Background The SARS-CoV-2 reverse-transcription polymerase chain reaction (RT-PCR) cycle threshold (Ct) has been used to estimate quantitative viral load, with the goal of targeting isolation precautions for individuals with COVID-19 and guiding public health interventions. However, variability in specimen quality can alter the Ct values obtained from SARS-CoV-2 clinical assays. We sought to define how variable nasopharyngeal (NP) swab quality impacts clinical SARS-CoV-2 test sensitivity. Methods We performed amplification of a human gene target (β-actin) in parallel with a clinical RT-PCR targeting the SARS-CoV-2 ORF1ab gene for 1282 NP specimens collected from patients with clinical concern for COVID-19. We evaluated the relationship between NP specimen quality, characterized by late Ct values for the human gene target β-actin Ct, and the probability of SARS-CoV-2 detection via logistic regression, as well as the linear relationship between SARS-CoV-2 and β-actin Ct. Results Low quality NP swabs are less likely to detect SARS-CoV-2 (odds ratio 0.607, 95%CI 0.487 to 0.753). We observed a positive linear relationship between SARS-CoV-2 and β-actin Ct values (slope 0.181, 95%CI 0.097 to 0.264), consistent with a reduction in detection of 0.181 cycles for each additional cycle of the β-actin target. COVID-19 disease severity was not associated with β-actin Ct values. Conclusions Variability in NP specimen quality significantly impacts the performance of clinical SARS-CoV-2 assays, and caution should be taken when interpreting quantitative SARS-CoV-2 Ct results. If unrecognized, low quality NP specimens, which are characterized by a low level of amplifiable human DNA target, may limit the successful application of SARS-CoV-2 Ct values to direct infection control and public health interventions.

BackgroundThe SARS-CoV-2 reverse-transcription polymerase chain reaction (RT-PCR) cycle of threshold (Ct) has been used to estimate quantitative viral load, with the goal of targeting isolation precautions for individuals with COVID-19 and guiding public health interventions. However, variability in specimen quality can alter the Ct values obtained from SARS-CoV-2 clinical assays. We sought to define how variable nasopharyngeal (NP) swab quality impacts clinical SARS-CoV-2 test sensitivity.MethodsWe performed amplification of a human gene target (β-actin) in parallel with a clinical RT-PCR targeting the SARS-CoV-2 ORF1ab gene for 1311 NP specimens collected from patients with clinical concern for COVID-19. We evaluated the relationship between NP specimen quality, characterized by high Ct values for the human gene target β-actin Ct, and the probability of SARS-CoV-2 detection via logistic regression, as well as the linear relationship between SARS-CoV-2 and β-actin Ct.ResultsLow quality NP swabs are less likely to detect SARS-CoV-2 (odds ratio 0.654, 95%CI 0.523 to 0.802). We observed a positive linear relationship between SARS-CoV-2 and β-actin Ct values (slope 0.169, 95%CI 0.092 to 0.247). COVID-19 disease severity was not associated with β-actin Ct values.ConclusionsVariability in NP specimen quality accounts for significant differences in the sensitivity of clinical SARS-CoV-2 assays. If unrecognized, low quality NP specimens, which are characterized by a low level of amplifiable human DNA target, may limit the application of SARS-CoV-2 Ct values to direct infection control and public health interventions.

Background Although SARS-CoV-2 predominantly targets the respiratory system, it has also been associated with vascular complications including stroke. Identifying COVID-19 patients at elevated risk for stroke can help inform target anticoagulation strategies. We sought to understand how symptoms and laboratory markers at presentation with COVID-19 relate to stroke risk. Methods We enrolled a cohort of 1324 subjects who were hospitalized with COVID-19 across six PennMedicine hospitals between April and August 2020 and performed retrospective, manual chart review to measure exposures including presenting symptoms and admission inflammatory markers. Data were organized with a REDCap database, and analyses were performed using R statistical software, with Bayesian binomial regression models fit using Stan Hamiltonian Monte Carlo via the “brms” package. Results Among 1324 subjects, 19 stroke events were observed within 30 days of COVID-19 diagnosis. Admission inflammatory markers, including C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), ferritin, and D-dimer, were poor predictors of stroke risk. Among presenting symptoms, including respiratory, gastrointestinal, dermatologic, and neurologic features of COVID-19 disease, only altered mental status documented on presentation (in 529 subjects) was significantly associated with stroke risk (odds ratio 6.06, 95% credible interval 2.16 - 18.7). Conclusion Inflammatory markers associated with COVID-19 disease severity did not discriminate patients at high versus low risk of stroke in this cohort. Altered mental status documented on presentation was significantly associated with incident stroke during COVID-19 disease. Disclosures Ebbing Lautenbach, MD, MPH, MSCE, Merck (Other Financial or Material Support, Member of Data and Safety Monitoring Board (DSMB)) Michael Z. David, MD PhD, GSK (Board Member)