Distinct Laboratory Test Result Profiles Between SARS-CoV-2 and Seasonable Influenza Infected Patients.

OBJECTIVE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses are contagious respiratory pathogens with similar symptoms but require different treatment and management strategies. This study investigated the differences in laboratory test result profiles between SARS-CoV-2 and influenza infected patients upon presentation to emergency department (ED). METHODS: Laboratory test results and demographic information from 723 influenza positive (2018/1/1 to 2020/3/15) and 1,281 SARS-CoV-2 positive (2020/3/11 to 2020/6/30) ED patients were retrospectively analyzed. The dataset was randomly divided into a training/validation set (2/3) and a test set (1/3) with the same SARS-CoV-2/influenza ratio. Four machine learning models in differentiating the laboratory profiles of RT-PCR confirmed SARS-CoV-2 and influenza positive patients were evaluated. The Shapley Additive Explanations technique was employed to visualize the impact of laboratory tests on the overall differentiation. Furthermore, the model performance was also evaluated in a new test dataset including 519 SARS-CoV-2 ED patients (2020/12/1 to 2021/2/28) and the previous influenza positive patients (2018/1/1 to 2020/3/15). RESULTS: A laboratory test result profile consisting of 15 blood tests, together with patient age, gender, and race can discriminate the two types of viral infections using a random forest (RF) model. The RF model achieved an area under the receiver operating characteristic curve (AUC) of 0.90 in the test set. Among the profile of 15 laboratory tests, the serum total calcium level exhibited the greatest contribution to the overall differentiation. Furthermore, the model achieved an AUC of 0.81 in a new test set. CONCLUSION: We developed a laboratory tests-based RF model differentiating SARS-CoV-2 from influenza, which may be useful for the preparedness of overlapping COVID-19 resurgence and future seasonal influenza.

Machine Learning Highlights Downtrending of COVID-19 Patients with a Distinct Laboratory Profile

Background New York City (NYC) experienced an initial surge and gradual decline in the number of SARS-CoV-2-confirmed cases in 2020. A change in the pattern of laboratory test results in COVID-19 patients over this time has not been reported or correlated with patient outcome. Methods We performed a retrospective study of routine laboratory and SARS-CoV-2 RT-PCR test results from 5,785 patients evaluated in a NYC hospital emergency department from March to June employing machine learning analysis. Results A COVID-19 high-risk laboratory test result profile (COVID19-HRP), consisting of 21 routine blood tests, was identified to characterize the SARS-CoV-2 patients. Approximately half of the SARS-CoV-2 positive patients had the distinct COVID19-HRP that separated them from SARS-CoV-2 negative patients. SARS-CoV-2 patients with the COVID19-HRP had higher SARS-CoV-2 viral loads, determined by cycle threshold values from the RT-PCR, and poorer clinical outcome compared to other positive patients without the COVID12-HRP. Furthermore, the percentage of SARS-CoV-2 patients with the COVID19-HRP has significantly decreased from March/April to May/June. Notably, viral load in the SARS-CoV-2 patients declined, and their laboratory profile became less distinguishable from SARS-CoV-2 negative patients in the later phase. Conclusions Our longitudinal analysis illustrates the temporal change of laboratory test result profile in SARS-CoV-2 patients and the COVID-19 evolvement in a US epicenter. This analysis could become an important tool in COVID-19 population disease severity tracking and prediction. In addition, this analysis may play an important role in prioritizing high-risk patients, assisting in patient triaging and optimizing the usage of resources.

Examining key factors of beginner's continuance intention in blended learning in higher education.

With the outbreak of the COVID-19 pandemic, blended learning became exceptionally widespread, especially in higher education. As a result, many college students became beginners in this learning method. To identify key factors that impact beginners' continuance intention in blended learning, this study surveyed 1845 first-year college students at a university in central China in the falls of 2020 and 2021 who used blended learning for the first time. Structural equation modeling was employed to verify a model that integrates intrinsic motivation and academic self-efficacy in the Expectation-Confirmation Model of Information System Continuance. The results show that performance expectancy, intrinsic motivation, and satisfaction significantly impact beginners' continuance intention in blended learning. Moreover, performance expectancy, intrinsic motivation, and confirmation significantly impact beginners' continuance intention through mediating variable satisfaction. Academic self-efficacy does not directly impact college students' continuance intention but indirectly impacts their continuance intention through intrinsic motivation. Finally, this study provides suggestions for educators to improve beginners' blended learning experience thus promoting their continuance intention in blended learning.

