Characterizing the Severity of SARS-CoV-2 Variants at a Single Pediatric Center.

Since March 2020, SARS-CoV-2 has plagued the world with COVID-19 and individuals of all ages have experienced varying symptoms of disease. Older adults were experiencing more severe disease compared to children and were prioritized by vaccination efforts. While biologic therapies and vaccinations were implemented, there were changes in public health restrictions with subsequent surges resulting in more infected children. During these surges there was a rise of different SARS-CoV-2 variants with the dominant variant initially alpha (B.1.1.7 and other Pango lineages) and epsilon (B.1.427/B.1.429) in early 2021 and a dramatic shift to delta (B.1.617.2 and other Pango lineages) by mid-summer 2021. In this study we aimed to characterize the clinical severity and host factors associated with disease by SARS-CoV-2 variant and evaluate if there are differences in disease severity by circulating variant. We retrospectively included all individuals 0-25 years of age who presented to our center and had a positive SARS-CoV-2 RT-PCR, SARS-CoV-2 variant mutation testing, and documented clinical notes from 1 January 2021 through 31 December 2021. We identified 745 individuals who met inclusion criteria and found the delta variant was associated with severe/critical disease compared to the other variants studied. The results of the model showed that underlying respiratory disease and diabetes were risk factors for progression to severe disease. These insights are important when evaluating public health measures and treatment options for children as more variants arise.

Statistical Modeling for Practical Pooled Testing During the COVID-19 Pandemic

Pooled testing offers an efficient solution to the unprecedented testing demands of the COVID-19 pandemic, despite their potentially lower sensitivity and increased costs to implementation in certain settings. Assessments of this trade-off typically assume the underlying infection statuses of pooled specimens to be independent and identically distributed. Yet, in the context of COVID-19, these assumptions are often violated: testing done on networks (housemates, spouses, co-workers) captures individuals with correlated infection statuses and risk, while infection risk varies substantially across time, place and individuals. Neglecting dependencies and heterogeneity may bias established optimality grids and induce a sub-optimal implementation of the procedure. As a lesson learned from this pandemic, this paper highlights the necessity of integrating field sampling information with statistical modeling to efficiently optimize pooled testing. Using real data, we show that (a) greater gains can be achieved at low logistical cost by exploiting natural correlations (nonindependence) between samples-allowing improvements in sensitivity and efficiency of up to 30% and 90%, respectively;and (b) these gains are robust despite substantial heterogeneity across pools (nonidentical). Our modeling results complement and extend the observations of Barak et al. (Sci. Transl. Med. 13 (2021) 1–8) who report an empirical sensitivity well beyond expectations. Finally, we provide an interactive tool for selecting an optimal pool size using contextual information.11

Evaluation of a Rapid and Accessible Reverse Transcription-Quantitative PCR Approach for SARS-CoV-2 Variant of Concern Identification.

The ability to distinguish between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) is of ongoing interest due to differences in transmissibility, responses to vaccination, clinical prognosis, and therapy. Although detailed genetic characterization requires whole-genome sequencing (WGS), targeted nucleic acid amplification tests can serve a complementary role in clinical settings, as they are more rapid and accessible than sequencing in most laboratories. We designed and analytically validated a two-reaction multiplex reverse transcription-quantitative PCR (RT-qPCR) assay targeting spike protein mutations L452R, E484K, and N501Y in reaction 1 and del69-70, K417N, and T478K in reaction 2. This assay had 95 to 100% agreement with WGS for 502 upper respiratory tract swab samples collected between 26 April 2021 and 1 August 2021, consisting of 43 Alpha, 2 Beta, 20 Gamma, 378 Delta, and 59 non-VOC infections. Validation in a separate group of 230 WGS-confirmed Omicron variant samples collected in December 2021 and January 2022 demonstrated 100% agreement. This RT-qPCR-based approach can be implemented in clinical laboratories already performing SARS-CoV-2 nucleic acid amplification tests to assist in local epidemiological surveillance and clinical decision-making.

Post-Vaccination Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infections and Incidence of the Presumptive B.1.427/B.1.429 Variant Among Healthcare Personnel at a Northern California Academic Medical Center.

