Recent studies have provided insights into the pathogenesis of coronavirus disease 2019 (COVID-19) 1 – 4 . However, the longitudinal immunological correlates of disease outcome remain unclear. Here we serially analysed immune responses in 113 patients with moderate or severe COVID-19. Immune profiling revealed an overall increase in innate cell lineages, with a concomitant reduction in T cell number. An early elevation in cytokine levels was associated with worse disease outcomes. Following an early increase in cytokines, patients with moderate COVID-19 displayed a progressive reduction in type 1 (antiviral) and type 3 (antifungal) responses. By contrast, patients with severe COVID-19 maintained these elevated responses throughout the course of the disease. Moreover, severe COVID-19 was accompanied by an increase in multiple type 2 (anti-helminths) effectors, including interleukin-5 (IL-5), IL-13, immunoglobulin E and eosinophils. Unsupervised clustering analysis identified four immune signatures, representing growth factors (A), type-2/3 cytokines (B), mixed type-1/2/3 cytokines (C), and chemokines (D) that correlated with three distinct disease trajectories. The immune profiles of patients who recovered from moderate COVID-19 were enriched in tissue reparative growth factor signature A, whereas the profiles of those with who developed severe disease had elevated levels of all four signatures. Thus, we have identified a maladapted immune response profile associated with severe COVID-19 and poor clinical outcome, as well as early immune signatures that correlate with divergent disease trajectories.

CLIA-certified laboratories were enrolled through the IMPACT biorepository study 15. In the IMPACT study, biospecimens including blood, nasopharyngeal swabs, saliva, urine and stool samples were collected at study enrolment (baseline denotes the first time point) and longitudinally on average every 3 to 7 days (serial time points). The detailed demographics and clinical characteristics of these 98 participants are shown in Extended Data Table 1. Plasma and peripheral blood mononuclear cells (PBMCs) were isolated from whole blood, and plasma was used for titre measurements of SARS-CoV-2 spike S1 protein-specific IgG and IgM antibodies (anti-S1-IgG and-IgM) and cytokine or chemokine measurements. Freshly isolated PBMCs were stained and analysed by flow cytometry 15. We obtained longitudinal serial time-point samples from a subset of these 98 study participants (n = 48; information in Extended Data Table 1). To compare the immune phenotypes between sexes, two sets of data analyses were performed in parallel-baseline and longitudinal, as described below. As a control group, healthcare workers (HCWs) from Yale-New Haven Hospital were enrolled who were uninfected with COVID-19. Demographics and background information for the HCW group and the demographics of HCWs for cytokine assays and flow cytometry assays for the primary analyses are in Extended Data Table 1. Demographic data, time-point information of the samples defined by the days from the symptom onset (DFSO) in each patient, treatment information, and raw data used to generate figures and tables is in Supplementary Table 1. Baseline analysis The baseline analysis was performed on samples from the first time point of patients who met the following criteria: not in intensive care unit (ICU), had not received tocilizumab, and had not received high doses of corticosteroids (prednisone equivalent of more than 40 mg) before the first sample collection date. This patient group, cohort A, consisted of 39 patients (17 male and 22 female) (Extended Data Tables 1, 2). Intersex and transgender individuals were not represented in this study. Figures 1-4 represent analyses of baseline raw values obtained from patients in cohort A. In cohort A patients, male and female patients were matched in terms of age, body mass index (BMI), and DFSO at the first time point sample collection (Extended Data Fig. 1a). However, there were significant differences in age and BMI between HCW controls and patients (patients had higher age and BMI values) (Extended Data Table 1), and therefore an age-and BMI-adjusted difference-indifferences analysis was also performed in parallel (Extended Data Table 3). Longitudinal analysis As parallel secondary analyses, we performed longitudinal analysis on a total patient cohort (cohort B) to evaluate the difference in immune response over the course of the disease between male and female patients. Cohort B included all patient samples from cohort A (including several time-point samples from the cohort A patients) as well as an additional 59 patients who d...