Integrative metabolomic and proteomic signatures define clinical outcomes in severe COVID-19.

The coronavirus disease-19 (COVID-19) pandemic has ravaged global healthcare with previously unseen levels of morbidity and mortality. In this study, we performed large-scale integrative multi-omics analyses of serum obtained from COVID-19 patients with the goal of uncovering novel pathogenic complexities of this disease and identifying molecular signatures that predict clinical outcomes. We assembled a network of protein-metabolite interactions through targeted metabolomic and proteomic profiling in 330 COVID-19 patients compared to 97 non-COVID, hospitalized controls. Our network identified distinct protein-metabolite cross talk related to immune modulation, energy and nucleotide metabolism, vascular homeostasis, and collagen catabolism. Additionally, our data linked multiple proteins and metabolites to clinical indices associated with long-term mortality and morbidity. Finally, we developed a novel composite outcome measure for COVID-19 disease severity based on metabolomics data. The model predicts severe disease with a concordance index of around 0.69, and shows high predictive power of 0.83-0.93 in two independent datasets.

Measurements of SARS-CoV-2 antibody dissociation rate constant by chaotrope-free biolayer interferometry in serum of COVID-19 convalescent patients.

Kinetics measurements of antigen-antibody binding interactions are critical to understanding the functional efficiency of SARS-CoV-2 antibodies. Previously reported chaotrope-based avidity assays that rely on artificial disruption of binding do not reflect the natural binding kinetics. This study developed a chaotrope- and label-free biolayer interferometry (BLI) assay for the real-time monitoring of receptor binding domain (RBD) binding kinetics with SARS-CoV-2 spike protein in convalescent COVID-19 patients. An improved conjugation biosensor probe coated with streptavidin-polysaccharide (SA-PS) led to a six-fold increase of signal intensities and two-fold reduction of non-specific binding (NSB) compared to streptavidin only probe. Furthermore, by utilizing a separate reference probe and biotin-human serum albumin (B-HSA) blocking process to subtracted NSB signal in serum, this BLI biosensor can measure a wide range of the dissociation rate constant (koff), which can be measured without knowledge of the specific antibody concentrations. The clinical utility of this improved BLI kinetics assay was demonstrated by analyzing the koff values in sera of 24 pediatric (≤18 years old) and 63 adult (>18 years old) COVID-19 convalescent patients. Lower koff values for SARS-CoV-2 serum antibodies binding to RBD were measured in samples from children. This rapid, easy to operate and chaotrope-free BLI assay is suitable for clinical use and can be readily adapted to characterize SARS-CoV-2 antibodies developed by COVID-19 patients and vaccines.

Efficient Neutralization of SARS-CoV-2 Omicron and Other VOCs by a Broad Spectrum Antibody 8G3 (preprint)

Numerous mutations in the spike protein of SARS-CoV-2 B.1.1.529 Omicron variant pose a crisis for antibody-based immunotherapies. The efficacy of emergency use authorized (EUA) antibodies that developed in early SARS-CoV-2 pandemic seems to be in flounder. In this work, we examined the Omicron variant neutralization using an early B cell antibody repertoire as well as several EUA antibodies in pseudovirus and authentic virus systems. More than half of the antibodies in the repertoire that showed good activity against WA1/2020 previously had completely lost neutralizing activity against Omicron, while antibody 8G3 from our early B cell repertoire displayed non-regressive activity. EUA antibodies Etesevimab, Casirivimab, Imdevimab and Bamlanivimab neutralized authentic WA1/2020 virus with low half maximal inhibitory concentration (IC50) values, but were entirely desensitized by Omicron. Only Sotrovimab targeting the non-ACE2 overlap epitope showed activity but with a dramatic decrease. Interestingly, antibody 8G3 efficiently neutralized Omicron in pseudovirus and authentic virus systems. 8G3 also showed excellent activity against other variants of concern (VOCs). Collectively, our results suggest that neutralizing antibodies with breadth remains broad neutralizing activity in tackling SARS-CoV-2 infection despite the universal evasion from EUA antibodies by Omicron variant.

