Metagenomic next-generation sequencing to characterize etiologies of non-malarial fever in a cohort living in a high malaria burden area of Uganda

BackgroundCauses of non-malarial fevers in sub-Saharan Africa remain understudied. We hypothesized that metagenomic next-generation sequencing (mNGS), which allows for broad genomic-level detection of infectious agents in a biological sample, can systematically identify potential causes of non-malarial fevers. Methods and FindingsThe 212 participants in this study were of all ages and were enrolled in a longitudinal malaria cohort in eastern Uganda. Between December 2020 and August 2021, respiratory swabs and plasma samples were collected at 313 study visits where participants presented with fever and were negative for malaria by microscopy. Samples were analyzed using CZ ID, a web-based platform for microbial detection in mNGS data. Overall, viral pathogens were detected at 123 of 313 visits (39%). SARS-CoV-2 was detected at 11 visits, from which full viral genomes were recovered from nine. Other prevalent viruses included Influenza A (14 visits), RSV (12 visits), and three of the four strains of seasonal coronaviruses (6 visits). Notably, 11 influenza cases occurred between May and July 2021, coinciding with when the Delta variant of SARS-CoV-2 was circulating in this population. The primary limitation of this study is that we were unable to estimate the contribution of bacterial microbes to non-malarial fevers, due to the difficulty of distinguishing bacterial microbes that were pathogenic from those that were commensal or contaminants. ConclusionsThese results revealed the co-circulation of multiple viral pathogens likely associated with fever in the cohort during this time period. This study illustrates the utility of mNGS in elucidating the multiple causes of non-malarial febrile illness. A better understanding of the pathogen landscape in different settings and age groups could aid in informing diagnostics, case management, and public health surveillance systems.

Reconstructing the SARS-CoV-2 epidemic in eastern Uganda through longitudinal serosurveillance in a malaria cohort

ImportanceEstimating the true burden of SARS-CoV-2 infection has been difficult in sub-Saharan Africa due to asymptomatic infections and inadequate testing capacity. Antibody responses from serologic surveys can provide an estimate of SARS-CoV-2 exposure at the population level. ObjectiveTo estimate SARS-CoV-2 seroprevalence, attack rates, and re-infection in eastern Uganda using serologic surveillance from 2020 to early 2022. DesignPlasma samples from participants in the Program for Resistance, Immunology, Surveillance, and Modeling of Malaria in Uganda (PRISM) Border Cohort were obtained at four sampling intervals: October-November 2020; March-April 2021; August-September 2021; and February-March 2022. Setting: Tororo and Busia districts, Uganda. Participants1,483 samples from 441 participants living in 76 households were tested. Each participant contributed up to 4 time points for SARS-CoV-2 serology, with almost half of all participants contributing at all 4 time points, and almost 90% contributing at 3 or 4 time points. Information on SARS-CoV-2 vaccination status was collected from participants, with the earliest reported vaccinations in the cohort occurring in May 2021. Main Outcome(s) and Measure(s)The main outcomes of this study were antibody responses to the SARS-CoV-2 spike protein as measured with a bead-based serologic assay. Individual-level outcomes were aggregated to population-level SARS-CoV-2 seroprevalence, attack rates, and boosting rates. Estimates were weighted by the local age distribution based on census data. ResultsBy the end of the Delta wave and before widespread vaccination, nearly 70% of the study population had experienced SARS-CoV-2 infection. During the subsequent Omicron wave, 85% of unvaccinated, previously seronegative individuals were infected for the first time, and [~]50% or more of unvaccinated, already seropositive individuals were likely re-infected, leading to an overall 96% seropositivity in this population. Our results suggest a lower probability of re-infection in individuals with higher pre-existing antibody levels. We found evidence of household clustering of SARS-CoV-2 seroconversion. We found no significant associations between SARS-CoV-2 seroconversion and gender, household size, or recent Plasmodium falciparum malaria exposure. Conclusions and RelevanceFindings from this study are consistent with very high infection rates and re-infection rates for SARS-CoV-2 in a rural population from eastern Uganda throughout the pandemic.

