In serology, each sample is typically tested individually, one antigen at a time. This is costly and time consuming. Serology techniques should ideally allow recurrent measurements in parallel in small sample volumes and be inexpensive and fast. Here we show that mass cytometry can be used to scale up multiplexed serology testing by leveraging polystyrene beads uniformly loaded with combinations of stable isotopes. We generated 18,480 unique isotopically barcoded beads to simultaneously detect, in a single tube with 924 serum samples, the levels of immunoglobulins G and M against 19 proteins from SARS-CoV-2 (a total of 36,960 tests in 400 nl of sample volume and 30 μl of reaction volume). As a rapid, high-throughput and cost-effective technique, serology by mass cytometry may contribute to the effective management of public health emergencies originating from infectious diseases.

Long COVID (LC), a type of post-acute sequelae of SARS-CoV-2 infection (PASC), occurs after at least 10% of SARS-CoV-2 infections, yet its etiology remains poorly understood. Here, we used multiple "omics" assays (CyTOF, RNAseq/scRNAseq, Olink) and serology to deeply characterize both global and SARS-CoV-2-specific immunity from blood of individuals with clear LC and non-LC clinical trajectories, 8 months following infection and prior to receipt of any SARS-CoV-2 vaccine. Our analysis focused on deep phenotyping of T cells, which play important roles in immunity against SARS-CoV-2 yet may also contribute to COVID-19 pathogenesis. Our findings demonstrate that individuals with LC exhibit systemic inflammation and immune dysregulation. This is evidenced by global differences in T cell subset distribution in ways that imply ongoing immune responses, as well as by sex-specific perturbations in cytolytic subsets. Individuals with LC harbored increased frequencies of CD4+ T cells poised to migrate to inflamed tissues, and exhausted SARS-CoV-2-specific CD8+ T cells. They also harbored significantly higher levels of SARS-CoV-2 antibodies, and in contrast to non-LC individuals, exhibited a mis-coordination between their SARS-CoV-2-specific T and B cell responses. RNAseq/scRNAseq and Olink analyses similarly revealed immune dysregulatory mechanisms, along with non-immune associated perturbations, in individuals with LC. Collectively, our data suggest that proper crosstalk between the humoral and cellular arms of adaptive immunity has broken down in LC, and that this, perhaps in the context of persistent virus, leads to the immune dysregulation, inflammation, and clinical symptoms associated with this debilitating condition.

AbstractTo understand the roles of acute-phase viral dynamics and host immune responses in post-acute sequelae of SARS-CoV-2 infection (PASC), we enrolled 136 participants within 5 days of their first positive SARS-CoV-2 real-time PCR test. Participants self-collected up to 21 nasal specimens within the first 28 days post-symptom onset; interviewer-administered questionnaires and blood samples were collected at enrollment, days 9, 14, 21, 28, and month 4 and 8 post-symptom onset. Defining PASC as the presence of any COVID-associated symptom at their 4-month visit, we compared viral markers (quantity and duration of nasal viral RNA load, infectious viral load, and plasma N-antigen level) and host immune markers (IL-6, IL-10, TNF-α, IFN-α, IFN-γ, MCP, IP-10, and Spike IgG) over the acute period. Compared to those who fully recovered, those reporting PASC demonstrated significantly higher maximum levels of SARS-CoV-2 RNA and N-antigen, burden of RNA and infectious viral shedding, and lower Spike-specific IgG levels within 9 days post-illness onset. No significant differences were identified among a panel of host immune markers. Our results suggest early viral dynamics and the associated host immune responses play a role in the pathogenesis of PASC, highlighting the importance of understanding early biological markers in the natural history of PASC.