BACKGROUND: Although mRNA-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines report >90% efficacy, breakthrough infections occur. Little is known about their effectiveness against SARS-CoV-2 variants, including the highly prevalent B.1.427/B.1.429 variant. METHODS: In this quality improvement project, we collected demographic and clinical information from post-vaccine SARS-CoV-2 cases (PVSCs), defined as healthcare personnel (HCP) with positive SARS-CoV-2 nucleic acid amplification test after receiving ≥1 vaccine dose. Available specimens were tested for L452R, N501Y, and E484K mutations using reverse-transcription polymerase chain reaction. Mutation prevalence was compared among unvaccinated, early post-vaccinated (≤14 days after dose 1), partially vaccinated (positive test >14 days after dose 1 and <14 days after dose 2), and fully vaccinated (>14 days after dose 2) PVSCs. RESULTS: From December 2020 to April 2021, ≥23 090 HCP received ≥1 dose of an mRNA-based SARS-CoV-2 vaccine, and 660 HCP cases of SARS-CoV-2 occurred, of which 189 were PVSCs. Among the PVSCs, 114 (60.3%), 49 (25.9%), and 26 (13.8%) were early post-vaccination, partially vaccinated, and fully vaccinated, respectively. Of 261 available samples from vaccinated and unvaccinated HCP, 103 (39.5%), including 42 PVSCs (36.5%), had the L452R mutation presumptive of B.1.427/B.1.429. When adjusted for community prevalence of B.1.427/B.1.429, PVSCs did not have significantly elevated risk of B.1.427/B.1.429 compared with unvaccinated HCP. CONCLUSIONS: Most PVSCs occurred prior to expected onset of full, vaccine-derived immunity. Presumptive B.1.427/B.1.429 was not more prevalent in post-vaccine cases than in unvaccinated SARS-CoV-2 HCP. Continued infection control measures, particularly <14 days post-vaccination, and continued variant surveillance in PVSCs are imperative to control future SARS-CoV-2 surges.

Direct comparison of antibody responses to four SARS-CoV-2 vaccines in Mongolia.

Different SARS-CoV-2 vaccines are approved in various countries, but few direct comparisons of the antibody responses they stimulate have been reported. We collected plasma specimens in July 2021 from 196 Mongolian participants fully vaccinated with one of four COVID-19 vaccines: Pfizer/BioNTech, AstraZeneca, Sputnik V, and Sinopharm. Functional antibody testing with a panel of nine SARS-CoV-2 viral variant receptor binding domain (RBD) proteins revealed marked differences in vaccine responses, with low antibody levels and RBD-ACE2 blocking activity stimulated by the Sinopharm and Sputnik V vaccines in comparison to the AstraZeneca or Pfizer/BioNTech vaccines. The Alpha variant caused 97% of infections in Mongolia in June and early July 2021. Individuals who recover from SARS-CoV-2 infection after vaccination achieve high antibody titers in most cases. These data suggest that public health interventions such as vaccine boosting, potentially with more potent vaccine types, may be needed to control COVID-19 in Mongolia and worldwide.

Multiplex SARS-CoV-2 Genotyping Reverse Transcriptase PCR for Population-Level Variant Screening and Epidemiologic Surveillance.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with concerning phenotypic mutations is of public health interest. Genomic surveillance is an important tool for a pandemic response, but many laboratories do not have the resources to support population-level sequencing. We hypothesized that a nucleic acid amplification test (NAAT) to genotype mutations in the viral spike protein could facilitate high-throughput variant surveillance. We designed and analytically validated a one-step multiplex allele-specific reverse transcriptase PCR (RT-qPCR) to detect three nonsynonymous spike protein mutations (L452R, E484K, N501Y). Assay specificity was validated with next-generation whole-genome sequencing. We then screened a large cohort of SARS-CoV-2-positive specimens from our San Francisco Bay Area population. Between 1 December 2020 and 1 March 2021, we screened 4,049 unique infections by genotyping RT-qPCR, with an assay failure rate of 2.8%. We detected 1,567 L452R mutations (38.7%), 34 N501Y mutations (0.84%), 22 E484K mutations (0.54%), and 3 (0.07%) E484K plus N501Y mutations. The assay had perfect (100%) concordance with whole-genome sequencing of a validation subset of 229 specimens and detected B.1.1.7, B.1.351, B.1.427, B.1.429, B.1.526, and P.2 variants, among others. The assay revealed the rapid emergence of the L452R variant in our population, with a prevalence of 24.8% in December 2020 that increased to 62.5% in March 2021. We developed and clinically implemented a genotyping RT-qPCR to conduct high-throughput SARS-CoV-2 variant screening. This approach can be adapted for emerging mutations and immediately implemented in laboratories already performing NAAT worldwide using existing equipment, personnel, and extracted nucleic acid.

SARS-CoV-2 Nucleocapsid Plasma Antigen for Diagnosis and Monitoring of COVID-19.

