Prevalence, Patterns, and Predictors of SARS-CoV-2 RNA and Culturable Virus in Tears of a Case-Ascertained Household Cohort.

PURPOSE: To investigate the prevalence, patterns, and predictors of SARS-CoV-2 RNA and culturable virus in tears of a case-ascertained household cohort. DESIGN: Prospective, longitudinal case-ascertained household cohort identified through convenience sampling. METHODS: This analysis was restricted to individuals who were non-hospitalized, symptomatic, and tested positive for SARS-CoV-2 by nasal RT-PCR. Tears and anterior nasal biospecimens were serially collected throughout the acute period. Tears specimens were collected by the study staff using Schirmer test strips, and nasal specimens were self-collected. For both, SARS-CoV-2 RNA was quantified using qRT-PCR, and culturable virus was detected using presence of cytopathic effect (CPE) in tissue culture; positive CPE was confirmed by a qRT-PCR step. A series of cross-sectional unadjusted analyses were performed investigating the relationship between different sociodemographic determinants and biological factors associated with tears RNA positivity. RESULTS: Among the 83 SARS-CoV-2 infected participants, 10 (12%) had at least one RNA-positive tears specimen. Amongst these 10, 5 (50%) had concurrent presence of culturable virus, at a median of 7 days postsymptom onset (IQR: 4-7 days) (absolute range: 4-8 days). CONCLUSIONS: In this longitudinal cohort, we found evidence of culturable virus in the tears of a small proportion of nonhospitalized SARS-CoV-2 infected individuals. Current public health infection precautions do not account for transmission via tears, so these findings may improve our understanding of potential sources of SARS-CoV-2 transmission and contribute to developing future guidelines.

Viral Determinants of Acute COVID-19 Symptoms in a Nonhospitalized Adult Population in the Pre-Omicron Era.

Background: The influence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA level and presence of infectious virus on symptom occurrence is poorly understood, particularly among nonhospitalized individuals. Methods: The study included 85 nonhospitalized, symptomatic adults, who were enrolled from September 2020 to November 2021. Data from a longitudinal cohort studied over 28 days was used to analyze the association of individual symptoms with SARS-CoV-2 viral RNA load, or the presence or level of infectious (culturable) virus. Presence of infectious virus and viral RNA load were assessed daily, depending on specimen availability, and amount of infectious virus was assessed on the day of maximum RNA load. Participants were surveyed for the start and end dates of 31 symptoms at enrollment and at days 9, 14, 21, and 28; daily symptom presence was determined analytically. We describe symptoms and investigate their possible association with viral determinants through a series of single or pooled (multiple days across acute period) cross-sectional analyses. Results: There was an association between viral RNA load and the same-day presence of many individual symptoms. Additionally, individuals with infectious virus were more than three times as likely to have a concurrent fever than individuals without infectious virus, and more than two times as likely to have concurrent myalgia, chills, headache, or sore throat. Conclusions: We found evidence to support the association of viral RNA load and infectious virus on some, but not all symptoms. Fever was most strongly associated with the presence of infectious virus; this may support the potential for symptom-based isolation guidance for COVID-19.

Longitudinal and quantitative fecal shedding dynamics of SARS-CoV-2, pepper mild mottle virus, and crAssphage.

Wastewater-based epidemiology (WBE) emerged during the coronavirus disease 2019 (COVID-19) pandemic as a scalable and broadly applicable method for community-level monitoring of infectious disease burden. The lack of high-resolution fecal shedding data for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) limits our ability to link WBE measurements to disease burden. In this study, we present longitudinal, quantitative fecal shedding data for SARS-CoV-2 RNA, as well as for the commonly used fecal indicators pepper mild mottle virus (PMMoV) RNA and crAss-like phage (crAssphage) DNA. The shedding trajectories from 48 SARS-CoV-2-infected individuals suggest a highly individualized, dynamic course of SARS-CoV-2 RNA fecal shedding. Of the individuals that provided at least three stool samples spanning more than 14 days, 77% had one or more samples that tested positive for SARS-CoV-2 RNA. We detected PMMoV RNA in at least one sample from all individuals and in 96% (352/367) of samples overall. CrAssphage DNA was detected in at least one sample from 80% (38/48) of individuals and was detected in 48% (179/371) of all samples. The geometric mean concentrations of PMMoV and crAssphage in stool across all individuals were 8.7 × 104 and 1.4 × 104 gene copies/milligram-dry weight, respectively, and crAssphage shedding was more consistent for individuals than PMMoV shedding. These results provide us with a missing link needed to connect laboratory WBE results with mechanistic models, and this will aid in more accurate estimates of COVID-19 burden in sewersheds. Additionally, the PMMoV and crAssphage data are critical for evaluating their utility as fecal strength normalizing measures and for source-tracking applications. IMPORTANCE This research represents a critical step in the advancement of wastewater monitoring for public health. To date, mechanistic materials balance modeling of wastewater-based epidemiology has relied on SARS-CoV-2 fecal shedding estimates from small-scale clinical reports or meta-analyses of research using a wide range of analytical methodologies. Additionally, previous SARS-CoV-2 fecal shedding data have not contained sufficient methodological information for building accurate materials balance models. Like SARS-CoV-2, fecal shedding of PMMoV and crAssphage has been understudied to date. The data presented here provide externally valid and longitudinal fecal shedding data for SARS-CoV-2, PMMoV, and crAssphage which can be directly applied to WBE models and ultimately increase the utility of WBE.