Comprehensive Evaluation of ACE2-Fc Combination with Neutralization Antibody on Broad Protection against SARS-CoV-2 and Its Variants (preprint)

Emerging SARS-CoV-2 variants are threatening the efficacy of antibody therapies. Combination treatments including ACE2-Fc have been developed to overcome the evasion of neutralizing antibodies (NAbs) in individual cases. Here we conducted a comprehensive evaluation of this strategy by combining ACE2-Fc with NAbs of diverse epitopes on the RBD. NAb+ACE2-Fc combinations efficiently neutralized HIV-based pseudovirus carrying the spike protein of the Delta or Omicron variants, achieving a balance between efficacy and breadth. In an antibody escape assay using replication-competent VSV-SARS-CoV-2-S, all the combinations had no escape after fifteen passages. By comparison, all the NAbs without combo with ACE2-Fc had escaped within six passages. Further, the VSV-S variants escaped from NAbs were neutralized by ACE2-Fc, revealing the mechanism of NAb+ACE2-Fc combinations survived after fifteen passages. We finally examined ACE2-Fc neutralization against pseudovirus variants resistant to the therapeutic antibodies that have currently been in clinic. Our results suggest ACE2-Fc is a universal combination partner to combat SARS-CoV-2 variants including Delta and Omicron.

Automatic Patient-Level Detection of Coronavirus Disease (COVID-19) Using Convolutional Neural Network from Lung CT Scans

The outbreak of 2019 novel coronavirus (COVID-19) has caused more than 176 million confirmed cases by June 14, 2021, and this number will continue to grow. Automatic and accurate COVID-19 detection/evaluation from the computed tomography (CT) scans is of great significance for COVID-19 diagnosis and treatment. Due to individual variations of patients and the influx of a large number of patients, the current clinical practices remain subject to shortcomings of potential high-risk and time-consumption issues from radiologists. In this paper, we propose a computer aided detection system to relieve the clinical physicians from tediously reading the CT images of COVID-19 patients. Particularly, a COVID-19 detection network (COVIDNet) is proposed using deep convolutional neural networks (DCNNs) for patient-level COVID-19 detection to distinguish infected and non-infected patients. The underlying method complementarily and comprehensively extract multi-level interplane volumetric correlation features of typical ground glass opacities (GGOs) lesions using 3D multi-Scale Network (MSN). To cover more GGO lesion features and reduce intra-class differences, a Phase Ensemble (PE) is proposed for aggregation of different phases in one CT scan. The proposed method is evaluated on a clinically established COVID-19 database with five-fold cross-validation. Experimental results show that the proposed framework achieves classification performance with the specificity of 1.0000, sensitivity of 0.9700, accuracy of 0.9850, precision of 1.0000, and Area Under the Curve (AUC) of 0.9980. All of these indicate that our method enables an efficient, accurate and reliable patient-level COVID-19 detection for clinical diagnosis. This can significantly improve the work efficiency of clinical physicians on COVID-19 patient diagnosis and evaluation in hospitals and clinics. Impact statement—The proposed method can automatically and accurately distinguish the COVID-19 patients from patient-level CT scan images. On a clinically established large-scale COVID-19 database with five-fold cross-validation, the experimental results show that the proposed framework achieves a classification performance with the specificity of 1.0000, sensitivity of 0.9700, accuracy of 0.9850, precision of 1.0000, and Area Under the Curve (AUC) of 0.9980. It can relieve the clinical physicians from tediously reading the CT images of COVID-19 patients. Thus, it can significantly improve the work efficiency of clinical physicians on COVID-19 patient diagnosis and evaluation in hospitals and clinics, particularly in the pandemic period of COVID-19.

