomatic at the time of sample collection. Whole-genome sequencing provided evidence the same virus was carried over time. Positive test results following negative results had Ct values >29.5 and were not associated with virus culture. Droplet digital polymerase chain reaction results were positive in 5.6% of negative specimens collected from patients with confirmed or clinically suspected COVID-19.Conclusions: Low Ct values in SARS-CoV-2 diagnostic tests were associated with virus growth in cell culture. Symptomatic patients with prolonged viral RNA shedding can also be infectious.

Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant of concern (VOC) B.1.617.2 (Delta) displaced B.1.1.7 (Alpha) and is associated with increases in coronavirus disease 2019 (COVID-19) cases, greater transmissibility, and higher viral RNA loads, but data are lacking regarding the infectious virus load and antiviral antibody levels in the nasal tract. Methods: Whole genome sequencing, cycle threshold (Ct) values, infectious virus, anti-SARS-CoV-2 immunoglobulin G (IgG) levels, and clinical chart reviews were combined to characterize SARS-CoV-2 lineages circulating in the National Capital Region between January and September 2021 and differentiate infections in vaccinated and unvaccinated individuals by the Delta, Alpha, and B.1.2 (the predominant lineage prior to Alpha) variants. Results: The Delta variant displaced the Alpha variant to constitute 99% of the circulating lineages in the National Capital Region by August 2021. In Delta infections, 28.5% were breakthrough cases in fully vaccinated individuals compared to 4% in the Alpha infected cohort. Breakthrough infections in both cohorts were associated with comorbidities, but only Delta infections were associated with a significant increase in the median days after vaccination. More than 74% of Delta samples had infectious virus compared to <30% from the Alpha cohort. The recovery of infectious virus with both variants was associated with low levels of local SARS-CoV-2 IgG. Conclusions: Infection with the Delta variant was associated with more frequent recovery of infectious virus in vaccinated and unvaccinated individuals compared to the Alpha variant but was not associated with an increase in disease severity in fully vaccinated individuals. Infectious virus was correlated with the presence of low amounts of antiviral IgG in the nasal specimens.

The increase in SARS-CoV-2 infections in December 2021 was driven primarily by the Omicron variant, which largely displaced the Delta over a three-week span. Outcomes from infection with Omicron remain uncertain. We evaluated whether clinical outcomes and viral loads differed between Delta and Omicron infections during the period when both variants were co-circulating. In this retrospective observational cohort study, remnant clinical specimens, positive for SARS-CoV-2 after standard of care testing at the Johns Hopkins Microbiology Laboratory, between the last week of November and the end of December 2021, were used for whole viral genome sequencing. Cycle threshold values (Ct) for viral RNA, the presence of infectious virus, and levels of respiratory IgG were measured, and clinical outcomes were obtained. Differences in each measure were compared between variants stratified by vaccination status. The Omicron variant displaced Delta during the study period and constituted 95% of the circulating lineages by the end of December 2021. Patients with Omicron infections (N = 1,119) were more likely to be vaccinated compared to patients with Delta (N = 908), but were less likely to be admitted (0.33 CI 0.21–0.52), require ICU level care (0.38 CI 0.17–0.87), or succumb to infection (0.26 CI 0.06–1.02) regardless of vaccination status. There was no statistically significant difference in Ct values based on the lineage regardless of the vaccination status. Recovery of infectious virus in cell culture was reduced in boosted patients compared to fully vaccinated without a booster and unvaccinated when infected with the Delta lineage. However, in patients with Omicron infections, recovery of infectious virus was not affected by vaccination. Compared to Delta, Omicron was more likely to cause breakthrough infections of vaccinated individuals, yet admissions were less frequent. Admitted patients might develop severe disease comparable to Delta. Efforts for reducing Omicron transmission are required as, though the admission risk might be lower, the increased numbers of infections cause large numbers of hospitalizations. NIH/NIAID Center of Excellence in Influenza Research and Surveillance contract HHS N2772201400007C, Johns Hopkins University, Maryland department of health, Centers for Disease Control and Prevention contract 75D30121C11061, and The Modeling Infectious Diseases in Healthcare Network (MInD) under awards U01CK000589.