Association of Culturable-Virus Detection and Household Transmission of SARS-CoV-2, California and Tennessee, 2020-2022.

From 2 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) household transmission studies (enrolling April 2020 to January 2022) with rapid enrollment and specimen collection for 14 days, 61% (43/70) of primary cases had culturable virus detected ≥6 days post-onset. Risk of secondary infection among household contacts tended to be greater when primary cases had culturable virus detected after onset. Regardless of duration of culturable virus, most secondary infections (70%, 28/40) had serial intervals <6 days, suggesting early transmission. These data examine viral culture as a proxy for infectiousness, reaffirm the need for rapid control measures after infection, and highlight the potential for prolonged infectiousness (≥6 days) in many individuals.

Social Network Analysis of Ebola Virus Disease During the 2014 Outbreak in Sukudu, Sierra Leone.

Background: Transmission by unreported cases has been proposed as a reason for the 2013-2016 Ebola virus (EBOV) epidemic decline in West Africa, but studies that test this hypothesis are lacking. We examined a transmission chain within social networks in Sukudu village to assess spread and transmission burnout. Methods: Network data were collected in 2 phases: (1) serological and contact information from Ebola cases (n = 48, including unreported); and (2) interviews (n = 148), including Ebola survivors (n = 13), to identify key social interactions. Social links to the transmission chain were used to calculate cumulative incidence proportion as the number of EBOV-infected people in the network divided by total network size. Results: The sample included 148 participants and 1522 contacts, comprising 10 social networks: 3 had strong links (>50% of cases) to the transmission chain: household sharing (largely kinship), leisure time, and talking about important things (both largely non-kin). Overall cumulative incidence for these networks was 37 of 311 (12%). Unreported cases did not have higher network centrality than reported cases. Conclusions: Although this study did not find evidence that explained epidemic decline in Sukudu, it excluded potential reasons (eg, unreported cases, herd immunity) and identified 3 social interactions in EBOV transmission.

Evaluation of Severe Acute Respiratory Syndrome Coronavirus 2 Nucleocapsid Antigen in the Blood as a Diagnostic Test for Infection and Infectious Viral Shedding.

Background: SARS-CoV-2 nucleocapsid antigen can be detected in plasma, but little is known about its performance as a diagnostic test for acute SARS-CoV-2 infection or infectious viral shedding among nonhospitalized individuals. Methods: We used data generated from anterior nasal and blood samples collected in a longitudinal household cohort of SARS-CoV-2 cases and contacts. Participants were classified as true positives if polymerase chain reaction (PCR) positive for SARS-CoV-2 and as true negatives if PCR negative and seronegative. Infectious viral shedding was determined by the cytopathic effect from viral culture. Stratified by 7 days after symptom onset, we constructed receiver operating characteristic (ROC) curves to describe optimized accuracy (Youden index), optimized sensitivity, and specificity. Results: Of 80 participants, 58 (73%) were true positives while 22 (27%) were true negatives. Using the manufacturer's cutoff of 1.25 pg/mL for evaluating infection, sensitivity was higher from 0 to 7 days (77.6% [95% confidence interval {CI}, 64%-88.2%]) than from 8 to 14 days (43.2% [95% CI, 31.1%-54.5%]) after symptom onset; specificity was unchanged at 100% (95% CI, 88.1%-100%). This test had higher sensitivity (100% [95% CI, 88.4%-100%]) and lower specificity (65% [95% CI, 40.8%-84.6%]) for infectious viral shedding as compared with infection, particularly within the first week of symptom onset. Although the presence of N-antigen correlated with infectious viral shedding (r = 0.63; P < .01), sensitivity still declined over time. Additional cutoffs from ROC curves were identified to optimize sensitivity and specificity. Conclusions: We found that this SARS-CoV-2 N-antigen test was highly sensitive for detecting early but not late infectious viral shedding, making it a viable screening test for community-dwelling individuals to inform isolation practices.