Rapid, robust, and sustainable antibody responses to mRNA COVID-19 vaccine in convalescent COVID-19 individuals.

Longitudinal studies are needed to evaluate the SARS-CoV-2 mRNA vaccine antibody response under real-world conditions. This longitudinal study investigated the quantity and quality of SARS-CoV-2 antibody response in 846 specimens from 350 patients, comparing BNT162b2-vaccinated individuals (19 previously diagnosed with COVID-19, termed RecoVax; and 49 never diagnosed, termed NaiveVax) with 122 hospitalized unvaccinated (HospNoVax) and 160 outpatient unvaccinated (OutPtNoVax) COVID-19 patients. NaiveVax experienced delay in generating SARS-CoV-2 total antibodies (TAb) and surrogate neutralizing antibodies (SNAb) after the first vaccine dose (D1) but rapid increase in antibody levels after the second dose (D2). However, these never reached RecoVax's robust levels. In fact, NaiveVax TAb and SNAb levels decreased 4 weeks after D2. For the most part, RecoVax TAb persisted, after reaching maximal levels 2 weeks after D2, but SNAb decreased significantly about 6 months after D1. Although NaiveVax avidity lagged behind that of RecoVax for most of the follow-up periods, NaiveVax did reach similar avidity by about 6 months after D1. These data suggest that 1 vaccine dose elicits maximal antibody response in RecoVax and may be sufficient. Also, despite decreasing levels in TAb and SNAb over time, long-term avidity may be a measure worth evaluating and possibly correlating to vaccine efficacy.

Broad ultra-potent neutralization of SARS-CoV-2 variants by monoclonal antibodies specific to the tip of RBD (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) continue to wreak havoc across the globe. Higher transmissibility and immunologic resistance of VOCs bring unprecedented challenges to epidemic extinguishment. Here we describe a monoclonal antibody, 2G1, that neutralizes all current VOCs and has surprising tolerance to mutations adjacent to or within its interaction epitope. Cryo-electron microscopy structure showed that 2G1 bound to the tip of receptor binding domain (RBD) of spike protein with small contact interface but strong hydrophobic effect, which resulted in nanomolar to sub-nanomolar affinities to spike proteins. The epitope of 2G1 on RBD partially overlaps with ACE2 interface, which gives 2G1 ability to block interaction between RBD and ACE2. The narrow binding epitope but high affinity bestow outstanding therapeutic efficacy upon 2G1 that neutralized VOCs with sub-nanomolar IC50 in vitro. In SARS-CoV-2 and Beta- and Delta-variant-challenged transgenic mice and rhesus macaque models, 2G1 protected animals from clinical illness and eliminated viral burden, without serious impact to animal safety. Mutagenesis experiments suggest that 2G1 could be potentially capable of dealing with emerging SARS-CoV-2 variants in future. This report characterized the therapeutic antibodies specific to the tip of spike against SARS-CoV-2 variants and highlights the potential clinical applications as well as for developing vaccine and cocktail therapy.

Cytokine signatures of end organ injury in COVID-19.

Increasing evidence has shown that Coronavirus disease 19 (COVID-19) severity is driven by a dysregulated immunologic response. We aimed to assess the differences in inflammatory cytokines in COVID-19 patients compared to contemporaneously hospitalized controls and then analyze the relationship between these cytokines and the development of Acute Respiratory Distress Syndrome (ARDS), Acute Kidney Injury (AKI) and mortality. In this cohort study of hospitalized patients, done between March third, 2020 and April first, 2020 at a quaternary referral center in New York City we included adult hospitalized patients with COVID-19 and negative controls. Serum specimens were obtained on the first, second, and third hospital day and cytokines were measured by Luminex. Autopsies of nine cohort patients were examined. We identified 90 COVID-19 patients and 51 controls. Analysis of 48 inflammatory cytokines revealed upregulation of macrophage induced chemokines, T-cell related interleukines and stromal cell producing cytokines in COVID-19 patients compared to the controls. Moreover, distinctive cytokine signatures predicted the development of ARDS, AKI and mortality in COVID-19 patients. Specifically, macrophage-associated cytokines predicted ARDS, T cell immunity related cytokines predicted AKI and mortality was associated with cytokines of activated immune pathways, of which IL-13 was universally correlated with ARDS, AKI and mortality. Histopathological examination of the autopsies showed diffuse alveolar damage with significant mononuclear inflammatory cell infiltration. Additionally, the kidneys demonstrated glomerular sclerosis, tubulointerstitial lymphocyte infiltration and cortical and medullary atrophy. These patterns of cytokine expression offer insight into the pathogenesis of COVID-19 disease, its severity, and subsequent lung and kidney injury suggesting more targeted treatment strategies.

