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Few studies have measured COVID-19 seroprevalence in North Carolina, especially among rural, Black, and Hispanic/Latino communities that have been heavily affected. Antibody results show high rates of COVID-19 among industrial livestock operation workers and their household members.

SARS-CoV-2 circulating variants coupled with waning immunity pose a significant threat to the long-term care (LTC) population. Our objective was to measure salivary IgG antibodies in residents and staff of an LTC facility to (1) evaluate IgG response in saliva post-natural infection and vaccination and (2) assess its feasibility to describe the seroprevalence over time.We performed salivary IgG sampling of all residents and staff who agreed to test in a 150-bed skilled nursing facility during three seroprevalence surveys between October 2020 and February 2021. The facility had SARS-CoV-2 outbreaks in May 2020 and November 2020, when 45 of 138 and 37 of 125 residents were infected, respectively; they offered two Federal vaccine clinics in January 2021. We evaluated quantitative IgG in saliva to the Nucleocapsid (N), Spike (S), and Receptor-binding domain (RBD) Antigens of SARS-CoV-2 over time post-infection and post-vaccination.One hundred twenty-four residents and 28 staff underwent saliva serologic testing on one or more survey visits. Over three surveys, the SARS-CoV-2 seroprevalence at the facility was 49%, 64%, and 81%, respectively. IgG to S, RBD, and N Antigens all increased post infection. Post vaccination, the infection naïve group did not have a detectable N IgG level, and N IgG levels for the previously infected did not increase post vaccination (p 0.001). Fully vaccinated subjects with prior COVID-19 infection had significantly higher RBD and S IgG responses compared with those who were infection-naïve prior to vaccination (p 0.001 for both).Positive SARS-COV-2 IgG in saliva was concordant with prior infection (Anti N, S, RBD) and vaccination (Anti S, RBD) and remained above positivity threshold for up to 9 months from infection. Salivary sampling is a non-invasive method of tracking immunity and differentiating between prior infection and vaccination to inform the need for boosters in LTC residents and staff.

BackgroundOral fluid (hereafter, saliva) is a non-invasive and attractive alternative to blood for SARS-CoV-2 IgG testing; however, the heterogeneity of saliva as a matrix poses challenges for immunoassay performance.ObjectivesTo optimize performance of a magnetic microparticle-based multiplex immunoassay (MIA) for SARS-CoV-2 IgG measurement in saliva, with consideration of: i) threshold setting and validation across different MIA bead batches; ii) sample qualification based on salivary total IgG concentration; iii) calibration to U.S. SARS-CoV-2 serological standard binding antibody units (BAU); and iv) correlations with blood-based SARS-CoV-2 serological and neutralizing antibody (nAb) assays.MethodsThe salivary SARS-CoV-2 IgG MIA included 2 nucleocapsid (N), 3 receptor-binding domain (RBD), and 2 spike protein (S) antigens. Gingival crevicular fluid (GCF) swab saliva samples were collected before December, 2019 (n=555) and after molecular test-confirmed SARS-CoV-2 infection from 113 individuals (providing up to 5 repeated-measures; n=398) and used to optimize and validate MIA performance (total n=953). Combinations of IgG responses to N, RBD and S and total salivary IgG concentration (μg/mL) as a qualifier of nonreactive samples were optimized and validated, calibrated to the U.S. SARS-CoV-2 serological standard, and correlated with blood-based SARS-CoV-2 IgG ELISA and nAb assays.ResultsThe sum of signal to cutoff (S/Co) to all seven MIA SARS-CoV-2 antigens and disqualification of nonreactive saliva samples with ≤15 μg/mL total IgG led to correct classification of 62/62 positives (sensitivity [Se]=100.0%; 95% confidence interval [CI]=94.8%, 100.0%) and 108/109 negatives (specificity [Sp]=99.1%; 95% CI=97.3%, 100.0%) at 8-million beads coupling scale and 80/81 positives (Se=98.8%; 95% CI=93.3%, 100.0%] and 127/127 negatives (Sp=100%; 95% CI=97.1%, 100.0%) at 20-million beads coupling scale. Salivary SARS-CoV-2 IgG crossed the MIA cutoff of 0.1 BAU/mL on average 9 days post-COVID-19 symptom onset and peaked around day 30. Among n=30 matched saliva and plasma samples, salivary SARS-CoV-2 MIA IgG levels correlated with corresponding-antigen plasma ELISA IgG (N: ρ=0.67, RBD: ρ=0.76, S: ρ=0.82; allpppConclusionsA salivary SARS-CoV-2 IgG MIA produced consistently high Se (>98.8%) and Sp (>99.1%) across two bead coupling scales and correlations with nAb responses that were similar to blood-based SARS-CoV-2 IgG ELISA data. This non-invasive salivary SARS-CoV-2 IgG MIA could increase engagement of vulnerable populations and improve broad understanding of humoral immunity (kinetics and gaps) within the evolving context of booster vaccination, viral variants and waning immunity.