Shared within-host SARS-CoV-2 variation in households

BackgroundThe limited variation observed among SARS-CoV-2 consensus sequences makes it difficult to reconstruct transmission linkages in outbreak settings. Previous studies have recovered variation within individual SARS-CoV-2 infections but have not yet measured the informativeness of within-host variation for transmission inference. MethodsWe performed tiled amplicon sequencing on 307 SARS-CoV-2 samples from four prospective studies and combined sequence data with household membership data, a proxy for transmission linkage. ResultsConsensus sequences from households had limited diversity (mean pairwise distance, 3.06 SNPs; range, 0-40). Most (83.1%, 255/307) samples harbored at least one intrahost single nucleotide variant (iSNV; median: 117; IQR: 17-208), when applying a liberal minor allele frequency of 0.5% and prior to filtering. A mean of 15.4% of within-host iSNVs were recovered one day later. Pairs in the same household shared significantly more iSNVs (mean: 1.20 iSNVs; 95% CI: 1.02-1.39) than did pairs in different households infected with the same viral clade (mean: 0.31 iSNVs; 95% CI: 0.28-0.34), a signal that increases with increasingly liberal thresholds. ConclusionsAlthough only a subset of within-host variation is consistently shared across likely transmission pairs, shared iSNVs may augment the information in consensus sequences for predicting transmission linkages.

TNFα-producing CD4+ T cells dominate the SARS-CoV-2-specific T cell response in COVID-19 outpatients and are associated with durable antibodies

SARS-CoV-2-specific CD4+ T cells are likely important in immunity against COVID-19, but our understanding of CD4+ longitudinal dynamics following infection and specific features that correlate with the maintenance of neutralizing antibodies remains limited. We characterized SARS-CoV-2-specific CD4+ T cells in a longitudinal cohort of 109 COVID-19 outpatients. The quality of the SARS-CoV-2-specific CD4+ response shifted from cells producing IFN{gamma} to TNF+ from five days to four months post-enrollment, with IFN{gamma}-IL21-TNF+ CD4+ T cells the predominant population detected at later timepoints. Greater percentages of IFN{gamma}-IL21-TNF+ CD4+ T cells on day 28 correlated with SARS-CoV-2 neutralizing antibodies measured seven months post-infection ({rho}=0.4, P=0.01). mRNA vaccination following SARS-CoV-2 infection boosted both IFN{gamma} and TNF producing, spike protein-specific CD4+ T cells. These data suggest that SARS-CoV-2-specific, TNF-producing CD4+ T cells may play an important role in antibody maintenance following COVID-19.

Magnitude and breadth of neutralizing antibody responses elicited by SARS-CoV-2 infection or vaccination

Multiple SARS-CoV-2 variants that possess mutations associated with increased transmission and antibody escape have arisen over the course of the current pandemic. While the current vaccines have largely been effective against past variants, the number of mutations found on the Omicron (B.1.529) spike appear to diminish the efficacy of pre-existing immunity. Using pseudoparticles expressing the spike of several SARS-CoV-2 variants, we evaluated the magnitude and breadth of the neutralizing antibody response over time in naturally infected and in mRNA-vaccinated individuals. We observed that while boosting increases the magnitude of the antibody response to wildtype (D614), Beta, Delta and Omicron variants, the Omicron variant was the most resistant to neutralization. We further observed that vaccinated healthy adults had robust and broad antibody responses while responses were relatively reduced in vaccinated pregnant women, underscoring the importance of learning how to maximize mRNA vaccine responses in pregnant populations. Findings from this study show substantial heterogeneity in the magnitude and breadth of responses after infection and mRNA vaccination and may support the addition of more conserved viral antigens to existing SARS-CoV-2 vaccines. One Sentence SummaryDiminished efficacy of pre-existing immunity to highly mutated SARS-CoV-2 variants.

