"Multicenter Observational Study to Assess the Incidence and Features of COVID-19 and the Response to COVID-19 Vaccination in Fragile Patients"
Conditions: SARS CoV 2 Infection, Vaccination; Infection, Breakthrough Infection
Sponsor: University of Bologna

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
 
COVID-19 AND FRAGILE PATIENTS 
Multicenter observational study to assess the incidence and features of COVID-19 and the 
response to COVID-19 vaccination in fragile patients 
 
 
 
STUDY PROTOCOL 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
 
History of Changes 
   
Version    
Date   
Created/Modified by   
1.0 
 15.04.2021 
 Maddalena Giannella (UNIBO), Natascia Caroccia 
(UNIBO)  
1.1 
 28.09.2021 
Maddalena Giannella (UNIBO), Natascia Caroccia 
(UNIBO), Federica Arbizzani (UNIBO)   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
PROTOCOL SIGNATURE PAGE 
 
Title:  “COVID-19 AND FRAGILE PATIENTS Multicenter observational study to assess the 
incidence and features of COVID-19 and the response to COVID-19 vaccination in fragile patients” 
 
Protocol Code: ORCHESTRA-WP4 
 
 
 
 
 
PI of the Coordinating Center: 
Maddalena Giannella 
Document: 
Protocol 
Version: 
1.0  
Release date: 
15-APR-2021 
Number of pages: 
42 
 
 
 
 
 
 
Maddalena Giannella 
signature 
 
date 
 
 
 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
 
PROTOCOL SIGNATURE PAGE 
 
Title:  “COVID-19 AND FRAGILE PATIENTS Multicenter observational study to assess the 
incidence and features of COVID-19 and the response to COVID-19 vaccination in fragile patients” 
 
Protocol Code: ORCHESTRA-WP4 
 
 
 
 
 
PI of the Coordinating Center: 
Maddalena Giannella 
Document: 
Protocol 
Version: 
1.1  
Release date: 
28-SEP-2021 
Number of pages: 
56 
 
 
 
 
 
 
Maddalena Giannella 
signature 
 
date 
 
 
 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
 
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. Objectives 
8. Study design 
9. Patient cohorts 
9.1. University of Bologna (UNIBO) – Italy 
9.2. AOU of Parma – Italy 
9.3. Servicio Andaluz de Salud (SAS) – Spain 
9.4. University of Verona (UNIVR) – Italy 
9.5 Regione del Veneto (REG VEN)– Italy 
9.6 Catholics Bishops Conference of India, Society for Medical Education (CBCI) – India 
9.7 Universidad de Buenos Aires (UBA) – Argentina 
9.8 PENTA Foundation ONLUS – Italy 
9.8.1 Italian cohorts 
9.8.2 EPPICC cohort plus Brazilian pregnant women cohort  
9.8.3 EPICO cohort 
9.8.4 Bahia cohort 
9.9 University College Dublin (UCD) – Ireland 
9.10 Luxembourg Institute of Health (LIH) – Luxembourg 
9.11 Translational Health Science and Technology Institute (THSTI) – India 
9.12 ZIKAlliance (UMCG & UKHD) 
9.13 Centre de Recherches Medicales de Lambaréné (CERMEL) – Gabon 
10. Inclusion and exclusion criteria 
11. Study procedures 
12. Study variables 
13. Statistical analysis 
14. Regulatory, ethical, and study oversight considerations 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
14.1. Regulatory and ethical aspects 
14.2. Financial disclosure 
14.3. Informed consent process 
14.4. Data protection 
14.5. Description of the data collected 
14.6 Data quality assurance 
14.7. Source documents 
14.8. Study and site closure 
14.9. Publication policy 
14.10 Amendments or any other modification 
15. Add-on substudies 
15.1 Pregnant Women (PW) Cohort for evaluation of risks of congenital malformations and 
other adverse pregnancy outcomes after Zika virus infection (part of ZIKAlliance) - SARS-
CoV-2 sub study. 
15.2 Children (CH) cohort for the evaluation of developmental and neurological 
abnormalities in infants born to mothers residing in areas with Zika virus transmission 
during pregnancy (part of ZIKALLIANCE) - SARS-CoV-2 sub study. 
15.3 Epidemiologic study of respiratory infections by novel coronavirus (SARS-CoV-2) in 
the paediatric population 
15.4 UNIVR: Rete Oncologica Veneta (ROV) 
15.5 UNIVR: LTCF-COVID19 
15.6 UNIBO: CONTRAST study 
16. References 
 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
3. PROTOCOL SUMMARY 
 
