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1.
Curr Protoc ; 2(10): e521, 2022 Oct.
Article in English | MEDLINE | ID: covidwho-2047527

ABSTRACT

Antibody detection assays are essential for evaluating immunity of individuals against a given virus, and this has been particularly relevant during the COVID-19 pandemic. Current serology assays either require a laboratory setting and take >1 hr (i.e., enzyme-linked immunosorbent assay [ELISA]) or are rapid but only qualitative in nature and cannot accurately track antibody levels over time (i.e., lateral flow assay [LFA]). Therefore, there is a need for development of a rapid and simple but also quantitative assay that can evaluate antibody levels in patients accurately over time. We have developed an assay that uses a split nanoluciferase fused to the spike or nucleocapsid proteins of the SARS-CoV-2 virus to enable luminescent-based detection of spike- or nucleocapsid-binding antibodies in serum, plasma, and whole blood samples. The resulting approach is simple, rapid, and quantitative and is highly amenable to low-/medium-throughput scale using plate-based assays, high-throughput scale using robotics, and point-of-care applications. In this article, we describe how to perform the assay in a laboratory setting using a plate reader or liquid-handling robotics and in a point-of-care setting using a handheld, battery-powered luminometer. Together, these assays allow antibody detection to be easily performed in multiple settings by simplifying and reducing assay time in a laboratory or clinical environment and by allowing for antibody detection in point-of-care, nonlaboratory settings. © 2022 Wiley Periodicals LLC. Basic Protocol: SARS-CoV-2 antibody detection using the split-luciferase assay on a medium-throughput scale with a laboratory luminometer Alternate Protocol 1: High-throughput-based protocol for SARS-CoV-2 antibody detection using a robotic platform Alternate Protocol 2: Point-of-care-based protocol for SARS-CoV-2 antibody detection using a handheld luminometer Support Protocol: Determining positive/negative cutoffs for test samples and standardizing the assay between days.


Subject(s)
Biosensing Techniques , COVID-19 , Antibodies, Viral/analysis , COVID-19/diagnosis , Clinical Laboratory Techniques/methods , Humans , Luciferases , Nucleocapsid Proteins , Pandemics , SARS-CoV-2 , Sensitivity and Specificity
2.
JCI Insight ; 7(13)2022 07 08.
Article in English | MEDLINE | ID: covidwho-1932894

ABSTRACT

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.


Subject(s)
COVID-19 , COVID-19/complications , Humans , Immunoglobulin G , SARS-CoV-2 , Post-Acute COVID-19 Syndrome
3.
Cell ; 184(2): 323-333.e9, 2021 01 21.
Article in English | MEDLINE | ID: covidwho-956964

ABSTRACT

The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic, and pre-symptomatic carriers of the virus. CRISPR diagnostics can augment gold-standard PCR-based testing if they can be made rapid, portable, and accurate. Here, we report the development of an amplification-free CRISPR-Cas13a assay for direct detection of SARS-CoV-2 from nasal swab RNA that can be read with a mobile phone microscope. The assay achieved ∼100 copies/µL sensitivity in under 30 min of measurement time and accurately detected pre-extracted RNA from a set of positive clinical samples in under 5 min. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity and directly quantified viral load using enzyme kinetics. Integrated with a reader device based on a mobile phone, this assay has the potential to enable rapid, low-cost, point-of-care screening for SARS-CoV-2.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , Cell Phone/instrumentation , Optical Imaging/methods , RNA, Viral/analysis , Viral Load/methods , Animals , COVID-19 Nucleic Acid Testing/economics , COVID-19 Nucleic Acid Testing/instrumentation , CRISPR-Cas Systems , Cell Line , Coronavirus Nucleocapsid Proteins/genetics , Humans , Nasopharynx/virology , Optical Imaging/instrumentation , Phosphoproteins/genetics , Point-of-Care Testing , RNA Interference , RNA, Viral/genetics , Sensitivity and Specificity , Viral Load/economics , Viral Load/instrumentation
4.
EClinicalMedicine ; 27: 100518, 2020 Oct.
Article in English | MEDLINE | ID: covidwho-730421

ABSTRACT

BACKGROUND: Most data on the clinical presentation, diagnostics, and outcomes of patients with COVID-19 have been presented as case series without comparison to patients with other acute respiratory illnesses. METHODS: We examined emergency department patients between February 3 and March 31, 2020 with an acute respiratory illness who were tested for SARS-CoV-2. We determined COVID-19 status by PCR and metagenomic next generation sequencing (mNGS). We compared clinical presentation, diagnostics, treatment, and outcomes. FINDINGS: Among 316 patients, 33 tested positive for SARS-CoV-2; 31 without COVID-19 tested positive for another respiratory virus. Among patients with additional viral testing (27/33), no SARS-CoV-2 co-infections were identified. Compared to those who tested negative, patients with COVID-19 reported longer symptoms duration (median 7d vs. 3d, p < 0.001). Patients with COVID-19 were more often hospitalized (79% vs. 56%, p = 0.014). When hospitalized, patients with COVID-19 had longer hospitalizations (median 10.7d vs. 4.7d, p < 0.001) and more often developed ARDS (23% vs. 3%, p < 0.001). Most comorbidities, medications, symptoms, vital signs, laboratories, treatments, and outcomes did not differ by COVID-19 status. INTERPRETATION: While we found differences in clinical features of COVID-19 compared to other acute respiratory illnesses, there was significant overlap in presentation and comorbidities. Patients with COVID-19 were more likely to be admitted to the hospital, have longer hospitalizations and develop ARDS, and were unlikely to have co-existent viral infections. FUNDING: National Center for Advancing Translational Sciences, National Heart Lung Blood Institute, National Institute of Allergy and Infectious Diseases, Chan Zuckerberg Biohub, Chan Zuckerberg Initiative.

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