Development and Implementation of Dried Blood Spot-based COVID-19 Serological Assays for Epidemiologic Studies

Serological surveillance studies of infectious diseases provide population-level estimates of infection and antibody prevalence, generating crucial insight into population-level immunity, risk factors leading to infection, and effectiveness of public health measures. These studies traditionally rely on detection of pathogen-specific antibodies in samples derived from venipuncture, an expensive and logistically challenging aspect of serological surveillance. During the COVID-19 pandemic, guidelines implemented to prevent the spread of SARS-CoV-2 infection made collection of venous blood logistically difficult at a time when SARS-CoV-2 serosurveillance was urgently needed. Dried blood spots (DBS) have generated interest as an alternative to venous blood for SARS-CoV-2 serological applications due to their stability, low cost, and ease of collection; DBS samples can be self-generated via fingerprick by community members and mailed at ambient temperatures. Here, we detail the development of four DBS-based SARS-CoV-2 serological methods and demonstrate their implementation in a large serological survey of community members from 12 cities in the East Bay region of the San Francisco metropolitan area using at-home DBS collection. We find that DBS perform similarly to plasma/serum in enzyme-linked immunosorbent assays and commercial SARS-CoV-2 serological assays. In addition, we show that DBS samples can reliably detect antibody responses months post-infection and track antibody kinetics after vaccination. Implementation of DBS enabled collection of valuable serological data from our study population to investigate changes in seroprevalence over an eight-month period. Our work makes a strong argument for the implementation of DBS in serological studies, not just for SARS-CoV-2, but any situation where phlebotomy is inaccessible.

Characterization of Canine Influenza Virus A (H3N2) Circulating in Dogs in China from 2016 to 2018

Avian H3N2 influenza virus follows cross-host transmission and has spread among dogs in Asia since 2005. After 2015–2016, a new H3N2 subtype canine influenza epidemic occurred in dogs in North America and Asia. The disease prevalence was assessed by virological and serological surveillance in dogs in China. Herein, five H3N2 canine influenza virus (CIV) strains were isolated from 1185 Chinese canine respiratory disease samples in 2017–2018; these strains were on the evolutionary branch of the North American CIVs after 2016 and genetically far from the classical canine H3N2 strain discovered in China before 2016. Serological surveillance showed an HI antibody positive rate of 6.68%. H3N2 was prevalent in the coastal areas and northeastern regions of China. In 2018, it became the primary epidemic strain in the country. The QK01 strain of H3N2 showed high efficiency in transmission among dogs through respiratory droplets. Nevertheless, the virus only replicated in the upper respiratory tract and exhibited low pathogenicity in mice. Furthermore, highly efficient transmission by direct contact other than respiratory droplet transmission was found in a guinea pig model. The low-level replication in avian species other than ducks could not facilitate contact and airborne transmission in chickens. The current results indicated that a novel H3N2 virus has become a predominant epidemic strain in dogs in China since 2016 and acquired highly efficient transmissibility but could not be replicated in avian species. Thus, further monitoring is required for designing optimal immunoprophylactic tools for dogs and estimating the zoonotic risk of CIV in China.

374. Need to Improve Minority Representation through COVID-19 Community Research Partnership

Background Minorities are often unrepresented in research, which limits equity in healthcare advances. The racial and ethnic disparities in outcomes of individuals infected with COVID-19 highlight the importance of inclusivity in research to improve public health measures. Methods We performed a descriptive analysis of the racial and ethnic distribution of children enrolled in our COVID-19 Community Research Partnership (CRP) study, a syndromic and serological surveillance study of children aged 2 – 17 years receiving care at three healthcare systems spanning North and South Carolina. Syndromic surveillance involved daily symptom reporting using a web-based monitoring application. Participants consenting to serological surveillance were mailed at-home tests sampling finger prick capillary blood. In-person and electronic recruitment efforts were conducted in English and Spanish. At one of the study sites, we compared the racial/ethnic distribution of enrolled children to the racial/ethnic distribution of all children who received care at the same site during the same timeframe. We compared the racial/ethnic distribution of participants who ultimately submitted samples for serological testing compared to those who consented to serologic testing. Results At total of1630 children were enrolled from April 2, 2021 – June 8, 2021. Most children were > 5 years old, 50.2% were female, and 88.5% were from mostly urban counties (Table 1). Of enrolled children, 4.2% were Hispanic, 8.2% were black, and 81.6% were white (Table 2). Among 135,355 unique children who received care at the institution during the same time, 12.4% were Hispanic, 23.0% were black, and 63.1% were white. Of 1552 participants who consented to serologic testing, 4.4% were Hispanic, 8.1% were black, and 81.8% were white (Table 3). To date, 242 children submitted serologic samples; 4.1% were Hispanic, 5.0% were black, and 85.5% were white. Table 1. Characteristics of enrolled children in COVID-19 surveillance study Table 2. Racial and Ethnic distribution of children enrolled in the study compared to target population Table 3. Racial and ethnic distribution of children who participated in serology testing Conclusion Despite efforts to recruit a diverse group of children, the proportion of minorities enrolled in our COVID-19 surveillance study underrepresents the targeted population. Ongoing efforts will work to identify barriers and facilitators to research participation among minority families. Disclosures Amina Ahmed, MD, Nothing to disclose

