Serum Of COVID-19 Patients Research Articles

SARS-CoV-2 infection impairs NK cell functions via activation of the LLT1-CD161 axis.

Natural killer (NK) cells plays a pivotal role in the control of viral infections, and their function depend on the balance between their activating and inhibitory receptors. The immune dysregulation observed in COVID-19 patients was previously associated with downregulation of NK cell numbers and function, yet the mechanism of inhibition of NK cell functions and the interplay between infected cells and NK cells remain largely unknown. In this study we show that SARS-CoV-2 infection of airway epithelial cells can directly influence NK cell phenotype and functions in the infection microenvironment. NK cells were co-cultured with SARS-CoV-2 infected epithelial cells, in a direct contact with A549ACE2/TMPRSS2 cell line or in a microenvironment of the infection in a 3D ex vivo human airway epithelium (HAE) model and NK cell surface expression of a set of most important receptors (CD16, NKG2D, NKp46, DNAM-1, NKG2C, CD161, NKG2A, TIM-3, TIGIT, and PD-1) was analyzed. We observed a selective, in both utilized experimental models, significant downregulation the proportion of CD161 (NKR-P1A or KLRB1) expressing NK cells, and its expression level, which was followed by a significant impairment of NK cells cytotoxicity level against K562 cells. What is more, we confirmed that SARS-CoV-2 infection upregulates the expression of the ligand for CD161 receptor, lectin-like transcript 1 (LLT1, CLEC2D or OCIL), on infected epithelial cells. LLT1 protein can be also detected not only in supernatants of SARS-CoV-2 infected A549ACE2/TMPRSS2 cells and HAE basolateral medium, but also in serum of COVID-19 patients. Finally, we proved that soluble LLT1 protein treatment of NK cells significantly reduces i) the proportion of CD161+ NK cells, ii) the ability of NK cells to control SARS-CoV-2 infection in A549ACE2/TMPRSS2 cells and iii) the production of granzyme B by NK cells and their cytotoxicity capacity, yet not degranulation level. We propose a novel mechanism of SARS-CoV-2 inhibition of NK cell functions via activation of the LLT1-CD161 axis.

Plasmablasts in previously immunologically naïve COVID-19 patients express markers indicating mucosal homing and secrete antibodies cross-reacting with SARS-CoV-2 variants and other beta-coronaviruses.

Antigen-specific class-switched antibodies are detected at the same time or even before IgM in serum of non-vaccinated individuals infected with SARS-CoV-2. These derive from the first wave of plasmablasts formed. Hence, the phenotype and specificity of plasmablasts can reveal information about early B-cell activation. Here we have analyzed B cells and plasmablasts circulating in blood of COVID-19 patients not previously exposed to SARS-CoV-2 during and after disease. We find that during infection with the original Wuhan strain, plasmablasts in blood produce IgA1, IgG1, and IgM, and that most express CCR10 and integrin β1, only some integrin β7, while the majority lack CCR9. Plasmablast-secreted antibodies are reactive to the spike (S) and nucleocapsid (N) proteins of the Wuhan strain as well as later variants of concern, but also bind S proteins from endemic and non-circulating betacoronaviruses. In contrast, after recovery, antibodies produced from memory B cells target variants of SARS-CoV-2 and SARS-CoV-1 but compared to previously non-infected individuals do not show increased binding to endemic coronaviruses. This suggests that the early antibody response to a large extent stems from pre-existing cross-reactive class-switched memory B cells, and that although newly formed memory cells target the novel SARS-CoV-2 virus the numbers of broadly cross-reactive memory B cells do not increase extensively. The observations give insight into the role of pre-existing memory B cells in early antibody responses to novel pathogens and may explain why class-switched antibodies are detected early in the serum of COVID-19 patients.

