ORCHESTRA WP2 - Follow-up of Covid-19 Long Term Sequelae
Conditions: COVID-19 Pneumonia, COVID-19 Respiratory Infection, COVID-19 Acute Bronchitis, COVID-19 Acute Respiratory Distress Syndrome, COVID-19 Lower Respiratory Infection, Covid19, Corona Virus Infection, Coronavirus, Coronavirus Infections, SARS-CoV2 Infection, SARS-CoV-2 Acute Respiratory Disease, SARS-Associated Coronavirus, SARS Pneumonia, SARS (Severe Acute Respiratory Syndrome), SARS (Disease), Coronavirus Pneumonia, Coronavirus Disease 2019, Coronavirus Sars-Associated
Sponsor: Azienda Ospedaliera Universitaria Integrata Verona

 
 
 
 
 
1 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
 
 
 
 
 
 
 
 
FOLLOW-UP OF COVID-19 LONG-TERM SEQUELAE 
STUDY PROTOCOL 
 
 
 
Università degli Studi di Verona (UNIVR) 
 
 
 
 
 
Connecting European Co-
horts to Increase Common 
and Effective Response to 
SARS- CoV-2 Pandemic 

 
 
 
 
 
2 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
Project Classification   
Project Acronym:    
ORCHESTRA   
Project Title:   
Connecting European Cohorts to Increase Common and Effec-
tive Response to SARS- CoV-2 Pandemic   
Coordinator:   
UNIVR   
Grant Agreement Number:   
101016167   
Funding Scheme:   
Horizon 2020   
Start:   
1st December 2020   
Duration:   
36 months   
Website:   
www.orchestra-cohort.eu   
Email:   
info@orchestra.eu   
   
Document Classification   
Title:   
FOLLOW-UP OF COVID-19 LONG-TERM SEQUELAE - 
STUDY PROTOCOL 
Lead Beneficiary:   
UNIVR  
Co-Leader: 
INSERM, UHC 
Other Involved   
Beneficiaries:  
INSERM, UNIBO, SAS, HMGU, CINECA, UANTWERPEN, 
AP-HP, LIH, UHC, PENTA, UBA, REG VEN, RER-ASSR, 
FCRM, UMCG 
Nature:   
Protocol  
Submission Date:   
09.03.2021  
Status:   
Final  
Version:  
 0.1 
Author(s):  
 Elisa Gentiloltti, Fulvia Mazzaferii, Elda Righi, Evelina Tac-
conelli (UNIVR), Giorgia Tomassini 

 
 
 
 
 
3 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
  
  
History of Changes   
Version    
Date   
Created/Modified by   
0.1   
 05.03.2021 
 Elisa Gentilotti (UNIVR), Fulvia Mazzaferri (UNIVR), 
Elda Righi (UNIVR), Evelina Tacconelli (UNIVR), Gior-
gia Tomassini (UNIVR) 
0.2 
 07.06.2021 
 Lidia Del Piccolo (UNIVR), Maddalena Marcanti, Evelina 
Tacconelli (UNIVR) 
0.3 
12.07.2021 
Gaia Maccarrone (UNIVR), Evelina Tacconelli (UNIVR), 
Giorgia Tomassini (UNIVR)  
   
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
4 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
 
 
 
2. TABLE OF CONTENTS 
1. Title page 
2. Table of contents 
3. Protocol summary 
3.1 Synopsis 
3.2 Schedule of assessments (SoA) 
4. List of Abbreviations 
5. Background 
6. Study rationale 
7. Study design 
8. Objectives  
9. Patient cohorts 
9.1. University of Verona (UNIVR) – Italy 
9.2. The French Covid-19 cohort 
9.3. The Regional Agency for Health and Social Care of Emilia-Romagna Region  
9.4. Lean European Open Survey on SARS-CoV-2 Infected Patients” (LEOSS)  
9.5. COVID-HOME study  
9.6. Regione del Veneto - Italy  
9.7. Fondation Congolaise pour la Recherche Médicale (FCRM)- Republic of The Congo 
9.8. ZIKAction 
10. Inclusion and exclusion criteria 
11. Recruitment strategy 
12. Study procedures 
13. Statistical analysis 
14. Regulatory, ethical, and study oversight considerations 
14.1. Regulatory and ethical aspects 
14.2. Financial disclosure 
14.3. Informed consent process 
14.4. Data protection 
14.5. Data quality assurance 
14.6. Source documents 
14.7. Study and site start and closure 
14.8. Publication policy 
15. References 
 
 

 
 
 
 
 
5 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
3. PROTOCOL SUMMARY 
3.1 Synopsis 
Steering Committee  
 
Evelina Tacconelli, UNIVR 
Elda Righi, UNIVR 
Fulvia Mazzaferri, UNIVR 
Maddalena Giannella, UNIBO   
Jade Gosh, AP-HP 
Cedric Laouenan, INSERM  
Janne Vehreschild / Luise Wolf, UHC  
Adriana Tami, UMCG  
Jesus Rodrìguez Baño, SAS  
Maria Luisa Moro, RER-ASSR 
Francine Ntoumi, FCRM 
Michele Mongillo, REG VEN (Elena Narne, Az. 
Zero) 
Surbhi Malothra, UANTWERPEN  
Alex Friedrich, UMGC  
Carlo Giaquinto, PENTA  
Paulo Roberto Abrão Ferreira, UNIFESP, Brazil  
Gabriel Levy Hara, UBA, Argentina  
Vidhya Krishnamoorthy, THSTI, India Mouli 
Natchu, CBCI, India  
Principal investigator 
Evelina Tacconelli, UNIVR 
Co-principal investigators 
Elisa Gentilotti, UNIVR  
Study design 
Multicenter, observational, prospective cohort study 
enrolling hospitalized and non-hospitalized patients with 
a diagnosed SARS-CoV-2 infection. 
Study population 
Patients of all age and any comorbidity with 
previous diagnosis of SARS-CoV-2 infection 
admitted to or treated as inpatients or outpatients in 
university and non-university hospitals or medical 
practices. 
Inclusion criteria 
 
Any age 
Any comorbidity 

 
 
 
 
 
6 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
Laboratory confirmed SARS-CoV-2 infection by 
PCR diagnosis from nasopharynx, oropharynx, 
bronchoalveolar lavage, stool, or blood. Rapid tests 
are an acceptable alternative. 
Person (or attorney or deputy who has been author-
ized to make the decision for patients who lack ca-
pacity) consent to participate 
Exclusion criteria 
Lack of consent to participate 
ORCHESTRA Partners 
Università degli Studi di Verona (UNIVR); 
Alma Mater Studiorum – Università di Bologna 
(UNIBO); 
Institut National de la Sante et de la Recherche Me-
dicale (INSERM); 
Servicio Andaluz de Salud (SAS); 
Consorzio Interuniversitario (CINECA); 
Luxembourg Institute of Health (LIH); 
Assistance Publique Hopitaux de Paris (AP-HP); 
Regione Emilia Romagna (RER-ASSR); 
Fundacion Privada Instituto de Salud Global Barce-
lona (ISGLOBAL); 
Ludwig-Maximilians-Universitaet   Muenchen   
(LMU   MUENCHEN); 
Universiteit Antwerpen (UANTWERPEN); 
Helmholtz Zentrum Muenchen Deutsches For-
schungszentrumfuer Gesundheit und Umwelt 
GMBH (HMGU); 
Klinikum der Universitaet zu Koeln (UHC); 
Fondazione PENTA – for the treatment and care of 
children with HIV and related diseases - ONLUS 
(PENTA); 
Universitaet Stuttgart (USTUTT); 
Centre de Recherches Medicales DE Lambaréné 
(CERMEL); 
Regionalny Urad Verejneho Zdravotnictva so si-
idlom v Banskejbytrici(RAPH BB); 
Charité – Universitaetsmedizin Berlin (CHARITÉ); 
Academisch Ziekenhuis Groningen (UMCG); 

 
 
 
 