Interplay between nuclear factor erythroid 2-related factor 2 and inflammatory mediators in COVID-19-related liver injury.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 is a global pandemic and poses a major threat to human health worldwide. In addition to respiratory symptoms, COVID-19 is usually accompanied by systemic inflammation and liver damage in moderate and severe cases. Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that regulates the expression of antioxidant proteins, participating in COVID-19-mediated inflammation and liver injury. Here, we show the novel reciprocal regulation between NRF2 and inflammatory mediators associated with COVID-19-related liver injury. Additionally, we describe some mechanisms and treatment strategies.

TOP-Plus Is a Versatile Biosensor Platform for Monitoring SARS-CoV-2 Antibody Durability.

BACKGROUND: Low initial severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody titers dropping to undetectable levels within months after infection have raised concerns about long-term immunity. Both the antibody levels and the avidity of the antibody-antigen interaction should be examined to understand the quality of the antibody response. METHODS: A testing-on-a-probe "plus" panel (TOP-Plus) was developed to include a newly developed avidity assay built into the previously described SARS-CoV-2 TOP assays that measured total antibody (TAb), surrogate neutralizing antibody (SNAb), IgM, and IgG on a versatile biosensor platform. TAb and SNAb levels were compared with avidity in previously infected individuals at 1.3 and 6.2 months after infection in paired samples from 80 patients with coronavirus disease 2019 (COVID-19). Sera from individuals vaccinated for SARS-CoV-2 were also evaluated for antibody avidity. RESULTS: The newly designed avidity assay in this TOP panel correlated well with a reference Bio-Layer Interferometry avidity assay (r = 0.88). The imprecision of the TOP avidity assay was <10%. Although TAb and neutralization activity (by SNAb) decreased between 1.3 and 6.2 months after infection, the antibody avidity increased significantly (P < 0.0001). Antibody avidity in 10 SARS-CoV-2 vaccinated individuals (median: 28 days after vaccination) was comparable to the measured antibody avidity in infected individuals (median: 26 days after infection). CONCLUSIONS: This highly precise and versatile TOP-Plus panel with the ability to measure SARS-CoV-2 TAb, SNAb, IgG, and IgM antibody levels and avidity of individual sera on one sensor can become a valuable asset in monitoring not only patients infected with SARS-CoV-2 but also the status of individuals' COVID-19 vaccination response.

Postconvalescent SARS-CoV-2 IgG and Neutralizing Antibodies are Elevated in Individuals with Poor Metabolic Health.