Infectious viral shedding of SARS-CoV-2 Delta following vaccination: A longitudinal cohort study.

The impact of vaccination on SARS-CoV-2 infectiousness is not well understood. We compared longitudinal viral shedding dynamics in unvaccinated and fully vaccinated adults. SARS-CoV-2-infected adults were enrolled within 5 days of symptom onset and nasal specimens were self-collected daily for two weeks and intermittently for an additional two weeks. SARS-CoV-2 RNA load and infectious virus were analyzed relative to symptom onset stratified by vaccination status. We tested 1080 nasal specimens from 52 unvaccinated adults enrolled in the pre-Delta period and 32 fully vaccinated adults with predominantly Delta infections. While we observed no differences by vaccination status in maximum RNA levels, maximum infectious titers and the median duration of viral RNA shedding, the rate of decay from the maximum RNA load was faster among vaccinated; maximum infectious titers and maximum RNA levels were highly correlated. Furthermore, amongst participants with infectious virus, median duration of infectious virus detection was reduced from 7.5 days (IQR: 6.0-9.0) in unvaccinated participants to 6 days (IQR: 5.0-8.0) in those vaccinated (P = 0.02). Accordingly, the odds of shedding infectious virus from days 6 to 12 post-onset were lower among vaccinated participants than unvaccinated participants (OR 0.42 95% CI 0.19-0.89). These results indicate that vaccination had reduced the probability of shedding infectious virus after 5 days from symptom onset.

Magnitude and Determinants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Household Transmission: A Longitudinal Cohort Study.

BACKGROUND: Households have emerged as important venues for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission. Little is known, however, regarding the magnitude and determinants of household transmission in increasingly vaccinated populations. METHODS: From September 2020 to January 2022, symptomatic nonhospitalized individuals with SARS-CoV-2 infection by RNA detection were identified within 5 days of symptom onset; all individuals resided with at least 1 other SARS-CoV-2-uninfected household member. These infected persons (cases) and their household members (contacts) were subsequently followed with questionnaire-based measurement and serial nasal specimen collection. The primary outcome was SARS-CoV-2 infection among contacts. RESULTS: We evaluated 42 cases and their 74 household contacts. Among the contacts, 32 (43%) became infected, of whom 5 (16%) were asymptomatic; 81% of transmissions occurred by 5 days after the case's symptom onset. From 21 unvaccinated cases, 14-day cumulative incidence of SARS-CoV-2 infection among contacts was 18/40 (45% [95% confidence interval {CI}, 29%-62%]), most of whom were unvaccinated. From 21 vaccinated cases, 14-day cumulative incidence of SARS-CoV-2 infection was 14/34 (41% [95% CI, 25%-59%]) among all contacts and 12/29 (41% [95% CI, 24%-61%]) among vaccinated contacts. At least 1 comorbid condition among cases and 10 or more days of RNA detection in cases were associated with increased risk of infection among contacts. CONCLUSIONS: Among households including individuals with symptomatic SARS-CoV-2 infection, both vaccinated-to-vaccinated and unvaccinated-to-unvaccinated transmission of SARS-CoV-2 to household contacts was common. Because vaccination alone did not notably reduce risk of infection, household contacts will need to employ additional interventions to avoid infection.

A post-outbreak assessment of exposure proximity and Ebola virus disease-related stigma among community members in Kono District, Sierra Leone: A cross-sectional study.

