Study design
Pa-COVID-19 is a prospective observational cohort study. All patients diagnosed with COVID-19 at Charite - Universitätsmedizin Berlin are eligible for inclusion. The protocol was developed in accordance with and extends upon the standardized protocol for the rapid, coordinated clinical investigation of severe acute infections by pathogens of public health interest published by the International Severe Acute Respiratory and Emerging Infection Consortium (ISARIC) [20].
We aimed to establish a study protocol that can serve as blueprint and central platform for harmonized deep clinical, molecular, and immunological phenotyping studies in COVID-19 patients in Germany. The protocol supports data sharing efforts through adherence to international data harmonization standards. Data are collected longitudinally from patients with confirmed COVID-19 three times per week during their hospitalization and at follow-up visits. Data include epidemiological and demographic parameters, medical history and potential risk factors, documentation of standard of care procedures, and clinical course, including different patterns of organ involvement, quality of care, morbidity, and quality of life. Moreover, extensive serial high-quality bio-sampling consisting of various sample types with deep molecular, immunological, and virological phenotyping through single-cell- and bulk- multi-omics analysis is performed.
Here, we provide a comprehensive protocol for the interdisciplinary characterization of the syndrome caused by infection with SARS-CoV-2, a newly-emerged zoonotic virus and poorly characterized human pathogen. Therefore, the sample size is not prospectively determined. Recruitment of participants will depend on the emergence and spread of the disease in Berlin, Germany, and on the number of patients presenting at Charite - Universitätsmedizin Berlin. With the evolving outbreak, recruitment in other study centers will be considered to facilitate the establishment of a comprehensive clinical and molecular database. The study has no set end date.
Inclusion criteria
Proven infection with SARS-CoV-2 (positive pathogen testing). Willingness to participate in the study.
Exclusion criteria
Refusal to participate by patient, parent, or appropriate legal representative.
Any conditions that prohibit supplemental blood sampling.
Data collection
Harmonized scalable dataset/electronic Case Report Form (eCRF)
This protocol outlines the methodology of the Berlin prospective cohort for capturing data of COVID-19. The parameters were selected and adapted to local standards through a multidisciplinary expert review board consisting of clinical researchers at Charité, Department of Infectious Diseases and Respiratory Medicine, Institute of Virology, Departments of Anaesthesiology and Operative Intensive Care Medicine, Department of Psychosomatic Medicine, Department of Pediatrics, Departments of Cardiology, Department of Neurology and Center for Stroke Research, Department of Nephrology and Medical Intensive Care, Department of Endocrinology and Metabolism, Department of Medicine, Division of Gastroenterology, Infectious Diseases and Rheumatology, Department of Hepatology and Gastroenterology, Institute of Tropical Medicine and International Health, Central Biobank Charité (ZeBanC), and the Clinical Study Centre at the Berlin Institute of Health, based on available preliminary data and in accordance with the WHO supported case report form (CRF) proposed by the International Severe Acute Respiratory and Emerging Infection Consortium (ISARIC). Items of the ISARIC–CRF were translated to German using the standardized Functional Assessment of Chronic Illness Therapy (FACIT) translation methodology [21]. Data elements were prioritized into three categories (core data, recommended data, and supplemental data elements, see eCRF, supplementary file 1) to allow the adjustment of data collection to different healthcare and research resources. The latest version of the eCRF is available up-to-date at the study website https://studycenter.charite.de/covid_19/.
In addition to medical history and history of present illness, we aim for detailed longitudinal documentation of (i) clinical symptoms, (ii) vital signs and blood gases, (iii) laboratory parameters (including inflammation markers, cardiac enzymes, coagulation parameters, etc.), (iv) imaging data (e.g., X-ray, echocardiography, ultrasound, CT scans, and MRI scans), (v) microbiological and virological tests, (vi) supplementary diagnostic data (e.g., bronchoscopy and lumbar puncture), (vi) concomitant medication, and (vii) details of clinical interventions (mechanical ventilation, organ replacement therapy, etc.) (Table 1). Patients can also be included in Pa-COVID-19 if they participate in other clinical studies, e.g., in interventional clinical trials. In these patients, trial participation and application of any study medication will be documented in the eCRF. During in-patient stay, the patient-reported health status will be measured using tailored short forms from the Patient-Reported Outcome Measurement Information System® (PROMIS), targeting for precise assessments of dyspnoea and physical function [22]. Overall outcomes will be evaluated using clinician as well as patient-reported health data, including the WHO 7 category ordinal scale [23] at study visits until day 15 and at discharge from hospital. Quality of life, health status, and performance in activities of daily living will be assessed using the Barthel Index and an adapted version of the PROMIS®-29 Profile at discharge, 6 weeks, 3 months, 6 months, and 12 months after inclusion. In addition, lung function, blood gases, cardiac function, renal function, occurrence of Post-Intensive Care Syndrome, neuropsychiatric function, exercise capacity, and cognitive function will be assessed in selected patient groups at follow-up visits [24].
Table 1 Study procedures: each visit during the patient's hospital stay will register as a new visit We will also recruit a representative cohort of outpatients with mild symptoms of COVID-19, in whom study visits and biosamplings are only performed at enrolment (V1) and at day 15 (V7) plus follow-up, provided that no hospitalization becomes necessary during the course of disease. Telephone interviews will be performed with outpatients to assess patient-reported health status between V1 and V7.