The concentrations of essential/toxic elements in serum of COVID-19 patients are not directly related to the severity of the disease

BackgroundIn recent months, the current COVID-19 pandemic has generated thousands of studies directly or indirectly related with this disease and/or the coronavirus SARS-CoV-2 causing the infection. On August 22, 2022, the database PUBMED included 287,639 publications containing the term COVID-19. However, in spite of the importance of trace elements in human health, including the immune system, data on the levels of metals/metalloids in COVID-19 patients is very limited. MethodsThe concentrations of As, Cd, Cr, Cu, Hg, Fe, Mg, Mn, Pb, Se, V and Zn were determined by inductively coupled plasma-mass spectrometry (ICP-MS) in 126 serum samples of individuals infected with SARS-CoV-2, as well as in 88 samples of non-infected individuals. Participants were divided into four groups: i) individuals COVID-19 positive (COVID-19 +) with an asymptomatic infection course; ii) individuals suffering mild COVID-19; iii) individuals suffering severe COVID-19, and iv) individuals COVID-19 negative (COVID-19-) (control group). The occurrence of the analyzed metals/metalloids was evaluated along with the biochemical profile, including blood cell counts, lipids, proteins and crucial enzymes. ResultsSerum levels of Mg, V, Cr, Cu, Cd, and Pb were higher in COVID-19 positive patients than those in the control group. Although no significant differences were observed between the different groups of patients, the concentrations of Cd, Pb, V and Zn showed a tendency to be higher in individuals with severe COVID-19 than in those showing mild symptoms or being asymptomatic. Arsenic and Hg were rarely detected, regardless if the subjects were infected by SARS-CoV-2, or not. The current results did not show significant differences in the levels of the rest of analyzed elements according to the severity of the disease (asymptomatic, mild and severe). ConclusionsIn spite of the results here obtained, we highlight the need to reduce the exposure to Cd, Pb and V to minimize the potential adverse health outcomes after COVID-19 infection. On the other hand, although a protective role of essential elements was not found, Mg and Cu concentrations were higher in severe COVID-19 patients than in non-infected individuals.

Inexpensive High-Throughput Multiplexed Biomarker Detection Using Enzymatic Metallization with Cellphone-Based Computer Vision.

Multiplexed biomarker detection can play a critical role in reliable and comprehensive disease diagnosis and prediction of outcome. Enzyme-linked immunosorbent assay (ELISA) is the gold standard method for immunobinding-based biomarker detection. However, this is currently expensive, limited to centralized laboratories, and usually limited to the detection of a single biomarker at a time. We present a low-cost, smartphone-based portable biosensing platform for high-throughput, multiplexed, sensitive, and quantitative detection of biomarkers from single, low-volume drops (<1 μL) of clinical samples. Biomarker binding to spotted capture antigens is converted, via enzymatic metallization, to the localized surface deposition of amplified, dry-stable, silver metal spots whose darkness is proportional to biomarker concentration. A custom smartphone application is developed, which uses real-time computer vision to enable easy optical detection of the deposited metal spots and sensitive and reproducible quantification of the biomarkers. We demonstrate the use of this platform for high-throughput, multiplexed detection of multiple viral antigen-specific antibodies from convalescent COVID-19 patient serum as well as vaccine-elicited antibody responses from uninfected vaccine-recipient serum and show that distinct multiplexed antibody fingerprints are observed among them.

Gas Chromatography-Mass Spectrometry Technology: Application in the Study of Inflammatory Mechanism in COVID-19 Patients.

SARS-CoV-2 infection in the human body induces a severe storm of inflammatory factors. However, its specific mechanism is still not clear. Gas chromatography–mass spectrometry (GC–MS) technology is expected to explain the possible mechanism of the disease by detecting differential metabolites. 15 COVID-19 patients and healthy controls were included in this study. Immune indicators such as hs CRP and cytokines were detected to reflect the level of inflammation in patients with COVID-19. The distribution of lymphocytes and subpopulations in peripheral whole blood were detected using flow cytometry to assess the immune function of COVID-19 patients. The expression of differential metabolites in serum was analyzed using GC–MS non-targeted metabolomics. The results showed that hs CRP, IL-5/6/8/10 and IFN-α in the serum of COVID-19 patients increased to varying degrees, and CD3/4/8+ T lymphocytes decreased. Additionally, 53 metabolites in the serum of COVID-19 patients were up regulated, 18 metabolites were down regulated, and 8 metabolites remained unchanged. Increased Cholesterol, Lactic Acid and 1-Monopalmitin may be the mechanism that causes excessive inflammation in COVID-19 patients. The increase of D-Allose may be involved in the process of lymphocyte decrease. In conclusion, the significance of our study is to reveal the possible mechanism of inflammatory response in patients with COVID-19 from the perspective of metabolomics. This provided a new idea for the treatment of COVID-19.

