key: cord-0890576-a74u6mws
authors: Horton, Daniel B; Barrett, Emily S; Roy, Jason; Gennaro, Maria Laura; Andrews, Tracy; Greenberg, Patricia; Bruiners, Natalie; Datta, Pratik; Ukey, Rahul; Velusamy, Senthil K; Fine, Daniel; Honnen, William J; Yin, Yue Sandra; Pinter, Abraham; Brooks, Andrew; Tischfield, Jay; Hussain, Sabiha; Jagpal, Sugeet; Swaminathan, Shobha; Parmar, Veenat; Reilly, Nancy; Gaur, Sunanda; Panettieri, Jr., Reynold A; Carson, Jeffrey L; Blaser, Martin J
title: Determinants and dynamics of SARS-CoV-2 infection in a diverse population: 6-month evaluation of a prospective cohort study
date: 2021-08-13
journal: J Infect Dis
DOI: 10.1093/infdis/jiab411
sha: 00100c41ac5cb705f074dc9a9f8ce582e1bda21d
doc_id: 890576
cord_uid: a74u6mws

BACKGROUND: We studied risk factors, antibody responses, and symptoms of SARS-CoV-2 infection in a diverse, ambulatory population. METHODS: A prospective cohort (n=831, including 548 hospital-based healthcare workers) previously undiagnosed with SARS-CoV-2 infection was followed for six months with serial testing (SARS-CoV-2 PCR, specific IgG) and surveys. RESULTS: 93 participants (11.2%) tested SARS-CoV-2-positive; 14 (15.1%) were asymptomatic and 24 (25.8%), severely symptomatic. Healthcare workers were more likely to become infected (14.2% vs. 5.3%, aOR 2.1, 95% CI 1.4-3.3) and have severe symptoms (29.5% vs. 6.7%). IgG antibodies were detected after 79% of asymptomatic infections, 89% with mild-moderate symptoms, and 96% with severe symptoms. IgG trajectories after asymptomatic infection (slow increases) differed from symptomatic infections (early peaks within 2 months). Most participants (92%) had persistent IgG responses (median 171 days). In multivariable models, IgG titers were positively associated with symptom severity, certain comorbidities, and hospital work. Dyspnea, altered smell and taste, and other neurologic changes persisted for ≥120 days in ≥10% of affected participants. Participants with prolonged symptoms (generally more severely symptomatic) had higher antibody levels. CONCLUSIONS: In a prospective, ethnically diverse cohort, symptom severity correlated with the magnitude and trajectory of IgG production. Symptoms frequently persisted for many months after infection.

Nonetheless, asymptomatic infections may be accompanied by subclinical abnormalities in laboratory tests and lung imaging [6] . Important questions remain about long-term clinical and immunologic consequences of asymptomatic infections.

Most persons infected by SARS-CoV-2 develop antibodies against the virus [7] . However, immune responses vary considerably, with a minority of infected people not producing detectable antibodies [8] .

The magnitude of humoral immune responses may be proportional to illness severity [9, 10] . The duration and trajectory of humoral immunity also remains unclear; some studies report substantial declines in antibody responses within a few months [11, 12] while others report persistent responses over many months [8, 13, 14] . One challenge in interpreting these studies is differences in study populations: most studies have focused on hospitalized, convalescent, and referred patients previously diagnosed with SARS-CoV-2 infection, raising questions about selection bias and generalizability. Few prospective studies have systematically evaluated long-term antibody trends and associated factors among diverse, previously undiagnosed populations of individuals across a spectrum of illness severity, including asymptomatic infections [15] .

A c c e p t e d M a n u s c r i p t 7 We characterized the incidence of and risk factors for SARS-CoV-2 infection in a prospective cohort of ambulatory, previously undiagnosed healthcare workers (HCWs) and non-HCWs recruited early in the U.S. pandemic and followed over 6 months. The study was conducted in New Jersey (NJ), an ethnically diverse state hit particularly hard by the spring 2020 COVID-19 surge [1, 16] . We further examined dynamics and correlates of anti-SARS-CoV-2 antibodies and persistence of symptoms up to 6 months post-infection.