Nucleocapsid antibody positivity as a marker of past SARS-CoV-2 infection in population serosurveillance studies: impact of variant, vaccination, and choice of assay cut-off

Serological surveillance studies sometimes use presence of anti-nucleocapsid antibody as a marker of natural SARS-CoV-2 infection. We explore seroconversion rates and antibody titres following Alpha and Delta variant infections, and vaccine breakthrough infections. We find lower seroconversion rates particularly following Alpha-variant vaccine breakthrough infections. We re-evaluate assay performance with a mix of past waned infections and recent breakthrough infections, that is relevant to current serological surveillance.

Implementing Serosurveys in India: Experiences, Lessons Learned, and Recommendations

Serological surveillance for vaccine-preventable diseases, such as measles and rubella, can provide direct measures of population immunity across age groups, identify gaps in immunity, and document changes in immunity over time. Rigorously conducted, representative household serosurveys provide high-quality estimates with minimal bias. However, they can be logistically challenging, expensive, and have higher refusal rates than vaccine coverage surveys. This article shares lessons learned through implementing nine measles and rubella household serosurveys in five districts in India—the challenges faced, the potential impact on results, and recommendations to facilitate the conduct of serosurveys. Specific lessons learned arose from challenges related to community mobilization owing to lack of cooperation in certain settings and populations, limitations of outdated census information, nonresponse due to refusal or unavailability during survey enumeration and enrollment, data collection issues, and specimen collection and handling issues. Although some experiences are specific to serosurveys in India, these lessons are generalizable to other household surveys, particularly vaccination coverage and serosurveys conducted in low- and middle-income settings.

Serological Surveillance of Rabies in Free-Range and Captive Common Vampire Bats Desmodus rotundus

The control of vampire bat rabies (VBR) in Brazil is based on the culling of Desmodus rotundus and the surveillance of outbreaks caused by D. rotundus in cattle and humans in addition to vaccination of susceptible livestock. The detection of anti-rabies antibodies in vampire bats indicates exposure to the rabies virus, and several studies have reported an increase of these antibodies following experimental infection. However, the dynamics of anti-rabies antibodies in natural populations of D. rotundus remains poorly understood. In this study, we took advantage of recent outbreaks of VBR among livestock in the Sao Paulo region of Brazil to test whether seroprevalence in D. rotundus reflects the incidence of rabies in nearby livestock populations. Sixty-four D. rotundus were captured during and after outbreaks from roost located in municipalities belonging to three regions with different incidences of rabies in herbivores. Sixteen seropositive bats were then kept in captivity for up to 120 days, and their antibodies and virus levels were quantified at different time points using the rapid fluorescent focus inhibition test (RFFIT). Antibody titers were associated with the occurrence of ongoing outbreak, with a higher proportion of bats showing titer >0.5 IU/ml in the region with a recent outbreak. However, low titers were still detected in bats from regions reporting the last outbreak of rabies at least 3 years prior to sampling. This study suggests that serological surveillance of rabies in vampire bats can be used as a tool to evaluate risk of outbreaks in at risk populations of cattle and human.

Seroprevalence of anti-SARS-CoV-2 IgG and IgA antibodies before the launch of COVID-19 vaccination in Kazakhstan