Quantitative detection of RAS and KKS peptides in COVID-19 patient serum by stable isotope dimethyl labeling LC-MS.

Angiotensin and kinin metabolic pathways are reported to be altered by many diseases, including COVID-19. Monitoring levels of these peptide metabolites is important for understanding mechanisms of disease processes. In this paper, we report dimethyl labeling of amines in peptides by addition of formaldehyde to samples and deutero-formaldehyde to internal standards to generate nearly identical isotopic standards with 4 m/z units larger per amine group than the corresponding analyte. We apply this approach to rapid, multiplexed, absolute LC-MS/MS quantitation of renin angiotensin system (RAS) and kallikrein-kinin system (KKS) peptides in human blood serum. Limits of detection (LODs) were obtained in the low pg mL-1 range with 3 orders of magnitude dynamic ranges, appropriate for determinations of normal and elevated levels of the target peptides in blood serum and plasma. Accuracy is within ±15% at concentrations above the limit of quantitation, as validated by spike-recovery in serum samples. Applicability was demonstrated by measuring RAS and KKS peptides in serum from COVID-19 patients, but is extendable to any class of peptides or other small molecules bearing reactive -NH2 groups.

Cardiovascular-related proteomic changes in ECFCs exposed to the serum of COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection significantly affects the cardiovascular system, causing vascular damage and thromboembolic events in critical patients. Endothelial dysfunction represents one of the first steps in response to COVID-19 that might lead to cardiovascular complications and long-term sequelae. However, despite the enormous efforts in the last two years, the molecular mechanisms involved in such processes remain poorly understood. Herein, we analyzed the protein changes taking place in endothelial colony forming cells (ECFCs) after the incubation with the serum from individuals infected with COVID-19, whether asymptomatic or critical patients, by application of a label free-quantitative proteomics approach. Specifically, ECFCs from healthy individuals were incubated ex-vivo with the serum of either COVID-19 negative donors (PCR-/IgG-, n:8), COVID-19 asymptomatic donors at different infective stages (PCR+/ IgG-, n:8and PCR-/IgG+, n:8), or hospitalized critical COVID-19 patients (n:8), followed by proteomics analysis. In total, 590 proteins were differentially expressed in ECFCs in response to all infected serums. Predictive analysis highlighted several proteins like CAPN5, SURF4, LAMP2 or MT-ND1, as highly discriminating features between the groups compared. Protein changes correlated with viral infection, RNA metabolism or autophagy, among others. Remarkably, the angiogenic potential of ECFCs in response to the infected serums was impaired, and many of the protein alterations in response to the serum of critical patients were associated with cardiovascular-related pathologies.

Comparison of Some Biochemical Parameters in Covid-19 Patients and Vaccinated Individuals in Kirkuk/Iraq.

