Health inequities in SARS-CoV-2 infection, seroprevalence, and COVID-19 vaccination: Results from the East Bay COVID-19 study

Comprehensive data on transmission mitigation behaviors and both SARS-CoV-2 infection and serostatus are needed from large, community-based cohorts to identify COVID-19 risk factors and the impact of public health measures. We conducted a longitudinal, population-based study in the East Bay Area of Northern California. From July 2020-March 2021, approximately 5,500 adults were recruited and followed over three data collection rounds to investigate the association between geographic and demographic characteristics and transmission mitigation behavior with SARS-CoV-2 prevalence. We estimated the populated-adjusted prevalence of antibodies from SARS-CoV-2 infection and COVID-19 vaccination, and self-reported COVID-19 test positivity. Population-adjusted SARS-CoV-2 seroprevalence was low, increasing from 1.03% (95% CI: 0.50–1.96) in Round 1 (July-September 2020), to 1.37% (95% CI: 0.75–2.39) in Round 2 (October-December 2020), to 2.18% (95% CI: 1.48–3.17) in Round 3 (February-March 2021). Population-adjusted seroprevalence of COVID-19 vaccination was 21.64% (95% CI: 19.20–24.34) in Round 3, with White individuals having 4.35% (95% CI: 0.35–8.32) higher COVID-19 vaccine seroprevalence than individuals identifying as African American or Black, American Indian or Alaskan Native, Asian, Hispanic, two or more races, or other. No evidence for an association between transmission mitigation behavior and seroprevalence was observed. Despite >99% of participants reporting wearing masks individuals identifying as African American or Black, American Indian or Alaskan Native, Asian, Hispanic, two or more races, or other, as well as those in lower-income households, and lower-educated individuals had the highest SARS-CoV-2 seroprevalence and lowest vaccination seroprevalence. Results demonstrate that more effective policies are needed to address these disparities and inequities.

Occurrence of SARS-CoV-2 reinfections at regular intervals in Ecuador.

SARS-CoV-2 reinfection is defined as a new infection with a different virus variant in an individual who has already recovered from a previous episode of COVID-19. The first case of reinfection in the world was described in August 2020, since then, reinfections have increased over time and their incidence has fluctuated with specific SARS-CoV-2 variant waves. Initially, reinfections were estimated to represent less than 1% of total COVID-19 infections. With the advent of the Omicron variant, reinfections became more frequent, representing up to 10% of cases (based on data from developed countries). The frequency of reinfections in Latin America has been scarcely reported. The current study shows that in Ecuador, the frequency of reinfections has increased 10-fold following the introduction of Omicron, after 22 months of surveillance in a single center of COVID-19 diagnostics. Suspected reinfections were identified retrospectively from a database of RT-qPCR-positive patients. Cases were confirmed by sequencing viral genomes from the first and second infections using the ONT MinION platform. Monthly surveillance showed that the main incidence peaks of reinfections were reached within four to five months, coinciding with the increase of COVID-19 cases in the country, suggesting that the emergence of reinfections is related to higher exposure to the virus during outbreaks. This study performed the longest monitoring of SARS-CoV-2 reinfections, showing an occurrence at regular intervals of 4-5 months and confirming a greater propensity of Omicron to cause reinfections.

Health inequities in SARS-CoV-2 infection, seroprevalence, and COVID-19 vaccination: Results from the East Bay COVID-19 study.

Comprehensive data on transmission mitigation behaviors and both SARS-CoV-2 infection and serostatus are needed from large, community-based cohorts to identify COVID-19 risk factors and the impact of public health measures. We conducted a longitudinal, population-based study in the East Bay Area of Northern California. From July 2020-March 2021, approximately 5,500 adults were recruited and followed over three data collection rounds to investigate the association between geographic and demographic characteristics and transmission mitigation behavior with SARS-CoV-2 prevalence. We estimated the populated-adjusted prevalence of antibodies from SARS-CoV-2 infection and COVID-19 vaccination, and self-reported COVID-19 test positivity. Population-adjusted SARS-CoV-2 seroprevalence was low, increasing from 1.03% (95% CI: 0.50-1.96) in Round 1 (July-September 2020), to 1.37% (95% CI: 0.75-2.39) in Round 2 (October-December 2020), to 2.18% (95% CI: 1.48-3.17) in Round 3 (February-March 2021). Population-adjusted seroprevalence of COVID-19 vaccination was 21.64% (95% CI: 19.20-24.34) in Round 3, with White individuals having 4.35% (95% CI: 0.35-8.32) higher COVID-19 vaccine seroprevalence than individuals identifying as African American or Black, American Indian or Alaskan Native, Asian, Hispanic, two or more races, or other. No evidence for an association between transmission mitigation behavior and seroprevalence was observed. Despite >99% of participants reporting wearing masks individuals identifying as African American or Black, American Indian or Alaskan Native, Asian, Hispanic, two or more races, or other, as well as those in lower-income households, and lower-educated individuals had the highest SARS-CoV-2 seroprevalence and lowest vaccination seroprevalence. Results demonstrate that more effective policies are needed to address these disparities and inequities.

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 postinfection 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 8-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. IMPORTANCE Estimation of community-level antibody responses to SARS-CoV-2 from infection or vaccination is critical to inform public health responses. Traditional studies of antibodies rely on collection of blood via venipuncture, an invasive procedure not amenable to pandemic-related social-distancing measures. Dried blood spots (DBS) are an alternative to venipuncture, since they can be self-collected by study participants at home and do not require refrigeration for shipment or storage. However, DBS-based assays to measure antibody levels to SARS-CoV-2 have not been widely utilized. Here, we show that DBS are comparable to blood as a sampling method for antibody responses to SARS-CoV-2 infection and vaccination over time measured using four distinct serological assays. The DBS format enabled antibody surveillance in a longitudinal cohort where study participants self-collected samples, ensuring the participants' safety during an ongoing pandemic. Our work demonstrates that DBS are an excellent sampling method for measuring antibody responses whenever venipuncture is impractical.