BACKGROUND: Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid antigen in blood has been described, but the diagnostic and prognostic role of antigenemia is not well understood. This study aimed to determine the frequency, duration, and concentration of nucleocapsid antigen in plasma and its association with coronavirus disease 2019 (COVID-19) severity. METHODS: We utilized an ultrasensitive electrochemiluminescence immunoassay targeting SARS-CoV-2 nucleocapsid antigen to evaluate 777 plasma samples from 104 individuals with COVID-19. We compared plasma antigen to respiratory nucleic acid amplification testing (NAAT) in 74 individuals with COVID-19 from samples collected ±1 day of diagnostic respiratory NAAT and in 52 SARS-CoV-2-negative individuals. We used Kruskal-Wallis tests, multivariable logistic regression, and mixed-effects modeling to evaluate whether plasma antigen concentration was associated with disease severity. RESULTS: Plasma antigen had 91.9% (95% CI 83.2%-97.0%) clinical sensitivity and 94.2% (84.1%-98.8%) clinical specificity. Antigen-negative plasma samples belonged to patients with later respiratory cycle thresholds (Ct) when compared with antigen-positive plasma samples. Median plasma antigen concentration (log10 fg/mL) was 5.4 (interquartile range 3.9-6.0) in outpatients, 6.0 (5.4-6.5) in inpatients, and 6.6 (6.1-7.2) in intensive care unit (ICU) patients. In models adjusted for age, sex, diabetes, and hypertension, plasma antigen concentration at diagnosis was associated with ICU admission [odds ratio 2.8 (95% CI 1.2-6.2), P=.01] but not with non-ICU hospitalization. Rate of antigen decrease was not associated with disease severity. CONCLUSIONS: SARS-CoV-2 plasma nucleocapsid antigen exhibited comparable diagnostic performance to upper respiratory NAAT, especially among those with late respiratory Ct. In addition to currently available tools, antigenemia may facilitate patient triage to optimize intensive care utilization.

Ultra-sensitive Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Antigen Detection for the Diagnosis of Coronavirus Disease 2019 (COVID-19) in Upper Respiratory Samples.

An ultra-sensitive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid antigen assay (S-PLEX, MesoScale Diagnostics) was evaluated in 250 retrospective and 200 prospective upper respiratory specimens. In samples with cycle threshold <35, there was 95%-98% positive and 93%-96% negative percent agreement with reverse transcription-polymerase chain reaction. S-PLEX may provide a high-throughput alternative to nucleic acid-based testing for coronavirus disease 2019 (COVID-19) diagnosis.

Case-Control Study of Individuals with Discrepant Nucleocapsid and Spike Protein SARS-CoV-2 IgG Results.

BACKGROUND: Laboratory-based methods for SARS-CoV-2 antibody detection vary widely in performance. However, there are limited prospectively-collected data on assay performance, and minimal clinical information to guide interpretation of discrepant results. METHODS: Over a 2-week period, 1080 consecutive plasma samples submitted for clinical SARS-CoV-2 IgG testing were tested in parallel for anti-nucleocapsid IgG (anti-N, Abbott) and anti-spike IgG (anti-S1, EUROIMMUN). Chart review was conducted for samples testing positive or borderline on either assay, and for an age/sex-matched cohort of samples negative by both assays. CDC surveillance case definitions were used to determine clinical sensitivity/specificity and conduct receiver operating characteristics curve analysis. RESULTS: There were 52 samples positive by both methods, 2 positive for anti-N only, 34 positive for anti-S1 only, and 27 borderline for anti-S1. Of the 34 individuals positive for anti-S1 alone, 8 (24%) had confirmed COVID-19. No anti-S1 borderline cases were positive for anti-N or had confirmed/probable COVID-19. The anti-N assay was less sensitive (84.2% [95% CI 72.1-92.5%] vs 94.7% [95% CI 85.4-98.9%]) but more specific (99.2% [95% CI 95.5-100%] vs 86.9% [95% CI 79.6-92.3%]) than anti-S1. Abbott anti-N sensitivity could be improved to 96.5% with minimal effect on specificity if the index threshold was lowered from 1.4 to 0.6. CONCLUSION: Real-world concordance between different serologic assays may be lower than previously described in retrospective studies. These findings have implications for the interpretation of SARS-CoV-2 IgG results, especially with the advent of spike antigen-targeted vaccination, as a subset of patients with true infection are anti-N negative and anti-S1 positive.

Characteristics of Nursing Homes by COVID-19 Cases Among Staff: March to August 2020.