Corona virus2019(Covid-19) or severe Acute respiratory syndrome coronavirus-2 (SARS-Cov-2) has been reported by the World Health Organization (WHO) are global clinical biomarkers have been important in determining its severity, although its aspect have been well characterized, so the aim of this study is to investigate some common alteration in some biochemical parameters in serum of covid-19patients (hospitalized and non-hospitalized, vaccinated and control group. Study Design: The study conducted across sectional study in cohort of 167(40 hospitalized covid-19 whom paid a visit to Al-Shifaa-14 hospital, Kirkuk, from Iraq, 30 non-hospitalized covid-19, 67vaccinated individuals and 30 control healthy group), from december20,2021 till 20 March 2022. Their ages ranging from (20-75) years. The total 167 patients ,79 males and 88 were females. Age, comorbidities, and abnormalities in several clinical biomarkers have all been shown to be important in determining the severity of covid-19 illness in several investigations. Although the clinical aspects of COVID-19 have been well characterized, the assessment of changes in the most common biochemical indicators reported in COVID-19 patients is still lacking. So the study aimed to investigate common alterations in some biochemical parameters in the serum of COVID-19 patients (hospitalized and non-hospitalized), vaccinated individuals and control group. Sampling: Blood samples were collected from 167 individuals, then centrifuged to obtain serum for the biochemical tests that included B. urea and creatinine, Glutamic-pyruvic transaminase(GPT), Glutamic-oxaloacetic transaminase(GOT) and alkaline phosphatase (ALP). Nasopharyngeal swabs were collected from non-hospitalized of COVID-19 infection which was confirmed using real-time PCR technique. The study's findings revealed substantial variations in biochemical parameters between the examined groups in terms of creatinine (value &gt;0.001) and urea (value &gt;0.0001), as well as significant differences in GOT (value &gt;0.0001) and ALP (value &gt;0.0001). So this study can have concluded that patients infected with Covid-19 (hospitalized and infected outside the hospital- subsequently) have an increase in most biochemical parameters, however the effect was lower in the vaccinated group.

Detection of unusual high molecular form of albumin in blood serum of COVID-19 patients

Blood sera of 12 severe Covid-19 patients and 14 healthy human donors were subjected to original TCA-extraction/acetone-precipitation followed by SDS-PAAG electrophoresis and mass-spectrometry. 76 kDa protein was detected as one of the differentially expressed proteins in the samples of Covid-19 patients. This 76 kDa protein was identified with mass-spectrometry as human serum albumin. Such molecular form of albumin was absent in blood serum of healthy human donors. The potential ways of generation of the unusual form of human serum albumin and its probable diagnostic value were discussed. Keywords: acetone-precipitation, biomarker proteins, COVID-19, electrophoresis, mass-spectrometry, TCA-extraction, unusual form of human serum albumin

Evaluation of Interlukein-6 and Vitamin D in Patients with COVID-19

COVID-19 is a unique viral infectious illness that causes a variety of symptoms and health hazards, particularly to the respiratory system and has been declared a worldwide pandemic. The disease is characterized by a cytokine release in severe conditions. Interleukin-6 (IL-6), a proinflammatory cytokine, mediates an important immunomodulatory process. Also, vitamin D was identified to have a role in the innate immunity of individuals. Our study was designed to find the role of IL-6 and vitamin D in COVID-19 patients, as well as, to see whether there is a link between vitamin D deficiency and cytokine syndrome development. The study included 90 COVID-19 patients and 30 control people from Baghdad, Iraq. The age of the participants was non-significantly (P&gt;0.05) different between COVID-19 and control, so was the body mass index. IL-6 level was significantly (P&lt;0.05) higher in COVID-19 patients compared to control, while vitamin D level was significantly (P&lt;0.05) reduced in COVID-19 patients. Nevertheless, the results have shown no significant correlation between vitamin D and IL-6 in the serum of COVID-19 patients. Furthermore, both IL-6 and vitamin D exhibited an excellent sensitivity in the diagnosis of COVID-19.

Tumor biomarkers CEA, CA19.9, CA15.3 and AFP levels in the serum of patients with COVID-19

Early diagnosis is very important to reduce morbidity and mortality in COVID-19 infected patients. The aim of this study was to detect of tumor antigens CEA, CA19.9, CA15.3, and AFP and to compare their levels in the serum of 69 COVID-19 patients and 69 healthy individuals who did not have COVID-19. The levels of CEA, CA19.9, CA15.3, and AFP in the serum were measured using ELISA. The levels of the tumor biomarkers in the serum of COVID-19 patients were found to be 7.74 ± 4.65 ng/ml for CEA, 29.33 ± 16.35 U/ml for CA19.9, 23.24 ± 13.48 U/ml for CA15.3 and 7.46 ± 5.57 ng/ml for AFP, while in the serum of healthy control patients 9.73 ± 43.58 ng/ml for CEA, 20.66 ± 11.1 for CA19.9, 19.64 ± 10.99 U/ml for CA15.3, and 3.83 ± 9.20 ng/ml for AFP, indicating no differences in the levels of the studied tumor biomarkers in the two experimental groups. It is concluded that tumor biomarkers CEA, CA19.9, CA15.3, and AFP cannot be used as effective screening tools for patients with COVID-19.

