Undiagnosed COVID-19 in households with a child with mitochondrial disease

BackgroundThe impact of the COVID-19 pandemic on medically fragile populations, who are at higher risk of severe illness and sequelae, has not been well characterized. Viral infection is a major cause of morbidity in children with mitochondrial disease (MtD), and the COVID-19 pandemic represents an opportunity to study this vulnerable population. MethodsA convenience sampling cross-sectional serology study was conducted (October 2020 to June 2021) in households (N = 20) containing a child with MtD (N = 22). Samples (N = 83) were collected in the home using a microsampling apparatus and shipped to investigators. Antibodies against SARS-CoV-2 nucleocapsid (IgG), spike protein (IgG, IgM, IgA), and receptor binding domain (IgG, IgM, IgA) were determined by enzyme linked immunosorbent assay. ResultsWhile only 4.8% of participants were clinically diagnosed for SARS-CoV-2 infection, 75.9% of study participants were seropositive for SARS-CoV-2 antibodies. Most samples were IgM positive for spike or RBD (70%), indicating that infection was recent. This translated to all 20 families showing evidence of infection in at least one household member. For the children with MtD, 91% had antibodies against SARS-CoV-2 and had not experienced any adverse outcomes at the time of assessment. For children with recent infections (IgM+ only), serologic data suggest household members as a source. ConclusionsCOVID-19 was highly prevalent and undiagnosed in households with a child with MtD through the 2020-2021 winter wave of the pandemic. In this first major wave, children with MtD tolerated SARS-CoV-2 infection well, potentially due to household adherence to CDC recommendations for risk mitigation. FundingThis study was funded by the Intramural Research Program of the National Institutes of Health (HG200381-03). Clinical trial numberNCT04419870

SARS-CoV-2 Seroprevalence and Drug Use in Trauma Patients from Six Sites in the United States

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.

Rapidly increasing SARS-CoV-2 seroprevalence and limited clinical disease in three Malian communities: a prospective cohort study

BackgroundThe extent of SARS-CoV-2 exposure and transmission in Mali and the surrounding region is not well understood, although infection has been confirmed in nearly 14,000 symptomatic individuals and their contacts since the first case in March 2020. We aimed to estimate the cumulative incidence of SARS-CoV-2 in three Malian communities, and understand factors associated with infection. MethodsBetween 27 July 2020 and 29 January 2021, we collected blood samples along with demographic, social, medical and self-reported symptoms information from residents aged 6 months and older in three study communities at two study visits. SARS-CoV-2 antibodies were measured using a highly specific two-antigen ELISA optimized for use in Mali. We calculated cumulative adjusted seroprevalence for each site and evaluated factors associated with serostatus at each visit by univariate and multivariate analysis. FindingsOverall, 94.8% (2533/2672) of participants completed both study visits. A total of 50.3% (1343/2672) of participants were male, and 31.3% (837/2672) were aged <10 years, 27.6% (737/2672) were aged 10-17 years, and 41.1% (1098/2572) were aged [≥]18 years. The cumulative SARS-CoV-2 exposure rate was 58.5% (95% CI: 47.5 to 69.4). This varied between sites and was 73.4% (95% CI: 59.2 to 87.5) in the urban community of Sotuba, 53.2% (95% CI: 42.8 to 63.6) in the rural town of Bancoumana, and 37.1% (95% CI: 29.6 to 44.5) in the rural village of Doneguebougou. This equates to an infection rate of approximately 1% of the population every three days in the study communities between visits. Increased age and study site were associated with serostatus at both study visits. There was minimal difference in reported symptoms based on serostatus. InterpretationThe true extent of SARS-CoV-2 exposure in Mali is greater than previously reported and now approaches hypothetical herd immunity in urban areas. The epidemiology of the pandemic in the region may be primarily subclinical and within background illness rates. In this setting, ongoing surveillance and augmentation of diagnostics to characterize locally circulating variants will be critical to implement effective mitigation strategies like vaccines. FundingThis project was funded by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institute of Biomedical Imaging and Bioengineering, and National Cancer Institute.

SARS-CoV-2 seroassay optimization and performance in a population with high background reactivity in Mali

Serological tests are an indispensable tool to understand the epidemiology of the SARS-CoV-2 pandemic, particularly in areas where molecular diagnostics are limited. Poor assay performance may hinder the utility of these tests, including high rates of false-positivity previously reported in sub-Saharan Africa. From 312 Malian samples collected prior to 2020, we measured antibodies to the commonly tested SARS-CoV-2 antigens and four other betacoronaviruses by ELISA, and assessed functional cross-reactivity in a subset by SARS-CoV-2 pseudovirus neutralization assay. We then evaluated the performance of an ELISA developed in the US, using two-antigen SARS-CoV-2 spike protein and receptor-binding domain. To optimize test performance, we compared single and two-antigen approaches using existing assay cutoffs and population-specific cutoffs for Malian control samples (positive and negative). Background reactivity to SARS-CoV-2 antigens was common in pre-pandemic samples compared to US controls (43.4% (135/311) for spike protein, 22.8% (71/312) for RBD, and 33.9% (79/233) for nucleocapsid protein). SARS-CoV-2 reactivity correlated weakly with other betacoronavirus reactivity, varied between Malian communities, and increased with age. No pre-pandemic samples demonstrated functional activity. Regardless of the cutoffs applied, specificity improved using a two-antigen approach. Test performance was optimal using a two-antigen assay with population-specific cutoffs derived from ROC curve analysis [Sensitivity: 73.9% (51.6-89.8), Specificity: 99.4% (97.7-99.9)]. In the setting of high background reactivity, such as sub-Saharan Africa, SARS-CoV-2 serological assays need careful qualification is to characterize the epidemiology of disease, prevent unnecessary harm, and allocate resources for targeted control measures.