Background: Based on findings from other contexts, informed by intergroup contact theory, that more contact is associated with less stigma, we hypothesized that community members with greater exposure to cases of Ebola virus disease (EVD) were less likely to report EVD-related stigma towards EVD survivors. We assessed personal stigmatizing attitudes towards Ebola survivors, which reflects personal fear and judgement, as well as perceived stigma towards EVD survivors, which reflects an individual's perception of the attitudes of the community towards a stigmatized group. Methods: From September 2016 to July 2017, we conducted a cross-sectional, community-based study of EVD-related stigma among individuals who did not contract Ebola in four EVD-affected rural communities of Kono District, Sierra Leone. We identified individuals from all quarantined households and obtained a random sample of those who were unexposed. Exposed individuals either lived in a quarantined household or were reported to have been in contact with an EVD case. Our explanatory variable was proximity to an EVD case during the outbreak. Our primary outcome was stigma towards EVD survivors, measured by a 6-item adapted HIV-related stigma index validated in Zambia and South Africa, with 1 item reflecting personal stigmatizing attitudes and 5 items reflecting perceived community stigma. The 6-item EVD stigma index had good internal consistency (Cronbach's alpha=0.82). We used modified Poisson and negative binomial regression models, adjusting for potential confounders, to estimate the association between exposure proximity and EVD stigma. Results: We interviewed 538 participants aged 12 to 85 years. Most (57%) had been quarantined. Over one-third (39%) reported personal stigmatizing attitudes or perceived community stigma; the most frequently endorsed item was fear and judgment towards EVD survivors. Having contact with someone with EVD was significantly associated with a lower likelihood of perceived community stigma (prevalence ratio [PR], 0.26; 95% CI, 0.13-0.54) and personal stigmatizing attitudes (PR, 0.44; 95% CI, 0.29-0.65). In contrast, being quarantined was significantly associated with a higher likelihood of perceived community stigma (PR, 3.9; 95% CI, 1.5-10.1). Conclusions: In this cross-sectional study, we found evidence of an inverse relationship between EVD-related stigma and contact with an EVD case. This finding substantiates intergroup contact theory and may form the basis for anti-stigma interventions.

Association of Lower Exposure Risk With Paucisymptomatic/Asymptomatic Infection, Less Severe Disease, and Unrecognized Ebola Virus Disease: A Seroepidemiological Study.

Background: It remains unclear if there is a dose-dependent relationship between exposure risk to Ebola virus (EBOV) and severity of illness. Methods: From September 2016 to July 2017, we conducted a cross-sectional, community-based study of Ebola virus disease (EVD) cases and household contacts of several transmission chains in Kono District, Sierra Leone. We analyzed 154 quarantined households, comprising both reported EVD cases and their close contacts. We used epidemiological surveys and blood samples to define severity of illness as no infection, pauci-/asymptomatic infection, unrecognized EVD, reported EVD cases who survived, or reported EVD decedents. We determine seropositivity with the Filovirus Animal Nonclinical Group EBOV glycoprotein immunoglobulin G antibody test. We defined levels of exposure risk from 8 questions and considered contact with body fluid as maximum exposure risk. Results: Our analysis included 76 reported EVD cases (both decedents and survivors) and 421 close contacts. Among these contacts, 40 were seropositive (22 paucisymptomatic and 18 unrecognized EVD), accounting for 34% of the total 116 EBOV infections. Higher exposure risks were associated with having had EBOV infection (maximum risk: adjusted odds ratio [AOR], 12.1 [95% confidence interval {CI}, 5.8-25.4; trend test: P < .001) and more severe illness (maximum risk: AOR, 25.2 [95% CI, 6.2-102.4]; trend test: P < .001). Conclusions: This community-based study of EVD cases and contacts provides epidemiological evidence of a dose-dependent relationship between exposure risk and severity of illness, which may partially explain why pauci-/asymptomatic EBOV infection, less severe disease, and unrecognized EVD occurs.

Persistence, Magnitude, and Patterns of Postacute Symptoms and Quality of Life Following Onset of SARS-CoV-2 Infection: Cohort Description and Approaches for Measurement.

BACKGROUND: There is mounting evidence for the presence of postacute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (PASC), but there is limited information on the spectrum, magnitude, duration, and patterns of these sequelae as well as their influence on quality of life. METHODS: We assembled a cohort of adults with a documented history of SARS-CoV-2 RNA positivity at ≥2 weeks past onset of coronavirus disease 2019 (COVID-19) symptoms or, if asymptomatic, first positive test. At 4-month intervals, we queried physical and mental health symptoms and quality of life. RESULTS: Of the first 179 participants enrolled, 10 were asymptomatic during the acute phase of SARS-CoV-2 infection, 125 were symptomatic but not hospitalized, and 44 were symptomatic and hospitalized. During the postacute phase, fatigue, shortness of breath, concentration problems, headaches, trouble sleeping, and anosmia/dysgeusia were most common through 8 months of observation. Symptoms were typically at least somewhat bothersome and sometimes exhibited a waxing-and-waning course. Some participants experienced symptoms of depression, anxiety, and post-traumatic stress, as well as difficulties with performance of usual activities. The median visual analogue scale rating of general health was lower at 4 and 8 months compared with pre-COVID-19. Two clusters of symptom domains were identified. CONCLUSIONS: Many participants report bothersome symptoms following onset of COVID-19 with variable patterns of persistence and impact on quality of life. The substantial variability suggests the existence of multiple subphenotypes of PASC. A rigorous approach to the prospective measurement of symptoms and functional manifestations sets the stage for the next phase of research focusing on the pathophysiologic causes of the various subgroups of PASC.