Potential Predictive Value of miR-125b-5p, miR-155-5p and Their Target Genes in the Course of COVID-19.

PurposeThis study aimed to provide new biomarkers for predicting the disease course of COVID-19 by analyzing the dynamic changes of microRNA (miRNA) and its target gene expression in the serum of COVID-19 patients at different stages.MethodsSerum samples were collected from all COVID-19 patients at three time points: the acute stage, the turn-negative stage, and the recovery stage. The expression level of miRNA and the target mRNA was measured by Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR). The classification tree model was established to predict the disease course, and the prediction efficiency of independent variables in the model was analyzed using the receiver operating characteristic (ROC) curve.ResultsThe expression of miR-125b-5p and miR-155-5p was significantly up-regulated in the acute stage and gradually decreased in the turn-negative and recovery stages. The expression of the target genes CDH5, STAT3, and TRIM32 gradually down-regulated in the acute, turn-negative, and recovery stages. MiR-125b-5p, miR-155-5p, STAT3, and TRIM32 constituted a classification tree model with 100% accuracy of prediction and AUC >0.7 for identification and prediction in all stages.ConclusionMiR-125b-5p, miR-155-5p, STAT3, and TRIM32 could be useful biomarkers to predict the time nodes of the acute, turn-negative, and recovery stages of COVID-19.

SARS-CoV-2 Nucleocapsid Protein is Associated With Lower Testosterone Levels: An Experimental Study.

The ongoing COVID-19 pandemic represents an extra burden in the majority of public and private health systems worldwide beyond the most pessimistic expectations, driving an urgent rush to develop effective vaccines and effective medical treatments against the SARS-CoV-2 pandemic. The Nucleocapsid structural viral protein is remarkably immunogenic and hugely expressed during infection. High IgG antibodies against Nucleocapsid protein (N protein) levels were detected in the serum of COVID-19 patients, confirming its pivotal antigen role for a T lymphocyte response in a vaccine microenvironment. Currently, adverse events associated with immunizations have raised some degree of concern, irrespective of its huge benefits in dealing with disease severity and decreasing mortality and morbidity. This hitherto study evaluates histological changes in rats’ testes, epididymis, prostate, and seminal vesicles and analyzes hormone levels after solely N protein inoculation. Therefore, we exposed a group of Lewis rats to weekly injections of the recombinant N protein for 28 days, while a control group was inoculated with a buffer solution. The N group revealed a more significant number of spermatozoa. Spermatozoa in the seminiferous tubules were counted in twenty 400 × microscopy fields (mean of 9.2 vs. 4.6 in the control group; p < 0,01), but significantly lower testosterone levels (mean of 125.70 ng/dl vs. 309,00 ng/dl in the control group; p < 0,05) were found. No other histological and biochemical changes were displayed. Conclusively, these data suggest testicular hormonal imbalance mediated by the SARS-CoV-2 N protein that could be linked to reported post-COVID-19 syndrome hypogonadism. More relevant research might be performed to confirm this viral antigen’s deleterious mechanism in the human testicular microenvironment, particular in Leydig cell function.

COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants

As global vaccination campaigns against SARS-CoV-2 proceed, there is particular interest in the longevity of immune protection, especially with regard to increasingly infectious virus variants. Neutralizing antibodies (Nabs) targeting the receptor binding domain (RBD) of SARS-CoV-2 are promising correlates of protective immunity and have been successfully used for prevention and therapy. As SARS-CoV-2 variants of concern (VOCs) are known to affect binding to the ACE2 receptor and by extension neutralizing activity, we developed a bead-based multiplex ACE2-RBD inhibition assay (RBDCoV-ACE2) as a highly scalable, time-, cost-, and material-saving alternative to infectious live-virus neutralization tests. By mimicking the interaction between ACE2 and the RBD, this serological multiplex assay allows the simultaneous analysis of ACE2 binding inhibition to the RBDs of all SARS-CoV-2 VOCs and variants of interest (VOIs) in a single well. Following validation against a classical virus neutralization test and comparison of performance against a commercially available assay, we analyzed 266 serum samples from 168 COVID-19 patients of varying severity. ACE2 binding inhibition was reduced for ten out of eleven variants examined compared to wild-type, especially for those displaying the E484K mutation such as VOCs beta and gamma. ACE2 binding inhibition, while highly individualistic, positively correlated with IgG levels. ACE2 binding inhibition also correlated with disease severity up to WHO grade 7, after which it reduced.