Favipiravir for treatment of outpatients with asymptomatic or uncomplicated COVID-19: a double-blind randomized, placebo-controlled, phase 2 trial

BackgroundFavipiravir is an oral, RNA-dependent RNA polymerase inhibitor with in vitro activity against SARS-CoV2. Despite limited data, favipiravir is administered to patients with COVID-19 in several countries. MethodsWe conducted a phase 2 double-blind randomized controlled outpatient trial of favipiravir in asymptomatic or mildly symptomatic adults with a positive SARS-CoV2 RT-PCR within 72 hours of enrollment. Participants were randomized 1:1 to receive placebo or favipiravir (1800 mg BID Day 1, 800mg BID Days 2-10). The primary outcome was SARS-CoV-2 shedding cessation in a modified intention-to-treat (mITT) cohort of participants with positive enrollment RT-PCRs. Using SARS-CoV-2 deep sequencing, we assessed favipiravirs impact on mutagenesis. ResultsFrom July 8, 2020 - March 23, 2021, we randomized 149 participants with 116 included in the mITT cohort. The participants mean age was 43 years (SD 12.5) and 57 (49%) were women. We found no difference in time to shedding cessation by treatment arm overall (HR 0.76 favoring placebo, 95% confidence interval [CI] 0.48 - 1.20) or in sub-group analyses (age, sex, high-risk comorbidities, seropositivity or symptom duration at enrollment). We observed no difference in time to symptom resolution (initial: HR 0.84, 95% CI 0.54 - 1.29; sustained: HR 0.87, 95% CI 0.52 - 1.45). We detected no difference in accumulation of transition mutations in the viral genome during treatment. ConclusionsOur data do not support favipiravir use at commonly used doses in outpatients with uncomplicated COVID-19. Further research is needed to ascertain if higher doses of favipiravir are effective and safe for patients with COVID-19. Trial registration numberNCT04346628 SummaryIn this phase 2 double-blind randomized controlled outpatient trial of favipiravir in asymptomatic or uncomplicated patients with COVID-19, we found no difference in time to shedding cessation or time to symptom resolution by treatment arm.

Long Term Accuracy of SARS-CoV-2 Interferon-γ Release Assay and its Application in Household Investigation

BackgroundAn immunodiagnostic assay that sensitively detects a cell-mediated immune response to SARS-CoV-2 is needed for epidemiological investigation and for clinical assessment of T cell-mediated immune response to vaccines, particularly in the context of emerging variants that might escape antibody responses. MethodsThe performance of a whole blood interferon-gamma (IFN-{gamma}) release assay (IGRA) for the detection of SARS-CoV-2 antigen-specific CD4 and CD8 T cells was evaluated in COVID-19 convalescents tested serially up to 10 months post-infection and in healthy blood donors. SARS-CoV-2 IGRA was applied in contacts of households with index cases. Freshly collected blood in the lithium heparin tube was left unstimulated, stimulated with a SARS-CoV-2 peptide pool, and stimulated with mitogen. ResultsThe overall sensitivity and specificity of IGRA were 84.5% (153/181; 95% confidence interval [CI] 79.0-89.0) and 86.6% (123/142; 95% CI;80.0-91.2), respectively. The sensitivity declined from 100% (16/16; 95% CI 80.6-100) at 0.5-month post-infection to 79.5% (31/39; 95% CI 64.4-89.2) at 10 months post-infection (P<0.01). The IFN-{gamma} response remained relatively robust at 10 months post-infection (3.8 vs. 1.3 IU/mL, respectively). In 14 households, IGRA showed a positivity rate of 100% (12/12) and 65.2% (15/23), and IgG of 50.0% (6/12) and 43.5% (10/23) in index cases and contacts, respectively, exhibiting a difference of +50% (95% CI +25.4-+74.6) and +21.7% (95% CI, +9.23-+42.3), respectively. Either IGRA or IgG was positive in 100% (12/12) of index cases and 73.9% (17/23) of contacts. ConclusionsThe SARS-CoV-2 IGRA is a useful clinical diagnostic tool for assessing cell-mediated immune response to SARS-CoV-2. Key pointsSARS-CoV-2 immunodiagnostics are needed to identify infected individuals in order to understand the transmission dynamics of emerging variants and to assess vaccine response. Interferon-gamma release assay maintains sensitivity 10 months post-infection in convalescents and detects more household contacts than IgG.