Triangulating Evidence to Infer Pathways that Influence Ebola Virus Disease-Related Stigma and Clinical Findings among Survivors: An Observational Cohort Study.

Visible signs of disease can evoke stigma while stigma contributes to depression and mental illness, sometimes manifesting as somatic symptoms. We assessed these hypotheses among Ebola virus disease (EVD) survivors, some of whom experienced clinical sequelae. Ebola virus disease survivors in Liberia were enrolled in an observational cohort study starting in June 2015 with visits every 6 months. At baseline and 18 months later, a seven-item index of EVD-related stigma was administered. Clinical findings (self-reported symptoms and abnormal findings) were obtained at each visit. We applied the generalized estimating equation method to assess the bidirectional concurrent and lagged associations between clinical findings and stigma, adjusting for age, gender, educational level, referral to medical care, and HIV serostatus as confounders. When assessing the contribution of stigma to later clinical findings, we restricted clinical findings to five that were also considered somatic symptoms. Data were obtained from 859 EVD survivors. In concurrent longitudinal analyses, each additional clinical finding increased the adjusted odds of stigma by 18% (95% CI: 1.11, 1.25), particularly palpitations, muscle pain, joint pain, urinary frequency, and memory loss. In lagged associations, memory loss (adjusted odds ratio [AOR]: 4.6; 95% CI: 1.73, 12.36) and anorexia (AOR: 4.17; 95% CI: 1.82, 9.53) were associated with later stigma, but stigma was not significantly associated with later clinical findings. Stigma was associated with select symptoms, not abnormal objective findings. Lagged associations between symptoms and later stigma substantiate the possibility of a pathway related to visible symptoms identified by community members and leading to fear of contagion.

Rapid implementation of a cohort for the study of post-acute sequelae of SARS-CoV-2 infection/COVID-19.

BACKGROUND: As the coronavirus disease 2019 (COVID-19) pandemic continues and millions remain vulnerable to infection with severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), attention has turned to characterizing post-acute sequelae of SARS-CoV-2 infection (PASC). METHODS: From April 21 to December 31, 2020, we assembled a cohort of consecutive volunteers who a) had documented history of SARS-CoV-2 RNA-positivity; b) were ≥ 2 weeks past onset of COVID-19 symptoms or, if asymptomatic, first test for SARS-CoV-2; and c) were able to travel to our site in San Francisco. Participants learned about the study by being identified on medical center-based registries and being notified or by responding to advertisements. At 4-month intervals, we asked participants about physical symptoms that were new or worse compared to the period prior to COVID-19, mental health symptoms and quality of life. We described 4 time periods: 1) acute illness (0-3 weeks), 2) early recovery (3-10 weeks), 3) late recovery 1 (12-20 weeks), and 4) late recovery 2 (28-36 weeks). Blood and oral specimens were collected at each visit. RESULTS: We have, to date, enrolled 179 adults. During acute SARS-CoV-2 infection, 10 had been asymptomatic, 125 symptomatic but not hospitalized, and 44 symptomatic and hospitalized. In the acute phase, the most common symptoms were fatigue, fever, myalgia, cough and anosmia/dysgeusia. During the post-acute phase, fatigue, shortness of breath, concentration problems, headaches, trouble sleeping and anosmia/dysgeusia were the most commonly reported symptoms, but a variety of others were endorsed by at least some participants. Some experienced symptoms of depression, anxiety, and post-traumatic stress, as well as difficulties with ambulation and performance of usual activities. The median visual analogue scale value rating of general health was lower at 4 and 8 months (80, interquartile range [IQR]: 70-90; and 80, IQR 75-90) compared to prior to COVID-19 (85; IQR 75-90). Biospecimens were collected at nearly 600 participant-visits. CONCLUSION: Among a cohort of participants enrolled in the post-acute phase of SARS-CoV-2 infection, we found many with persistent physical symptoms through 8 months following onset of COVID-19 with an impact on self-rated overall health. The presence of participants with and without symptoms and ample biological specimens will facilitate study of PASC pathogenesis. Similar evaluations in a population-representative sample will be needed to estimate the population-level prevalence of PASC.