 
7 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
Centre Informatique National de l’Enseignement 
Superieur  (CINES); 
Universidad de Oviedo (UNIOVI); 
Universidad de Buenos Aires (UBA); 
Institutul National de  Sanatate Publica  (INSP); 
Regione del Veneto (REG VEN); 
Fondation Congolaise pour la Recherche Medicale 
(FCRM); 
Translational Health Science and Technology Insti-
tute (THSTI), Faridabad, Haryana, India; 
Catholics Bishops Conference of India, Society for 
Medical Education (CBCI), Bangalore, India; 
Escola Paulista de Medicina -Universidade Federal 
de São Paulo, Brazil. 
Study rationale 
The present study aims at harmonising follow-up 
strategies across the participating cohorts to allow a 
standardized collection of data on characteristics 
and determinants of COVID-19 long-term sequelae. 
Primary objective 
To describe characteristics of COVID-19 sequelae, 
including type, rate, and length through clinical, la-
boratory, and radiological assessments 
To investigate valuable, confounder-adjusted, asso-
ciations between COVID-19 sequelae and COVID-
19 severity, comorbidities, aetiology (SARS-CoV-2 
variants), COVID treatment (including monoclonal 
antibodies), and trends in SARS-CoV-2 antibodies 
To describe the rate, the aetiology (SARS-CoV-2 
variants), severity, and clinical determinants of 
COVID-19 re-infections 
Secondary objective 
To compare the time course of the immunological re-
sponse of the population with sequelae with the im-
munological response of the population without se-
quelae. 
To investigate immunological patterns related to spe-
cific long-term sequelae. 
To investigate possible associations of SARS-CoV-2 
variants with COVID-19 severity, time course of the 
immunological response, and long-term sequelae. 

 
 
 
 
 
8 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
To describe the rate and severity of sequelae and im-
munological trends of COVID-19 in patients vac-
cinated against SARS-CoV-2. 
To describe the time course of intestinal and pulmo-
nary microbiome after SARS-CoV-2 infection. 
To investigate possible associations of long-term se-
quelae with hypercoagulability 
To describe the relationship between risk perception 
and adherence to preventative measures over time, 
including vaccine acceptance. 
To describe the use of health care services among 
SARS-CoV-2 patients. 
To describe the relationship between risk perception 
of reinfection and the adherence to preventative 
measures over time after the SARS-CoV-2 infection, 
including vaccine acceptance. 
To describe the use of health care services among pa-
tients recovered from SARS-CoV-2. 
To identify human and viral genetic markers indica-
tive of disease severity by doing genetic and epige-
netic analysis in COVID-19 patients. 
Time schedule 
 
Patients will be followed-up for up to 18 months af-
ter the SARS-CoV-2 infection diagnosis. 
 
 
3.2 Schedule of assessments (SoA) 
Table 1 – Schedule of follow-up Assessments 
 
  
COVID-19  
(2 weeks1  
± 2 weeks)  
3 months1  
± 1 month  
6 months1  
± 1 month  
12 months1  
± 1 month  
18 months1  
± 2 months  
Screening/baseline  
   
   
   
   
   
Inclusion criteria1 
  
  
  
  
  
Demographics2  
  
   
   
  
   
Healthcare setting3  
  
   
   
   
   
Length of hospital stay, days  
  
   
   
   
   
ICU admission  
  
   
   
   
   
Medical history4  
  
   
   
   
   
Treatment  
   
   
   
   
   
Comorbidity management5  
X  
X  
X  
X  
X  
Anti-COVID therapy6  
X  
   
   
   
   
Antibiotic therapy7 
 
 
 
 
 

 
 
 
 
 
9 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
Oxygen therapy8 
X  
X*  
X*  
X*  
X*  
SARS-CoV-2 vaccination9  
X  
X  
X  
X  
X  
Clinical assessment  
   
   
   
   
   
Relevant medical new events10 
X  
X  
X  
X  
X  
COVID-19 symptom11onset  
X  
   
   
   
   
COVID-19 symptom end  
X  
X*  
X*  
X*  
X*  
COVID severity12 
X  
   
   
   
   
SOFA score  
X  
   
   
   
   
Vital signs13 
X  
X  
X  
X  
X  
Physical examination14 
X  
X  
X  
X  
X  
12-lead electrocardiography  
X  
X  
X  
X  
X  
6-minute walking test  
X  
X  
X  
X  
X  
DLCO (diffusing capacity for car-
bon monoxide)  
X  
X  
X  
X  
X  
Pulmonary function test15  
X  
X  
X  
X  
X  
Questionnaires  
   
   
   
   
   
Functional status16  
X  
X  
X  
X  
X  
Respiratory impairment17 
X  
X  
X  
X  
X  
Mental health18 
X  
X  
X  
X  
X  
Perceived risk of re-infection/ad-
mission/re-admission19  
  
X  
X  
X  
X  
X  
Adherence to main preventative 
non-pharmacological measures20 
X  
X  
X  
X  
X  
SARS-CoV-2 vaccination: ac-
ceptance/non-acceptance and rea-
sons21 
X  
X  
X  
X  
X  
Imaging  
   
   
   
   
   
Lung ultrasound  
X  
X  
X*  
X  
X*  
X-ray  
X  
X  
X*  
X*  
X*  
High-resolution CT scan  
X  
X  
X*  
X*  
X*  
Cardiac ultrasound  
X  
X  
X*  
X  
X*  
Cardiac MRI 22 
X21  
X21  
X*  
X21  
X*  
Biochemistry  
   
   
   
   
   
Blood tests23 
X  
X  
X*  
X  
X*  
Arterial blood gas test 
(pO2/pCO2/pH)  
X  
X  
X*  
X*  
X*  
Urine tests24 
X  
X  
X*  
X  
X*  
Immunology  
   
   
   
  
   
Immune - serology and type I 
IFNs autoantibodies 
X  
X  
X  
X  
X  
Immune - cytokine and chemokine 
X  
X 35 
X 35 
X 35 
X * 
Immune - cellular 
X  
X 35 
X 35 
X 35 
X * 
Microbiological tests-NP swabs  
   
   
   
   
   
Viral variant and metagenomics 
sequencing   
X  
X*,34  
X*,34  
X*,34  
X*  
EDTA whole blood 
 
 
 
 
 
Genetic and epigenetic analysis 
X 
X 
X 
X 
X 
Stool Sample (faeces or rectal 
swab) 
 
 
 
 
 
Metagenomic sequencing 
X 
 
X 
 
X 
Adjunctive variables for specific fragile populations  
HIV  
  
  
  
  
  
HIV-infection status25  
X  
X  
X  
X  
X  
HIV-Infection therapy26 
X  
X  
X  
X  
X  
Assessment of adherence to fol-
low-up visits and antiretroviral 
therapy  
X  
X  
X  
X  
X  

 
 
 
 
 
10 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
Elderly   
  
  
  
  
  
Cognitive status27 
X  
X  
X  
X  
X  
Pregnant women/new mother  
  
  
  
  
  
History of positive SARS-CoV-2 
molecular test on amniotic fluid or 
breast milk28  
  
  
  
  
  
History of detection of micro-
thrombotic disease on placenta tis-
sue or umbelical cord tissue  
  
  
  
  
  
Children  
  
  
  
  
  
History of positive SARS-CoV-2 
molecular test on amniotic fluid or 
breast milk28 
  
  
  
  
  
Biometric paramethers29 
X  
X  
X  
X  
X  
Transplant  
  
  
  
  
  
Transplant general information30  
  
  
  
  
  
Graft function31 
X  
X  
X  
X  
X  
Immunosuppressive regimen32 
X  
X  
X  
X  
X  
Onco-haematology  
  
  
  
  
  
Assessment of adherence to onco-
logic follow-up visits and therapy  
X  
X  
X  
X  
X  
Assessment of progression of the 
disease and relapse  
X  
X  
X  
X  
X  
Assessment of adverse events33 
X  
X  
X  
X  
X  
 
 
Footnotes 
Modular data capture according to level of commitment (level I, level II, level III).  
Level I 
Assessments in level I are mandatory 
Level II 
Customized according to the feasibility of each cohort 
 
*   Reassessed only if outside the normal ranges at the previous assessment or if clinically indicated 
1. Day 0: first positive SARS-CoV-2 test 
2. Demographics: age (years), sex, ethnic group (African, Asian, European, Latin America...), educa-
tion (no formal education, lower than college, college or higher), cigarette smoking (never-smoker, 
former smoker, current smoker), usual residence (home, long-term care facility, public dormitory, 
prison, homeless), current occupation (student, unemployed with no benefits, unemployed with ben-
efits, employed, self-employed, informal worker) 
3. Healthcare setting: (a) outpatient (b) non-intensive care unit (c) intensive care unit.  
4. Medical history: cardiovascular diseases (hypertension, coronary artery disease, congestive heart 
failure), diabetes (without insulin, with insulin), chronic respiratory disease (asthma, chronic ob-