Mapping a Pandemic: SARS-CoV-2 Seropositivity in the United States

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 10th and July 31st, 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.

Improved production of SARS-CoV-2 spike receptor-binding domain (RBD) for serology assays

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a commonly used antigen for serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Different versions of the RBD protein have been developed and utilized in assays, with higher sensitivity attributed to particular forms of the protein. To improve the yield of these high-sensitivity forms of RBD and support the increased demand for this antigen in serology assays, we investigated several protein expression variables including DNA elements such as promoters and signal peptides, cell culture expression parameters, and purification processes. Through this investigation, we developed a simplified and robust purification strategy that consistently resulted in high levels of the high-sensitivity form of RBD and demonstrated that a carboxyterminal tag is responsible for the increased sensitivity in the ELISA. These improved reagents and processes produce high-quality proteins which are functional in serology assays and can be used to investigate seropositivity to SARS-CoV-2 infection. Highlights: O_LIImproved yields of SARS-CoV-2 spike RBD through modification of DNA constructs and purification parameters C_LIO_LITwo versions of RBD show different sensitivity in serology assays C_LIO_LIYields of greater than 50 mg/l obtained under optimal conditions C_LIO_LIMagnetic bead purification technology improves throughput of protein production C_LI

D614G Spike Variant Does Not Alter IgG, IgM, or IgA Spike Seroassay Performance

Emergence of a new variant of spike protein (D614G) with increased infectivity and transmissibility has prompted many to analyze the potential role of this variant in the SARS-CoV-2 pandemic. When a new variant emerges, there is a concern regarding whether an individual exposed to one variant of a virus will have cross-reactive immune memory to the second variant. Accordingly, we analyzed the serologic reactivity of D614 (original) and G614 variant spike proteins. We found that antibodies from a high-incidence population in New York City reacted both toward the original D614 spike and the G614 spike variant. These data suggest that patients who have been exposed to either SARS-CoV-2 variant have humoral immunity that can respond against both variants. This is an important finding both for SARS-CoV-2 disease biology and for potential antibody-based therapeutics.

Serologic cross-reactivity of SARS-CoV-2 with endemic and seasonal Betacoronaviruses

In order to properly understand the spread of SARS-CoV-2 infection and development of humoral immunity, researchers have evaluated the presence of serum antibodies of people worldwide experiencing the pandemic. These studies rely on the use of recombinant proteins from the viral genome in order to identify serum antibodies that recognize SARS-CoV-2 epitopes. Here, we discuss the cross-reactivity potential of SARS-CoV-2 antibodies with the full spike proteins of four other Betacoronaviruses that cause disease in humans, MERS-CoV, SARS-CoV, HCoV-OC43, and HCoV-HKU1. Using enzyme-linked immunosorbent assays (ELISAs), we detected the potential cross-reactivity of antibodies against SARS-CoV-2 towards the four other coronaviruses, with the strongest cross-recognition between SARS-CoV-2 and SARS /MERS-CoV antibodies, as expected based on sequence homology of their respective spike proteins. Further analysis of cross-reactivity could provide informative data that could lead to intelligently designed pan-coronavirus therapeutics or vaccines.

Standardization of enzyme-linked immunosorbent assays for serosurveys of the SARS-CoV-2 pandemic using clinical and at-home blood sampling

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.

Optimizing high-yield production of SARS-CoV-2 soluble spike trimers for serology assays

The SARS-CoV-2 spike trimer is the primary antigen for several serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Until stable cell lines are developed to increase the titer of this secreted protein in mammalian cell culture, the low yield of spike protein produced from transient transfection of HEK293 cells will be a limiting factor for these assays. To improve the yield of spike protein and support the high demand for antigens in serology assays, we investigated several recombinant protein expression variables by altering the incubation temperature, harvest time, chromatography strategy, and final protein manipulation. Through this investigation, we developed a simplified and robust purification strategy that consistently yields 5 mg of protein per liter of expression culture for two commonly used forms of the SARS-CoV-2 spike protein. We show that these proteins form well-behaved stable trimers and are consistently functional in serology assays across multiple protein production lots. HighlightsO_LIImproved yields of SARS-CoV-2 spike protein through modification of expression and purification parameters C_LIO_LIYields of greater than 5 mg/l obtained for VRC spike under optimal conditions C_LIO_LISpike protein quality was validated by QC methods to ensure utility in serology assays C_LI