Study Protocol for DeCOmPRESS: Defining the Disease Course and Immune Profile of COVID-19 in the Immunosuppressed Patient

Study design

The DeCOmPRESS study will use AAV as a chronic autoimmune disease model and take advantage of the existing Irish clinical research infrastructures built around this rare disease. This is a prospective multi-site cohort sub-study of the Rare Kidney Disease (RKD) Biobank, with collection of clinical data and biospecimens as defined in the primary RKD protocol, accessible at https://www.tcd.ie/medicine/thkc/research/rare.php. The RKD Data Management Plan and RKD Registry Data Dictionary are accessible at https://www.tcd.ie/medicine/thkc/decompress/. Our key outcome measure is severe COVID-19 infection, defined as death, ICU admission and/or the need for high-flow (FiO₂>30%) oxygen, non-invasive ventilation, or invasive ventilation.

Research Timeline

The start date for this project was June 1^st, 2020 with an estimated timeframe of 24 months. Existing infrastructure supports the immediate implementation of this work.

Study Population – The RKD Biobank

The RKD Biobank cohort comprises over 850 members with systemic vasculitis. The three AAV phenotypes are microscopic polyangiitis (MPO), granulomatosis with polyangiitis (GPA), and eosinophilic granulomatosis with polyangiitis (EGPA). This bioresource, which includes about 70% of all known Irish AAV cases, has already supported many published studies. All recruits have provided biological samples during periods of remission and have consented to further sampling and data collection as envisaged for this project. All patients with systemic vasculitis enrolled in the RKD registry who develop probable or confirmed COVID-19 over the next 12–18 months will be included in the DeCOmPRESS study.

Based on modelling data, we anticipate that approximately half of our enrolled patients will develop COVID-19 over the entire duration of the pandemic, and we will capture 75% of these cases (n=306). Based on empiric data (current medication usage in our cohort), we expect that two-thirds (n=202) will meet our definition of exposed (immunosuppressed) and one third (n=104) will not. We expect 52% of cases in the non-immunosuppressed group to develop severe infection. This expected sample size will have 80% power to detect an OR=0.5 (i.e. absolute frequency of severe infection of 35% in immunosuppressed group) or smaller, with 2-tailed alpha of 0.05.

Exposure groups. The patient group for this study comprises RKD Biobank recruits with a diagnosis of systemic vasculitis, as defined by the RKD protocol, and a diagnosis of COVID-19 as confirmed by polymerase chain reaction (PCR) at the time of diagnosis and/or by retrospective serologic testing. Patients with a high clinical suspicion of COVID-19 but no timely PCR test will be included pending confirmatory serologic testing. To estimate the overall period prevalence of SARS-CoV-2 infection in this population, including asymptomatic infection, all RKD recruits will be sampled between September 2020 and March 2021 (i.e., 6 to 12 months after the beginning of the pandemic) to determine the presence of SARS-CoV-2 antibodies. Review of the RKD database will be used to determine type, use, dosage, and frequency of immunosuppressive medications at the time of COVID-19 onset. Immunosuppressive exposure is defined as: current use of >5 mg daily prednisolone or any dose of azathioprine, mycophenolate, avacopan or methotrexate; or rituximab and/or cyclophosphamide use within the previous 6 months. The control group will comprise patients on minimal or no immunosuppression, defined as ≤5 mg daily prednisolone and no other immunosuppressant exposure, as defined above.

Study procedures

Patients with vasculitis previously recruited to the RKD Biobank will be identified, where possible, at the time of COVID-19 diagnosis. Patients will be treated at their local hospital or in the community and clinical data recorded in the RKD database. Blood samples will be obtained for immunophenotyping, serum cytokine measurements, and serologic analysis. This requires an efficient and responsive workflow to allow recruitment at short notice and outside office hours, something that the RKD infrastructure already allows for. However, due to delays in recognising cases or notifying study investigators, some patients might not be recruited until sometime after resolution of COVID-19; only convalescent immunophenotyping will be possible in these cases. A simplified workflow for the DeCOmPRESS study is described in Figure 1.

Figure 1. A simplified workflow of the DeCOmPRESS study.