The impact of pre-existing neutralizing antibodies (NAbs) titers on SARS-CoV-2 viral shedding dynamics in post-vaccination infection (PVI) are not well understood. We characterized viral shedding longitudinally in nasal specimens in relation to baseline (pre/peri-infection) serum neutralizing antibody titers in 125 participants infected with distinct SARS-CoV-2 variants. Among 68 participants who had received vaccinations, we quantified the effect of baseline serum NAb titers on maximum viral RNA titers and on the duration of infectivity. Baseline NAb titers were higher and efficiently targeted a broader range of variants in participants who received one or two monovalent ancestral booster vaccinations compared to those with a full primary vaccine series. In participants with Delta variant infections, baseline NAb titers targeting Delta were negatively correlated with maximum viral RNA copies. Per log10 increase in baseline NAb IC50, maximum viral load was reduced -2.43 (95% confidence interval [CI] -3.76, -1.11) log10 N copies and days of infectious viral shedding were reduced -2.79 [95% CI: -4.99, -0.60] days. By contrast, in those with Omicron infections (BA.1, BA.2, BA.4 or BA.5 lineages) baseline NAb responses against Omicron lineages did not predict viral outcomes. Our results provide robust estimates of the effect of baseline NAbs on the magnitude and duration of nasal viral replication after PVI (albeit with an unclear effect on transmission) and show how immune escape variants efficiently evade these modulating effects. ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND: Low cortisol concentrations have been reported in some people with Long COVID (LC), but more data from diverse cohorts are needed to validate this observation. A subset of people with LC present with symptoms resembling those of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). The objective of this study was to compare cortisol concentrations in those with and without Long COVID, with a particular focus on people experiencing ME/CFS-like Long COVID. METHODS: We measured plasma cortisol in 200 individuals 3-6 months following a SARS-CoV-2 infection. Banked biospecimens collected between 8 AM-12 PM were used. Participants met the case definition for Long COVID if they had >=1 COVID-attributed symptom at least 3 months after symptom onset. People who did not report any symptoms at least 3 months after symptom onset served as recovered controls. Adapting the 2015 Institute of Medicine criteria for ME/CFS, we further defined those with LC resembling ME/CFS (LC-ME). RESULTS: We found no difference in overall morning cortisol concentrations between people with LC (n=144) and those who fully recovered (n=56) (median 8.9 μg/dL vs. 8.8 μg/dL, p=0.97). Analyses of samples collected between 8-10 AM, however, revealed that, compared to those who fully recovered, cortisol concentrations were lower between 8-9 AM for those with LC-ME (median 8.2 vs. 14.8, p=0.02), but higher between 9-10 AM for those with severe LC (>=5 symptoms) (median 12.4 vs. 8.5, p=0.009) and those with LC-ME (median 13.7 vs. 8.5, p=0.02). CONCLUSION: We found no difference in overall morning plasma cortisol concentrations between those with and without Long COVID. Although our data could be suggestive of altered morning cortisol dynamics in a subset of people with Long COVID, longitudinal measures of cortisol in individuals with Long COVID will be critical to further inform the biology of the condition. ### Competing Interest Statement SGD reports consulting for Enanta Pharmaceuticals and Pfizer and reports research support from Aerium Therapeutics outside the submitted work. MJP has received consulting fees from Gilead Sciences, AstraZeneca, BioVie, Apellis Pharmaceuticals, and BioNTech and research support from Aerium Therapeutics and Shionogi, outside the submitted work. ### Funding Statement MJP is supported on K23AI157875. The LIINC clinical core is supported by the PolyBio Research Foundation, with additional funding from R01AI141003 (to TJH) and 1R01NS136197 (to MJP). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was approved by the UCSF Institutional Review Board. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.

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.