 
 
 
 
 
11 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
structive pulmonary disease, obstructive sleep apnoea, restrictive lung disease, pulmonary hyper-
tension), kidney disease (chronic with/without dialysis), liver disease other than cancer 
(HBV/HCV/HDV chronic viral hepatitis, other chronic disease, cirrhosis), metabolic disease, im-
munosuppressive conditions (solid organ transplant recipient, auto-immune diseases), cancer (solid 
cancer, haematological malignancies, type of primitive cancer/haematological malignancies, pres-
ence of metastases, if ongoing chemotherapy), mental or neurological disorders (psychiatric illness, 
anxiety disorder, mood disorder, psychotic disorder, Alzheimer disease, dementia other than Alz-
heimer, Parkinson’s disease, myasthenia gravis, epilepsy, stroke (with/without residual deficits, 
neuromuscular disease, multiple sclerosis), muscular dystrophy, amyotrophic lateral sclerosis); TB 
co-infection; other opportunistic co-infection (specify) for HIV population 
5. Comorbidity management: drug name and dose (to include only treatments taken regularly)  
6. Anti-COVID therapy: drug name, maintenance dose, and duration  
7. Antibiotic therapy: drug name, dose, duration, and type of treated infection 
8. Oxygen therapy: nasal prongs, face mask, face mask with reservoir, high-flow nasal cannula, non-
invasive ventilation, mechanical ventilation; numbers of O2 (L/min) provided (maximum reached) 
and fraction of inspired O2 (FiO2) provided (maximum reached)  
9. SARS-CoV-2 vaccination: vaccine name, date of administration 
10. Relevant new medical events or worsening of previous conditions, including deep venous throm-
bosis, pulmonary embolism, infections (including a new SARS-CoV-2-infection during follow-up), 
malignancies (type of cancer, overall stage). 
11. Symptoms: abdominal pain, ageusia/dysgeusia, anosmia, balance impairment, behaviour disorder, 
chest pain or chest tightness, confusion, cough, delirium, diarrhoea, disrupted sleep, dizziness, dysp-
noea, fatigue, fever (including low-grade fever), headache, hypothermia, impaired cognitive status, 
lethargy, loss of appetite, mood affective disorder, myalgia, nausea/vomiting, palpitation, phlegm, 
runny nose, sore throat, stuffed nose, syncope, wheeze. 
12. WHO Clinical Progression Scale  
13. Vital signs: dead/alive, blood pressure, body temperature, heart rate, respiratory rate, peripheral ox-
ygen saturation  
14. Physical examination: BMI, abdominal examination, pulmonary examination, cardiac examination, 
neurological examination, peripheral vascular examination 
15. Pulmonary function test: FEV1, FVC, FEV1/FVC, TLC, FRC, RV  
16. Questionnaires to address the functional status: Post-COVID-19 Functional Status (PCFS) Scale, 
Global Physical Activity, Questionnaire (GPAQ), Barthel Index, Medical Outcome Study Short 

 
 
 
 
 
12 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
Form (MOS SF)-36 Score, EuroQol five-dimension five-level (EQ-5D-5L) questionnaire, Clinical 
Frailty Scale (CFS), Basic Activity of Daily Living (BADL). 
17. Questionnaires to address the respiratory impairment: Saint George Respiratory Questionnaire 
(SGRQ), Transition Dyspnoea Index (TDI), mMRC (Modified Medical Research Council) Dyspnea 
Scale  
18. Questionnaires to address the mental health: Hospital Anxiety and Depression Scale (HADS), Kess-
ler Psychological Distress Scale (K10), Impact of Event Scale – Revised (IES-R), Resilience Scale 
for Adults (RSA) 
19. Perceived risk of re-infection on a scale 0-10 (no risk- very high risk); perceived risk of admis-
sion/re-admission on a scale 0-10 (no risk- very high risk) 
20.  Frequency mask-wearing (type of mask); frequency hand washing; respect of social distance; 
avoidance of social gathering 
21. Was the vaccine accepted? Why not accepted (lack of trust in efficacy and/or safety; not useful in 
the specific case; prefer someone else gets it before me) 
22. Cardiac MRI only if abnormal cardiac ultrasound  
23. Blood tests: White blood cell count, lymphocyte count, neutrophil count, platelets, sodium, potas-
sium, creatinine, glucose, bilirubin, alanine aminotransferase, aspartate aminotransferase, gamma 
glutamyl transpeptidase, albumin, lactate dehydrogenase, ferritin, creatine kinase, fibrinogen, INR, 
partial thromboplastin time, D-dimer, NT-pro-BNP, troponin, C-reactive protein (CRP), procalci-
tonin, venous lactate  
24. Urine tests: pH, concentration, protein, glucose, red blood, white blood cell count  
25. CD4 lymphocyte count; HIV-viral load; AIDS status  
26. HIV-therapy: drug name and dose (only ongoing treatment); previous switch to other regimens for 
virological failure  
27. Questionnaires to address cognitive status: Cognitive Failure Questionnaire (CFQ), Mini-Mental 
State Examination, Clinical Dementia rating Scale, Montreal Cognitive Assessment (MOCA). 
28. Results of SARS-CoV-2 molecular test on amniotic fluid  
29. Weight, height/length, cranial circumference, BMI 
30. Type of transplant (hearth, lung, kidney, liver, pancreas); single-combined; year of transplantation 
31. Graft function: good, impaired, failure, rejection acute-chronic, recurrence of underlying disease, 
other 
32. Immunosuppressive regimen: drug name and dose 
33. According to Common Terminology Criteria for Adverse Events (CTCAE) 

 
 
 
 
 
13 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
34. At least one of three timepoints (month 3, month 6, month 12) is required to perform metagenomics 
analysis. 
35. At least one of the three timepoints (month 3, month 6, month 12) is required. 
 
 

 
 
 
 
 
14 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
4. LIST OF ABBREVIATIONS  
 
AOUI - Azienda Ospedaliera Integrata Universitaria 
AP-HP - Public Assistance Hospital of Paris 
BADL - Basic Activity of Daily Living 
BMI - Body Mass Index 
CBCI - Catholics Bishops Conference of India, Society for Medical Education , Bangalore, India;  
CERMEL - Lambaréné Medical Research Center 
CFQ - Cognitive Failure Questionnaire 
CFS - Clinical Frailty Scale 
CHARITÉ - University Medicine Berlin 
CI - Confidance Interval 
CINECA - Interuniversity Consortium 
CINES - National IT Center for Higher Education confidence interval Cis 
COVID-19 - COronaVIrus Disease 19  
CRF - Clinical research form 
CRO - Clinical Research Associate 
CRP C-reactive protein 
CTCAE - According to Common Terminology Criteria for Adverse Events 
DLCO - Diffusion Lung CO 
ECG - Electrocardiograph 
eCRF - Electronic clinical research form 
EQ-5D-5L - EuroQol five-dimension five-level 
FCRM - Congolese Foundation for Medical Research 
FDA – Food and Drug Administration 
FEV - Forced Expiratory Volume 
FRC – Functional Residual Capacity 
FVC - Forced Vital Capcity 
GHI - Good Clinical Practice,  
GMBH HMGU - Helmholtz Zentrum Muenchen German Research Center for Health and Environ-
ment 
GPAQ - Global Physical Activity, Questionnaire 
HADS - Anxiety and Depression Scale 
HCW - Health Care Workers   
HIV - Human Immunodeficiency Virus 
HMGU – Helmholtz Zentrum Munchen 
ICF - Informal Consent Form 
ICH - Conference on Harmonisation 
ICMJE -International Committee of Medical Journal Editors 
ICU - Intensive Care Unit 
IES-R - Impact of Event Scale – Revised 
INR - International Normalized Ratio 
INSERM - Institut National de la Santé et de la Recherche Médicale  
INSP - National Institute of Public Health 
IQR - Interquartile Range 
IRB – Institutional Review Board 
IRB/IEC – Institutional review board/inidipendent ethics 
ISGLOBAL-Barcelona Private Foundation Global Health Institut 
K10 - Kessler Psychological Distress Scale 
LCTF – Long term care facilities 