PURPOSE: Comorbidities making up metabolic syndrome (MetS), such as obesity, type 2 diabetes, and chronic cardiovascular disease can lead to increased risk of coronavirus disease-2019 (COVID-19) with a higher morbidity and mortality. SARS-CoV-2 antibodies are higher in severely or critically ill COVID-19 patients, but studies have not focused on levels in convalescent patients with MetS, which this study aimed to assess. METHODS: This retrospective study focused on adult convalescent outpatients with SARS-CoV-2 positive serology during the COVID-19 pandemic at NewYork Presbyterian/Weill Cornell. Data collected for descriptive and correlative analysis included SARS-COV-2 immunoglobin G (IgG) levels and history of MetS comorbidities from April 17, 2020 to May 20, 2020. Additional data, including SARS-CoV-2 IgG levels, body mass index (BMI), hemoglobin A1c (HbA1c) and lipid levels were collected and analyzed for a second cohort from May 21, 2020 to June 21, 2020. SARS-CoV-2 neutralizing antibodies were measured in a subset of the study cohort. RESULTS: SARS-CoV-2 IgG levels were significantly higher in convalescent individuals with MetS comorbidities. When adjusted for age, sex, race, and time duration from symptom onset to testing, increased SARS-CoV-2 IgG levels remained significantly associated with obesity (P < 0.0001). SARS-CoV-2 IgG levels were significantly higher in patients with HbA1c ≥6.5% compared to those with HbA1c <5.7% (P = 0.0197) and remained significant on multivariable analysis (P = 0.0104). A positive correlation was noted between BMI and antibody levels [95% confidence interval: 0.37 (0.20-0.52) P < 0.0001]. Neutralizing antibody titers were higher in COVID-19 individuals with BMI ≥ 30 (P = 0.0055). CONCLUSION: Postconvalescent SARS-CoV-2 IgG and neutralizing antibodies are elevated in obese patients, and a positive correlation exists between BMI and antibody levels.

Association of Age With SARS-CoV-2 Antibody Response.

Importance: Accumulating evidence suggests that children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are more likely to manifest mild symptoms and are at a lower risk of developing severe respiratory disease compared with adults. It remains unknown how the immune response in children differs from that of adolescents and adults. Objective: To investigate the association of age with the quantity and quality of SARS-CoV-2 antibody responses. Design, Setting, and Participants: This cross-sectional study used 31 426 SARS-CoV-2 antibody test results from pediatric and adult patients. Data were collected from a New York City hospital from April 9 to August 31, 2020. The semiquantitative immunoglobin (Ig) G levels were compared between 85 pediatric and 3648 adult patients. Further analysis of SARS-CoV-2 antibody profiles was performed on sera from 126 patients aged 1 to 24 years. Main Outcomes and Measures: SARS-CoV-2 antibody positivity rates and IgG levels were evaluated in patients from a wide range of age groups (1-102 years). SARS-CoV-2 IgG level, total antibody (TAb) level, surrogate neutralizing antibody (SNAb) activity, and antibody binding avidity were compared between children (aged 1-10 years), adolescents (aged 11-18 years), and young adults (aged 19-24 years). Results: Among 31 426 antibody test results (19 797 [63.0%] female patients), with 1194 pediatric patients (mean [SD] age, 11.0 [5.3] years) and 30 232 adult patients (mean [SD] age, 49.2 [17.1] years), the seroprevalence in the pediatric (197 [16.5%; 95% CI, 14.4%-18.7%]) and adult (5630 [18.6%; 95% CI, 18.2%-19.1%]) patient populations was similar. The SARS-CoV-2 IgG level showed a negative correlation with age in the pediatric population (r = -0.45, P < .001) and a moderate but positive correlation with age in adults (r = 0.24, P < .001). Patients aged 19 to 30 years exhibited the lowest IgG levels (eg, aged 25-30 years vs 1-10 years: 99 [44-180] relative fluorescence units [RFU] vs 443 [188-851] RFU). In the subset cohort aged 1 to 24 years, IgG, TAb, SNAb and avidity were negatively correlated with age (eg, IgG: r = -0.51; P < .001). Children exhibited higher median (IQR) IgG levels, TAb levels, and SNAb activity compared with adolescents (eg, IgG levels: 473 [233-656] RFU vs 191 [82-349] RFU; P < .001) and young adults (eg, IgG levels: 473 [233-656] RFU vs 85 [38-150] RFU; P < .001). Adolescents also exhibited higher median (IQR) TAb levels, IgG levels, and SNAb activity than young adults (eg, TAb levels: 961 [290-2074] RFU vs 370 [125-697]; P = .006). In addition, children had higher antibody binding avidity compared with young adults, but the difference was not significant. Conclusions and Relevance: The results of this study suggest that SARS-CoV-2 viral specific antibody response profiles are distinct in different age groups. Age-targeted strategies for disease screening and management as well as vaccine development may be warranted.