Patients from designated cohorts are recruited at local hospital sites and clinical data recorded in REDCap. Serum and EDTA plasma samples are taken for immunophenotyping and serum cytokine analysis at the Central Pathology Laboratory in St. James's Hospital. Repeat sampling at 3 and 6 months for convalescent antibody testing. Data will be incorporated into the ADAPT RDF data engine for subsequent analysis.

Sample and data collection

All participants in the DeCOmPRESS study will also be recruited to the RKD Biobank, either previously or at the time of COVID-19 diagnosis. Potential recruits presenting with COVID-19 at participating clinical sites will be reported in a de-identified manner as soon as possible to the DeCOmPRESS study team. There are two primary ways to identify suitable subjects for the DeCOmPRESS study:

Upon identification of an eligible DeCOmPRESS study subject, the study team will follow the steps below:

Clinical data collection. Clinical data for the DeCOmPRESS study will be captured in an eCRF format using the REDCap platform. REDCap is a secure electronic data capture web platform that stores pseudonymised clinical data for the RKD biobank. The RKD registry database is hosted by Trinity College Dublin (TCD) Research-IT and access permissions are managed by the Principal Investigator (MAL). Comprehensive guidelines for completing the REDCap COVID-19 instrument are available in the RKD Clinical Data Entry standard operating procedure, and video tutorials for REDCap data entry are also available (accessible at www.tcd.ie/medicine/thkc/research/rare.php).

Registration fields are referred to as ‘instruments’ in REDCap. The detailed COVID-19 instrument records information about COVID-19 symptom onset, diagnosis, clinical features, management, and outcomes. Clinical data entry will be completed as soon as possible after patient recruitment to the study and no more than 7 days after biospecimen sampling. Particular attention will be paid to the COVID-19 instrument and prior and current vasculitis encounter medication entries. Patients will be classified as either exposed or unexposed based on immunosuppressant use.

Blood sample analysis

Immunophenotyping and serum cytokine analysis will be carried out at the central Pathology Laboratory (CPL) at St. James’ Hospital Dublin. Serum (minimum 5 ml) and plasma (minimum 5 ml) samples will be taken no more than 24 hours, and ideally within 4 hours, prior to analysis. Immune cell populations (percentages and counts of leukocytes) will be determined by flow cytometry. Serum will be archived to allow subsequent batch measurement of cytokines by ELISA and ELLA Multiplex cytokine assay. Cytokine analysis of historical (obtained during stable vasculitis remission before 1^st September 2019), active infection, and convalescent samples will be performed, and compared both within and across participants, to determine the influence of immunosuppression, as well as COVID-19 disease severity, on cytokine levels before, during, and after COVID-19 infection. Additional details of immunophenotyping and serum cytokine analysis procedures are available in the DeCOmPRESS Study Manual (accessible at https://www.tcd.ie/medicine/thkc/decompress/).

Serology. All RKD subjects will be tested for antibodies (IgG, IgM and IgA) to SARS-CoV-2 -spike protein and receptor-binding domain of the spike protein using an in-house developed assay based on work from Mount Sinai¹⁰. The samples to be tested will be obtained:

A pilot series of patients with PCR-confirmed infection and 20 unselected RKD recruits with samples before and during COVID pandemic were tested for the purpose of this report. We will review patient-reported symptoms suggestive of possible COVID-19, logged via a customised smartphone app within the prior 12 months, and link this to the serological data. This will allow a comprehensive assessment of the impact of immunosuppressive therapy on the antibody response to SARS-CoV-2.

Integrated data analysis

Data analysis will employ the ADAPT Resource Description Framework (RDF) data integration engine and multivariable logistic regression with adjustment for demographics, comorbidities, and vasculitis disease activity¹¹. Information from the multiple DeCOmPRESS data streams integrated into ADAPT’s analysis networks will be stored in a “triplestore” database to facilitate analysis and data sharing. This is a purpose-built database for the storage and retrieval of triples through semantic queries. Semantic triples codify statements about the data in the form of subject–predicate–object expressions. Data from various sources will be formatted appropriately to allow efficient uplift into the triplestore. The primary analysis outcomes will examine differences in immunophenotypes and cytokine profiles, clinical characteristics and outcomes between immunosuppressed and non-immunosuppressed patients. Additional exploratory analyses will include:

