Search

The extent of SARS-CoV-2 infection throughout the United States population is currently unknown. High quality serology is a key tool to understanding the spread of infection, immunity against the virus, and correlates of protection. Limited validation and testing of serology assays used for serosurveys can lead to unreliable or misleading data, and clinical testing using such unvalidated assays can lead to medically costly diagnostic errors and improperly informed public health decisions. Estimating prevalence and clinical decision making is highly dependent on specificity. Here, we present an optimized ELISA-based serology protocol from antigen production to data analysis. This protocol defines thresholds for IgG and IgM for determination of seropositivity with estimated specificity well above 99%. Validation was performed using both traditionally collected serum and dried blood on mail-in blood sampling kits, using archival (pre-2019) negative controls and known PCR-diagnosed positive patient controls. Minimal cross-reactivity was observed for the spike proteins of MERS, SARS1, OC43 and HKU1 viruses and no cross reactivity was observed with anti-influenza A H1N1 HAI titer during validation. This strategy is highly specific and is designed to provide good estimates of seroprevalence of SARS-CoV-2 seropositivity in a population, providing specific and reliable data from serosurveys and clinical testing which can be used to better evaluate and understand SARS-CoV-2 immunity and correlates of protection.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is characterized by respiratory distress, multiorgan dysfunction, and, in some cases, death. The pathological mechanisms underlying COVID-19 respiratory distress and the interplay with aggravating risk factors have not been fully defined. Lung autopsy samples from 18 patients with fatal COVID-19, with symptom onset-to-death times ranging from 3 to 47 days, and antemortem plasma samples from 6 of these cases were evaluated using deep sequencing of SARS-CoV-2 RNA, multiplex plasma protein measurements, and pulmonary gene expression and imaging analyses. Prominent histopathological features in this case series included progressive diffuse alveolar damage with excessive thrombosis and late-onset pulmonary tissue and vascular remodeling. Acute damage at the alveolar-capillary barrier was characterized by the loss of surfactant protein expression with injury to alveolar epithelial cells, endothelial cells, respiratory epithelial basal cells, and defective tissue repair processes. Other key findings included impaired clot fibrinolysis with increased concentrations of plasma and lung plasminogen activator inhibitor-1 and modulation of cellular senescence markers, including p21 and sirtuin-1, in both lung epithelial and endothelial cells. Together, these findings further define the molecular pathological features underlying the pulmonary response to SARS-CoV-2 infection and provide important insights into signaling pathways that may be amenable to therapeutic intervention.

In comparison to the general patient population, trauma patients show higher level detections of bloodborne infectious diseases, such as Hepatitis and Human Immunodeficiency Virus. In comparison to bloodborne pathogens, the prevalence of respiratory infections such as SARS-CoV-2 and how that relates with other variables, such as drug usage and trauma type, is currently unknown in trauma populations. Here, we evaluated SARS-CoV-2 seropositivity and antibody isotype profile in 2,542 trauma patients from six Level-1 trauma centers between April and October of 2020 during the first wave of the COVID-19 pandemic. We found that the seroprevalence in trauma victims 18-44 years old (9.79%, 95% confidence interval/CI: 8.33 11.47) was much higher in comparison to older patients (45-69 years old: 6.03%, 4.59-5.88; 70+ years old: 4.33%, 2.54 – 7.20). Black/African American (9.54%, 7.77 – 11.65) and Hispanic/Latino patients (14.95%, 11.80 – 18.75) also had higher seroprevalence in comparison, respectively, to White (5.72%, 4.62 7.05) and Non-Latino patients (6.55%, 5.57 – 7.69). More than half (55.54%) of those tested for drug toxicology had at least one drug present in their system. Those that tested positive for narcotics or sedatives had a significant negative correlation with seropositivity, while those on anti-depressants trended positive. These findings represent an important consideration for both the patients and first responders that treat trauma patients facing potential risk of respiratory infectious diseases like SARS-CoV-2.

Asymptomatic SARS-CoV-2 infection and delayed implementation of diagnostics have led to poorly defined viral prevalence rates. To address this, we analyzed seropositivity in US adults who have not previously been diagnosed with COVID-19. Individuals with characteristics that reflect the US population (n = 11,382) and who had not previously been diagnosed with COVID-19 were selected by quota sampling from 241,424 volunteers (ClinicalTrials.gov NCT04334954). Enrolled participants provided medical, geographic, demographic, and socioeconomic information and 9,028 blood samples. The majority (88.7%) of samples were collected between May 10(th) and July 31(st), 2020. Samples were analyzed via ELISA for anti-Spike and anti-RBD antibodies. Estimation of seroprevalence was performed by using a weighted analysis to reflect the US population. We detected an undiagnosed seropositivity rate of 4.6% (95% CI: 2.6 – 6.5%). There was distinct regional variability, with heightened seropositivity in locations of early outbreaks. Subgroup analysis demonstrated that the highest estimated undiagnosed seropositivity within groups was detected in younger participants (ages 18-45, 5.9%), females (5.5%), Black/African American (14.2%), Hispanic (6.1%), and Urban residents (5.3%), and lower undiagnosed seropositivity in those with chronic diseases. During the first wave of infection over the spring/summer of 2020 an estimate of 4.6% of adults had a prior undiagnosed SARS-CoV-2 infection. These data indicate that there were 4.8 (95% CI: 2.8-6.8) undiagnosed cases for every diagnosed case of COVID-19 during this same time period in the United States, and an estimated 16.8 million undiagnosed cases by mid-July 2020.