Early immune responses have long-term associations with clinical, virologic, and immunologic outcomes in patients with COVID-19

The great majority of SARS-CoV-2 infections are mild and uncomplicated, but some individuals with initially mild COVID-19 progressively develop more severe symptoms. Furthermore, mild to moderate infections are an important contributor to ongoing transmission. There remains a critical need to identify host immune biomarkers predictive of clinical and virologic outcomes in SARS-CoV-2-infected patients. Leveraging longitudinal samples and data from a clinical trial of Peginterferon Lambda for treatment of SARS-CoV-2 infected outpatients, we used host proteomics and transcriptomics to characterize the trajectory of the immune response in COVID-19 patients within the first 2 weeks of symptom onset. We define early immune signatures, including plasma levels of RIG-I and the CCR2 ligands (MCP1, MCP2 and MCP3), associated with control of oropharyngeal viral load, the degree of symptom severity, and immune memory (including SARS-CoV-2-specific T cell responses and spike (S) protein-binding IgG levels). We found that individuals receiving BNT162b2 (Pfizer-BioNTech) vaccine had similar early immune trajectories to those observed in this natural infection cohort, including the induction of both inflammatory cytokines (e.g. MCP1) and negative immune regulators (e.g. TWEAK). Finally, we demonstrate that machine learning models using 8-10 plasma protein markers measured early within the course of infection are able to accurately predict symptom severity, T cell memory, and the antibody response post-infection.

Divergent early antibody responses define COVID-19 disease trajectories

A damaging inflammatory response is strongly implicated in the pathogenesis of severe COVID-19 but mechanisms contributing to this response are unclear. In two prospective cohorts, early non-neutralizing, afucosylated, anti-SARS-CoV-2 IgG predicted progression from mild, to more severe COVID-19. In contrast to the antibody structures that predicted disease progression, antibodies that were elicited by mRNA SARS-CoV-2 vaccines were low in Fc afucosylation and enriched in sialylation, both modifications that reduce the inflammatory potential of IgG. To study the biology afucosylated IgG immune complexes, we developed an in vivo model which revealed that human IgG-Fc{gamma}R interactions can regulate inflammation in the lung. Afucosylated IgG immune complexes induced inflammatory cytokine production and robust infiltration of the lung by immune cells. By contrast, vaccine elicited IgG did not promote an inflammatory lung response. Here, we show that IgG-Fc{gamma}R interactions can regulate inflammation in the lung and define distinct lung activities associated with the IgG that predict severe COVID-19 and protection against SARS-CoV-2. One Sentence SummaryDivergent early antibody responses predict COVID-19 disease trajectory and mRNA vaccine response and are functionally distinct in vivo.

SARS-CoV-2 subgenomic RNA kinetics in longitudinal clinical samples

BackgroundGiven the persistence of viral RNA in clinically recovered COVID-19 patients, subgenomic RNAs (sgRNA) have been reported as potential molecular viability markers for SARS-CoV-2. However, few data are available on their longitudinal kinetics, compared with genomic RNA (gRNA), in clinical samples. MethodsWe analyzed 536 samples from 205 patients with COVID-19 from placebo-controlled, outpatient trials of Peginterferon Lambda-1a (Lambda; n=177) and favipiravir (n=359). Nasal swabs were collected at three time points in the Lambda (Day 1, 4 and 6) and favipiravir (Day 1, 5, and 10) trials. N-gene gRNA and sgRNA were quantified by RT-qPCR. To investigate the decay kinetics in vitro, we measured gRNA and sgRNA in A549ACE2+ cells infected with SARS-CoV-2, following treatment with remdesivir or DMSO control. ResultsAt six days in the Lambda trial and ten days in the favipiravir trial, sgRNA remained detectable in 51.6% (32/62) and 49.5% (51/106) of the samples, respectively. Cycle threshold (Ct) values for gRNA and sgRNA were highly linearly correlated (Pearsons r=0.87) and the rate of increase did not differ significantly in Lambda (1.36 cycles/day vs 1.36 cycles/day; p = 0.97) or favipiravir (1.03 cycles/day vs 0.94 cycles/day; p=0.26) trials. From samples collected 15-21 days after symptom onset, sgRNA was detectable in 48.1% (40/83) of participants. In SARS-CoV-2 infected A549ACE2+ cells treated with remdesivir, the rate of Ct increase did not differ between gRNA and sgRNA. ConclusionsIn clinical samples and in vitro, sgRNA was highly correlated with gRNA and did not demonstrate different decay patterns to support its application as a viability marker. SummaryWe observed prolonged detection of subgenomic RNA in nasal swabs and equivalent decay rates to genomic RNA in both longitudinal nasal swabs and in remdesivir-treated A549ACE2+ cells infected with SARS-CoV-2. Taken together, these findings suggest that subgenomic RNA from SARS-CoV-2 is comparably stable to genomic RNA and that its detection is therefore not a more reliable indicator of replicating virus.

