GoKawiilStudy design and participant recruitment
IMPACC is a prospective longitudinal study that enrolled >1,000 hospitalized patients with COVID-19, as previously described27,32,55,56,57,58. Participants 18 years and older were recruited from 20 hospitals across 15 academic institutes within the USA (Fig. 1a). All participants were confirmed to be SARS-CoV-2-positive by reverse transcription PCR (RT–PCR) testing and no participants were vaccinated for SARS-CoV-2 at time of enrolment. Nasal swabs, blood, and endotracheal aspirate (for ventilated patients) were collected within 72 h of hospital admission (visit 1) and on days 4, 7, 14, 21 and 28 post-hospital admission in addition to convalescent samples at 3, 6, 9 and 12 months. Additionally, nasal swabs, blood and endotracheal aspirate were also collected (when possible) within 24 h and 96 h of escalation to intensive care unit-level care or when a participant was readmitted to the hospital >48 h after discharge. In the IMPACC dataset, these samples were indicated as ‘escalation visits’, and in this Article, the escalation visit samples were only used for the longitudinal GAMM and the CyTOF cellular association analyses to minimize effects of more frequent sampling for critically ill participants. Participants were characterized into one of five trajectory groups based on latent class mixed modelling of a seven-point ordinal scale that characterized degree of respiratory illness and reflected acute COVID-19 severity24. Similarly, participants were clustered into 4 PRO groups from latent class mixed modelling trajectories of convalescent survey responses collected at 3, 6, 9 and 12 months32. The modelling used participant responses from the EQ-5D-5L59, health recovery score (visual analogue scale of 1–100 to indicate overall physical and mental function compared to pre-COVID function), and the following PROMIS forms (https://commonfund.nih.gov/promis): PROMIS Item Bank v2.0—Physical Function, PROMIS Item Bank v2.0—Cognitive Function60, PROMIS Scale v1.2—Global Health Mental 2a61, PROMIS Item Bank v1.0—Psychosocial Illness Impact-Positive—Short Form 8a61, and PROMIS Pool v1.0—Dyspnea Time Extension62. All PROMIS measures were scored and standardized following PROMIS standardized instructions. Clinical characteristics and demographics for the entire cohort are reported in Supplementary Table 1.
Ethics statement
The Department of Health and Human Services Office for Human Research Protections (OHRP) and the National Institute of Allergy and Infectious Diseases (NIAID) concurred that the IMPACC study qualified for public health surveillance exemption. The study protocol was sent for review to each site’s institutional review board (IRB), with twelve sites conducting as a public health surveillance study, and three sites integrating the IMPACC study into IRB-approved protocols (The University of Texas at Austin, IRB 2020-04-0117; University of California San Francisco, IRB 20-30497; Case Western Reserve University, IRB STUDY20200573) with participants providing informed consent. Participants enrolled at sites operating as a public health surveillance study were provided information sheets describing the study including the samples to be collected and plans for analysis and data de-identification. Participants who requested not to participate after review of the study plan and information were not enrolled. Participants were not compensated while hospitalized but were subsequently compensated for outpatient visits and surveys. This study was registered at clinicaltrials.gov (NCT04378777) and followed the strengthening the reporting of observational studies in epidemiology (STROBE) guidelines (Extended Data Fig. 1a).
Sample processing and assays
Samples were processed as previously described27,32,55,56,57,58, with the sample protocol extensively documented in the IMPACC study design and protocol paper55. In brief, 10 ml of blood and nasal swabs were collected at each visit, with blood processed within 6 h of collection. Blood was collected in both a 2.5 ml Greiner Vacuette CAT Serum Separating Tube (SST) (454243P) for serum and a 7.5 ml Sarstedt Venous blood collection monovette EDTA (NC9453456) for whole blood, PBMCs and plasma. The SST was kept vertical at room temperature for at least 30 min before centrifuging at room temperature for 10 min at 1,000g. Serum was then aliquoted at 100 μl for downstream assays.
From the EDTA tube, it was briefly inverted to mix before aliquoting 270 μl of whole blood for CyTOF. The 270 μl CyTOF aliquot was added directly to the Maxpar Direct Immune Profiling Assay tube (MDIPA antibodies listed in Supplementary Table 9) and incubated for 30 min at room temperature. After incubation, 410 μl of Smart Tube Prot1 Stabilizer (SmartTube) was added with a 10 min incubation at room temperature before storage at –80 °C until shipment to the respective processing core. The remaining blood was centrifuged at room temperature for 10 min at 1,000g before aliquoting and storing 500 μl of plasma at –80 °C for proteomic and metabolomics. PBMCs were then isolated from the remaining sample using the SepMate and Lymphoprep system (StemCell) following manufacturer protocol and as previously described55. PBMCs were then stored at 2.5 × 105 cells in 200 μl of RLT Buffer (Qiagen) and β-mercaptoethanol at –80 °C.
Interior nasal turbinate swabs (herein referred to as nasal swabs) were collected and stored in 1 ml of Zymo-DNA/RNA shield reagent (Zymo Research), before RNA was extracted twice in parallel from 250 μl of sample and purified with the KingFisher Flex sample purification system (ThermoFisher) and the quick DNA-RNA MagBead kit (Zymo Research). The duplicated RNA was pooled and aliquoted at 20 μl for the downstream assays (SARS-CoV-2 RT–qPCR and RNA-seq).
When participants were ventilated, an endotracheal aspirate was also collected in a 40 cm3 Argyle specimen trap and was processed within 2 h of collection. First, 500 μl of 1:1 diluted endotracheal aspirate with Maxpar PBS (Ca2+ and Mg2+ free) was mixed with 500 μl of DNA/RNA shield in a Zymo tube with lysis beads and subsequently stored at –80 °C for bulk RNA-seq.
Collected samples were then shipped and processed for nasal, PBMC, and endotracheal aspirate RNA-seq, plasma proteomics, serum cytokine PEA, serum EBV and CMV antibody titres, whole-blood CyTOF, and plasma metabolomics at their respective processing cores as previously described27,32,55,56,57,58. Each assay is described in brief below with additional technical details in the prior publications27,32,55,56,57,58, and a list of analytes evaluated from the PBMC RNA-seq, nasal RNA-seq, serum cytokines and chemokines, and plasma metabolomics assays is reported in Supplementary Table 8.
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