Persistent symptoms among some individuals who develop COVID-19 have led to the hypothesis that SARS-CoV-2 might, in some form or location, persist for long periods following acute infection.1Proal AD VanElzakker MB Aleman S et al.SARS-CoV-2 reservoir in post-acute sequelae of COVID-19 (PASC).Nat Immunol. 2023; 24: 1616-1627Crossref PubMed Scopus (25) Google Scholar, 2Davis HE McCorkell L Vogel JM Topol EJ Long COVID: major findings, mechanisms and recommendations.Nat Rev Microbiol. 2023; 21: 133-146Crossref PubMed Scopus (896) Google Scholar Studies on SARS-CoV-2 persistence to date, however, have been limited by small and non-representative study populations, short durations since acute infection, unclear documentation of vaccination and reinfection histories, and the absence of a true negative comparator group to assess assay specificity (appendix p 2). To address these limitations, we evaluated the presence of SARS-CoV-2 antigens in once-thawed plasma from a well characterised group of 171 adults (appendix pp 3, 9) at several timepoints in the 14 months following RNA-confirmed SARS-CoV-2 infection, most of whom were studied before vaccination or reinfection (so-called pandemic-era participants).3Peluso MJ Kelly JD Lu S et al.Persistence, magnitude, and patterns of postacute symptoms and quality of life following onset of SARS-CoV-2 infection: cohort description and approaches for measurement.Open Forum Infect Dis. 2021; 9ofab640PubMed Google Scholar To understand the specificity of our findings, we compared them to 250 adults (appendix pp 3, 9) whose plasma was collected before 2020, who, by definition, were not infected with SARS-CoV-2 (pre-pandemic era). We used the Simoa (Quanterix) single molecule array detection platform to measure SARS-CoV-2 spike, S1, and nucleocapsid antigens (appendix p 4).4Wu D Milutinovic MD Walt DR Single molecule array (Simoa) assay with optimal antibody pairs for cytokine detection in human serum samples.Analyst. 2015; 140: 6277-6282Crossref Google Scholar, 5Swank Z Senussi Y Manickas-Hill Z et al.Persistent circulating severe acute respiratory syndrome coronavirus 2 spike is associated with post-acute coronavirus disease 2019 sequelae.Clin Infect Dis. 2023; 76: e487-e490Crossref PubMed Scopus (134) Google Scholar Of 660 pandemic-era specimens tested, 61 (9·2%) specimens from 42 participants (25% of the group), had one or more detectable SARS-CoV-2 antigens (figure A). The most commonly detected antigen was spike (n=33, 5·0%), followed by S1 (n=15, 2·3%) and N (n=15, 2·3%). Compared with the pre-pandemic era participants who had 2% assay positivity, detection of any SARS-CoV-2 antigen was significantly more frequent among the pandemic-era participants at all three timepoints in the post-acute phase of infection (figure B–E). The absolute difference in SARS-CoV-2 plasma antigen prevalence was +10·6% (95% CI +5·0 to +16·2) at 3·0–6·0 months post-onset of COVID-19; +8·7% (+3·1 to +14·3) at 6·1–10·0 months; and +5·4% (+0·42 to +10·3) at 10·1–14·1 months (appendix p 11).FigureSARS-CoV-2 antigen positivity in plasma among participants in the post-acute phase of COVID-19 in comparison with pre-pandemic participantsShow full caption(A) Quantitative measurement of concentrations of SARS-CoV-2 spike, S1, and nucleocapsid antigens in plasma among all pandemic-era participants during the post-acute phase of SARS-CoV-2 infection. Dashed lines indicate limit of quantification for each antigen. (B–E) Prevalence of SARS-CoV-2 antigen positivity in plasma among participants in the post-acute phase of COVID-19 compared with pre-pandemic participants. P-values are derived from Fisher's two-sided exact tests. (B) Prevalence of any SARS-CoV-2 antigen positivity (spike, S1, or nucleocapsid). (C) Prevalence of spike antigen presence. (D) Prevalence of nucleocapsid antigen presence. (E) Prevalence of S1 antigen presence.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Quantitative measurement of concentrations of SARS-CoV-2 spike, S1, and nucleocapsid antigens in plasma among all pandemic-era participants during the post-acute phase of SARS-CoV-2 infection. Dashed lines indicate limit of quantification for each antigen. (B–E) Prevalence of SARS-CoV-2 antigen positivity in plasma among participants in the post-acute phase of COVID-19 compared with pre-pandemic participants. P-values are derived from Fisher's two-sided exact tests. (B) Prevalence of any SARS-CoV-2 antigen positivity (spike, S1, or nucleocapsid). (C) Prevalence of spike antigen presence. (D) Prevalence of nucleocapsid antigen presence. (E) Prevalence of S1 antigen presence. Compared with those not hospitalised, participants who required hospitalisation for acute COVID-19 were nearly twice as likely to have SARS-CoV-2 antigens detected (prevalence ratio 1·97, 95% CI 1·11 to 3·48), an absolute difference of +18·4% (95% CI +0·3 to +36·5). Among participants not hospitalised, those with worse self-reported health during acute COVID-19 had greater post-acute antigen detection (appendix pp 7, 10). These findings suggest the influence of the acute phase of infection in establishing a persistent SARS-CoV-2 reservoir. Coupled with a 2024 study of replication-competent virus in blood during acute infection,6Platt A Singh M Stein S Soherwardi S de Wit E Chertow DS Replication-competent virus detected in blood of a fatal COVID-19 case.Ann Intern Med. 2024; 177: 113-115Crossref PubMed Scopus (2) Google Scholar our findings suggest that SARS-CoV-2 might seed distal sites through the bloodstream and establish protected reservoirs in some sites. Alternatively, more severe acute infection could be a marker of higher inoculum in sites of primary infection, which then have a greater chance of evading immune clearance. Because our measurement of SARS-CoV-2 is via immunoreactivity, it cannot be assumed that every signal is specific for SARS-CoV-2 antigen. Antigens from related pathogens or the host can theoretically cross-react, differing from the detection of nucleic acid for which specificity is typically complete and direct analyte evaluation by sequencing is possible. Therefore, understanding the specificity of any immune-based assay purporting to detect SARS-CoV-2 antigens is crucial and requires a large true negative group to precisely estimate false positivity. The 98% specificity that we documented for our Simoa assay is high but imperfect, so caution is needed if interpreting individual-level results. To mitigate concerns that vaccination against SARS-CoV-2 or recent reinfections could affect interpretation of positive results,1Proal AD VanElzakker MB Aleman S et al.SARS-CoV-2 reservoir in post-acute sequelae of COVID-19 (PASC).Nat Immunol. 2023; 24: 1616-1627Crossref PubMed Scopus (25) Google Scholar we studied specimens largely collected before these occurrences. Most samples were collected before the emergence of the Delta and Omicron SARS-CoV-2 variants, when reinfections became common.7Ma KC Dorabawila V León TM et al.Trends in laboratory-confirmed SARS-CoV-2 reinfections and associated hospitalizations and deaths among adults aged ≥18 years – 18 US jurisdictions, September 2021–December 2022.MMWR Morb Mortal Wkly Rep. 2023; 72: 683-689Crossref Scopus (7) Google Scholar Although not without limitations (appendix p 14), our data provide strong evidence that SARS-CoV-2, in some form or location, persists for up to 14 months following acute SARS-CoV-2 infection (appendix p 13). This persistence is influenced by the events of acute infection. These findings motivate an urgent research agenda regarding the clinical manifestations of SARS-CoV-2 persistence, specifically whether it is causally related to either post-acute chronic symptoms (eg, fatigue, pain, and cognitive difficulty) or discrete incident complications (eg, cardiovascular events). MJP reports consulting for Gilead Sciences and AstraZeneca and reports research support from Aerium Therapeutics outside the submitted work. SGD reports consulting for Enanta Pharmaceuticals and Pfizer and reports research support from Aerium Therapeutics outside the submitted work. DRW has a financial interest in Quanterix Corporation, a company that develops an ultra-sensitive digital immunoassay platform and is an inventor of the Simoa technology, reports being a founder of Quanterix Corporation, and also serves on its Board of Directors. DRW's interests were reviewed and are managed by Mass General Brigham and Harvard University in accordance with their conflict of interest policies. All other authors declare no competing interests. This work was supported with funding from the PolyBio Research Foundation for the LIINC Clinical Core and was supported by NIH/NIAID 3R01AI141003–03S1, NIH/NIAID R01AI158013, NIH/NIAID K23AI134327, and NINDS 1R01NS136197-01. 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The mechanisms of postacute medical conditions and unexplained symptoms after SARS-CoV-2 infection [Long Covid (LC)] are incompletely understood. There is growing evidence that viral persistence, immune dysregulation, and T cell dysfunction may play major roles. We performed whole-body positron emission tomography imaging in a well-characterized cohort of 24 participants at time points ranging from 27 to 910 days after acute SARS-CoV-2 infection using the radiopharmaceutical agent [ 18 F]F-AraG, a selective tracer that allows for anatomical quantitation of activated T lymphocytes. Tracer uptake in the postacute COVID-19 group, which included those with and without continuing symptoms, was higher compared with prepandemic controls in many regions, including the brain stem, spinal cord, bone marrow, nasopharyngeal and hilar lymphoid tissue, cardiopulmonary tissues, and gut wall. T cell activation in the spinal cord and gut wall was associated with the presence of LC symptoms. In addition, tracer uptake in lung tissue was higher in those with persistent pulmonary symptoms specifically. Increased T cell activation in these tissues was also observed in many individuals without LC. Given the high [ 18 F]F-AraG uptake detected in the gut, we obtained colorectal tissue for in situ hybridization of SARS-CoV-2 RNA and immunohistochemical studies in a subset of five participants with LC symptoms. We identified intracellular SARS-CoV-2 single-stranded spike protein–encoding RNA in rectosigmoid lamina propria tissue in all five participants and double-stranded spike protein–encoding RNA in three participants up to 676 days after initial COVID-19, suggesting that tissue viral persistence could be associated with long-term immunologic perturbations.