Standardized and optimized preservation, extraction and quantification techniques for detection of fecal SARS-CoV-2 RNA

COVID-19 patients shed SARS-CoV-2 viral RNA in their stool, sometimes well after they have cleared their respiratory infection. This feature of the disease may be significant for patient health, epidemiology, and diagnosis. However, to date, methods to preserve stool samples from COVID patients, and to extract and quantify viral RNA concentration have yet to be optimized. We sought to meet this urgent need by developing and benchmarking a standardized protocol for the fecal detection of SARS-CoV-2 RNA. We test three preservative conditions for their ability to yield detectable SARS-CoV-2 RNA: OMNIgene-GUT, Zymo DNA/RNA shield kit, and the most common condition, storage without any preservative. We test these in combination with three extraction kits: the QIAamp Viral RNA Mini Kit, Zymo Quick-RNA Viral Kit, and MagMAX Viral/Pathogen Kit. Finally, we also test the utility of two detection methods, ddPCR and RT-qPCR, for the robust quantification of SARS-CoV-2 viral RNA from stool. We identify that the Zymo DNA/RNA shield collection kit and the QiaAMP viral RNA mini kit yield more detectable RNA than the others, using both ddPCR and RT-qPCR assays. We also demonstrate key features of experimental design including the incorporation of appropriate controls and data analysis, and apply these techniques to effectively extract viral RNA from fecal samples acquired from COVID-19 outpatients enrolled in a clinical trial. Finally, we recommend a comprehensive methodology for future preservation, extraction and detection of RNA from SARS-CoV-2 and other coronaviruses in stool.

Inflammatory but not respiratory symptoms associated with ongoing upper airway viral replication in outpatients with uncomplicated COVID-19

BackgroundThe vast majority of SARS-CoV-2 infections are uncomplicated and do not require hospitalization, but contribute to ongoing transmission. Our understanding of the clinical course of uncomplicated COVID-19 remains limited. MethodsWe detailed the natural history of uncomplicated COVID-19 among 120 outpatients enrolled in a randomized clinical trial of Peginterferon Lambda. We characterized symptom trajectory and clusters using exploratory factor analysis, assessed predictors of symptom resolution and cessation of oropharyngeal viral shedding using Cox proportional hazard models, and evaluated associations between symptoms and viral shedding using mixed effects linear models. ResultsHeadache, myalgias and chills peaked at day 4 after symptom onset; cough peaked on day 9. Two distinct symptom cluster trajectories were identified; one with mild, upper respiratory symptoms, and the other with more severe and prolonged inflammatory symptoms. The median time to symptom resolution from earliest symptom onset was 17 days (95% CI 14-18). Neither enrollment SARS-CoV-2 IgG levels (Hazard ratio [HR] 1.88, 95% CI 0.84-4.20) nor oropharyngeal viral load at enrollment (HR 1.01, 95% CI 0.98-1.05) were significantly associated with the time to symptom resolution. The median time to cessation of viral shedding was 10 days (95% CI 8-12), with higher SARS-CoV-2 IgG levels at enrollment associated with hastened resolution of viral shedding (HR 3.12, 95% CI 1.4-6.9, p=0.005). Myalgia, joint pains, and chills were associated with a significantly greater odds of oropharyngeal SARS-CoV-2 RNA detection. ConclusionsIn this outpatient cohort, inflammatory symptoms peaked early and were associated with ongoing SARS-CoV-2 replication. SARS-CoV-2 antibody levels were associated with more rapid viral shedding cessation, but not with time to symptom resolution. These findings have important implications for COVID-19 screening approaches and clinical trial design.