Identifying Immunological and Clinical Predictors of COVID-19 Severity and Sequelae by Mathematical Modeling.

Since its emergence as a pandemic in March 2020, coronavirus disease (COVID-19) outcome has been explored via several predictive models, using specific clinical or biochemical parameters. In the current study, we developed an integrative non-linear predictive model of COVID-19 outcome, using clinical, biochemical, immunological, and radiological data of patients with different disease severities. Initially, the immunological signature of the disease was investigated through transcriptomics analysis of nasopharyngeal swab samples of patients with different COVID-19 severity versus control subjects (exploratory cohort, n=61), identifying significant differential expression of several cytokines. Accordingly, 24 cytokines were validated using a multiplex assay in the serum of COVID-19 patients and control subjects (validation cohort, n=77). Predictors of severity were Interleukin (IL)-10, Programmed Death-Ligand-1 (PDL-1), Tumor necrosis factors-α, absolute neutrophil count, C-reactive protein, lactate dehydrogenase, blood urea nitrogen, and ferritin; with high predictive efficacy (AUC=0.93 and 0.98 using ROC analysis of the predictive capacity of cytokines and biochemical markers, respectively). Increased IL-6 and granzyme B were found to predict liver injury in COVID-19 patients, whereas interferon-gamma (IFN-γ), IL-1 receptor-a (IL-1Ra) and PD-L1 were predictors of remarkable radiological findings. The model revealed consistent elevation of IL-15 and IL-10 in severe cases. Combining basic biochemical and radiological investigations with a limited number of curated cytokines will likely attain accurate predictive value in COVID-19. The model-derived cytokines highlight critical pathways in the pathophysiology of the COVID-19 with insight towards potential therapeutic targets. Our modeling methodology can be implemented using new datasets to identify key players and predict outcomes in new variants of COVID-19.

Innovative FO-SPR Label-free Strategy for Detecting Anti-RBD Antibodies in COVID-19 Patient Serum and Whole Blood.

The ongoing COVID-19 pandemic has emphasized the urgent need for rapid, accurate, and large-scale diagnostic tools. Next to this, the significance of serological tests (i.e., detection of SARS-CoV-2 antibodies) also became apparent for studying patients’ immune status and past viral infection. In this work, we present a novel approach for not only measuring antibody levels but also profiling of binding kinetics of the complete polyclonal antibody response against the receptor binding domain (RBD) of SARS-CoV-2 spike protein, an aspect not possible to achieve with traditional serological tests. This fiber optic surface plasmon resonance (FO-SPR)-based label-free method was successfully accomplished in COVID-19 patient serum and, for the first time, directly in undiluted whole blood, omitting the need for any sample preparation. Notably, this bioassay (1) was on par with FO-SPR sandwich bioassays (traditionally regarded as more sensitive) in distinguishing COVID-19 from control samples, irrespective of the type of sample matrix, and (2) had a significantly shorter time-to-result of only 30 min compared to >1 or 4 h for the FO-SPR sandwich bioassay and the conventional ELISA, respectively. Finally, the label-free approach revealed that no direct correlation was present between antibody levels and their kinetic profiling in different COVID-19 patients, as another evidence to support previous hypothesis that antibody-binding kinetics against the antigen in patient blood might play a role in the COVID-19 severity. Taking all this into account, the presented work positions the FO-SPR technology at the forefront of other COVID-19 serological tests, with a huge potential toward other applications in need for quantification and kinetic profiling of antibodies.