Serial bio-sampling, molecular and immunological phenotyping, and systematic biobanking
The molecular basis of the widely variable clinical course of COVID-19 is unknown. Many risk factors for severe disease, prognostic biomarkers, or correlates of protective immunity remain unclear. Several reports have indicated a central role for improper immune responses in COVID-19-associated lung damage and severe disease [25]. To elucidate the immunological basis for different courses of COVID-19 and the development of natural immunity, we have devised a detailed sequential deep phenotyping strategy, to decipher the composition and activation state of cellular and humoral components of the host immune response to SARS-CoV-2, employing high-resolution technologies including single-cell RNA-Sequencing and Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq), T-cell receptor and B-cell receptor sequencing, Cytometry by Time-of-Flight (CyTOF), quantitative plasma proteomics, epigenetic profiling, as well as hypothesis-driven investigation and validation of individual biomarkers by conventional immunoassays.
As scientific data on clinical, cellular, and molecular aspects of COVID-19 are rapidly evolving, we will preserve high-quality biosamples, including cryopreserved immune cells, through a large-scale biobanking effort. An overview of the sample flow is shown in Fig. 1. Blood samples are collected three times per week in hospitalized patients, alongside blood drawing for routine laboratory analysis. Biobanking is organized in close cooperation with central biobank of Charité/BIH (ZeBanC) according to standard operational procedures (SOPs) and under certified conditions.
To ensure maximal safety for medical staff involved in this clinical study, SOPs for clinical sample handling are defined. Personal safety equipment for laboratory staff includes tightly closed protective gown, face shield or protective glasses, surgical face mask, and protective gloves. All blood samples are handled under a bio-safety level (BSL) 2 hoods and primary tubes are discharged. Analyses of infectious specimen will be handled according to the current regulation under BSL2 safety conditions.
Post-mortem analysis
Post-mortem analysis under strict safety considerations will be considered for patients succumbing to COVID-19 in this study. A detailed SOP has been devised at our institution. Autopsy will provide in-depth insights into viral tropism and organ pathologies of severe disease. Additional analysis of infected tissue using spectral confocal microscopy will provide insights into the pathology of fatal COVID-19. Samples will be stored for future analysis.
Data management and storage
Clinical and laboratory data will be collected from patient charts, through patient interviews or from examinations and submitted online to a purpose-built eCRF using the electronic data capture system SecuTrial®. All data will be pseudonymized using a six-digit alphanumerical patient code and the online records kept will not include any information that allows patient identification. An enrolment log list will be used to match patient codes in the database to individual patients to record clinical outcomes and supply any missing data points. The enrolment log and study data will be kept separately. Access to the database is protected by username and password. The electronic database set up in this study is suitable for the use in interventional trials and can, therefore, rapidly be modified for future clinical testing of therapeutic agents.
Quality assurance and control measures
A detailed data dictionary will define data to be collected on eCRF. Quality checks will be built into the data management system, including the possibility to generate queries, to ensure standardization and validity of the data collected. Any information that is not available to the investigators will be considered as missing. No assumptions will be made for missing data. Data monitoring of a randomly selected subset of collected data will be performed in the course of the study. Discussion of data collection techniques throughout the course of the study with investigators will ensure data quality and allow for adjustments.
Data storing, access, and sharing
Access to samples for additional analyses, open access, and data sharing via the Global Research Collaboration for Infectious Disease Preparedness (GloPID-R) will be governed by a research committee comprising the clinical lead investigators and scientific investigators for this study. It is a fundamental principle of this study that all contributors and researchers who have access to samples commit to unrestricted data sharing to provide timely insights into this new disease. In addition, all clinical investigators contributing to this research efforts will be given full recognition for their efforts and the opportunity to access data and samples. Data management adheres to the FAIR data principles, stating that all data collected shall be findable, accessible, interoperable, and re-usable [26]. Samples collected will be used for the purpose of this study as stated in the protocol and consented for future use.
Ethics and registration
This study is conducted in compliance with the principles laid down in the 1964 Declaration of Helsinki and its later amendments. Where applicable, the principles of Good Clinical Practice (International Council for Harmonization, ICH 1996) and other applicable regulations and guidelines will be used to guide procedures and considerations. The study was reviewed and approved by the Charité Ethics Committee (EA2/066/20). All patients enrolled will give written informed consent in person, or by a legal guardian. Pa-COVID-19 is registered at the German Clinical Trials Register and WHO International Clinical Trials Registry Platform (DRKS00021688).
Medical management of participants in this study must never be compromised by study procedures. At all times, priority will be given to samples required for medical management and day-to-day care of patients. Study blood samples will be taken together with routine blood sampling for clinical monitoring to minimize the individual risk connected with phlebotomy or manipulation of intravascular catheters. There is no direct benefit for patients participating in the study, but results from the study might improve clinical care in future patients and benefit public health.
Specific ethical consideration includes the recruitment of critically ill patients who might not be able to consent, the strong moral obligation to participate in a public health emergency, keeping the balance between public health and research, and the potential risk for research staff involved in this study.
The enrolment of patients, who are unable to consent, is a common challenge in critical care, acute research, and minors. The process follows a clear legal framework with consent being obtained from legal guardians as soon as possible. In addition, all efforts will be made to obtain informed consent from patients retrospectively after recovery. For patients refusing retrospective informed consent, all data and biosamples will be deleted. For patients succumbing to COVID-19 before informed consent can be obtained, all data will be anonymized.
The patient information clearly outlines potential benefits and limitations. The autonomy and well-being of the individual patient and the medically indicated procedures and treatments are always paramount. All staff is trained in infection control measures and has ready access to appropriate personal protective equipment. Dedicated research staff will be available to support the study activities.