New-Onset IgG Autoantibodies in Hospitalized Patients with COVID-19

Coronavirus Disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), is associated with a wide range of clinical manifestations, including autoimmune features and autoantibody production. We developed three different protein arrays to measure hallmark IgG autoantibodies associated with Connective Tissue Diseases (CTDs), Anti-Cytokine Antibodies (ACA), and anti-viral antibody responses in 147 hospitalized COVID-19 patients in three different centers. Autoantibodies were identified in approximately 50% of patients, but in <15% of healthy controls. When present, autoantibodies largely targeted autoantigens associated with rare disorders such as myositis, systemic sclerosis and CTD overlap syndromes. Anti-nuclear antibodies (ANA) were observed in [~]25% of patients. Patients with autoantibodies tended to demonstrate one or a few specificities whereas ACA were even more prevalent, and patients often had antibodies to multiple cytokines. Rare patients were identified with IgG antibodies against angiotensin converting enzyme-2 (ACE-2). A subset of autoantibodies and ACA developed de novo following SARS-CoV-2 infection while others were transient. Autoantibodies tracked with longitudinal development of IgG antibodies that recognized SARS-CoV-2 structural proteins such as S1, S2, M, N and a subset of non-structural proteins, but not proteins from influenza, seasonal coronaviruses or other pathogenic viruses. COVID-19 patients with one or more autoantibodies tended to have higher levels of antibodies against SARS-CoV-2 Nonstructural Protein 1 (NSP1) and Methyltransferase (ME). We conclude that SARS-CoV-2 causes development of new-onset IgG autoantibodies in a significant proportion of hospitalized COVID-19 patients and are positively correlated with immune responses to SARS-CoV-2 proteins.

Peginterferon Lambda-1a for treatment of outpatients with uncomplicated COVID-19: a randomized placebo-controlled trial

Type III interferons have been touted as promising therapeutics in outpatients with coronavirus disease 2019 (COVID-19). We conducted a randomized placebo-controlled trial in 120 patients with mild to moderate COVID-19 to determine whether a single, 180 mcg subcutaneous dose of Peginterferon Lambda-1a (Lambda) could shorten the duration of viral shedding (primary endpoint) or symptoms (secondary endpoint, NCT04331899). In both the 60 patients receiving Lambda and the 60 receiving placebo, the median time to cessation of viral shedding was 7 days (hazard ratio [HR] = 0.81; 95% confidence interval [CI] 0.56 to 1.19). Symptoms resolved in 8 and 9 days in Lambda and placebo, respectively (HR 0.94; 95% CI 0.64 to 1.39). At enrollment; 41% of subjects were SARS-CoV-2 IgG seropositive; compared to placebo, lambda tended to delay shedding cessation in seronegatives (aHR 0.66, 95% CI 0.39-1.10) and to hasten shedding cessation in seropositives (aHR 1.58, 95% CI 0.88-2.86; p for interaction = 0.03). Liver transaminase elevations were more common in the Lambda vs. placebo arm (15/60 vs 5/60; p = 0.027). In this study, a single dose of subcutaneous Peginterferon Lambda-1a neither shortened the duration of SARS-CoV-2 viral shedding nor improved symptoms in outpatients with uncomplicated COVID-19.

Symptomatic SARS-CoV-2 infections display specific IgG Fc structures

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused a public health crisis that is exacerbated by our poor understanding of correlates of immunity. SARS-CoV-2 infection can cause Coronavirus Disease 2019 (COVID-19), with a spectrum of symptoms ranging from asymptomatic carriage to life threatening pneumonia and cytokine dysregulation [1-3]. Although antibodies have been shown in a variety of in vitro assays to promote coronavirus infections through mechanisms requiring interactions between IgG antibodies and Fc gamma receptors (Fc{gamma}Rs), the relevance of these observations to coronavirus infections in humans is not known [4-7]. In light of ongoing clinical trials examining convalescent serum therapy for COVID-19 patients and expedited SARS-CoV-2 vaccine testing in humans, it is essential to clarify the role of antibodies in the pathogenesis of COVID-19. Here we show that adults with PCR-diagnosed COVID-19 produce IgG antibodies with a specific Fc domain repertoire that is characterized by reduced fucosylation, a modification that enhances interactions with the activating Fc{gamma}R, Fc{gamma}RIIIa. Fc fucosylation was reduced when compared with SARS-CoV-2-seropositive children and relative to adults with symptomatic influenza virus infections. These results demonstrate an antibody correlate of symptomatic SARS-CoV-2 infections in adults and have implications for novel therapeutic strategies targeting Fc{gamma}RIIIa pathways.