Evaluation of Ortho VITROS and Roche Elecsys S and NC Immunoassays for SARS-CoV-2 Serosurveillance Applications.

SARS-CoV-2 seroprevalence studies are instrumental in monitoring epidemic activity and require well-characterized, high-throughput assays, and appropriate testing algorithms. The U.S. Nationwide Blood Donor Seroprevalence Study performed monthly cross-sectional serological testing from July 2020 to December 2021, implementing evolving testing algorithms in response to changes in pandemic activity. With high vaccine uptake, anti-Spike (S) reactivity rates reached >80% by May 2021, and the study pivoted from reflex Roche anti-nucleocapsid (NC) testing of Ortho S-reactive specimens to parallel Ortho S/NC testing. We evaluated the performance of the Ortho NC assay as a replacement for the Roche NC assay and compared performance of parallel S/NC testing on both platforms. Qualitative and quantitative agreement of Ortho NC with Roche NC assays was evaluated on preselected S/NC concordant and discordant specimens. All 190 Ortho S+/Roche NC+ specimens were reactive on the Ortho NC assay; 34% of 367 Ortho S+/Roche NC- specimens collected prior to vaccine availability and 43% of 37 Ortho S-/Roche NC+ specimens were reactive on the Ortho NC assay. Performance of parallel S/NC testing using Ortho and Roche platforms was evaluated on 200 specimens collected in 2019 and 3,903 study specimens collected in 2021. All 200 pre-COVID-19 specimens tested negative on the four assays. Cross-platform agreement between Roche and Ortho platforms was 96.4% (3,769/3,903); most discordant results had reactivity close to the cutoffs on the alternate assays. These findings, and higher efficiency and throughput, support the use of parallel S/NC testing on either Roche or Ortho platforms for large serosurveillance studies. IMPORTANCE Seroprevalence studies like the U.S. Nationwide Blood Donor Seroprevalence Study (NBDS) have been critical in monitoring SARS-CoV-2 epidemic activity. These studies rely on serological assays to detect antibodies indicating prior infection. It is critical that the assays and testing algorithms used in seroprevalence studies have adequate performance (high sensitivity, high specificity, ability to discriminate vaccine-induced and infection-induced antibodies, etc.), as well as appropriate characteristics to support large-scale studies, such as high throughput and low cost. In this study we evaluated the performance of Ortho's anti-nucleocapsid assay as a replacement for the Roche anti-nucleocapsid assay and compared performance of parallel anti-spike and anti-nucleocapsid testing on both platforms. These data demonstrate similar performance of the Ortho and Roche anti-nucleocapsid assays and that parallel anti-spike and anti-nucleocapsid testing on either platform could be used for serosurveillance applications.

How do we form and coordinate a national serosurvey of SARS-CoV-2 within the blood collection industry?

BACKGROUND: A national serosurvey of U.S. blood donors conducted in partnership with the Centers for Disease Control and Prevention (CDC) was initiated to estimate the prevalence of SARS-CoV-2 infections and vaccinations. METHODS: Beginning in July 2020, the Nationwide Blood Donor Seroprevalence Study collaborated with multiple blood collection organizations, testing labs, and leadership from government partners to capture, test, and analyze approximately 150,000 blood donation specimens per month in a repeated, cross-sectional seroprevalence survey. RESULTS: A CDC website (https://covid.cdc.gov/covid-data-tracker/#nationwide-blood-donor-seroprevalence) provided stratified, population-level results to public health professionals and the general public. DISCUSSION: The study adapted operations as the pandemic evolved, changing specimen flow and testing algorithms, and collecting additional data elements in response to changing policies on universal blood donation screening and administration of SARS-CoV-2 spike-based vaccines. The national serosurvey demonstrated the utility of serosurveillance testing of residual blood donations and highlighted the role of the blood collection industry in public-private partnerships during a public health emergency.

Evaluation of Commercially Available High-Throughput SARS-CoV-2 Serologic Assays for Serosurveillance and Related Applications.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serosurveys can estimate cumulative incidence for monitoring epidemics, requiring assessment of serologic assays to inform testing algorithm development and interpretation of results. We conducted a multilaboratory evaluation of 21 commercial high-throughput SARS-CoV-2 serologic assays using blinded panels of 1,000 highly characterized specimens. Assays demonstrated a range of sensitivities (96%-63%), specificities (99%-96%), and precision (intraclass correlation coefficient 0.55-0.99). Durability of antibody detection was dependent on antigen and immunoglobulin targets; antispike and total Ig assays demonstrated more stable longitudinal reactivity than antinucleocapsid and IgG assays. Assays with high sensitivity, specificity, and durable antibody detection are ideal for serosurveillance, but assays demonstrating waning reactivity are appropriate for other applications, including correlation with neutralizing activity and detection of anamnestic boosting by reinfections. Assay performance must be evaluated in context of intended use, particularly in the context of widespread vaccination and circulation of SARS-CoV-2 variants.