Industrial livestock operations (ILOs), particularly processing facilities, emerged as centers of coronavirus disease 2019 (COVID-19) outbreaks in spring 2020. Confirmed cases of COVID-19 underestimate true prevalence. To investigate prevalence of antibodies against SARS-CoV-2, we enrolled 279 participants in North Carolina from February 2021 to July 2022: 90 from households with at least one ILO worker (ILO), 97 from high-ILO intensity areas (ILO neighbors – ILON), and 92 from metropolitan areas (Metro). Participants provided a saliva swab we analyzed for SARS-CoV-2 IgG using a multiplex immunoassay. Prevalence of infection-induced IgG (positive for nucleocapsid and receptor binding domain) was higher among ILO (63%) compared to ILON (42.9%) and Metro (48.7%) participants (prevalence ratio [PR] =1.38; 95% confidence interval [CI]: 1.06, 1.80; ref. ILON and Metro combined). Prevalence of infection-induced IgG was also higher among ILO participants compared to an Atlanta healthcare worker cohort (PR=2.45, 95% CI: 1.8, 3.3) and a general population cohort in North Carolina (PRs 6.37-10.67). Infection-induced IgG prevalence increased over the study period. Participants reporting not masking in public in the past two weeks had higher infection-induced IgG prevalence (78.6%) compared to participants reporting masking (49.3%) (PR=1.59; 95% CI: 1.19, 2.13). Lower education, more people per bedroom, Hispanic/Latino ethnicity, and more contact with people outside the home were also associated with higher infection-induced IgG prevalence. Similar proportions of ILO (51.6%), ILON (48.4%), and Metro (55.4%) participants completed the COVID-19 primary vaccination series; median completion was more than four months later for ILO compared to ILON and Metro participants.ImportanceFew studies have measured COVID-19 seroprevalence in North Carolina, especially among rural, Black, and Hispanic/Latino communities that have been heavily affected. Antibody results show high rates of COVID-19 among industrial livestock operation workers and their household members. Antibody results add to evidence of health disparities in COVID-19 by socioeconomic status and ethnicity. Associations between masking and physical distancing with antibody results also add to evidence of the effectiveness of these prevention strategies. Delays in the timing of receipt of COVID-19 vaccination reinforce the importance of dismantling vaccination barriers, especially for industrial livestock operation workers and their household members.

In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and "to develop, validate, improve, and implement serological testing and associated technologies" (https://www.cancer.gov/research/key-initiatives/covid-19/coronavirus-research-initiatives/serological-sciences-network). SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology standard reference material and first WHO international standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. SeroNet institutions reported development of a total of 27 enzyme-linked immunosorbent assay (ELISA) methods, 13 multiplex assays, and 9 neutralization assays and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. In conclusion, SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons. IMPORTANCE SeroNet institutions have developed or implemented 61 diverse COVID-19 serological assays and are collaboratively working to harmonize these assays using reference materials to establish standardized reporting units. This will facilitate clinical interpretation of serology results and cross-comparison of research data.

PurposeArsenic exposure and micronutrient deficiencies may alter immune reactivity to influenza vaccination in pregnant women, transplacental transfer of maternal antibodies to the fetus, and maternal and infant acute morbidity. The Pregnancy, Arsenic, and Immune Response (PAIR) Study is a longitudinal pregnancy and birth cohort designed to assess whether arsenic exposure and micronutrient deficiencies alter maternal or newborn immunity and acute morbidity following maternal seasonal influenza vaccination during pregnancy.ParticipantsWe enrolled 784 pregnant women in rural Gaibandha District in northern Bangladesh between October 2018 and March 2019. Women received a quadrivalent seasonal inactivated influenza vaccine at enrollment in the late first or early second trimester between 11 and 17 weeks of gestational age. Follow-up included up to 13 visits between enrollment and three months postpartum as well as weekly telephone surveillance to ascertain influenza-like illness and other acute morbidity symptoms in women and infants. Tube well drinking water and urine specimens were collected to assess arsenic exposure. Of 784 women who enrolled, 736 (93.9%) delivered live births and 551 (70.3%) completed follow-up visit to three months postpartum.Findings to DateArsenic was ≥0.02 µg/L in 97.9% of water specimens collected from participants at enrollment. The medians (interquartile ranges) of water and urinary arsenic were 5.1 (0.5-25.1) µg/L and 33.1 (19.6-56.5) µg/L, respectively. Water and urinary arsenic were strongly correlated (Spearman’s ρ=0.72) among women with water arsenic ≥ median but weakly correlated (ρ=0.18) among women with water arsenic < median.Future PlansThe PAIR Study is well positioned to examine the effects of low-moderate arsenic exposure and micronutrient deficiencies on immune outcomes in women and infants.RegistrationNCT03930017