Study design and population. As described [17] , the Rutgers Corona Cohort (RCC) is a prospective, university-based observational cohort of HCWs and non-HCW comparators recruited and consented March 24-April 7, 2020, across two campuses (Newark and New Brunswick/Piscataway). Eligibility criteria included: (1) age ≥20 years; (2) not pregnant or breastfeeding; (3) no recent (prior 30 days) urgent care or emergency department (ED) visits, hospitalizations, operations, or changes in prescribed medicines; (4) no previously diagnosed SARS-CoV-2 infection/COVID- 19; and (5) no fever at the baseline visit. Eligibility for HCWs required: (1) ≥20 hours of weekly hospital work; (2) roles with regular patient exposure (e.g., physicians, nurses, technicians, respiratory therapists); and (3) regular direct patient contact (≥3 patients/shift). Eligibility for a comparator group of non-HCWs required: (1) work as faculty, staff, or students at Rutgers for ≥20 hours weekly; and (2) no patient contact. Hospital-based employees without direct patient care responsibilities were not eligible for enrollment. All study activities were approved by the Rutgers Institutional Review Board (Pro2020000679) and all participants provided electronic informed consent prior to engaging in study activities.

A c c e p t e d M a n u s c r i p t Additional information about SARS-CoV-2-positive participants was obtained through follow-up surveys, telephone calls, and medical chart review; symptoms were assessed using all available data sources.

Among symptomatic participants, overall symptom severity was assessed using the question: "Please consider any past or present COVID-19 symptoms when answering the following question: Overall, when these symptoms were at their worst, how bad or bothersome were they?" Responses options included: mild, moderate, severe, and very severe. Based on the distribution of responses, we categorized symptom severity as asymptomatic, mild-moderate, or severe.

All study data were managed using REDCap electronic data capture tools hosted at Rutgers Robert Wood Johnson Medical School [19] .

A c c e p t e d M a n u s c r i p t 9 SARS-CoV-2 assays. SARS-CoV-2 assays were conducted at all study visits under FDA-approved EUA#200090 at Infinity Biologix® (Piscataway NJ), as described [17, 18] . In brief, total RNA was extracted from OPS using nucleic acid-binding paramagnetic beads (Chemagic Viral DNA/RNA 300 Kit H96).

Reverse transcriptase-PCR (RT-PCR) was performed in triplicate for three SARS-CoV-2 genomic regions: nucleocapsid (N), spike protein (S), and ORF1ab. Positive and negative assay controls were used.

We used an in-house developed ELISA platform for antibody binding to two portions of SARS-CoV-2 spike protein (S1 subunit receptor-binding domain (RBD), full-length S2 subunit) [20] . Detection of antigen-bound antibodies used combined alkaline phosphatase-conjugated anti-human IgA, IgM, and IgG secondary antibodies, or anti-human IgG antibody alone, at 1:2,000 dilution (Supplementary Methods, Supplementary Table 1) . For participants with OD 405 ≥1 positive IgM/G/A (total) antibody test and/or positive PCR, anti-RBD IgG titers were determined. Seropositivity was defined as: total antibody levels OD 405 ≥0.7 across ≥2 timepoints or ≥1.0 once; or IgG titers ≥1:80 across ≥2 timepoints or ≥1:320 once. We chose RBD as our solid-phase antigen in our assay, because it is a preferred target of neutralizing antibodies [21] .

Comprehensive metabolic panels were analyzed on baseline plasma samples, and cell counts were analyzed on whole blood samples from all visits, using standard clinical assays (Beckman Coulter).

A c c e p t e d M a n u s c r i p t testing (IgM/G/A or IgG only). Comparisons of characteristics between SARS-CoV-2-infected anduninfected participants and between HCWs and non-HCWs used chi-square or Fisher exact testing (categorical data) and t-tests or Wilcoxon rank sum testing (continuous data), as appropriate. Trends were evaluated across levels of symptom severity using Cochran-Armitage tests (categorical data) and

Jonckheere-Terpstra tests (continuous data).

