Evaluation of a multiplexed oligonucleotide ligation assay for SARS-CoV-2 variant identification.

BACKGROUND: SARS-CoV-2 variant surveillance informs vaccine composition and decisions to de-authorize antibody therapies. Though detailed genetic characterization requires whole-genome sequencing, targeted mutation analysis may complement pandemic surveillance efforts. METHODS: This study investigated the qualitative performance of a multiplex oligonucleotide ligation assay targeting 19 spike mutations using 192 whole genome sequenced upper respiratory samples representing SARS-CoV-2 variants of concern. RESULTS: Initial valid results were obtained from 95.8% [95% confidence interval (CI): 92.0 - 98.2; 184/192] of samples. All eight invalid samples were valid on repeat testing. When comparing SARS-CoV-2 oligonucleotide ligase assay SARS-CoV-2 variant calls with whole genome sequencing, overall positive percent agreement was 100% (95% CI: 98.1 - 100.0; 192/192), as was the positive and negative percent agreement for each of the tested variants; Gamma, Delta, Omicron BA.1, BA.2, and BA.4/BA.5. CONCLUSIONS: This multiplexed oligonucleotide ligation assays demonstrated accurate SARS-CoV-2 variant typing compared to whole genome sequencing. Such an approach has the potential to provide improved turnaround compared to sequencing and more detailed mutation coverage than RT-qPCR.

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.

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.

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 Neutralization Resistance Mutations in Patient with HIV/AIDS, California, USA.

We report persistent severe acute respiratory syndrome coronavirus 2 infection in a patient with HIV/AIDS; the virus developed spike N terminal domain and receptor binding domain neutralization resistance mutations. Our findings suggest that immunocompromised patients can harbor emerging variants of severe acute respiratory syndrome coronavirus 2.

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.

Plasma as an alternative COVID-19 diagnostic specimen in a hospitalized patient negative for SARS-CoV-2 by nasopharyngeal swab.

We present the case of an inpatient with pneumonia and repeatedly negative nasopharyngeal SARS-CoV-2 testing. In such challenging cases, alternative diagnostic options include lower respiratory tract and plasma SARS-CoV-2 RNA testing, of which the latter may be particularly useful where bronchoscopy is deferred due to clinical factors or transmission risk.

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.

High Frequency of SARS-CoV-2 RNAemia and Association With Severe Disease.

BACKGROUND: Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in blood, also known as RNAemia, has been reported, but its prognostic implications are poorly understood. This study aimed to determine the frequency of SARS-CoV-2 RNA in plasma and its association with coronavirus disease 2019 (COVID-19) clinical severity. METHODS: An analytical cross-sectional study was performed in a single-center tertiary care institution and included consecutive inpatients and outpatients with confirmed COVID-19. The prevalence of SARS CoV-2 RNAemia and the strength of its association with clinical severity variables were examined and included intensive care unit (ICU) admission, invasive mechanical ventilation, and 30-day all-cause mortality. RESULTS: Paired nasopharyngeal and plasma samples were included from 85 patients. The median age was 55 years, and individuals with RNAemia were older than those with undetectable SARS-CoV-2 RNA in plasma (63 vs 50 years; P = .04). Comorbidities were frequent including obesity (37.6%), hypertension (30.6%), and diabetes mellitus (22.4%). RNAemia was detected in 28/85 (32.9%) of patients, including 22/28 (78.6%) who required hospitalization. In models adjusted for age, RNAemia was detected more frequently in individuals who developed severe disease including ICU admission (32.1 vs 14.0%; P = .04) and invasive mechanical ventilation (21.4% vs 3.5%; P = .02). All 4 deaths occurred in individuals with detectable RNAemia. An additional 121 plasma samples from 28 individuals with RNAemia were assessed longitudinally, and RNA was detected for a maximum duration of 10 days. CONCLUSIONS: This study demonstrated a high proportion of SARS-CoV-2 RNAemia, and an association between RNAemia and clinical severity suggesting the potential utility of plasma viral testing as a prognostic indicator for COVID-19.