3.1 Synopsis 
Steering Committee 
Maddalena Giannella, UNIBO 
Umberto Maggiore, AOU Parma 
Jesus Rodrìguez Baño, SAS 
Elisa Cordero, SAS 
Evelina Tacconelli, UNIVR 
Anna Maria Azzini, UNIVR 
Elda Righi, UNIVR 
Mauro Krampera, UNIVR 
Marco Cipolli, UNIVR 
Michele Milella, UNIVR 
Maurizio Nordio, REG VEN  
Fiorella Gastaldon, REG VEN 
Umberto Cillo, REG VEN 
Gino Gerosa, REG VEN 
Mara Cananzi, REG VEN 
Salvatore Piano, REG VEN 
Elisa Benetti, REG VEN 
Patrizia Burra, REG VEN 
Monica Loy, REG VEN 
Lucrezia Furian, REG VEN  
Antonella D’Arminio Monforte, ICONA 
Adriana Tami, UMCG, Netherlands 
Thomas Jaenisch, UKHD, Germany 
Rejko Krüger, LIH, Luxembourg 
Carlo Giaquinto, PENTA  
Isadora C. Siqueira, PENTA 
Francesco Lapi, PENTA 
Jeannie Collins, PENTA 
Cecilia Liberati, PENTA 
Alfredo Tagarro, PENTA 
Marthe Le Prevost, PENTA 
Gariel Levy Hara, UBA, Argentina  
Patrick Mallon, UCD, Ireland 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
Shinjini Bhatnagar, THSTI, India  
Jyothi Miriam Idiculla, CBCI, India 
Vineeta Shobha, CBCI, India 
Agnadji Selidji, CERMEL, Gabon   
Study Rationale 
Clinical spectrum and outcome of COVID-19 
is extremely variable. Older age and multiple 
comorbidities have been associated with 
worse outcome. However, the vulnerability 
to the SARS-CoV-2 infection, the clinical 
spectrum of COVID-19 disease, and the 
long-term morbidity and mortality in several 
types of fragile populations have to be 
reported yet. 
In addition, patients with fragile conditions 
are normally excluded from pivotal studies 
assessing the safety and efficacy of 
vaccinations. It is therefore paramount, 
particularly in the current pandemic situation, 
to address the question of immunogenic 
response to vaccines and safety in settings 
at high risk for altered immune competence 
and/or safety profiles.  
Study objectives 
● To describe the 
characteristics of 
COVID-19 and its sequelae in several 
types of fragile populations. 
● To 
describe 
the 
rate, 
the etiology 
(SARS-CoV-2 variants), the severity of 
COVID-19, and the rate of re-infections 
in several types of fragile patients. 
● To evaluate the immunologic response 
to the COVID-19 vaccination in selected 
fragile populations. 
● To evaluate the safety of COVID-19 
vaccination 
in 
selected 
fragile 
populations. 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
● Identify human genetic markers and 
epigenetic characteristics in frail patients 
during post-vaccination monitoring and 
in case of breakthrough infections as 
well as to analyse the gut microbiome in 
such patients 
Study population 
Patients of all age and any comorbidity 
included 
in 
an 
ORCHESTRA 
fragile 
population cohort  
Inclusion criteria 
 