Use of US Blood Donors for National Serosurveillance of Severe Acute Respiratory Syndrome Coronavirus 2 Antibodies: Basis for an Expanded National Donor Serosurveillance Program.

BACKGROUND: The Recipient Epidemiology and Donor Evaluation Study-IV-Pediatric (REDS-IV-P) Epidemiology, Surveillance and Preparedness of the Novel SARS-CoV-2 Epidemic (RESPONSE) seroprevalence study conducted monthly cross-sectional testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in blood donors in 6 US metropolitan regions to estimate the extent of SARS-CoV-2 infections over time. METHODS: During March-August 2020, approximately ≥1000 serum specimens were collected monthly from each region and tested for SARS-CoV-2 antibodies using a well-validated algorithm. Regional seroprevalence estimates were weighted based on demographic differences compared with the general population. Seroprevalence was compared with reported coronavirus disease 2019 (COVID-19) case rates over time. RESULTS: For all regions, seroprevalence was <1.0% in March 2020. New York, New York, experienced the biggest increase (peak seroprevalence, 15.8% in May). All other regions experienced modest increases in seroprevalence (1%-2% in May-June to 2%-4% in July-August). Seroprevalence was higher in younger, non-Hispanic black, and Hispanic donors. Temporal increases in donor seroprevalence correlated with reported case rates in each region. In August, 1.3-5.6 estimated cumulative infections (based on seroprevalence data) per COVID-19 case were reported to the Centers for Disease Control and Prevention. CONCLUSIONS: Increases in seroprevalence were found in all regions, with the largest increase in New York. Seroprevalence was higher in non-Hispanic black and Hispanic than in non-Hispanic white blood donors. SARS-CoV-2 antibody testing of blood donor samples can be used to estimate the seroprevalence in the general population by region and demographic group. The methods derived from the RESPONSE seroprevalence study served as the basis for expanding SARS-CoV-2 seroprevalence surveillance to all 50 states and Puerto Rico.

Social Behavior, Community Composition, Pathogen Strain, and Host Symbionts Influence Fungal Disease Dynamics in Salamanders.

The emerging fungal pathogen, Batrachochytrium dendrobatidis (Bd), which can cause a fatal disease called chytridiomycosis, is implicated in the collapse of hundreds of host amphibian species. We describe chytridiomycosis dynamics in two co-occurring terrestrial salamander species, the Santa Lucia Mountains slender salamander, Batrachoseps luciae, and the arboreal salamander, Aneides lugubris. We (1) conduct a retrospective Bd-infection survey of specimens collected over the last century, (2) estimate present-day Bd infections in wild populations, (3) use generalized linear models (GLM) to identify biotic and abiotic correlates of infection risk, (4) investigate susceptibility of hosts exposed to Bd in laboratory trials, and (5) examine the ability of host skin bacteria to inhibit Bd in culture. Our historical survey of 2,866 specimens revealed that for most of the early 20th century (~1920-1969), Bd was not detected in either species. By the 1990s the proportion of infected specimens was 29 and 17% (B. luciae and A. lugubris, respectively), and in the 2010s it was 10 and 17%. This was similar to the number of infected samples from contemporary populations (2014-2015) at 10 and 18%. We found that both hosts experience signs of chytridiomycosis and suffered high Bd-caused mortality (88 and 71% for B. luciae and A. lugubris, respectively). Our GLM revealed that Bd-infection probability was positively correlated with intraspecific group size and proximity to heterospecifics but not to abiotic factors such as precipitation, minimum temperature, maximum temperature, mean temperature, and elevation, or to the size of the hosts. Finally, we found that both host species contain symbiotic skin-bacteria that inhibit growth of Bd in laboratory trials. Our results provide new evidence consistent with other studies showing a relatively recent Bd invasion of amphibian host populations in western North America and suggest that the spread of the pathogen may be enabled both through conspecific and heterospecific host interactions. Our results suggest that wildlife disease studies should assess host-pathogen dynamics that consider the interactions and effects of multiple hosts, as well as the historical context of pathogen invasion, establishment, and epizootic to enzootic transitions to better understand and predict disease dynamics.

Estimated US Infection- and Vaccine-Induced SARS-CoV-2 Seroprevalence Based on Blood Donations, July 2020-May 2021.