Sensitive tracking of circulating viral RNA through all stages of SARS-CoV-2 infection.

BACKGROUNDCirculating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA may represent a more reliable indicator of infection than nasal RNA, but quantitative reverse transcription PCR (RT-qPCR) lacks diagnostic sensitivity for blood samples.METHODSA CRISPR-augmented RT-PCR assay that sensitively detects SARS-CoV-2 RNA was employed to analyze viral RNA kinetics in longitudinal plasma samples from nonhuman primates (NHPs) after virus exposure; to evaluate the utility of blood SARS-CoV-2 RNA detection for coronavirus disease 2019 (COVID-19) diagnosis in adults cases confirmed by nasal/nasopharyngeal swab RT-PCR results; and to identify suspected COVID-19 cases in pediatric and at-risk adult populations with negative nasal swab RT-qPCR results. All blood samples were analyzed by RT-qPCR to allow direct comparisons.RESULTSCRISPR-augmented RT-PCR consistently detected SARS-CoV-2 RNA in the plasma of experimentally infected NHPs from 1 to 28 days after infection, and these increases preceded and correlated with rectal swab viral RNA increases. In a patient cohort (n = 159), this blood-based assay demonstrated 91.2% diagnostic sensitivity and 99.2% diagnostic specificity versus a comparator RT-qPCR nasal/nasopharyngeal test, whereas RT-qPCR exhibited 44.1% diagnostic sensitivity and 100% specificity for the same blood samples. This CRISPR-augmented RT-PCR assay also accurately identified patients with COVID-19 using one or more negative nasal swab RT-qPCR results.CONCLUSIONResults of this study indicate that sensitive detection of SARS-CoV-2 RNA in blood by CRISPR-augmented RT-PCR permits accurate COVID-19 diagnosis, and can detect COVID-19 cases with transient or negative nasal swab RT-qPCR results, suggesting that this approach could improve COVID-19 diagnosis and the evaluation of SARS-CoV-2 infection clearance, and predict the severity of infection.TRIAL REGISTRATIONClinicalTrials.gov. NCT04358211.FUNDINGDepartment of Defense, National Institute of Allergy and Infectious Diseases, National Institute of Child Health and Human Development, and the National Center for Research Resources.

Testing-on-a-probe biosensors reveal association of early SARS-CoV-2 total antibodies and surrogate neutralizing antibodies with mortality in COVID-19 patients.

The association of mortality with the early humoral response to SARS-CoV-2 infection within the first few days after onset of symptoms (DAOS) has not been thoroughly investigated partly due to a lack of sufficiently sensitive antibody testing methods. Here we report two sensitive and automated testing-on-a-probe (TOP) biosensor assays for SARS-CoV-2 viral specific total antibodies (TAb) and surrogate neutralizing antibodies (SNAb), which are suitable for clinical use. The TOP assays employ an RBD-coated quartz probe using a Cy5-Streptavidin-polysacharide conjugate to improve sensitivity and minimize interference. Disposable cartridges containing pre-dispensed reagents require no liquid manipulation or fluidics during testing. The TOP-TAb assay exhibited higher sensitivity in the 0-7 DAOS window than a widely used FDA-EUA assay. The rapid and automated TOP-SNAb correlated well with two well-established SARS-CoV-2 virus neutralization tests. The clinical utility of the TOP assays was demonstrated by evaluating early antibody responses in 120 SARS-CoV-2 RT-PCR positive adult hospitalized patients. Higher TAb and SNAb positivity rates and more robust antibody responses at patient's initial hospital presentation were seen in inpatients who survived COVID-19 than those who died in the hospital. Survival analysis using the Cox Proportional Hazards Model showed that patients who had negative TAb and/or SNAb at initial hospital presentation were at a higher risk of in-hospital mortality. Furthermore, TAb and SNAb levels at presentation were inversely associated with SARS-CoV-2 viral load based on concurrent RT-PCR testing. Overall, the sensitive and automated TAb and SNAb assays allow the detection of early SARS-CoV-2 antibodies which associate with mortality.