 
 
 
 
 
15 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
LEOSS - Lean European Open Survey on SARS-CoV-2 Infected Patients   
LIH-Luxembourg Institute of Health 
LMU   MUENCHEN - Ludwig-Maximilians-University Munich 
LTCF – Long Term Care 
LURM Laboratorio Universitario di Ricerca Medica 
mMRC - Modified Medical Research Council 
MOS SF - Medical Outcome Study Short Form (MOS SF)-36 Score,  
MRI - Magnetic Resonance Imaging 
MRI - Magnetic Resonance Imaging 
PCFS - Post-COVID-19 Functional Status Scale, 
PCR- polymerase chain reaction  
PENTA foundation – for the treatment and care of children with HIV and related diseases - ONLUS 
USTUTT - University of Stuttgart 
PI – Principal Investigator  
RAPH BB - Regional Office of Public Health with its seat in Banská Bystrica 
REG VEN-Veneto region  
RER-ASSR Emilia Romagna region 
RT-PCR - reverse transcriptase–polymerase chain reaction   
RV-Residual volume 
SAS-Andalusian Health Service 
SGRQ - Saint George Respiratory Questionnaire 
SoA - Schedule of Assessment  
SOFA-Sequential[Sepsis-Related]Organ Failure Assessment Score 
TDI - Transition Dyspnoea Index 
THSTI - Translational Health Science and Technology Institute, Faridabad, Haryana, India;  
TLC – Total Lung Capacity 
UANTWERPEN - University of Antwerpen 
UBA - Universidad de Buenos Aires   
UHC - Clinic of the University of Cologne 
UMCG - University Medical Center Groningen   
UNIBO - University of Bologna 
UNIOVI - University of Oviedo  
UNIVR– University of Verona  
WGS – Whole genome sequencing 
WES – Whole exome sequencing 
WP – Work Package 
 
 
 
 
 
 

 
 
 
 
 
16 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
5. BACKGROUND 
The ongoing COVID-19 pandemic has created a global public health emergency that is challenging 
societies, health care systems and national economies worldwide [1]. Since the beginning of the 
pandemic, our knowledge of transmission dynamics [2], clinical presentation [3], long-term sequelae 
[4], and risk factors for disease progression [5-7] has been increasing steadily. Evidences on the efficacy 
of treatment strategies coming from clinical trials [8] have been obtained and preventive measures such 
as social distancing and vaccination campaigns, have been put in place. Nonetheless, there is still an 
urgent need for a more standardized research activity, connecting multiple countries and settings and 
focusing on the different aspects of this pandemic. High-quality data and biological samples collection 
coming from large multicentric cohorts are urgently required and have not yet been implemented in an 
international level. Evidence-based knowledge is required to enable rapid and effective decision-
making, with particular attention to the transferability of the collected information. 
COVID-19 can result in prolonged illness, even in young adults and children without underlying chronic 
medical conditions. In a telephone survey conducted by the Centers for Disease Control and Prevention 
among a random sample of 292 adults (≥18 years) who had a positive outpatient test result for SARS-
CoV-2 by RT-PCR, 35% of 274 symptomatic respondents reported not having returned to their usual 
state of health 2 weeks or more after testing [9]. The burden of COVID-19 long-term sequelae and the 
exact underlying pathophysiology mechanisms remain unknown. Results from the follow-up of large 
cohorts are particularly needed to fully understand the characteristics and risk factors for SARS-CoV-2 
infection long-term consequences. 
From the point of view of the social science, this study will allow investigating two largely unexplored 
issues. On one hand, there is a need to dig into the determinants of adherence to preventative strategies, 
including non-pharmacological measures and vaccines. The lack of knowledge is particularly large 
among individuals who have experienced SARS-CoV-2 infection due to the possibility of re-infection 
and of infection transmission to others. On the other hand, there is a lack of knowledge on the use of 
health system resources SARS-CoV-2 infection implies across different settings and resources availa-
bility: length of hospital stay, frequency of follow-up visits, for example, have been projected at the start 
of the pandemic but the actual figures constitute unexplored information. 
The present study is part of ORCHESTRA project, a three-year international research project aimed at 
tackling the coronavirus pandemic. ORCHESTRA provides an innovative approach to learn from the 
pandemic SARS-CoV-2 crisis, derive recommendations to further management of COVID-19 and be 
prepared for the possible future pandemic waves. The ORCHESTRA project aims to deliver sound sci-
entific evidence for the prevention and treatment of the infections caused by SARS-CoV-2 assessing 

 
 
 
 
 
17 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
epidemiological, clinical, microbiological, and genotypic aspects of population, environment and socio-
economic features. The project builds upon existing, and new largescale population cohorts in Europe 
(France, Germany, Spain, Italy, Belgium, Romania, Netherlands, Luxemburg, and Slovakia) and non-
European countries (India, Perú, Ecuador, Colombia, Venezuela, Argentina, Brazil, Congo and Gabon) 
including SARS-CoV-2 infected and non-infected individuals of all ages and conditions. The primary 
aim of ORCHESTRA is the creation of a new pan-European cohort applying homogenous protocols for 
data collection, data sharing, sampling, and follow-up, which can rapidly advance the knowledge on the 
control and management of the COVID-19. ORCHESTRA will include SARS-CoV-2-negative individ-
uals and thereby enable a prospective follow-up and an analysis of vaccination response. The cohort 
will involve all patients with SARS-CoV-2 infections including fragile individuals (children, elderly, 
transplanted, oncological, HIV infected, and those with Parkinson disease or rheumatologic disease) if 
followed up in one of the study Partners center. Within ORCHESTRA project, the Work Package 2 
(WP2) aims to assess long-term sequelae in recovered COVID-19 individuals. The scope of the present 
protocol is to describe the procedures of the long-term follow-up of COVID-19 patients enrolled in 
ORCHESTRA cohorts. 
 
6. STUDY RATIONALE 
The present protocol, in accordance with the objectives of ORCHESTRA project - Work Package 2, 
aims at investigating the characteristics and determinants of COVID-19 long-term sequelae. This goal 
will be reached through the harmonization of follow-up strategies across the participating cohorts to 
allow a standardized collection of data on COVID-19 long-term sequelae. The result will be a platform 
including a set of data and biomaterials from large scale international cohorts, that will be uniformly 
recorded, prospectively tracked and analysed with the ultimate goal of providing evidence which will 
contribute to the optimization and improvement of the management of COVID-19 sequelae and to their 
prevention.  
The follow-up will be organized in multiple levels of tests according to the capability of each cohort and 
will include questionnaires to collect demographic, epidemiological and clinical data, physical exami-
nation, radiological exams and biological sampling. The long-term follow-up will also allow the assess-
ment of long-term immunological response to SARS-CoV-2 infection and its association to different 
treatment strategies, including monoclonal antibodies and vaccination.  
 

 
 
 
 
 
18 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
7.  STUDY DESIGN 
This is a multicenter, observational, prospective cohort study investigating COVID-19 sequelae in hos-
pitalised and non-hospitalised patients up to 18 months after the diagnosis of SARS-CoV-2 infection. 
The present study is conceived, at the coordinating center of UNIVR, as an extension of the study “Bi-
obanca associata a banca dati dei casi di COVID 19 gestiti presso l'Azienda Ospedaliera Universitaria 
di Verona” (COVID-19-VR), which was started in April 2020 at the Verona University Hospital to 
assess the long-term effects of COVID-19 on mental, respiratory, and functional status through the ad-
ministration of questionnaires (CESC 2577). Patients will be recruited in multiple European and non-
European countries, accounting for the participation of approximately 10000 individuals in the prospec-
tive follow-up data collection. 
Recording of clinical data, administration of questionnaires, collection of biological samples and 
imaging will take place at fixed time-points to allow a comprehensive follow-up of COVID-19 patients. 
The follow-up will include two levels of assessments: the first one is mandatory, the second one will be 
customized according to the feasibility of each cohort. A detailed overview of the schedule of the study 
visits and the clinical variables to be recorded is shown in Table 1. An ad hoc database will be provided 
to each COVID-19 cohort involved to allow homogeneous and standardised data collection.  
 