Importance: People who have been infected with or vaccinated against SARS-CoV-2 have reduced risk of subsequent infection, but the proportion of people in the US with SARS-CoV-2 antibodies from infection or vaccination is uncertain. Objective: To estimate trends in SARS-CoV-2 seroprevalence related to infection and vaccination in the US population. Design, Setting, and Participants: In a repeated cross-sectional study conducted each month during July 2020 through May 2021, 17 blood collection organizations with blood donations from all 50 US states; Washington, DC; and Puerto Rico were organized into 66 study-specific regions, representing a catchment of 74% of the US population. For each study region, specimens from a median of approximately 2000 blood donors were selected and tested each month; a total of 1 594 363 specimens were initially selected and tested. The final date of blood donation collection was May 31, 2021. Exposure: Calendar time. Main Outcomes and Measures: Proportion of persons with detectable SARS-CoV-2 spike and nucleocapsid antibodies. Seroprevalence was weighted for demographic differences between the blood donor sample and general population. Infection-induced seroprevalence was defined as the prevalence of the population with both spike and nucleocapsid antibodies. Combined infection- and vaccination-induced seroprevalence was defined as the prevalence of the population with spike antibodies. The seroprevalence estimates were compared with cumulative COVID-19 case report incidence rates. Results: Among 1 443 519 specimens included, 733 052 (50.8%) were from women, 174 842 (12.1%) were from persons aged 16 to 29 years, 292 258 (20.2%) were from persons aged 65 years and older, 36 654 (2.5%) were from non-Hispanic Black persons, and 88 773 (6.1%) were from Hispanic persons. The overall infection-induced SARS-CoV-2 seroprevalence estimate increased from 3.5% (95% CI, 3.2%-3.8%) in July 2020 to 20.2% (95% CI, 19.9%-20.6%) in May 2021; the combined infection- and vaccination-induced seroprevalence estimate in May 2021 was 83.3% (95% CI, 82.9%-83.7%). By May 2021, 2.1 SARS-CoV-2 infections (95% CI, 2.0-2.1) per reported COVID-19 case were estimated to have occurred. Conclusions and Relevance: Based on a sample of blood donations in the US from July 2020 through May 2021, vaccine- and infection-induced SARS-CoV-2 seroprevalence increased over time and varied by age, race and ethnicity, and geographic region. Despite weighting to adjust for demographic differences, these findings from a national sample of blood donors may not be representative of the entire US population.

CpG-creating mutations are costly in many human viruses.

Mutations can occur throughout the virus genome and may be beneficial, neutral or deleterious. We are interested in mutations that yield a C next to a G, producing CpG sites. CpG sites are rare in eukaryotic and viral genomes. For the eukaryotes, it is thought that CpG sites are rare because they are prone to mutation when methylated. In viruses, we know less about why CpG sites are rare. A previous study in HIV suggested that CpG-creating transition mutations are more costly than similar non-CpG-creating mutations. To determine if this is the case in other viruses, we analyzed the allele frequencies of CpG-creating and non-CpG-creating mutations across various strains, subtypes, and genes of viruses using existing data obtained from Genbank, HIV Databases, and Virus Pathogen Resource. Our results suggest that CpG sites are indeed costly for most viruses. By understanding the cost of CpG sites, we can obtain further insights into the evolution and adaptation of viruses.

Batrachochytrium dendrobatidis infection in amphibians predates first known epizootic in Costa Rica.

Emerging infectious diseases are a growing threat to biodiversity worldwide. Outbreaks of the infectious disease chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), are implicated in the decline and extinction of numerous amphibian species. In Costa Rica, a major decline event occurred in 1987, more than two decades before this pathogen was discovered. The loss of many species in Costa Rica is assumed to be due to Bd-epizootics, but there are few studies that provide data from amphibians in the time leading up to the proposed epizootics. In this study, we provide new data on Bd infection rates of amphibians collected throughout Costa Rica, in the decades prior to the epizootics. We used a quantitative PCR assay to test for Bd presence in 1016 anuran museum specimens collected throughout Costa Rica. The earliest specimen that tested positive for Bd was collected in 1964. Across all time periods, we found an overall infection rate (defined as the proportion of Bd-positive individuals) of 4%. The number of infected individuals remained relatively low across all species tested and the range of Bd-positive specimens was shown to be geographically constrained up until the 1980s; when epizootics are hypothesized to have occurred. After that time, infection rate increased three-fold, and the range of specimens tested positive for Bd increased, with Bd-positive specimens collected across the entire country. Our results suggest that Bd dynamics in Costa Rica are more complicated than previously thought. The discovery of Bd's presence in the country preceding massive declines leads to a number of different hypotheses: 1) Bd invaded Costa Rica earlier than previously known, and spread more slowly than previously reported; 2) Bd invaded multiple times and faded out; 3) an endemic Bd lineage existed; 4) an earlier Bd lineage evolved into the current Bd lineage or hybridized with an invasive lineage; or 5) an earlier Bd lineage went extinct and a new invasion event occurred causing epizootics. To help visualize areas where future studies should take place, we provide a Bd habitat suitability model trained with local data. Studies that provide information on genetic lineages of Bd are needed to determine the most plausible spatial-temporal, host-pathogen dynamics that could best explain the epizootics resulting in amphibian declines in Costa Rica and throughout Central America.