● 
Any age 
● 
Any comorbidity 
● 
Person (or attorney or deputy who has 
been authorized to make the decision for 
patients who lack capacity) consent to 
participate or appropriate local waiver of 
consent. 
Exclusion criteria 
Patients did not agree to participate. 
Study design 
Multicenter, 
multinational 
observational 
study aimed at building several types of 
fragile populations in which to assess the 
rate and the clinical spectrum of COVID-19, 
with particular interest in long-term follow-up, 
and at monitoring clinical and immunological 
response after SARS-CoV-2 vaccination in 
fragile subjects. 
For long-term follow-up of fragile patients 
with COVID-19, a predefined schedule of 
assessment is planned in alignment with 
Work Package (WP) 2. Patients already 
diagnosed with COVID-19 can be included if 
baseline data according to WP2 protocol 
have been collected, and if follow-up visits 
are possible. 
For assessing the clinical and immunological 
response to vaccination, patients will be 
prospectively evaluated at baseline (first and 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
second vaccination doses), and at multiple 
timepoints after vaccination with clinical and 
immunological assessments. 
Vaccines 
Approved COVID-19 vaccines 
Follow-up 
 
Patients will be followed-up for up to 18 
months after the SARS-CoV-2 infection 
diagnosis or up to 12 months after 
vaccination. 
ORCHESTRA Partners 
Università degli Studi di Verona (UNIVR); 
Alma Mater Studiorum – Università di 
Bologna (UNIBO); 
Azienda Ospedaliera-Universitaria di Parma 
(AOU Parma); 
Institut National de la Sante et de la 
Recherche Medicale (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 Barcelona (ISGLOBAL); 
Ludwig-Maximilians-Universitaet   
Muenchen (LMU   MUENCHEN); 
Universiteit Antwerpen (UANTWERPEN); 
Helmholtz Zentrum Muenchen Deutsches 
Forschungszentrumfuer 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); 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
Regionalny Urad Verejneho Zdravotnictva 
so siidlom v Banskejbytrici(RAPH BB); 
Charité – Universitaetsmedizin Berlin 
(CHARITÉ); 
Academisch Ziekenhuis Groningen 
(UMCG); 
Centre Informatique National de 
l’Enseignement Superieur (CINES); 
Universidad de Buenos Aires (UBA); 
Institutul National de Sanatate Publica  
(INSP); 
Regione del Veneto (REG VEN); 
Translational Health Science and 
Technology Institute (THSTI), Faridabad, 
Haryana, India; 
Catholics Bishops Conference of India, 
Society for Medical Education (CBCI), 
Bangalore, India. 
 
 
 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
 
3.2 
Schedule of assessments (SoA) 
 
Table 1. Schedule of assessments for long-term follow-up in COVID-19 patients.  
 
COVID-19 
(2 weeks1 
± 2 weeks) 
3 months1 
(± 1 month) 
6 months1 
(± 1 month) 
12 months1 
(± 2 months) 
18 months1 
(± 3 months) 
Screening/baseline 
 
 
 
 
 
Inclusion criteria1 
X 
 
 
 
 
Demographics2 
X 
 
 
 
 
Healthcare setting3 
X 
 
 
 
 
Length of hospital stay, days 
X 
 
 
 
 
ICU admission 
X 
 
 
 
 
Medical history4 
X 
 
 
 
 
Treatment 
 
 
 
 
 
Comorbidity management5 
X 
X 
X 
X 
X 
Anti-COVID therapy6 
X 
 
 
 
 
Antibiotic therapy 
X 
X 
X 
X 
    X 
Oxygen therapy7 
X 
  X* 
  X* 
  X* 
X* 
SARS-CoV28 and other 
respiratory pathogen 
vaccination9 
X 
X 
X 
X 
X 
Clinical assessment 
 
 
 
 
 
Relevant medical new events10 
X 
X 
X 
X 
X 
COVID-19 symptom11 onset 
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 carbon 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 
Risk perception and 
behaviour19, 20  
 