Serological tests play an important role in the fight against Coronavirus Disease 2019 (COVID-19), including monitoring the dynamic immune response after vaccination, identifying past infection and determining community infection rate. Conventional methods for serological testing, such as enzyme-linked immunosorbent assays and chemiluminescence immunoassays, provide reliable and sensitive antibody detection but require sophisticated laboratory infrastructure and/or lengthy assay time. Conversely, lateral flow immunoassays are suitable for rapid point-of-care tests but have limited sensitivity. Here, we describe the development of a rapid and sensitive magnetofluidic immuno-PCR platform that can address the current gap in point-of-care serological testing for COVID-19. Our magnetofluidic immuno-PCR platform automates a magnetic bead-based, single-binding, and one-wash immuno-PCR assay in a palm-sized magnetofluidic device and delivers results in ∼30 min. In the device, a programmable magnetic arm attracts and transports magnetically-captured antibodies through assay reagents pre-loaded in a companion plastic cartridge, and a miniaturized thermocycler and a fluorescence detector perform immuno-PCR to detect the antibodies. We evaluated our magnetofluidic immuno-PCR with 108 clinical serum/plasma samples and achieved 93.8% (45/48) sensitivity and 98.3% (59/60) specificity, demonstrating its potential as a rapid and sensitive point-of-care serological test for COVID-19.

BackgroundSustained molecular detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in the upper respiratory tract (URT) in mild to moderate coronavirus disease 2019 (COVID-19) is common. We sought to identify host and immune determinants of prolonged SARS-CoV-2 RNA detection.MethodsNinety-five symptomatic outpatients self-collected midturbinate nasal, oropharyngeal (OP), and gingival crevicular fluid (oral fluid) samples at home and in a research clinic a median of 6 times over 1–3 months. Samples were tested for viral RNA, virus culture, and SARS-CoV-2 and other human coronavirus antibodies, and associations were estimated using Cox proportional hazards models.ResultsViral RNA clearance, as measured by SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR), in 507 URT samples occurred a median (interquartile range) 33.5 (17–63.5) days post–symptom onset. Sixteen nasal-OP samples collected 2–11 days post–symptom onset were virus culture positive out of 183 RT-PCR-positive samples tested. All participants but 1 with positive virus culture were negative for concomitant oral fluid anti-SARS-CoV-2 antibodies. The mean time to first antibody detection in oral fluid was 8–13 days post–symptom onset. A longer time to first detection of oral fluid anti-SARS-CoV-2 S antibodies (adjusted hazard ratio [aHR], 0.96; 95% CI, 0.92–0.99; P = .020) and body mass index (BMI) ≥25 kg/m2 (aHR, 0.37; 95% CI, 0.18–0.78; P = .009) were independently associated with a longer time to SARS-CoV-2 viral RNA clearance. Fever as 1 of first 3 COVID-19 symptoms correlated with shorter time to viral RNA clearance (aHR, 2.06; 95% CI, 1.02–4.18; P = .044).ConclusionsWe demonstrate that delayed rise of oral fluid SARS-CoV-2-specific antibodies, elevated BMI, and absence of early fever are independently associated with delayed URT viral RNA clearance.

We evaluated the durability of IgG responses specific to SARS-CoV-2 nucleocapsid (N), receptor binding domain (RBD), and spike (S) antigens in saliva up to 8 months after RT-PCR-confirmed COVID-19 using a multiplex salivary assay. We estimated a half-life of 64 days (d) (95% CI: 49, 80 d) for N, 100 d for RBD (95% CI: 58, 141 d), and 148 d (95% CI: 62, 238 d) for S IgG responses in saliva, consistent with half-life estimates previously reported in blood. Saliva can serve as an alternative to blood to monitor humoral immune responses on a large scale following SARS-CoV-2 infection and vaccination for surveillance and assessment of population immunity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of an ongoing pandemic that has infected over 36 million and killed over 1 million people. Informed implementation of government public health policies depends on accurate data on SARS-CoV-2 immunity at a population scale. We hypothesized that detection of SARS-CoV-2 salivary antibodies could serve as a noninvasive alternative to serological testing for monitoring of SARS-CoV-2 infection and seropositivity at a population scale., Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of an ongoing pandemic that has infected over 36 million and killed over 1 million people. Informed implementation of government public health policies depends on accurate data on SARS-CoV-2 immunity at a population scale. We hypothesized that detection of SARS-CoV-2 salivary antibodies could serve as a noninvasive alternative to serological testing for monitoring of SARS-CoV-2 infection and seropositivity at a population scale. We developed a multiplex SARS-CoV-2 antibody immunoassay based on Luminex technology that comprised 12 CoV antigens, mostly derived from SARS-CoV-2 nucleocapsid (N) and spike (S). Saliva and sera collected from confirmed coronavirus disease 2019 (COVID-19) cases and from the pre-COVID-19 era were tested for IgG, IgA, and IgM to the antigen panel. Matched saliva and serum IgG responses (n = 28) were significantly correlated. The salivary anti-N IgG response resulted in the highest sensitivity (100%), exhibiting a positive response in 24/24 reverse transcription-PCR (RT-PCR)-confirmed COVID-19 cases sampled at >14 days post-symptom onset (DPSO), whereas the salivary anti-receptor binding domain (RBD) IgG response yielded 100% specificity. Temporal kinetics of IgG in saliva were consistent with those observed in blood and indicated that most individuals seroconvert at around 10 DPSO. Algorithms employing a combination of the IgG responses to N and S antigens result in high diagnostic accuracy (100%) by as early as 10 DPSO. These results support the use of saliva-based antibody testing as a noninvasive and scalable alternative to blood-based antibody testing.