Background The emerging SARS-CoV-2 variant of concern (VOC) B.1.6.17.2 (Delta) quickly displaced the B.1.1.7 (Alpha) and is associated with increases in COVID-19 cases nationally. The Delta variant has been associated with greater transmissibility and higher viral RNA loads in both unvaccinated and fully vaccinated individuals. Data is lacking regarding the infectious virus load in Delta infected individuals and how that compares to individuals infected with other SARS-CoV-2 lineages. Methods Whole genome sequencing of 2,785 clinical isolates was used to characterize the prevalence of SARS-CoV-2 lineages circulating in the National Capital Region between January and July 2021. Clinical chart reviews were performed for the Delta, Alpha, and B.1.2 (a control predominant lineage prior to both VOCs) variants to evaluate disease severity and outcome and Cycle threshold values (Cts) were compared. The presence of infectious virus was determined using Vero-TMPRSS2 cells and anti-SARS-CoV-2 IgG levels were determined from upper respiratory specimen. An analysis of infection in unvaccinated and fully vaccinated populations was performed. Results The Delta variant displaced the Alpha variant to constitute 88.2% of the circulating lineages in the National Capital Region by July, 2021. The Delta variant associated with increased breakthrough infections in fully vaccinated individuals that were mostly symptomatic when compared to the Alpha breakthrough infections, though it is important to note there was a significantly longer period of time between vaccination and infection with Delta infections. The recovery of infectious virus on cell culture was significantly higher with the Delta variant compared to Alpha in both vaccinated and unvaccinated groups. The impact of vaccination on reducing the recovery of infectious virus from clinical samples was only observed with Alpha variant infections but was strongly associated with low localized SARS-CoV-2 IgG for both variants. A comparison of Ct values showed a significant decrease in the Delta compared to Alpha with no significant differences between unvaccinated and vaccinated groups. Conclusions Our data indicate that the Delta variant is associated with increased infectious virus loads when compared to the Alpha variant and decreased upper respiratory antiviral IgG levels. Measures to reduce transmission in addition to increasing vaccinations rates have to be implemented to reduce Delta variant spread. Funding NIH/NIAID Center of Excellence in Influenza Research and Surveillance contract HHS N2772201400007C, Johns Hopkins University, Maryland department of health, Centers for Disease Control and Prevention contract 75D30121C11061.

With the approach of respiratory virus season in the Northern Hemisphere, clinical microbiology and public health laboratories will need rapid diagnostic assays to distinguish severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from influenza virus and respiratory syncytial virus (RSV) infections for diagnosis and surveillance. In this study, the clinical performance of the Xpert Xpress SARS-CoV-2/Flu/RSV test (Cepheid, Sunnyvale, CA, USA) for nasopharyngeal swab specimens was evaluated in four centers: Johns Hopkins Medical Microbiology Laboratory, Northwell Health Laboratories, NYC Public Health Laboratory, and Los Angeles County/University of Southern California (LAC+USC) Medical Center. A total of 319 nasopharyngeal swab specimens, positive for SARS-CoV-2 (n = 75), influenza A virus (n = 65), influenza B virus (n = 50), or RSV (n = 38) or negative (n = 91) by the standard-of-care nucleic acid amplification tests at each site, were tested using the Cepheid panel test. The overall positive percent agreement for the SARS-CoV-2 target was 98.7% (n = 74/75), and the negative agreement was 100% (n = 91), with all other analytes showing 100% total agreement (n = 153). Standard-of-care tests to which the Cepheid panel was compared included the Cepheid Xpert Xpress SARS-CoV-2, Cepheid Xpert Xpress Flu/RSV, GenMark ePlex respiratory panel, BioFire respiratory panel 2.1 and v1.7, DiaSorin Simplexa COVID-19 Direct, and Hologic Panther Fusion SARS-CoV-2 assays. The Xpert Xpress SARS-CoV-2/Flu/RSV test showed high sensitivity and accuracy for all analytes included in the test. This test will provide a valuable clinical diagnostic and public health solution for detecting and differentiating SARS-CoV-2, influenza A and B virus, and RSV infections during the current respiratory virus season.