BackgroundCOVID-19 exposure in Central Asia appears underestimated and SARS-CoV-2 seroprevalence data are urgently needed to inform ongoing vaccination efforts and other strategies to mitigate the regional pandemic. Here, we assessed the prevalence of anti-SARS-CoV-2 antibody-mediated immunity in a heterogeneous cohort of public university employees in Karaganda, Kazakhstan.MethodsAsymptomatic subjects (n=100) were randomly recruited prior to their first COVID-19 vaccination. Questionnaires were administered to capture a range of demographic and clinical characteristics. Nasopharyngeal swabs were collected for SARS-CoV-2 RT-qPCR testing. Serological assays were performed to detect spike (S)-reactive IgG and IgA.ResultsAnti-S IgG and IgA seropositivity rates among SARS-CoV-2 PCR-negative participants (n=100) were 42% and 53%, respectively, and 61% of subjects tested positive for at least one of the antibodies. Serologically confirmed history of COVID-19 was associated with self-reported history of respiratory illness since March 2020 (p<0.001).ConclusionsSARS-CoV-2 exposure in this cohort is ∼15-fold higher compared to the reported all-time national and regional COVID-19 prevalence. Continuous serological surveillance is critical for understanding the COVID-19 transmission dynamics and should be nationally implemented to better inform the public health response in Central Asia.

Serological Surveillance of Influenza D Virus in Ruminants and Swine in West and East Africa, 2017–2020

Influenza D virus (IDV) was first isolated in 2011 in Oklahoma, USA from pigs presenting with influenza-like symptoms. IDV is known to mainly circulate in ruminants, especially cattle. In Africa, there is limited information on the epidemiology of IDV, although the virus has likely circulated in the region since 2012. In the present study, we investigated the seropositivity of IDV among domestic ruminants and swine in West and East Africa from 2017 to 2020. Serum samples were analyzed using the hemagglutination inhibition (HI) assay. Our study demonstrated that IDV is still circulating in Africa, with variations in seropositivity among countries and species. The highest seropositivity was detected in cattle (3.9 to 20.9%). Our data highlights a need for extensive surveillance of IDV in Africa in order to better understand the epidemiology of the virus in the region.

Serological Detection of SARS-CoV-2 Antibodies in Naturally-Infected Mink and Other Experimentally-Infected Animals

The recent emergence of SARS-CoV-2 in humans from a yet unidentified animal reservoir and the capacity of the virus to naturally infect pets, farmed animals and potentially wild animals has highlighted the need for serological surveillance tools. In this study, the luciferase immunoprecipitation systems (LIPS), employing the spike (S) and nucleocapsid proteins (N) of SARS-CoV-2, was used to examine the suitability of the assay for antibody detection in different animal species. Sera from SARS-CoV-2 naturally-infected mink (n = 77), SARS-CoV-2 experimentally-infected ferrets, fruit bats and hamsters and a rabbit vaccinated with a purified spike protein were examined for antibodies using the SARS-CoV-2 N and/or S proteins. From comparison with the known neutralization status of the serum samples, statistical analyses including calculation of the Spearman rank-order-correlation coefficient and Cohen’s kappa agreement were used to interpret the antibody results and diagnostic performance. The LIPS immunoassay robustly detected the presence of viral antibodies in naturally infected SARS-CoV-2 mink, experimentally infected ferrets, fruit bats and hamsters as well as in an immunized rabbit. For the SARS-CoV-2-LIPS-S assay, there was a good level of discrimination between the positive and negative samples for each of the five species tested with 100% agreement with the virus neutralization results. In contrast, the SARS-CoV-2-LIPS-N assay did not consistently differentiate between SARS-CoV-2 positive and negative sera. This study demonstrates the suitability of the SARS-CoV-2-LIPS-S assay for the sero-surveillance of SARS-CoV-2 infection in a range of animal species.

Surveillance of the rabies-related lyssavirus, Mokola in non-volant small mammals in South Africa

The reservoir host of Mokola virus (MOKV), a rabies-related lyssavirus species endemic to Africa, remains unknown. Only sporadic cases of MOKV have been reported since its first discovery in the late 1960s, which subsequently gave rise to various reservoir host hypotheses. One particular hypothesis focusing on non-volant small mammals (e.g. shrews, sengis and rodents) is buttressed by previous MOKV isolations from shrews (Crocidura sp.) and a single rodent (Lophuromys sikapusi). Although these cases were only once-off detections, it provided evidence of the first known lyssavirus species has an association with non-volant small mammals. To investigate further, retrospective surveillance was conducted in 575 small mammals collected from South Africa. Nucleic acid surveillance using a pan-lyssavirus quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay of 329 brain samples did not detect any lyssavirus ribonucleic acid (RNA). Serological surveillance using a micro-neutralisation test of 246 serum samples identified 36 serum samples that were positive for the presence of MOKV neutralising antibodies (VNAs). These serum samples were all collected from Gerbilliscus leucogaster (Bushveld gerbils) rodents from Meletse in Limpopo province (South Africa). Mokola virus infections in Limpopo province have never been reported before, and the high MOKV seropositivity of 87.80% in these gerbils may indicate a potential rodent reservoir.