Studies have demonstrated that at least 20% of individuals infected with SARS-CoV-2 remain asymptomatic 1 – 4 . Although most global efforts have focused on severe illness in COVID-19, examining asymptomatic infection provides a unique opportunity to consider early immunological features that promote rapid viral clearance. Here, postulating that variation in the human leukocyte antigen ( HLA ) loci may underly processes mediating asymptomatic infection, we enrolled 29,947 individuals, for whom high-resolution HLA genotyping data were available, in a smartphone-based study designed to track COVID-19 symptoms and outcomes. Our discovery cohort ( n = 1,428) comprised unvaccinated individuals who reported a positive test result for SARS-CoV-2. We tested for association of five HLA loci with disease course and identified a strong association between HLA-B*15:01 and asymptomatic infection, observed in two independent cohorts. Suggesting that this genetic association is due to pre-existing T cell immunity, we show that T cells from pre-pandemic samples from individuals carrying HLA-B*15:01 were reactive to the immunodominant SARS-CoV-2 S-derived peptide NQKLIANQF. The majority of the reactive T cells displayed a memory phenotype, were highly polyfunctional and were cross-reactive to a peptide derived from seasonal coronaviruses. The crystal structure of HLA-B*15:01–peptide complexes demonstrates that the peptides NQKLIANQF and NQKLIANAF (from OC43-CoV and HKU1-CoV) share a similar ability to be stabilized and presented by HLA-B*15:01. Finally, we show that the structural similarity of the peptides underpins T cell cross-reactivity of high-affinity public T cell receptors, providing the molecular basis for HLA-B*15:01 -mediated pre-existing immunity.