To identify explanatory baseline and early exposure characteristics associated with likelihood of SARS-CoV-2 infection, multivariable logistic regression models were fitted with elastic net penalty for regularization, permitting selection from many variables ( Table 1 ) while avoiding overfitting due to penalties on regression coefficients. Separate models were applied to all participants and to HCWs, the latter including HCW-specific variables (e.g., role, PPE use) ( Table 1) . Models accounted for time-varying exposures (e.g., sick contacts, patient care metrics) over the first study month, during the first surge's peak [22] ; data after SARS-CoV-2 diagnosis were excluded to limit bias from factors resulting from infection. Table 1 . Missing data was imputed using multiple imputation with chained equations based on 50 imputed data sets [23] .

Presence and persistence of symptoms over time was graphed using Kaplan-Meier plots and summarized by the median, 75 th and 90 th percentiles.

Analyses were performed using SAS 9.4, R 4.0.3, and Stata 16.1.

We enrolled 831 participants (548 HCWs, 283 non-HCWs) (Supplementary Figure 1) ; 722 (86.9%) completed a 26-week visit, and 758 (91.2%) completed at least 5 of 6 study visits. Overall, 71% of participants completed at least 12 of 16 follow-up questionnaires. Two-thirds of participants were female, and half were <40 years old ( Table 1 A c c e p t e d M a n u s c r i p t 12 COVID-19 within one month of enrollment. Compared to non-HCWs, HCWs were younger, more racially diverse, more likely Newark-based, and more likely to report unprotected COVID-19 exposures at and outside work before diagnosis (Supplementary Table 2 ). Compared to eligible persons who did not enroll, enrolled participants were more likely to be of Hispanic/Latino ethnicity, have certain comorbidities (e.g., respiratory, autoimmune), be a HCW, and be recruited at the Newark campus, and less likely to be female and a HCW caring for patients with COVID-19 (Supplementary Table 3 ).

Ultimately, 93 participants (11.2%) tested positive for virus and/or antibodies, 86 (92.5%) within the first 2 months of the study, echoing trends more broadly in NJ and participating hospitals (Supplementary (Figure 1A) . Among HCWs, nursing role and Newark affiliation were most positively associated with infection, whereas work in ICUs or COVID-19 units was negatively associated with infection ( Figure 1B ). More extensive N95 use was reported among SARS-CoV-2-negative participants ( Table 1) .

Median follow-up after diagnosis was 171 days (IQR 158, 180). Not including 2 participants newly PCRpositive at week 26, overall seropositivity was lower among asymptomatic participants (IgG 79%) compared to mildly-moderately symptomatic (IgG 89%) and severely symptomatic participants (IgG 96%) (Supplementary Table 4) . At the final visit, IgG antibody prevalences among previously infected participants were: asymptomatic, IgG 69%; mildly-moderately symptomatic, IgG 83%; severely symptomatic, IgG 91%. Among antibody-positive participants (by total Ig or IgG) infected in the first wave with available samples at month 6, detectable antibodies persisted in most (67/73, 92%) participants, irrespective of symptom severity. Severe symptomatic illness was most strongly associated with higher IgG titers over time (coefficient 1.03, 95% CI 0.71, 1.36) (Figures 2-3, Supplementary Table   4, Supplementary Figure 5) . Other factors associated with higher IgG titers included several comorbidities and HCW status; factors associated with lower IgG titers included Asian race, smoking, and working on-site within one month after enrollment (Figure 3) .