Th1, Th2 and Th17 inflammatory pathways synergistically predict cardiometabolic protein expression in serum of COVID-19 patients.

A predominant source of complication in SARS-CoV-2 patients arises from a severe systemic inflammation that can lead to tissue damage and organ failure. The high inflammatory burden of this viral infection often results in cardiovascular comorbidities. A better understanding of the interaction between immune pathways and cardiovascular proteins might inform medical decisions and therapeutic approaches. In this study we hypothesized that helper T-cell inflammatory pathways (Th1, Th2 and Th17) synergistically correlate with cardiometabolic proteins in serum of COVID-19 patients. We found that Th1, Th2 and Th17 cytokines and chemokines are able to predict expression of 186 cardiometabolic proteins profiled by Olink proteomics.

Untargeted metabolomics of COVID-19 patient serum reveals potential prognostic markers of both severity and outcome

IntroductionThe diagnosis of COVID-19 is normally based on the qualitative detection of viral nucleic acid sequences. Properties of the host response are not measured but are key in determining outcome. Although metabolic profiles are well suited to capture host state, most metabolomics studies are either underpowered, measure only a restricted subset of metabolites, compare infected individuals against uninfected control cohorts that are not suitably matched, or do not provide a compact predictive model.ObjectivesHere we provide a well-powered, untargeted metabolomics assessment of 120 COVID-19 patient samples acquired at hospital admission. The study aims to predict the patient’s infection severity (i.e., mild or severe) and potential outcome (i.e., discharged or deceased).MethodsHigh resolution untargeted UHPLC-MS/MS analysis was performed on patient serum using both positive and negative ionization modes. A subset of 20 intermediary metabolites predictive of severity or outcome were selected based on univariate statistical significance and a multiple predictor Bayesian logistic regression model was created.ResultsThe predictors were selected for their relevant biological function and include deoxycytidine and ureidopropionate (indirectly reflecting viral load), kynurenine (reflecting host inflammatory response), and multiple short chain acylcarnitines (energy metabolism) among others. Currently, this approach predicts outcome and severity with a Monte Carlo cross validated area under the ROC curve of 0.792 (SD 0.09) and 0.793 (SD 0.08), respectively. A blind validation study on an additional 90 patients predicted outcome and severity at ROC AUC of 0.83 (CI 0.74–0.91) and 0.76 (CI 0.67–0.86).ConclusionPrognostic tests based on the markers discussed in this paper could allow improvement in the planning of COVID-19 patient treatment.