Cryptic diversity of a widespread global pathogen reveals expanded threats to amphibian conservation.

Biodiversity loss is one major outcome of human-mediated ecosystem disturbance. One way that humans have triggered wildlife declines is by transporting disease-causing agents to remote areas of the world. Amphibians have been hit particularly hard by disease due in part to a globally distributed pathogenic chytrid fungus (Batrachochytrium dendrobatidis [Bd]). Prior research has revealed important insights into the biology and distribution of Bd; however, there are still many outstanding questions in this system. Although we know that there are multiple divergent lineages of Bd that differ in pathogenicity, we know little about how these lineages are distributed around the world and where lineages may be coming into contact. Here, we implement a custom genotyping method for a global set of Bd samples. This method is optimized to amplify and sequence degraded DNA from noninvasive skin swab samples. We describe a divergent lineage of Bd, which we call BdASIA3, that appears to be widespread in Southeast Asia. This lineage co-occurs with the global panzootic lineage (BdGPL) in multiple localities. Additionally, we shed light on the global distribution of BdGPL and highlight the expanded range of another lineage, BdCAPE. Finally, we argue that more monitoring needs to take place where Bd lineages are coming into contact and where we know little about Bd lineage diversity. Monitoring need not use expensive or difficult field techniques but can use archived swab samples to further explore the history-and predict the future impacts-of this devastating pathogen.

Pathogen invasion history elucidates contemporary host pathogen dynamics.

Amphibians, the most threatened group of vertebrates, are seen as indicators of the sixth mass extinction on earth. Thousands of species are threatened with extinction and many have been affected by an emerging infectious disease, chytridiomycosis, caused by the fungal pathogen, Batrachochytrium dendrobatidis (Bd). However, amphibians exhibit different responses to the pathogen, such as survival and population persistence with infection, or mortality of individuals and complete population collapse after pathogen invasion. Multiple factors can affect host pathogen dynamics, yet few studies have provided a temporal view that encompasses both the epizootic phase (i.e. pathogen invasion and host collapse), and the transition to a more stable co-existence (i.e. recovery of infected host populations). In the Sierra Nevada mountains of California, USA, conspecific populations of frogs currently exhibit dramatically different host/ Bd-pathogen dynamics. To provide a temporal context by which present day dynamics may be better understood, we use a Bd qPCR assay to test 1165 amphibian specimens collected between 1900 and 2005. Our historical analyses reveal a pattern of pathogen invasion and eventual spread across the Sierra Nevada over the last century. Although we found a small number of Bd-infections prior to 1970, these showed no sign of spread or increase in infection prevalence over multiple decades. After the late 1970s, when mass die offs were first noted, our data show Bd as much more prevalent and more spatially spread out, suggesting epizootic spread. However, across the ~400km2 area, we found no evidence of a wave-like pattern, but instead discovered multiple, nearly-simultaneous invasions within regions. We found that Bd invaded and spread in the central Sierra Nevada (Yosemite National Park area) about four decades before it invaded and spread in the southern Sierra Nevada (Sequoia and Kings Canyon National Parks area), and suggest that the temporal pattern of pathogen invasion may help explain divergent contemporary host pathogen dynamics.

Pathogen invasion and non-epizootic dynamics in Pacific newts in California over the last century.

Emerging infectious disease is a growing threat to global biodiversity. The infectious disease chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd) has led to the decline and extinction of hundreds of amphibian species. Severe Bd-caused epizootics have been documented in North, Central and South America-with many of the research focused on anurans. California, where Bd-related epizootics and amphibian declines have been reported, has some of the highest diversity of salamanders. After more than a decade since the first known epizootic in California, little is known about Bd disease dynamics in salamanders. Pacific newts (Genus: Taricha) are ideal study species because of their abundance, wide geographic range, occurrence in both aquatic and terrestrial habitats, and how little is known about Bd infection dynamics for this group. We conducted a retrospective study to determine the relationship between Pacific newts and the fungal pathogen. We tested 1895 specimens collected between 1889-2009 and found no evidence of Bd-infected Pacific newts until the late 1940's. Although we estimate that Bd emerged in this genus and rapidly spread geographically throughout California, we did not find evidence for epizootic dynamics. Bd infection prevalence and intensity, two measures commonly used to estimate dynamics, remained consistently low over time; suggesting Pacific newts may not be highly susceptible. Also, we found the timing of first Bd emergence in Pacific newts predate Bd emergence in other California salamander species. In addition, we found several environmental and anthropogenic factors correlated with Bd prevalence which may help explain Bd disease dynamics in the genus Taricha. Pacific newts may be a reservoir species that signal pathogen invasion into California salamanders, though further studies are needed.