Anti-nucleocapsid antibody levels and pulmonary comorbid conditions are linked to post-COVID-19 syndrome.

BACKGROUNDProlonged symptoms after SARS-CoV-2 infection are well documented. However, which factors influence development of long-term symptoms, how symptoms vary across ethnic groups, and whether long-term symptoms correlate with biomarkers are points that remain elusive.METHODSAdult SARS-CoV-2 reverse transcription PCR-positive (RT-PCR-positive) patients were recruited at Stanford from March 2020 to February 2021. Study participants were seen for in-person visits at diagnosis and every 1-3 months for up to 1 year after diagnosis; they completed symptom surveys and underwent blood draws and nasal swab collections at each visit.RESULTSOur cohort (n = 617) ranged from asymptomatic to critical COVID-19 infections. In total, 40% of participants reported at least 1 symptom associated with COVID-19 six months after diagnosis. Median time from diagnosis to first resolution of all symptoms was 44 days; median time from diagnosis to sustained symptom resolution with no recurring symptoms for 1 month or longer was 214 days. Anti-nucleocapsid IgG level in the first week after positive RT-PCR test and history of lung disease were associated with time to sustained symptom resolution. COVID-19 disease severity, ethnicity, age, sex, and remdesivir use did not affect time to sustained symptom resolution.CONCLUSIONWe found that all disease severities had a similar risk of developing post-COVID-19 syndrome in an ethnically diverse population. Comorbid lung disease and lower levels of initial IgG response to SARS-CoV-2 nucleocapsid antigen were associated with longer symptom duration.TRIAL REGISTRATIONClinicalTrials.gov, NCT04373148.FUNDINGNIH UL1TR003142 CTSA grant, NIH U54CA260517 grant, NIEHS R21 ES03304901, Sean N Parker Center for Allergy and Asthma Research at Stanford University, Chan Zuckerberg Biohub, Chan Zuckerberg Initiative, Sunshine Foundation, Crown Foundation, and Parker Foundation.

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.

Use of Outpatient-Derived COVID-19 Convalescent Plasma in COVID-19 Patients Before Seroconversion.

Background: Transfusion of COVID-19 convalescent plasma (CCP) containing high titers of anti-SARS-CoV-2 antibodies serves as therapy for COVID-19 patients. Transfusions early during disease course was found to be beneficial. Lessons from the SARS-CoV-2 pandemic could inform early responses to future pandemics and may continue to be relevant in lower resource settings. We sought to identify factors correlating to high antibody titers in convalescent plasma donors and understand the magnitude and pharmacokinetic time course of both transfused antibody titers and the endogenous antibody titers in transfused recipients. Methods: Plasma samples were collected up to 174 days after convalescence from 93 CCP donors with mild disease, and from 16 COVID-19 patients before and after transfusion. Using ELISA, anti-SARS-CoV-2 Spike RBD, S1, and N-protein antibodies, as well as capacity of antibodies to block ACE2 from binding to RBD was measured in an in vitro assay. As an estimate for viral load, viral RNA and N-protein plasma levels were assessed in COVID-19 patients. Results: Anti-SARS-CoV-2 antibody levels and RBD-ACE2 blocking capacity were highest within the first 60 days after symptom resolution and markedly decreased after 120 days. Highest antibody titers were found in CCP donors that experienced fever. Effect of transfused CCP was detectable in COVID-19 patients who received high-titer CCP and had not seroconverted at the time of transfusion. Decrease in viral RNA was seen in two of these patients. Conclusion: Our results suggest that high titer CCP should be collected within 60 days after recovery from donors with past fever. The much lower titers conferred by transfused antibodies compared to endogenous production in the patient underscore the importance of providing CCP prior to endogenous seroconversion.

Diagnosis of Dengue in a returning traveler from Pakistan suspected of COVID-19, California, USA.

Dengue and COVID-19 cocirculation presents a diagnostic conundrum for physicians evaluating patients with acute febrile illnesses, both in endemic regions and among returning travelers. We present a case of a returning traveler from Pakistan who, following repeated negative SARS-CoV-2 tests, was found to have a Dengue virus serotype 2 infection.

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.