371. Estimating SARS-CoV-2 Seroprevalence from Spent Blood Samples, January–March 2021

BackgroundMeasuring SARS-CoV-2 antibody prevalence in spent samples at serial time points can determine seropositivity in a diverse pool of individuals to inform understanding of trends as vaccinations are implemented.MethodsBlood samples collected for clinical testing and then discarded ("spent samples") were obtained from the clinical laboratory of a medical center in Atlanta. A convenience sample of spent samples from both inpatients (medical/surgical floors, intensive care, obstetrics) and outpatients (clinics and ambulatory surgery) were collected one day per week from January-March 2021. Samples were matched to clinical data from the electronic medical record. In-house single dilution serological assays for SARS-CoV-2 receptor binding domain (RBD) and nucleocapsid (N) antibodies were developed and validated using pre-pandemic and PCR-confirmed COVID-19 patient serum and plasma samples (Figure 1). ELISA optical density (OD) cutoffs for seroconversion were chosen using receiver operating characteristic analysis with areas under the curve for all four assays greater than 0.95 after 14 days post symptom onset. IgG profiles were defined as natural infection (RBD and N positive) or vaccinated (RBD positive, N negative). Figure 1. Nucleocapsid serology assay validation Single dilution serological assays for SARS-CoV-2 nucleocapsid antibodies were validated using pre-pandemic and PCR-confirmed COVID-19 patient serum and plasma samples. ELISA optical density (OD) cutoffs for seroconversion were chosen using receiver operating characteristic (ROC) analysis with areas under the curve (AUC) for all four assays greater than 0.95 after 14 days post symptom onset.ResultsA total of 2406 samples were collected from 2132 unique patients. Median age was 58 years (IQR 40-70), with 766 (36%) ≥ 65 years. The majority were female (1173, 55%), and 1341 (63%) were Black. Median Elixhauser comorbidity index was 5 (IQR 2-9). 210 (9.9%) patients ever had SARS-CoV-2 detected by PCR, and 191 (9.0%) received a COVID-19 vaccine within the health system. Nearly half (1186/2406, 49.3%) of samples were collected from inpatient units, 586 (24.4%) from outpatient labs, 403 (16.8%) from the emergency department, and 231 (9.6%) from infusion centers. Overall, 17.0% had the IgG natural infection profile, while 16.2% had a vaccination profile. Prevalence estimates for IgG due to natural infection ranged from 24.0% in week 2 to 9.7% in week 5, and for IgG due to vaccine from 4.4% in week 2 to 32.0% in week 6 (Table, Figure 2).Table. SARS-CoV-2 antibody seropositivity by week of sample collection for spent routine blood chemistry samples. RBD = receptor binding domain. N = nucleocapsid. Seropositivity defined by enzyme-linked immunoassay (ELISA) optical density cutoffs selected using receiver operating characteristic analysis with areas under the curve (AUC) for all four assays greater than 0.95 after 14 days post symptom onset. IgG defined as positive if both RBD and N seropositive.Figure 2. RBD and Nucleocapsid seropositivity to differentiate natural infection vs. vaccination by week of sample collection. RBD = receptor binding domain. N = nucleocapsid. Seropositivity defined by enzyme-linked immunoassay (ELISA) optical density cutoffs selected using receiver operating characteristic analysis with areas under the curve (AUC) for all four assays greater than 0.95 after 14 days post symptom onset.ConclusionEstimated SARS-CoV-2 IgG seroprevalence among patients at a medical center from January-March 2021 was 17% by natural infection, and 16% by vaccination. Weekly trends likely reflect community spread and vaccine uptake. Disclosures Daniel Graciaa, MD, MPH, MSc, Critica, Inc (Consultant)

Elevated SARS-Cov-2-Specific IgM Levels Indicate Clinically Unfavorable Outcomes in Patients with COVID-19: A Retrospective Cohort Study

BackgroundCOVID-19 outbreak began in Wuhan and pandemics occur. Although SARS-CoV-2-specific immunoglobulins have been detected in serum of COVID-19 patients, their dynamics and association with outcomes have not been fully characterized.MethodsThis retrospective cohort study investigated the association between SARS-CoV-2-specific immunoglobulins and clinical outcomes of COVID-19 patients. We recruited 137 participants who were diagnosed with COVID-19 in four wards of the Tongji Hospital in Wuhan, China. Among the 137 participants, 81 patients were recovered, 23 patients died, and 33 patients remained hospitalized by the end of the study. SARS-CoV-2-specific immunoglobulins were analyzed by chemiluminescence assays. Laboratory and radiological characteristics, and clinical outcomes were compared between the recovered group and the deceased group. Furthermore, a matched cohort study was conducted in which each non-survivor was matched to two recovered patients of similar age.ResultsSARS-CoV-2-specific IgM levels peaked in the fourth week after the onset of COVID-19, while serum IgG levels rose earlier and remained high up to the eighth week. In the age-matched cohort study, the serum IgM, but not IgG levels, were higher among the non-survivors than in the recovered group (P = 0.006). The area under the ROC curve for the IgM and IgG levels was 0.702 (95% CI: 0.560–0.845, P = 0.006) and 0.596 (95% confidence interval: 0.449–0.744, P = 0.194), respectively. We also showed that patients with COVID-19 who had high IgM or IgG levels (stratified according to best cut-off) exhibited significantly lower overall survival (Kaplan–Meier survival curves, P < 0.05).DiscussionThese results indicate the association between immunoglobulins and outcome in patients with COVID-19 and demonstrated that elevated serum IgM levels could indicate poor outcomes in patients with COVID-19. Further, the information about the profile of SARS-CoV-2-specific IgGs may be useful for the future epidemiological investigations of COVID-19 therapies.