A c c e p t e d M a n u s c r i p t 14 Among symptomatic infected participants, the median duration of most symptoms was ≤2 weeks except for neurologic changes besides altered smell and taste (e.g., brain fog, memory problems, visual disturbances; median 45 days), which were least prevalent among 15 symptoms measured (changes reported in 12% of participants) (Figure 4, Supplementary Figure 6) . Nonetheless, multiple symptoms were reported in ≥25% of affected individuals for ≥30 days, and ≥10% of affected individuals reported having ≥120 days of shortness of breath, chest congestion, loss of small and/or taste, and other neurologic changes (Figure 4, Supplementary Figure 6 ). About one-third (33/93, 35%) reported symptoms lasting 30 days or longer. Not surprisingly, symptom duration was correlated with symptom severity (r = 0.26, p = 0.03) and antibody titer ( Figure 5 ) (p=0.03).

This study represents a 6-month prospective cohort study of risk factors, humoral responses, and symptoms in ambulatory, previously undiagnosed, at-risk individuals, recruited from a diverse professional community affected early in the US pandemic. Over one in ten participants were SARS-CoV-2-infected, and most were followed for 5-6 months with excellent cohort retention. HCWs were more likely to become infected and have more severe illness. Among hospital workers, nurses were at greater risk for infection, whereas ICU and COVID-19 unit workers were at lower risk. Symptom severity and duration were associated with magnitude and trajectory of antibody responses. In contrast, most demographic characteristics, comorbidities (except hypertension), and laboratory criteria were not associated with antibody responses. Persons with asymptomatic infections had few changes in cell counts, lower seroconversion rates, and lower antibody levels. Multiple symptoms lasted one month or A c c e p t e d M a n u s c r i p t 15 longer in at least 25% of participants; neurologic changes besides altered smell or taste were less frequent (~1/8) but generally long-lasting in those reporting them.

We and others have reported increased risks of SARS-CoV-2 among HCWs [17, 22, 24] . In our cohort, professional role (e.g., nursing) was associated with greater risk. We also observed differences in illness severity between HCWs and others: HCWs were more likely to have severe symptoms and more robust antibody responses, consistent with other research suggesting higher risk of hospitalization among infected HCWs, particularly those with patient-facing roles, including nurses [25, 26] . In contrast to some [26] but consistent with other [27] studies, we observed lower infection rates among intensive care unit workers and even those on COVID-19 units, which may have related to more rigorous N95-mask usage. Unlike in other studies [28] , HCWs in our cohort were less likely to live in areas with higher rates of local transmission, and the increased rates observed were not well explained by outside exposures.

The excess rates of infection in Newark may have resulted from later implementation of universal masking in that hospital versus in New Brunswick [29] .

Our longitudinal cohort study contributes new insights into several aspects of the humoral response to SARS-CoV-2 infection. One is the correlation between rates of seropositivity and presence or severity of symptoms. Some studies have reported high rates of seropositivity after even asymptomatic or mild infections [6, 13] , while others have found a correlation between illness severity and seropositivity [9, 30] . Such distinctions may relate to differences in recruitment, since studies that recruited previously diagnosed or self-referred infected volunteers are subject to selection bias by excluding those with asymptomatic or milder, undiagnosed infection. This was not an issue with our prospective cohort study design, which minimized the influence of selection bias and enhanced the internal validity of our A c c e p t e d M a n u s c r i p t 16 findings. Second, we observed a strong positive correlation between antibody levels and symptom severity, which might be explained by stronger B cell activation in the context of excessive inflammation typically associated with severe COVID-19 [31] . Since our cohort comprised an ambulatory population with mostly mild infections not requiring hospitalization, our work expands on previous observations of positive correlations between strength of antibody responses and COVID-19 severity, which were obtained in studies including only hospitalized patients or clinically diagnosed convalescent cases [32] [33] [34] . Moreover, while our study does not address cellular immunity, it allows for indirect inferences about immune status, since no or weak anti-SARS-CoV-2 antibody responses are accompanied by low frequencies of antigen-specific T cells [35] . Third, prior reports of anti-SARS-CoV-2 antibody trajectories have been conflicting, with some studies reporting antibody declines and loss of detectable antibodies, particularly following asymptomatic infections [6, 11, 12] , and others showing antibody responses persisting for several months [8, 13, 14, 36] . In our study, most participants had sustained IgG up to 6 months after infection, irrespective of symptom severity. We also found severity-related differences in antibody trajectories, with slow, steady increases following asymptomatic infections compared to sharper rises and declines after symptomatic infections. These findings echo previous findings of sustained, non-declining antibody responses in those with milder infections [36] .