Alternative RNA extraction-free techniques for the real-time RT-PCR detection of SARS-CoV-2 in nasopharyngeal swab and sputum samples.

Standard diagnoses of SARS-CoV-2 infections are done by RNA extraction and real-time RT-PCR (rRT-PCR). However, the need for RNA extraction complicates testing due to increased processing time, high cost, and limited availability of commercial kits. Therefore, alternative methods for rRT-PCR detection of SARS-CoV-2 without RNA extraction were investigated. Nasopharyngeal and sputum samples were used to compare the sensitivity of three techniques: Trizol RNA extraction, thermal shock, and the direct use of samples with an RNase inhibitor. Direct, extraction-free use of primary samples plus the RNase inhibitor produced diagnostic values of 100 % sensitivity and specificity compared to standard protocols, and these findings were validated in a second, independent laboratory.

Genomic epidemiology of SARS-CoV-2 transmission lineages in Ecuador.

Characterisation of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic diversity through space and time can reveal trends in virus importation and domestic circulation and permit the exploration of questions regarding the early transmission dynamics. Here, we present a detailed description of SARS-CoV-2 genomic epidemiology in Ecuador, one of the hardest hit countries during the early stages of the coronavirus-19 pandemic. We generated and analysed 160 whole genome sequences sampled from all provinces of Ecuador in 2020. Molecular clock and phylogeographic analysis of these sequences in the context of global SARS-CoV-2 diversity enable us to identify and characterise individual transmission lineages within Ecuador, explore their spatiotemporal distributions, and consider their introduction and domestic circulation. Our results reveal a pattern of multiple international importations across the country, with apparent differences between key provinces. Transmission lineages were mostly introduced before the implementation of non-pharmaceutical interventions, with differential degrees of persistence and national dissemination.

Metagenome of a Bronchoalveolar Lavage Fluid Sample from a Confirmed COVID-19 Case in Quito, Ecuador, Obtained Using Oxford Nanopore MinION Technology.

We report the metagenome analysis of a bronchoalveolar lavage (BAL) fluid sample from a confirmed coronavirus disease 2019 (COVID-19) case in Quito, Ecuador. Sequencing was performed using MinION technology.

A Low-Cost SARS-CoV-2 rRBD ELISA to Detect Serostatus in Ecuadorian Population with COVID-19.

Laboratory diagnosis of the COVID-19 relies on RT-PCR to amplify specific fragments of SARS-CoV-2 genome. However, serological tests are required to determine the immune response elicited after infection. Here, we analyzed convalescent sera collected from positive individuals by RT-PCR to SARS-CoV-2 (n = 78), Zika (n = 20), dengue (n = 20), chikungunya (n = 54), intestinal parasites (n = 11), and HIV (n = 1), from different areas of Ecuador, with an in-house ELISA using a SARS-CoV-2 receptor binding domain recombinant (rRBD) antigen to detect IgG antibodies elicited by SARS-CoV-2 infection. Of the 78 samples positive for SARS-CoV-2 by RT-PCR, 73 showed high absorbance value compared with the cutoff and five were negative. All tested sera from other infections showed no reactivity. Sensitivity, specificity, positive predictive value, and negative predictive value were 93.6%, 100%, 100%, and 95.4%, respectively. This in-house anti-IgG rRBD ELISA offers an economic and simple alternative to determine IgG immune responses after SARS-CoV-2 infection.

Pre-existing T-cell immunity to SARS-CoV-2 in unexposed healthy controls in Ecuador, as detected with a COVID-19 Interferon-Gamma Release Assay.

BACKGROUND: Studies of T-cell immune responses against SARS-CoV-2 are important in understanding the immune status of individuals or populations. Here, we use a simple, cheap, and rapid whole blood stimulation assay - an Interferon-Gamma Release Assay (IGRA) - to study T-cell immunity to SARS-CoV-2 in convalescent COVID-19 patients and in unexposed healthy contacts from Quito, Ecuador. METHODS: Interferon-gamma (INF-γ) production was measured in the heparinized blood of convalescent and unexposed subjects after stimulation for 24 h with the SARS-CoV-2 Spike S1 protein, the Receptor Binding Domain (RBD) protein or the Nucleocapsid (NP) protein, respectively. The presence of IgG-RBD protein antibodies in both study groups was determined with an "in-house" ELISA. RESULTS: As measured with INF-γ production, 80% of the convalescent COVID-19 patients, all IgG-RBD seropositive, had a strong T-cell response. However, unexpectedly, 44% of unexposed healthy controls, all IgG-RBD seronegative, had a strong virus-specific T-cell response with the COVID-19 IGRA, probably because of prior exposure to common cold-causing coronaviruses or other viral or microbial antigens. CONCLUSION AND DISCUSSION: The high percentage of unexposed healthy subjects with a pre-existing immunity suggests that a part of the Ecuadorian population is likely to have SARS-CoV-2 reactive T-cells. Given that the IGRA technique is simple and can be easily scaled up for investigations where high numbers of patients are needed, this COVID-19 IGRA may serve to determine if the T-cell only response represents protective immunity to SARS-CoV-2 infection in a population-based study.