BACKGROUND In sub-Saharan Africa, increased antiretroviral therapy (ART) availability has improved survival after diagnosis of Kaposi sarcoma (KS) compared to the pre-ART era, but mortality among patients with KS is still considerably higher than HIV-infected persons without KS. Furthermore, among those patients with KS who are treated initially with ART without adjunct chemotherapy and who do survive, little is known about how well they function and feel — quality of life (QOL) — compared to those without KS. METHODS Among HIV-infected adults initiating ART in two prospective studies in Uganda, we compared those presenting with KS to those without KS. QOL was measured using the Medical Outcomes Survey-HIV instrument prior to ART initiation and at 16, 32, and 48 weeks thereafter; higher scores indicate better QOL. To ascertain the independent effect of KS versus non-KS on 11 domains of QOL and two summary scores, we created mixed effects models adjusted for directed acyclic graph-informed confounders. RESULTS We examined 224 participants with KS and 730 without KS, among whom 64% were women and median age was 34 years. Prior to ART initiation, participants had a median CD4+ T count of 159 cells/mm 3 and plasma HIV RNA of 5.1 log 10 copies/ml. In adjusted analyses prior to ART initiation, those with KS had lower mean scores in 8 of 11 QOL domains and both physical and mental health summary scores compared to those without KS. After 48 weeks of ART, those with KS had higher mean QOL scores compared those without KS in 4 domains and the mental health summary score, and lower scores in only one domain. There was no significant difference in 6 domains and the physical health summary score, CONCLUSIONS Amongst HIV-infected adults in East Africa, at time of ART initiation, those with KS had worse mean QOL compared to those without KS. Over the first year of ART, those with KS became comparable to or exceeded those without KS in most QOL domains. The findings indicate that some patients with KS can be treated with ART alone and further emphasize the need to predict those who will do well with ART alone versus those who need additional initial therapy.