16.8 million SARS-CoV-2 infections in the US went undiagnosed in the first 6 months of the pandemic compared to 3.5 million diagnosed infections., Elucidating seroprevalence in COVID-19 Symptoms of SARS-CoV-2 infection range from completely asymptomatic, to those of a common cold, to a drop in oxygen saturation and lung function, and death in some patients. To evaluate the proportion of the U.S. population who had an undiagnosed infection during the first wave of the COVID-19 pandemic, we measured antibody prevalence in study participants who had not previously been diagnosed with a SARS-CoV-2 infection. By mid-July of 2020, 16.8 million people had an undiagnosed SARS-CoV-2 infection, almost five times the rate of diagnosed infections., Asymptomatic SARS-CoV-2 infection and delayed implementation of diagnostics have led to poorly defined viral prevalence rates in the United States and elsewhere. To address this, we analyzed seropositivity in 9089 adults in the United States who had not been diagnosed previously with COVID-19. Individuals with characteristics that reflected the U.S. population (n = 27,716) were selected by quota sampling from 462,949 volunteers. Enrolled participants (n = 11,382) provided medical, geographic, demographic, and socioeconomic information and dried blood samples. Survey questions coincident with the Behavioral Risk Factor Surveillance System survey, a large probability-based national survey, were used to adjust for selection bias. Most blood samples (88.7%) were collected between 10 May and 31 July 2020 and were processed using ELISA to measure seropositivity (IgG and IgM antibodies against SARS-CoV-2 spike protein and the spike protein receptor binding domain). The overall weighted undiagnosed seropositivity estimate was 4.6% (95% CI, 2.6 to 6.5%), with race, age, sex, ethnicity, and urban/rural subgroup estimates ranging from 1.1% to 14.2%. The highest seropositivity estimates were in African American participants; younger, female, and Hispanic participants; and residents of urban centers. These data indicate that there were 4.8 undiagnosed SARS-CoV-2 infections for every diagnosed case of COVID-19, and an estimated 16.8 million infections were undiagnosed by mid-July 2020 in the United States.

The current interest in microanalysis has heightened over the past years with the development of capillary electrophoresis (CE) followed by the development of commercially available micro-fluidic devices such as micro -mixers, CE chips and micro-reaction vessels or plates. This has made basic micro-fluidic analysis more readily available and has extended their use to biomedical analysis, especially clinically relevant biomarkers and field studies. Here the advantages of such devices are their relative speed of analysis, lower reagent costs, smaller sample requirements, and the potential for high-throughput. These factors become important when special situations arise such as the analysis of precious, archival, or field samples, monitoring surgical procedures, assessing newborns, analyzing specific areas from biopsy materials, measuring the functional aspects of single cells isolated from biological fluids or monitoring contamination of environmental factors. The combination of antibody-mediated isolation techniques with micro and nano-scale electrokinetic separations has great potential for analyzing defined analytes in complex biological matrices. In chip-based formats, such systems can recover and measure up to 25 analytes in a reasonably rapid time frame. Further, such devices require sub-microlitre amounts of sample to perform the analyses. Coupling these devices to laser-induced fluorescence greatly enhances the sensitivity of the analysis allowing certain analytes such as protein, peptides, and toxins to be measured in the sub-picogram/mL range. Further, coupling micro-analysis to mass spectrometry adds in the characterization of many significant biomarkers. The combination of fast binding, bio-engineered antibodies requiring relatively short reaction time with rapid desorption and electrophoretic separation with online detection can make the analysis almost “real-time”. Today, chip-based analyses are performed on a variety of devices ranging from simple microsample plates, to micro-mixers, and chip-based CE, many of which are commercially available. However, more complicated devices such as the “lab-on-a-chip” still require intricate design and specialized facilities. These latter devices hold the potential for automation and involve procedures that utilize both chromatographic and electrophoretic driving mechanisms. Additionally, a lab-on-a-chip can involve the integration of hyphenated techniques in order to achieve the desired analysis, including the integration of a highly sensitive detection system capable of measurement in the femtomolar or attomolar range. The need for such sensitivity often arises from the extremely small sample size obtained for the analysis.

To evaluate the effect of using the ferumoxides-poly-l-lysine (PLL) complex for magnetic cell labeling on the long-term viability, function, metabolism, and iron utilization of mammalian cells.PLL was incubated with ferumoxides for 60 minutes, incompletely coating the superparamagnetic iron oxide (SPIO) through electrostatic interactions. Cells were coincubated overnight with the ferumoxides-PLL complex, and iron uptake, cell viability, apoptosis indexes, and reactive oxygen species formation were evaluated. The disappearance or the life span of the detectable iron nanoparticles in cells was also evaluated. The iron concentrations in the media also were assessed at different time points. Data were expressed as the mean +/- 1 SD, and one-way analysis of variance and the unpaired Student t test were used to test for significant differences.Intracytoplasmic nanoparticles were stained with Prussian blue when the ferumoxides-PLL complex had magnetically labeled the human mesenchymal stem and HeLa cells. The long-term viability, growth rate, and apoptotic indexes of the labeled cells were unaffected by the endosomal incorporation of SPIO, as compared with these characteristics of the nonlabeled cells. In nondividing human mesenchymal stem cells, endosomal iron nanoparticles could be detected after 7 weeks; however, in rapidly dividing cells, intracellular iron had disappeared by five to eight divisions. A nonsignificant transient increase in reactive oxygen species production was seen in the human mesenchymal stem and HeLa cell lines. Labeled human mesenchymal stem cells did not differentiate to other lineage. A significant increase in iron concentration was observed in both the human mesenchymal stem and HeLa cell media at day 7.Magnetic cellular labeling with the ferumoxides-PLL complex had no short- or long-term toxic effects on tumor or stem cells.