OBJECTIVE: To measure the association between nursing home (NH) characteristics and Coronavirus Disease 2019 (COVID-19) prevalence among NH staff. DESIGN: Retrospective cross-sectional study. SETTING AND PARTICIPANTS: Centers for Disease Control and Prevention COVID-19 database for US NHs between March and August 2020, linked to NH facility characteristics (LTCFocus database) and local COVID-19 prevalence (USA Facts). METHODS: We estimated the associations between NH characteristics, local infection rates, and other regional characteristics and COVID-19 cases among NH staff (nursing staff, clinical staff, aides, and other facility personnel) measured per 100 beds, controlling for the hospital referral regions in which NHs were located to account for local infection control practices and other unobserved characteristics. RESULTS: Of the 11,858 NHs in our sample, 78.6% reported at least 1 staff case of COVID-19. After accounting for local COVID-19 prevalence, NHs in the highest quartile of confirmed resident cases (413.5 to 920.0 cases per 1000 residents) reported 18.9 more staff cases per 100 beds compared with NHs that had no resident cases. Large NHs (150 or more beds) reported 2.6 fewer staff cases per 100 beds compared with small NHs (<50 beds) and for-profit NHs reported 0.8 fewer staff cases per 100 beds compared with nonprofit NHs. Higher occupancy and more direct-care hours per day were associated with more staff cases (0.4 more cases per 100 beds for a 10% increase in occupancy, and 0.7 more cases per 100 beds for an increase in direct-care staffing of 1 hour per resident day, respectively). Estimates associated with resident demographics, payer mix, or regional socioeconomic characteristics were not statistically significant. CONCLUSIONS AND IMPLICATIONS: These findings highlight the urgent need to support facilities with emergency resources such as back-up staff and protocols to reduce resident density within the facility, which may help stem outbreaks.

Strand-Specific Reverse Transcription PCR for Detection of Replicating SARS-CoV-2.

We developed an assay that detects minus-strand RNA as a surrogate for actively replicating severe acute respiratory syndrome coronavirus 2. We detected minus-strand RNA in 41 persons with coronavirus disease up to 30 days after symptom onset. This assay might inform clinical decision-making about patient infectiousness.

Defining the features and duration of antibody responses to SARS-CoV-2 infection associated with disease severity and outcome.

SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, can neutralize the virus. It is, however, unknown which features of the serological response may affect clinical outcomes of COVID-19 patients. We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness. Higher ratios of IgG antibodies targeting S1 or RBD domains of spike compared to nucleocapsid antigen were seen in outpatients who had mild illness versus severely ill patients. Plasma antibody increases correlated with decreases in viral RNAemia, but antibody responses in acute illness were insufficient to predict inpatient outcomes. Pseudovirus neutralization assays and a scalable ELISA measuring antibodies blocking RBD-ACE2 interaction were well correlated with patient IgG titers to RBD. Outpatient and asymptomatic individuals' SARS-CoV-2 antibodies, including IgG, progressively decreased during observation up to five months post-infection.

Large-Scale Testing of Asymptomatic Healthcare Personnel for Severe Acute Respiratory Syndrome Coronavirus 2.

Large-scale, 1-time testing of >12,000 asymptomatic healthcare personnel in California, USA, during April-June 2020 showed that prevalence of severe acute respiratory syndrome coronavirus 2 was low (<1%). Testing might identify asymptomatic and presymptomatic persons, including some with high viral burden, enabling prompt implementation of measures to limit nosocomial spread.

Performance of Nucleic Acid Amplification Tests for Detection of Severe Acute Respiratory Syndrome Coronavirus 2 in Prospectively Pooled Specimens.

Pooled nucleic acid amplification tests for severe acute respiratory syndrome coronavirus 2 could increase availability of testing at decreased cost. However, the effect of dilution on analytical sensitivity through sample pooling has not been well characterized. We tested 1,648 prospectively pooled specimens by using 3 nucleic acid amplification tests for severe acute respiratory syndrome coronavirus 2: a laboratory-developed real-time reverse transcription PCR targeting the envelope gene, and 2 commercially available Panther System assays targeting open reading frame 1ab. Positive percent agreement (PPA) of pooled versus individual testing ranged from 71.7% to 82.6% for pools of 8 and from 82.9% to 100.0% for pools of 4. We developed and validated an independent stochastic simulation model to estimate effects of dilution on PPA and efficiency of a 2-stage pooled real-time reverse transcription PCR testing algorithm. PPA was dependent on the proportion of tests with positive results, cycle threshold distribution, and assay limit of detection.