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.

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.

ABSTRACTBACKGROUNDPersistent symptoms among some persons who develop COVID-19 has led to the hypothesis that SARS-CoV-2 may, in some form or location, persist for long periods following acute infection. Several studies have shown data in this regard but are limited by non-representative and small study populations, short duration since acute infection, and lack of a true-negative comparator group to assess assay specificity.METHODSWe evaluated adults with RNA-confirmed COVID-19 at multiple time points following acute infection (pandemic-era participants) and adults with specimens collected prior to 2020 (pre-pandemic era). Using once-thawed plasma, we employed the Simoa® (Quanterix) single molecule array detection platform to measure SARS-CoV-2 spike, S1, and nucleocapsid antigens.RESULTSCompared to 250 pre-pandemic participants who had 2% assay positivity, detection of any SARS-CoV-2 antigen was significantly more frequent among 171 pandemic-era participants at three different time periods in the post-acute phase of infection. The absolute difference in SARS-CoV-2 plasma antigen prevalence was +11% (95% CI: +5.0% to +16%) at 3.0-6.0 months post-onset of COVID-19; +8.7% (95% CI: +3.1% to +14%) at 6.1 to 10.0 months; and +5.4% (95% CI: +0.42% to +10%) at 10.1-14.1 months. Hospitalization for acute COVID-19 and, among the non-hospitalized, worse self-reported health during acute COVID-19 were associated with greater post-acute phase antigen detection.CONCLUSIONSCompared to uninfected persons, there is an excess prevalence of SARS-CoV-2 antigenemia in SARS-CoV-2-infected individuals up to 14 months after acute COVID-19. These findings motivate an urgent research agenda regarding the short-term and long-term clinical manifestations of this viral persistence.

BACKGROUND The presence and reactivation of chronic viral infections, such as EBV, CMV, and HIV, have been proposed as potential contributors to long COVID (LC), but studies in well-characterized postacute cohorts of individuals with COVID-19 over a longer time course consistent with current case definitions of LC are limited.METHODS In a cohort of 280 adults with prior SARS-CoV-2 infection, we assessed the presence and types of LC symptoms and prior medical history (including COVID-19 history and HIV status) and performed serological testing for EBV and CMV using a commercial laboratory. We used covariate-adjusted binary logistic regression models to identify independent associations between variables and LC symptoms.RESULTS We observed that LC symptoms, such as fatigue and neurocognitive dysfunction, at a median of 4 months following initial diagnosis were independently associated with serological evidence suggesting recent EBV reactivation (early antigen–diffuse IgG positivity) or high nuclear antigen (EBNA) IgG levels but not with ongoing EBV viremia. Serological evidence suggesting recent EBV reactivation (early antigen–diffuse IgG positivity) was most strongly associated with fatigue (OR = 2.12). Underlying HIV infection was also independently associated with neurocognitive LC (OR = 2.5). Interestingly, participants who had serologic evidence of prior CMV infection were less likely to develop neurocognitive LC (OR = 0.52).CONCLUSION Overall, these findings suggest differential effects of chronic viral coinfections on the likelihood of developing LC and association with distinct syndromic patterns. Further assessment during the acute phase of COVID-19 is warranted.TRIAL REGISTRATION Long-term Impact of Infection with Novel Coronavirus; ClinicalTrials.gov NCT04362150.FUNDING This work was supported by NIH/National Institute of Allergy and Infectious Diseases grants (3R01AI141003-03S1, R01AI158013, and K24AI145806); the Zuckerberg San Francisco General Hospital Department of Medicine and Division of HIV, Infectious Diseases, and Global Medicine; and the UCSF-Bay Area Center for AIDS Research (P30-AI027763).