Rapid and accurate SARS-CoV-2 diagnostic testing is essential for controlling the ongoing COVID-19 pandemic. The current gold standard for COVID-19 diagnosis is real-time RT-PCR detection of SARS-CoV-2 from nasopharyngeal swabs. Low sensitivity, exposure risks to healthcare workers, and global shortages of swabs and personal protective equipment, however, necessitate the validation of new diagnostic approaches. Saliva is a promising candidate for SARS-CoV-2 diagnostics because (1) collection is minimally invasive and can reliably be self-administered and (2) saliva has exhibited comparable sensitivity to nasopharyngeal swabs in detection of other respiratory pathogens, including endemic human coronaviruses, in previous studies. To validate the use of saliva for SARS-CoV-2 detection, we tested nasopharyngeal and saliva samples from confirmed COVID-19 patients and self-collected samples from healthcare workers on COVID-19 wards. When we compared SARS-CoV-2 detection from patient-matched nasopharyngeal and saliva samples, we found that saliva yielded greater detection sensitivity and NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.consistency throughout the course of infection. Furthermore, we report less variability in self-sample collection of saliva. Taken together, our findings demonstrate that saliva is a viable and more sensitive alternative to nasopharyngeal swabs and could enable at-home self-administered sample collection for accurate large-scale SARS-CoV-2 testing.

Genome sequencing has become a powerful tool for studying emerging infectious diseases; however, genome sequencing directly from clinical samples without isolation remains challenging for viruses such as Zika, where metagenomic sequencing methods may generate insufficient numbers of viral reads. Here we present a protocol for generating coding-sequence complete genomes comprising an online primer design tool, a novel multiplex PCR enrichment protocol, optimised library preparation methods for the portable MinION sequencer (Oxford Nanopore Technologies) and the Illumina range of instruments, and a bioinformatics pipeline for generating consensus sequences. The MinION protocol does not require an internet connection for analysis, making it suitable for field applications with limited connectivity. Our method relies on multiplex PCR for targeted enrichment of viral genomes from samples containing as few as 50 genome copies per reaction. Viral consensus sequences can be achieved starting with clinical samples in 1-2 days following a simple laboratory workflow. This method has been successfully used by several groups studying Zika virus evolution and is facilitating an understanding of the spread of the virus in the Americas.

We measured severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA concentrations in primary sewage sludge in the New Haven, Connecticut, USA, metropolitan area during the Coronavirus Disease 2019 (COVID-19) outbreak in Spring 2020. SARS-CoV-2 RNA was detected throughout the more than 10-week study and, when adjusted for time lags, tracked the rise and fall of cases seen in SARS-CoV-2 clinical test results and local COVID-19 hospital admissions. Relative to these indicators, SARS-CoV-2 RNA concentrations in sludge were 0-2 d ahead of SARS-CoV-2 positive test results by date of specimen collection, 0-2 d ahead of the percentage of positive tests by date of specimen collection, 1-4 d ahead of local hospital admissions and 6-8 d ahead of SARS-CoV-2 positive test results by reporting date. Our data show the utility of viral RNA monitoring in municipal wastewater for SARS-CoV-2 infection surveillance at a population-wide level. In communities facing a delay between specimen collection and the reporting of test results, immediate wastewater results can provide considerable advance notice of infection dynamics. The progression of the COVID-19 pandemic has been monitored primarily by testing symptomatic individuals for the presence of SARS-CoV-2 RNA and counting the number of positive tests over time 1. However, in the United States and other countries, the spread of COVID-19 has commonly exceeded the testing capacity of public health systems. Moreover, test results are a lagging indicator of the pandemic's progression 2,3 , because testing is usually prompted by symptoms, which might take 2 weeks to present after infection 4 , and delays occur between the appearance of symptoms, testing and the reporting of test results. Monitoring sewage in a community's collection or treatment system has been used previously to provide early surveillance of disease prevalence at a population-wide level, notably for polio 5,6 , and might be similarly beneficial for the current COVID-19 pandemic. SARS-CoV-2 RNA is present in the stool of patients with COVID-19 (refs. 7-9) and in raw wastewater 10-12 , and increased RNA concentrations in raw wastewater have been recently associated with increases in reported COVID-19 cases 11. However, the utility of wastewater SARS-CoV-2 concentrations for tracking the progression of COVID-19 infections is poorly understood. In this study, we investigated how viral RNA concentrations in