 
8. OBJECTIVES  
Main objectives of the study are: 
• 
To describe characteristics of COVID-19 sequelae, including type, rate, and length through clinical, 
laboratory, and radiological assessments 
• 
To investigate valuable, confounder-adjusted, associations between COVID-19 sequelae and 
COVID-19 severity, comorbidities, aetiology (SARS-CoV-2 variants), COVID treatment (including 
monoclonal antibodies), and trends in SARS-CoV-2 antibodies 
• 
To describe the rate, the aetiology (SARS-CoV-2 variants), severity, and clinical determinants of 
COVID-19 re-infections 
 
Furthermore, data retrieved from COVID-19 cohorts will address the following objectives: 
• 
To compare the time course of the immunological response of the population with sequelae with the 
immunological response of the population without sequelae. 
• 
To investigate immunological patterns related to specific long-term sequelae. 

 
 
 
 
 
19 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
• 
To investigate possible associations of SARS-CoV-2 variants with COVID-19 severity, time course 
of the immunological response, and long-term sequelae. 
• 
To describe the rate and severity of sequelae and immunological trends of COVID-19 in patients 
vaccinated against SARS-CoV-2. 
• 
To describe the time course of intestinal and pulmonary microbiome after SARS-CoV-2 infection. 
• 
To investigate possible associations of long-term sequelae with hypercoagulability 
• 
To describe the relationship between risk perception of reinfection and the adherence to preventative 
measures over time after the SARS-CoV-2 infection, including vaccine acceptance. 
• 
To describe the use of health care services among patients recovered from SARS-CoV-2. 
• 
To identify human and viral genetic markers indicative of disease severity using WGS or WES 
followed by functional analysis of the most promising variants.  
 
9. PATIENT COHORTS 
A comprehensive and longitudinal research cohort will be established. A broad collection of fine-gran-
ular clinical and epidemiological data connected to collection of biological samples will help answer the 
most relevant questions in the context of the SARS-CoV-2 pandemic. By implementing an international 
cohort of SARS-CoV-2 infected patients, considering all age groups, with any comorbidity and socio-
economic background as well as any stage of disease severity and setting (in- and outpatients), answers 
regarding the epidemiology and optimal management of SARS-CoV-2 will be provided at local, re-
gional, national and international level. Besides the comprehensive gathering of data and biological 
samples, representative sub-cohorts such as patients with specific comorbidities, adolescents or children 
can be analysed. Long-term follow-up data will further support both insight into pathophysiological 
mechanisms as well as improve individualized patient management. The information provided will help 
in adapting hospital management and public health strategies and thereby in reducing further socioeco-
nomic damage. In long term perspective, the nation-wide collaboration of several stakeholders, the use 
and extension of existing and newly established infrastructures might serve as a fast response for coming 
global and national health challenges. The constitution of a “perpetual” cohort including not only pop-
ulation-based representatives but also substantial number of HCWs (high risk population in case of en-
demic event with high incidence of admission for infected individuals and therefore high risk of hospital 
spreading) and fragile population including pregnant women, children, elderly, immunocompromised 
subjects (transplant recipients, patients with onco-haematological malignancies or HIV-infection) and 
individual with neurological impairment enables to precisely define the target of vaccination trials based 

 
 
 
 
 
20 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
on severity of diseases in those population but also on the burden of diseases (in terms of delayed care 
as visits, chemotherapy or radiological assessment) in populations in need of periodic clinical evalua-
tions. COVID-19 fragile population cohorts will be assessed in collaboration with ORCHESTRA – 
Work Package 4, focusing on the prevalence, clinical spectrum and therapeutic management of COVID-
19 disease in established cohorts of fragile patients. Specific features of fragile populations to follow-
up is reported in table 1. 
Patients will be recruited within European and non-European cohorts participating to ORCHESTRA 
consortium. Data will be extracted according to the study protocol and the case report form in compli-
ance with local regulatory rules.  
A description of the cohorts is presented in the following sections. 
 
9.1. University of Verona (UNIVR) – Italy 
UNIVR will participate to the recruitment of COVID-19 general population and COVID-19 
fragile populations: HIV positive, solid organ transplanted, oncological (both solid cancer and 
haematological neoplasms) patients and elderly people resident in LTCFs (General nursing 
home; residential home; specialized LTCFs; mixed LTCFs, other LTCFs; overall number of 
beds; ownership of the facility: public, for profit, not for profit). Estimated number of COVID-
19 patients since the beginning of the pandemic: 66000 
 
9.2. The French Covid-19 cohort 
INSERM - Institut National de la Santé et de la Recherche Médicale 
National French cohort which follows more than 3000 hospitalized patients across France The 
objectives of this cohort are to describe: 1) clinical features of illness; 2) treatment used and 
their outcome; 3) virus replication, excretion and evolution in multiple sites; 4) host responses 
including innate and acquired immune responses; 5) host genetic variants associated with dis-
ease progression/severity. 
 
9.3.  Lean European Open Survey on SARS-CoV-2 Infected Patients (LEOSS)  
University Hospital of Cologne (UHC), Cologne- Germany 
International cohort of more than 2,500 SARS-CoV-2 infected patients from 250 study sites all 
over the world, implemented by the University Hospital of Cologne (UHC), Cologne- Germany 
since March 2020.  
 

 
 
 
 
 
21 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
9.4. COVID-HOME study  
University Medical Center Groningen (UMCG), Groningen, Netherlands 
Prospective cohort study of non-hospitalised COVID-19 patients. The COVID-HOME study 
focuses on the community and household with early, systematic and prospective long-term 
follow-up The parameters collected in this study, such as viral load, antibody response, cyto-
kine changes and clinical and laboratory parameters of patient evolution will allow the identi-
fication of independent factors/parameters determining evolution of disease, insight in other 
transmission routes than respiratory ones (such as sexual and faecal-oral), and immune re-
sponse dynamics in non-hospitalised patients.  
 
9.5. Fondation Congolaise pour la Recherche Médicale (FCRM) - Republic of The Congo 
FCRM will participate to the enrolment of a cohort of recovered COVID-19 individuals to 
assess long-term sequelae. 
 
9.6. ZIKAction 
Led by Penta, the ZIKAction research consortium brings together 14 partners across South 
and Central America, the Caribbean and Europe with the complementary goals of 1) develop-
ing a multidisciplinary multinational ready to-act network capable of rapidly addressing any 
maternal and paediatric research need arising from (re-)emerging infectious diseases including 
Zika virus and 2) conducting an interdisciplinary programme of research studies within this 
network to address key knowledge gaps relating to ZIKV epidemiology, natural history and 
pathogenesis, with a particular emphasis on maternal and child health. ZIKAction is funded by 
the European Union’s Horizon 2020 Programme. ZIKAction works closely with two other Eu-
ropean Union-funded consortia, ZikaPLAN and ZIKAlliance, to establish a Latin American 
and Caribbean network. Within this research consortium, the SARS-oV-2 sub-study will ad-
dress issues related to COVID-19 infection in pregnant women and children.  
9.6. Servicio Andaluz de Salud  (SAS) - Hospital Universitario Virgen Macarena (HUVM) 
SAS is a public body providing healthcare services to the 8.4 million inhabitants in Andalusia, 
Spain. Hospital Universitario Virgen Macarena (HUVM) belongs to SAS and is a reference 
900-beds hospital located in Seville. The Department of Infectious Diseases, Microbiology and 
Preventive Medicine has a vast experience in different fields of infectious diseases; it includes 

 
 
 
 
 
22 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
a multidisciplinary research group formed by basic scientists, clinical microbiologists, phar-
macists and infectious diseases researchers performing basic and clinical research in this field. 
It hosts the coordination of the Andalusian Network for Clinical Research in Infectious Dis-
eases (ANCRAID), the Spanish Network for Research in Infectious Diseases (REIPI) and is 
part of the Institute of Biomedicine of Seville (IBiS). 
9.7. Universidade Federal de São Paulo (UNIFESP) - Escola Paulista de Medicina  
A total of 1.652 patients diagnosed with COVID-19 confirmed by RT-PCR were hospitalized 
for at least 48 hours at Hospital São Paulo (HSP, UNIFESP), a 740-bed teaching hospital, lo-
cated in the city of São Paulo, Brazil, between March 1st 2020 and April 30th 2021. Nearly 
44.9% (N=723) of these patients were admitted to intensive care units and 1.152 were hospital 
discharged. UNIFESP will contribute with a retrospective and prospective cohort of COVID-
19 patients admitted to HSP and followed-up every 3 months, providing demographic, epide-
miological, clinical, imaging and laboratory data. 
9.8. University of Bologna (UNIBO) 
UNIBO has been involved in different research activities to tackle the diffusion of the virus. 
The Department of Medical and Surgical Sciences of UNIBO has been actively working, since 
the end of February 2020,  to  manage  the  COVID-19  pandemic  both  at  clinical  and  
research  level  in  close collaboration with the Infectious Diseases Clinical Unit of the Univer-
sity Hospital S.Orsola-Malpighi and the Public Healthcare Provider governance of the Emilia-
Romagna Region. 
10. INCLUSION AND EXCLUSION CRITERIA 
Inclusion criteria: 
• 
Any age 
• 
Any comorbidity 
• 
Laboratory confirmed SARS-CoV-2 infection by PCR diagnosis from nasopharynx, orophar-
ynx, bronchoalveolar lavage, stool, or blood. Rapid tests are an acceptable alternative. 
• 
Person (or attorney or deputy who has been authorized to make the decision for patients who 
lack capacity) consent to participate. 
Exclusion criteria: 
• 
Refusal to participate to the study. 