Some individuals do not return to baseline health following SARS-CoV-2 infection, leading to a condition known as long COVID. The underlying pathophysiology of long COVID remains unknown. Given that autoantibodies have been found to play a role in severity of SARS-CoV-2 infection and certain other post-COVID sequelae, their potential role in long COVID is important to investigate. Here, we apply a well-established, unbiased, proteome-wide autoantibody detection technology (T7 phage-display assay with immunoprecipitation and next-generation sequencing, PhIP-Seq) to a robustly phenotyped cohort of 121 individuals with long COVID, 64 individuals with prior COVID-19 who reported full recovery, and 57 pre-COVID controls. While a distinct autoreactive signature was detected that separated individuals with prior SARS-CoV-2 infection from those never exposed to SARS-CoV-2, we did not detect patterns of autoreactivity that separated individuals with long COVID from individuals fully recovered from COVID-19. These data suggest that there are robust alterations in autoreactive antibody profiles due to infection; however, no association of autoreactive antibodies and long COVID was apparent by this assay.

The associations between longitudinal dynamics and the breadth of SARS‐CoV‐2 neutralizing antibody (nAb) response with various Long COVID phenotypes before vaccination are not known. The capacity of antibodies to cross‐neutralize a variety of viral variants may be associated with ongoing pathology and persistent symptoms. We measured longitudinal neutralizing and cross‐neutralizing antibody responses to pre‐ and post‐SARS‐CoV‐2 Omicron variants in participants infected early in the COVID‐19 pandemic, before widespread rollout of SARS‐CoV‐2 vaccines. Cross‐sectional regression models adjusted for clinical covariates and longitudinal mixed‐effects models were used to determine the impact of the breadth and rate of decay of neutralizing responses on the development of Long COVID symptoms, as well as Long COVID phenotypes. We identified several novel relationships between SARS‐CoV‐2 antibody neutralization and the presence of Long COVID symptoms. Specifically, we show that, although nAb responses to the original, infecting strain of SARS‐CoV‐2 were not associated with Long COVID in cross‐sectional analyses, cross‐neutralization ID 50 levels to the Omicron BA.5 variant approximately 4 months following acute infection was independently and significantly associated with greater odds of Long COVID and with persistent gastrointestinal and neurological symptoms. Longitudinal modeling demonstrated significant associations in the overall levels and rates of decay of neutralization capacity with Long COVID phenotypes. A higher proportion of participants had antibodies capable of neutralizing Omicron BA.5 compared with BA.1 or XBB.1.5 variants. Our findings suggest that relationships between various immune responses and Long COVID are likely complex but may involve the breadth of antibody neutralization responses.

The etiologic mechanisms of post-acute medical morbidities and unexplained symptoms (Long COVID) following SARS-CoV-2 infection are incompletely understood. There is growing evidence that viral persistence and immune dysregulation may play a major role. We performed whole-body positron emission tomography (PET) imaging in a cohort of 24 participants at time points ranging from 27 to 910 days following acute SARS-CoV-2 infection using a novel radiopharmaceutical agent, [18F]F-AraG, a highly selective tracer that allows for anatomical quantitation of activated T lymphocytes. Tracer uptake in the post-acute COVID group, which included those with and without Long COVID symptoms, was significantly higher compared to pre-pandemic controls in many anatomical regions, including the brain stem, spinal cord, bone marrow, nasopharyngeal and hilar lymphoid tissue, cardiopulmonary tissues, and gut wall. Although T cell activation tended to be higher in participants imaged closer to the time of the acute illness, tracer uptake was increased in participants imaged up to 2.5 years following SARS-CoV-2 infection. We observed that T cell activation in spinal cord and gut wall was associated with the presence of Long COVID symptoms. In addition, tracer uptake in lung tissue was higher in those with persistent pulmonary symptoms. Notably, increased T cell activation in these tissues was also observed in many individuals without Long COVID. Given the high [18F]F-AraG uptake detected in the gut, we obtained colorectal tissue for in situ hybridization SARS-CoV-2 RNA and immunohistochemical studies in a subset of participants with Long COVID symptoms. We identified cellular SARS-CoV-2 RNA in rectosigmoid lamina propria tissue in all these participants, ranging from 158 to 676 days following initial COVID-19 illness, suggesting that tissue viral persistence could be associated with long-term immunological perturbations.