We cannot extrapolate our results to SARS-CoV-2 variants of concern (VOCs), since our cohort was recruited and followed in 2020 during earlier phases of the pandemic, prior to the emergence of several VOCs, including the highly transmissible Delta variant [37, 38] . Furthermore, given the relative reduction of protective immune responses against the Delta variant among previously infected persons [39, 40] , our data do not support changes in current recommendations for vaccinating those with history of prior infection.

A c c e p t e d M a n u s c r i p t 17 Our data on the duration of symptoms complement other reports of prolonged abnormalities in studies of previously infected subjects [41] [42] [43] [44] . Notably, we studied an ambulatory population with generally mild illnesses, most not requiring hospitalization, over a six-month timeframe that bracketed two infection surges in NJ. Of infected participants, about one-third reported symptoms lasting at least 30 days, and greater than 10% had persistent symptoms lasting for months, including fatigue, altered smell and taste, and shortness of breath. Notably, in contrast to other research [45] , we found that participants with longer duration of symptoms also had higher levels of antibodies over time, perhaps reflecting the correlation between symptom duration and symptom severity, although further study is warranted on the immune profiles among those with prolonged symptoms. Nonetheless, since mild illnesses are much more common than illnesses that required hospitalization, the frequency of prolonged symptoms in our cohort raises a cautionary note about post-SARS-CoV-2 sequelae.

Our study had multiple strengths. The prospective inclusion of generally healthy, ethnically diverse, previously undiagnosed participants followed longitudinally from the early phases of the USA pandemic, with exceptional retention, captured a range of clinical responses, including asymptomatic infections.

Most participants received no medical intervention for their illness (86%), allowing us to characterize the natural history of disease and biomarker trajectories in a predominantly untreated cohort.

Compared to results from hospitalized or convalescent cohorts, our findings may be more generalizable to the broader population of people with mild or asymptomatic infections [3, 4] , often undiagnosed and contributing to viral transmission [5] . Following participants at 6 time points over 6 months, with ≥5 months of follow-up data for most SARS-CoV-2-infected participants, enabled study of antibody This study also had limitations. The rapid enrollment of a highly motivated convenience cohort may have preferentially enriched the study population with persons who perceived themselves at higher risk for infection, such as people with underlying respiratory diseases (although not itself a risk factor in our analysis). Enrolled participants and eligible participants who did not enroll also differed in certain respects related to the populations recruited at each campus location (e.g., more healthcare workers and Hispanic/Latino participants in Newark). Nonetheless, the enrollment of persons not previously diagnosed with infection remains a strength of this study, and we do not believe that the enrollment procedures substantially affected the internal validity of our findings. Certain analyses were limited in statistical power due to smaller sample sizes of infected individuals. Less frequent sampling during periods of lower transmission may have missed some asymptomatic infections without seroconversion. Disease severity was based on self-report, but this classification correlated well with symptom burden, levels of care and treatments received, and antibody responses. Sources of infectious exposure among participants could not be known with certainty; some infected participants who reported infected household members before their diagnosis may still have been the source of infections for other household members. Finally, given the differences in infection risk and severity between HCWs and non-HCWs, findings from our study population may not fully generalize to all populations. a PCR-or antibody-positive for SARS-CoV-2 were classified as positive

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We thank the Rutgers Corona Cohort (RCC) participants serving on the frontlines of the pandemic in New Jersey.We acknowledge the work of the entire RCC study team, all based at Rutgers University: for conducting In-kind logistics support was provided by Marken/UPS and dfYoung.

A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t