Ethical approval and safety measures

DeCOmPRESS procedures are incorporated into the RKD ethical framework, with prior approval obtained in all RKD recruiting sites. All participants have provided consent for research projects deriving from the samples and clinical data. The DeCOmPRESS sub-project has received additional approval by the RKD Biobank Steering Committee and from the SJH/Tallaght Ethics Committee (REC: 2020-04 List 13 – Amendment (22)). The Health and Safety Authority has directed that university-based non-propagative work using SARS-CoV-2 positive samples should happen at containment level 2 using containment level 3 processes¹².

Informed consent

All patients previously enrolled to the RKD Biobank have already provided informed consent for their data and specimens to be used in studies like DeCOmPRESS. Full details are provided in the RKD Biobank protocol. Patient information leaflets are available to download via the following link: https://www.tcd.ie/medicine/thkc/assets/pdf/RKD-Vasculitis-Patient-PIL-ICF-Version-5-07AUG19.pdf. In cases of COVID-19 in a patient with vasculitis who has not already been recruited to the RKD biobank, standard RKD recruitment procedures will be followed.

Statistical analysis

Descriptive analysis of group characteristics will include number and proportion of cases for categorical variables, mean and standard deviation (SD) for normally distributed continuous variables, and median and inter-quartile range (IQR) for non-normally distributed continuous variables. When examining associations between immunosuppression exposure and COVID-19 outcomes, statistical methods will be employed to account for confounding in vasculitis disease activity, e.g. multivariable logistic regression with handing of disease activity as a stratification variable or interaction term. Additional control groups, within and outside the RKD registry, may be identified and included in the study, pending appropriate ethical approval.

Dissemination

A key objective of DeCOmPRESS is rapid and effective dissemination of research outputs. This has been achieved primarily in conjunction with the Vasculitis Ireland Network (VINE)¹³, and UK and Ireland Vasculitis Society in conjunction with frequent reviews of state-of-the-art emerging research of relevance to the DeCOmPRESS mission. These are disseminated via a Twitter feed, targeted email distribution lists (managed by the UKIVAS secretariat) and using the VINE website. The DeCOmPRESS study is still recruiting patients and data will be compiled and published 18–24 after project commencement. Initial subject data has contributed to a manuscript with additional UKIVAS patient data and has been submitted for peer review. Full details of the wide-reaching dissemination strategy are outlined in the Data Sharing Plan (accessible at https://www.tcd.ie/medicine/thkc/decompress/).

Strengths of this study

Experience and diversity of study team. The research team were able immediately mobilize the appropriate resources to begin this study on the proposed start date of June 1^st, 2020. Our core team and collaborators have a diverse expert knowledge in several areas of clinical and translational research, including immunology, epidemiology, autoimmune disease, and data analysis and integration. A patient representative from Vasculitis Awareness Ireland has also been included in our core research team to guide study design and will ensure that the best interests of patients are considered throughout the study, and that the research questions address issues of relevance to patients.

Access to patient resources via the RKD Biobank. The RKD Biobank comprises over 800 vasculitis patients, including about 70% of all prevalent Irish AAV cases. RKD Biobank recruits have already supported many studies^14–17 and provided consent to use their clinical data and biological samples for research. The Vasculitis Ireland Network¹³ has been recognised by the Department of Health as a centre of expert vasculitis care. Data collection standards are comprehensive and consistent across all clinical study sites.

Powerful data infrastructures. Data generated throughout the DeCOmPRESS study will be uplifted into ADAPT’s RDF data integration engine¹¹. This uses semantic web technology to create a normalised data pool that can be queried and rapidly analysed as a single logical dataset. This will allow rapidly harmonised dissemination of interoperable study data. We have generated a vasculitis-focused COVID-19 dataset, integrated into the RKD database, that adheres to FAIR data principles¹⁸. This was developed in collaboration with the FAIRVASC project, which seeks to merge multiple vasculitis registries across Europe, and is interoperable with international datasets. Finally, patients can monitor and report AAV and COVID-19 symptoms through a bespoke smartphone app, developed by Irish SME patientMpower.