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.

BACKGROUND Mechanisms underlying persistent cardiopulmonary symptoms following SARS-CoV-2 infection (post-acute sequelae of COVID-19 "PASC" or "Long COVID") remain unclear. This study sought to elucidate mechanisms of cardiopulmonary symptoms and reduced exercise capacity. METHODS We conducted cardiopulmonary exercise testing (CPET), cardiac magnetic resonance imaging (CMR) and ambulatory rhythm monitoring among adults > 1 year after confirmed SARS-CoV-2 infection in a post-COVID cohort, compared those with or without symptoms, and correlated findings with previously measured biomarkers. RESULTS Sixty participants (median age 53, 42% female, 87% non-hospitalized) were studied at median 17.6 months following SARS-CoV-2 infection. On CPET, 18/37 (49%) with symptoms had reduced exercise capacity (<85% predicted) compared to 3/19 (16%) without symptoms (p = 0.02). Adjusted peak VO2 was 5.2 ml/kg/min lower (95%CI 2.1-8.3; p = 0.001) or 16.9% lower percent predicted (95%CI 4.3-29.6; p = 0.02) among those with symptoms. Chronotropic incompetence was common. Inflammatory markers and antibody levels early in PASC were negatively correlated with peak VO2 more than 1 year later. Late-gadolinium enhancement on CMR and arrhythmias were absent. CONCLUSIONS Cardiopulmonary symptoms >1 year following COVID-19 were associated with reduced exercise capacity, which was associated with elevated inflammatory markers early in PASC. Chronotropic incompetence may explain exercise intolerance among some with cardiopulmonary Long COVID.

OBJECTIVE:As SARS-CoV-2 is known to invade neural cell mitochondria, a plasma system for quantifying central nervous system proteins in living humans was used to investigate neuropathogenic mechanisms of long-COVID-19. METHODS:SARS-CoV-2 proteins and mitochondrial proteins (MPs) in enriched plasma neuron-derived extracellular vesicles (NDEVs) and astrocyte-derived EVs (ADEVs) were quantified in resolved acute COVID-19 without post-acute sequelae of SARS-CoV-2 (PASC), PASC without neuropsychiatric manifestations (NP), PASC with NP and healthy controls. RESULTS:NDEV and ADEV mean levels of SARS-CoV-2 S1 and nucleocapsid (N) proteins were higher in all PASC sub-groups than controls, but only N levels were higher in PASC with than without NP. Exosome marker CD81-normalized NDEV mean levels of subunit 6 of MP respiratory chain complex I and subunit 10 of complex III, and neuroprotective MPs Humanin and mitochondrial open-reading frame of the 12S rRNA-c (MOTS-c) all were decreased significantly in PASC with NP but not in PASC without NP relative to controls. NDEV levels of MPs voltage-dependent anion-selective channel protein 1 (VDAC1) and N-methyl-D-aspartate receptor 1 (NMDAR1) were decreased in PASC without and with NP, whereas those of calcium channel MPs mitochondrial calcium uniporter (MCU), sodium/calcium exchanger (NCLX) and leucine zipper EF-hand containing transmembrane 1 protein (LETM1) were decreased only in PASC with NP. ADEV levels of MCU and NCLX only were increased in PASC without and with NP. INTERPRETATION:Abnormal NDEV and ADEV levels of SARS-CoV-2 N and S1 protein and MPs correlate with NP and may be biomarkers for long-COVID prognostics and therapeutic trials. ANN NEUROL 2022;91:772-781.