 
 
 
 
 
23 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
 
11. RECRUITMENT STRATEGY 
Inpatients will be recruited by the study team at the treating hospital. Outpatients will be recruited by 
the study team in the emergency rooms, outpatient clinics, and through the coordination with regional 
primary care networks, who will offer the opportunity to enter the study to patients with active or pre-
vious COVID-19. This allows a broad recruitment of cases and the rapid inclusion of additional study 
centers in regional hotspots. 
Patients will be followed up for up to 18 months after the SARS-CoV-2 infection diagnosis. The recruit-
ment can take place at any time between the SARS-CoV-2 infection diagnosis and the end of the 18-
month follow-up, provided that the patient ensures at least one follow-up visit. Discharged inpatients as 
well as recovered outpatients will be invited to participate by a phone call (which will be repeated once 
in case of no reply). The screening/baseline data collection (including the informed consent process) can 
be carried out on the same day of a follow-up visit.  
Before the first patient is recruited at a location, the responsible investigator ensures that all legal and 
regulatory requirements are met. Patients can revoke their consent to participate in the study without 
restriction at any time and at their own request, without giving reasons and without any consequences 
for their future treatment. In case of loss to follow-up, the previous follow-up assessments will be in-
cluded in the data analyses. Patients will be recruited until January 2023 and followed-up until June 
2023, allowing a partial follow-up for participants whose SARS-COV-2 infection occurred after January 
2022. 
 
12. STUDY PROCEDURES 
The assessments of this study are designed to improve the COVID-19 patient care in the context of 
routine clinical care. Considering the lack of standardized follow-up pathways for COVID-19 patients, 
the selection of assessments has been based on available evidence and according to the definition of 
good clinical practice.  
The organization of follow-up visits will be up to each center, according to the facilities and logistics of 
outpatient monitoring. 
 
There will be a modular data capture according to the level of commitment of each cohort. 
• 
Level I: 
Assessments in level I are mandatory 
• 
Level II: Customized according to the feasibility of each cohort  

 
 
 
 
 
24 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
 
The modular schedule of assessments is presented in Table 1. Overall, the follow-up will last 18 months. 
Day 0 corresponds to the time of the first positive SARS-CoV-2 test. The screening/baseline data col-
lection (including the informed consent process, as detailed in section 14.3) can be carried out on the 
same day of a follow-up visit (as detailed in section 11). Procedures conducted as part of the participant’s 
routine clinical management at the time of SARS-CoV-2 active infection and obtained before the signa-
ture of the informed consent form may be recorded, provided that the procedures meet the protocol-
specified criteria.  
Subsequent follow-up visits will occur at the following time-points: 3, 6, 2, 18 month(s). During each 
time-point, epidemiological data collection (SARS-CoV-2 vaccination status), treatment data collection 
(comorbidity management), clinical assessments (relevant medical new events, COVID-19 symptom 
assessment, physical examination, vital signs, 6-minute walking test), and the administration of ques-
tionnaires on functional status, respiratory impairment, and mental health will be performed as part of 
level I (mandatory) assessments, as well as the assessment of the SARS-CoV-2 immunological status 
(SARS-CoV-2 antibodies). A SARS-CoV-2 molecular test will be repeated only if it turned out positive 
at the previous follow-up visit. 
Self-administered questionnaires on symptoms, as listed in Table 1, will be completed daily by the par-
ticipant to provide timely data on the symptom length. The participant will assign a score based on 
symptom severity, which will be recorded daily in the self-administered diary: absent (0), mild (1), 
moderate (2) and severe (3).  
As detailed in Table 1, level II (imaging, biochemistry, pulmonary function tests, electrocardiography) 
will be performed according to the capability of each cohort and will be based on the clinical evaluation, 
according to the participant’s healthcare status. Imaging and biochemistry tests will be reassessed only 
if outside the normal ranges at the previous follow-up visit or if clinically indicated. During the follow-
up visit at month 12, cardiac and lung ultrasounds, blood and urine tests will be offered as part of the 
participant’s care. 
COVID-19 biological samples (including blood, naso-pharingeal swabs, urine, and stool) will be col-
lected and stored in the biobank of each participating center (according to local ethic commission rec-
ommendations). The Azienda Ospedaliera Integrata Universitaria (AOUI) of Verona developed a 
COVID-19-VR registry for biobanking COVID-19 biological samples to allow further research and 
national/international collaborations. As per protocol approved by the hospital Institutional Review 
Board (IRB 2577CESC), biological samples of COVID-19 patients are stored in the LURM (Laborato-

 
 
 
 
 
25 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
rio Universitario di Ricerca Medica). Test to be performed in a centralised laboratory (genomic, tran-
scriptomic, cytokine, viral analyses, PBMCs and genetic and epigenetic see Table 2) will be sent, fol-
lowing international regulation, to the University of Antwerp, INSERM, University of Bologna or 
HMGU. A dedicated protocol will be developed for expeditions procedures. Nasal swabs collected 
prospectively for diagnosis of SARS COVID-2 will be collected in the local laboratory and sent out to 
University of Antwerpen and INSERM. Stool simple for microbiological analysis will be sent out to 
University of Bologna (see Table 2). Whole blood samples for epigenetic and genetic analysis will be 
sent out to UNIBO, INSERM and Helmholtz Zentrum Munchen (HMGU). In-depth human genetic 
analysis will be conducted using WGS or whole exome sequencing (WES) followed by functional anal-
yses of the most promising variants. Genome-wide methylation analyses of COVID-19-positive patients 
in addition to a small number of control patients will enable differentiation of inherited and acquired 
genomic regulatory features through COVID-19 infection, which result in severe disease or an efficient 
clearing of infection through immune responses. 
 
Table 2 – Samples collected at SARS-CoV-2 diagnosis and at follow up. 
 
Sample 
Aliquot Sample type 
Volume 
Storage solu-
tion 
Storage 
temp. (°C) 
Shipping 
temp. 
(°C) 
Task 
Comment 
Partner 
PBMC 
1 or 2 
samples 
PBMC 
 
0.5 ml 
FBS/DMSO 
20% 
-70°C or be-
low or liquid 
nitrogen 
Dry ice 
Characterisa-
tion of T-cell 
immune re-
sponse 
 
UANT-
WERPEN 
Blood 
1 
EDTA 
plasma, but 
heparin 
plasma or se-
rum can also 
be used, if 
EDTA 
plasma is ab-
solutely una-
vailable 
350 µL 
EDTA plasma 
has to be pro-
cessed accord-
ing to the proto-
col provided be-
fore freezing 
(preferably at      
-80°C directly) 
Short term at 
-20 °C, long 
term at        
-70°C or be-
low 
Dry ice 
Cytokinome 
analysis 
Please pro-
cess and 
freeze 
within 2 
hours. 
UANT-
WERPEN 

 
 
 
 
 
26 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
2 
Se-
rum/plasma 
200 µL 
NA 
-20°C, long 
term at -
70°C or be-
low 
Dry ice 
Auto-anti-
bodies 
against type 
I IFNs 
If available 
INSERM 
3 
Serum 
100 µL 
NA 
-20°C, long 
term at -70 ° 
or below 
Dry ice 
Antibodies 
detection 
 
INSERM; 
UANT-
WERPEN 
Whole 
blood 
1 
Extracted 
DNA or 
whole blood 
4 µg if 
DNA, 2 
ml if 
whole 
blood 
NA 
-20°C/  -
80°C 
Dry ice 
NGS of 
COVID-19 
cohorts 
DNA could 
be ex-
tracted lo-
cally or at 
HMGU. 
INSERM/UN-
IBO 
2 
DNA or 
whole blood 
750 ng in 
45 µL if 
extracted 
DNA; 
otherwise 
1 aliquot 
TE buffer or wa-
ter if extracted 
DNA 
-20°C 
Dry ice 
Illumina 
EPIC DNA 
methylation 
DNA could 
be ex-
tracted lo-
cally or at 
HMGU. 
HMGU 
NP 
swab 
1 
NP swab 
400 µL 
 
TRIzol; RNA 
later; 
DNA/RNA 
shield 
 
-70°C or be-
low 
Dry ice 
Characterisa-
tion of viral 
markers 
 
Respiratory 
microbiome 
dynamics 
 
UANT-
WERPEN- 
INSERM 
Stool 
sample 
or rec-
tal 
swab 
1 
Stool (faecal 
swab if stool 
is unavaila-
ble) 
1-2 g 
RNA later if 
possible, other-
wise frozen. 
+4°C 
(up to 24 h) 
 
long term at 
-70°C or be-
low 
Dry ice 
Intestinal 
microbiome 
profiling 
 
UNIBO 
 
 
13. STATISTICAL ANALYSIS 
Sample size calculation. The number of inclusions will depend on the progress of the SARS-CoV-2 
pandemic, which is unknown at this time. Therefore, the number of patients who will be included cannot 
be determined in advance. However, depending on the timing of the project and the included cohorts, 
the expected enrollment is 10000 subjects. 
We will carry out comprehensive descriptive analyses taking into account sociodemographic factors and 
clinical courses. The frequency distributions of the characteristics will be given in absolute and relative 
numbers, median plus interquartile range (IQR) or mean values plus 95% confidence interval (CIs). 
Associations with specific treatment strategies, disease severity patterns and laboratory results will be 
analysed using chi-square tests, t-tests or Mann-Whitney tests, depending on the data. To evaluate po-
tential risk factors, multivariate regression models will be carried out. Outcome time analyses using Cox 
proportional-hazards regression models with time-dependent covariates will be performed to examine 

 
 
 
 
 
27 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
factors associated with each endpoint (including death). In addition, we will use cumulative incidence 
functions, such as the Fine-Gray subdistribution hazard regression model, to account for competing 
events (i.e., relocation, discharge against medical advice, etc.). For missing values, a different strategy 
to understand the causes and the significance for the analysis will be developed and a graduated proce-
dure for dealing with censorship and imputations via linked regressions will be developed. The signifi-
cance level is defined with a p-value <0.05. All statistical analyses will be carried out with STATA, 
Python and/or R statistics software by trained staff (epidemiologists, statisticians) using the latest anal-
ysis methods. 
 
14. REGULATORY, ETHICAL, AND STUDY OVERSIGHT CONSIDERATIONS 
 
14.1 Regulatory and ethical aspects 
The study protocol is designed and will be conducted to ensure adherence to the principles and 
procedures of Good Clinical Practice and to comply with Italian laws, as described in the following 
documents and accepted, with their signature, by the study investigators: 1. ICH harmonized tripartite 
guidelines for good clinical practice 1996.2. Directive 91/507 / EEC, The Rules Governing Medicinal 
Products in the European Community. 3. Legislative Decree No. 211 of 24 June 2003.4. Legislative 
Decree n.200 November 6, 2007.5. D.M. 21 December 2007.6. AIFA Determination March 20, 2008. 
All essential clinical documents will be kept to demonstrate the validity of the study and the integrity of 
the data collected.  
All the document and protocol, protocol amendments, ICF, and other relevant documents must be sub-
mitted by the principal investigator to the ethical committee of the promotor center and reviewed and 
approved by the ethical comittae before the study is initiated. The protocol will also be submitted to the 
local ethics committees of the participating centers by the centers themselves. 
14.2 Financial disclosure 
Finance and insurance are addressed in the Investigator and/or CRO agreements, as applicable.  
14.3 Informed consent process 
Participant’s informed consent/assent must be obtained and documented in accordance with local regu-
lations, ICH-GCP requirements, and the ethical principles that have their origin in the principles of the 

 
 
 
 
 
28 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
Declaration of Helsinki. Prior to obtaining informed consent, information should be given in a language 
and at a level of complexity understandable to the participant in both oral and written form by the In-
vestigator (or designee). 
Each participant will have the opportunity to discuss the study and its alternatives with the Investigator. 
Prior to participation in the study, the ICF should be signed and personally dated by the participant, or 
his/her legal representative. 
The participant or his/her legal representative must receive a copy of the signed and dated Informed 
Consent form. As part of the consent process, each participant must consent to direct access to his/her 
medical records for study-related monitoring, auditing, IRB/IEC review, and regulatory inspection. If 
the ICF is amended during the study, the Investigator must follow all applicable regulatory requirements 
pertaining to the approval of the amended Informed Consent form by the IRB/IEC and use of the 
amended form.  
The participant may withdraw their consent to participate in the study at any time. 
 
14.4 Data protection 
The participant must be informed that his/her personal study-related data will be used by the promotor 
in accordance with local data protection law. The level of disclosure must also be explained to the par-
ticipant who will be required to give consent for their data to be used as described in the informed 
consent. 
Participants will be assigned a unique identifier by the Promotor. Any participant records or datasets 
that are transferred to the sponsor will contain the identifier only; participant names or any information 
which would make the participant identifiable will not be transferred. 
The electronic case report form (eCRF) will be provided using RedCap® software of the University 
Hospital of Verona. Investigators from participating study sites log into the system with username and 
a safe password including letters, numbers, and symbols. 
Investigators will be informed about handling of their personal data and their IP and location during the 
registration process. 
The data collection could be performed also retrospectively after a patient case has been completed 
(treatment is finished or patient's death). This process will be compliant with all applicable European 

 
 
 
 
 
29 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
and German federal data protection regulations, including EU directive 2016/679 and the German DS-
GVO. 
 
14.5 Data collection 
Patient data will be collected through a specific eCRF created for the study, which will not contain 
personal data suitable for identifying the patient. The clinical and outcome information collected in re-
lation to the study will be limited to the objectives of the study, taking care to reduce as much as possible 
the burden for the patient and for the enrolling clinical center. Only the enrolling clinical center will 
have access to the patient's identity and will be able to contact him if the coordinating center needs to 
have further information or for follow-up checks during or after the closure of the study being analyzed 
and reporting. For each patient, a special unique digital identification code (barcode) will be provided. 
The code will consist of a three-character part to identify the recruiting center and a second part consist-
ing of 5 digits to identify the enlisted person. The code will be associated with the patient by the clinical 
center, which will keep a copy of it in the patient file and in the medical record. Each patient enrolled 
will have a specific study file with all ethical and clinical documentation. The manual for compiling the 
eCRF will be provided in the Trial Master File. 
The data will be entered directly into the patients' eCRF and the related clinical and outcome information 
will be digitized in a specific database that will be developed using the RedCap® data capturing plat-
form. The system has been successfully used in the network to which the centers are part of SOLIDAR-
ITY. RedCap®, installed on protected servers of the Integrated Hospital of Verona, is periodically up-
dated and allows you to create accounts for users, control their access for data entry, limit user privileges 
(access only to the data of your center) and closure of the validity of the account at the end of the study. 
 
14.6 Data quality assurance 
All participant data relating to the study will be recorded on printed, or eCRF unless transmitted to the 
Sponsor or designee electronically (eg, laboratory data). The Investigator is responsible for verifying 
that data entries are accurate and correct by physically or electronically signing the eCRF or paper CRF. 
The Investigator must maintain accurate documentation (source data) that supports the information 
entered in the eCRF or paper CRF. The Investigator must permit study-related monitoring, audits, 
IRB/IEC review, and regulatory agency inspections and provide direct access to source data documents. 

 
 
 
 
 
30 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
The Promotor is responsible for the data management of this study including quality checking of the 
data. 
 
14.7 Source Document 
All source documents must be accurate, clear, unambiguous, permanent, and capable of being audited. 
They should be made using some permanent form of recording (typing, printing, optical disc). They 
should not be obscured by correction fluid or have temporary attachments (such as removable self-stick 
notes). Source documents are original records in which raw data are first recorded. These may include 
hospital/clinic/general practitioner records, charts, diaries, x-rays, laboratory results, pharmacy records, 
care records, ECG or other printouts, questionnaires, or video, for example. Source documents should 
be kept in a secure, limited access area. 
 
14.8 Study and site closure 
The study start date is the date the clinical study will be open for the recruitment of participants. The 
first step for recruitment is informed consent and will be the start date of the study. 
The study centers will be closed upon completion of the study. A center will be considered closed when 
all required study documents and materials have been collected and a study closing visit has been 
performed in the center. 
 
14.9 Publication policy 
The PI is responsible for the final publication of data. Authors must satisfy all of the following ICMJE 
authorship criteria: 1. Substantial contributions to conception or design of the work; or the acquisition, 
analysis, or interpretation of data for the work; AND 2. Drafting the work or revising it critically for 
important intellectual content; AND 3. Final approval of the version to be published; AND 4. Agreement 
to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity 
of any part of the work are appropriately investigated and resolved. Data collection, general supervision 
of the research group, or overseeing the conduct of the study alone does not justify authorship. Publica-
tions will be planned by the PI and the scientific and statistical committees. Publication of partial or 

 
 
 
 
 
31 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
local data must be approved by the PI. A detailed publication policy agreement will be developed by 
Partners at the beginning of the study. 
 
14.10  Amendments or any other modification 
Modifications to the protocol will be made as amendment.  No other modality is allowed.  Any 
modification will be recorded in the "Clinical Study Report" Archiving documents. The principal 
investigator is responsible for archiving and storing the essential documents during all the period of 
study according by current legislation and GCP. 
 
 
15. REFERENCES 
1. Ranney ML, Griffeth V, Jha AK. Critical Supply Shortages - The Need for Ventilators and 
Personal Protective Equipment during the Covid-19 Pandemic. N Engl J Med. 2020 Apr 
30;382(18):e41. doi: 10.1056/NEJMp2006141.  
2. Rothe C, Schunk M, Sothmann P, Bretzel G, Froeschl G, Wallrauch C et al. Transmission of 
2019-nCoV Infection from an Asymptomatic Contact in Germany. New Engl J Med. 
2020;382(10):970-1. doi:10.1056/nejmc2001468.  
3. Docherty AB, Harrison EM, Green CA, Hardwick HE, Pius R, Norman L et al. Features of 20 
133 UK patients in hospital with covid-19 using the ISARIC WHO Clinical Characterisation 
Protocol: prospective observational cohort study. Bmj. 2020:m1985. doi:10.1136/bmj.m1985. 
4. Willi S, Lüthold R, Hunt A, Hänggi NV, Sejdiu D, Scaff C, Bender N, Staub K, Schlagenhauf 
P. COVID-19 Sequelae in adults aged less than 50 years: A Systematic Review. Travel Med 
Infect Dis. 2021 Feb 22:101995. doi: 10.1016/j.tmaid.2021.101995. Epub ahead of print. PMID: 
33631340; PMCID: PMC7898978. 
5. Mesas AE, Cavero-Redondo I, Álvarez-Bueno C, Sarriá Cabrera MA, Maffei de Andrade S, 
Sequí-Dominguez I, Martínez-Vizcaíno V. Predictors of in-hospital COVID-19 mortality: A 
comprehensive systematic review and meta-analysis exploring differences by age, sex and 
health conditions. PLoS One. 2020 Nov 3;15(11):e0241742. doi: 10.1371/jour-
nal.pone.0241742.  

 
 
 
 
 
32 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
6. Zeng ZY, Feng SD, Chen GP, Wu JN. Predictive value of the neutrophil to lymphocyte ratio 
for disease deterioration and serious adverse outcomes in patients with COVID-19: a prospec-
tive cohort study. BMC Infect Dis. 2021 Jan 18;21(1):80. doi: 10.1186/s12879-021-05796-3. 
PMID: 33461497; PMCID: PMC7812552. 
7. Dorjee K, Kim H, Bonomo E, Dolma R. Prevalence and predictors of death and severe disease 
in patients hospitalized due to COVID-19: A comprehensive systematic review and meta-anal-
ysis of 77 studies and 38,000 patients. PLoS One. 2020 Dec 7;15(12):e0243191. doi: 
10.1371/journal.pone.0243191.  
8. WHO Solidarity Trial Consortium, Pan H, Peto R, Henao-Restrepo AM, Preziosi MP, Sathiya-
moorthy V, Abdool Karim Q, Alejandria MM, Hernández García C, Kieny MP, Malekzadeh R, 
Murthy S, Reddy KS, Roses Periago M, Abi Hanna P, Ader F, Al-Bader AM, Alhasawi A, 
Allum E, Alotaibi A, Alvarez-Moreno CA, Appadoo S, Asiri A, Aukrust P, Barratt-Due A, 
Bellani S, Branca M, Cappel-Porter HBC, Cerrato N, Chow TS, Como N, Eustace J, García PJ, 
Godbole S, Gotuzzo E, Griskevicius L, Hamra R, Hassan M, Hassany M, Hutton D, Irmansyah 
I, Jancoriene L, Kirwan J, Kumar S, Lennon P, Lopardo G, Lydon P, Magrini N, Maguire T, 
Manevska S, Manuel O, McGinty S, Medina MT, Mesa Rubio ML, Miranda-Montoya MC, Nel 
J, Nunes EP, Perola M, Portolés A, Rasmin MR, Raza A, Rees H, Reges PPS, Rogers CA, 
Salami K, Salvadori MI, Sinani N, Sterne JAC, Stevanovikj M, Tacconelli E, Tikkinen KAO, 
Trelle S, Zaid H, Røttingen JA, Swaminathan S. Repurposed Antiviral Drugs for Covid-19 - 
Interim WHO Solidarity Trial Results. N Engl J Med. 2021 Feb 11;384(6):497-511. doi: 
10.1056/NEJMoa2023184. Epub 2020 Dec 2. PMID: 33264556; PMCID: PMC7727327.Ten-
forde  MW, Kim  SS, Lindsell  CJ,  et al; IVY Network Investigators; CDC COVID-19 Re-
sponse Team; IVY Network Investigators.  Symptom duration and risk factors for delayed re-
turn to usual health among outpatients with COVID-19 in a multistate health care systems net-
work: United States, March-June 2020.   MMWR Morb Mortal Wkly Rep. 2020;69(30):993-998. 
9. Akesson, J., S. Ashworth-Hayes, R. Hahn, R. D. Metcalfe, and I. Rasooly (2020). Fatalism, 
beliefs, and behaviors during the COVID-19 pandemic. Technical report, National Bureau of 
Economic Research. 
10. Abel, M., Byker, T., and Carpenter, J. (2021) Socially optimal mistakes? Debiasing COVID-19 
mortality risk perceptions and prosocial behavior, Journal of Economic Behavior & Organiza-
tion, 183: 456-480. 
11. Fetzer, T., L. Hensel, J. Hermle, and C. Roth (2020). Coronavirus perceptions and economic 
anxiety. Review of Economics and Statistics, 1-36. 

 
 
 
 
 
33 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020  research and innovation programme under grant 
agreement No 101016167 
 
12. Campos-Mercade, P., A. Meier, F. Schneider, and E.Wengström (2020). Prosociality predicts 
health behaviors during the COVID-19 pandemic. University of Zurich, Department of Eco-
nomics, Working Paper (346). 
13. Bartsch, S.M., et al., The Potential Health Care Costs And Resource Use Associated With 
COVID-19 In The United States. Health Affairs, 2020. 39(6): p. 927-935. 
14. Tan-Torres Edejer, T., et al., Projected health-care resource needs for an effective response to 
COVID-19 in 73 low-income and middle-income countries: a modelling study. Lancet Glob 
Health, 2020. 8(11): p. e1372-e1379. 
15. Recommendations 01/2020 on measures that supplement transfer tools to ensure compliance 
with the EU level of protectionof personal data, European data protection board.