 
 
 
 
SARS-CoV-2 vaccination: 
acceptance/non-acceptance 
and reasons21 
 
X 
 
X 
    X 
Socio-economic indicators 
 
X 
 
X 
    X 
Use of resources (COVID-19 
unrelated) 
 
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 
 
 
 
 
 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
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 
Microbiological tests 
 
 
 
 
 
SARS-CoV-2 molecular test 
in nasopharyngeal swab or 
tracheal aspirate or 
bronchoalveolar lavage or 
saliva 
 
X 
 
X* 
 
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 
follow-up visits and 
antiretroviral therapy 
 
X 
 
X 
 
X 
 
X 
 
X 
Elderly 
 
 
 
 
 
Cognitive status27 
X 
X 
X 
X 
X 
Pregnant women/new born 
 
 
 
 
 
History of positive SARS-
CoV-2 molecular test on 
amniotic fluid or breast milk28 
X 
 
 
 
 
History of detection 
of microthrombotic disease on 
placenta tissue or umbelical 
cord tissue 
X 
 
 
 
 
Children 
 
 
 
 
 
History of positive SARS-
CoV-2 molecular test on 
amniotic fluid or breast milk29 
X 
 
 
 
 
Biometric paramethers30 
X 
X 
X 
X 
X 
Transplant 
 
 
 
 
 
Transplant general 
information31 
X 
 
 
 
 
Graft function32 
X 
X 
X 
X 
X 
Immunosuppressive regimen33 
X 
X 
X 
X 
X 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
Onco-haematology 
 
 
 
 
 
Assessment of adherence to 
oncologic 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 
events34 
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...), 
education (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 benefits, 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 
obstructive pulmonary disease, obstructive sleep apnoea, restrictive lung disease, pulmonary 
hypertension), kidney disease (chronic with/without dialysis), liver disease other than cancer 
(HBV/HCV/HDV chronic viral hepatitis, other chronic disease, cirrhosis), metabolic disease, 
immunosuppressive conditions (solid organ transplant recipient, auto-immune diseases), cancer 
(solid cancer, haematological malignancies, type of primitive cancer/haematological 
malignancies, presence of metastases, if ongoing chemotherapy), mental or neurological disorders 
(psychiatric illness, anxiety disorder, mood disorder, psychotic disorder, Alzheimer disease, 
dementia other than Alzheimer, 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  

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
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 
thrombosis, 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, dyspnoea, 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 
oxygen 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 
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), 
Kessler 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 
admission/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 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
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, 
potassium, 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), procalcitonin, 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) 
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. 
 
 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
 
Table 2. Schedule of post-vaccination assessments 
 
1st dose 
2nd 
dose2 
3 months 
(± 1 month)* 
6 months 
(± 2 months)* 
12 months 
(± 3 months)* 
Baseline information1 
 
 
 
 
 
Underlying fragile condition 
X1 
X1 
 
 
 
Healthcare setting (i.e. LTCF) 
X1 
X1 
 
 
 
Comorbidities 
X1 
X1 
 
 
 
Concomitant medical treatments 
X1 
X1 
 
 
 
Past and/or new COVID-19 disease3 
X1 
X1 
X 
X 
          X 
Clinical evaluation 
 
 
 
 
 
Relevant medical new events4 
X 
X 
X 
X 
X 
Clinical symptoms suggestive of 
COVID-19 
X 
X 
X 
X 
X 
Lab assessment 
 
 
 
 
 
Whole blood count 
X 
X 
 
 
 
CD4/CD8 count 
X 
X 
 
 
 
IgG level 
X 
X 
 
 
 
C3 level 
X 
X 
 
 
 
Immunology 
 
 
 
 
 
Immune - serology and type I IFNs 
autoantibodies 
X  
X  
X  
X  
X  
Immune - cytokine and chemokine 
X  
X7 
X7 
X7 
X7 
Immune - cellular 
X  
X7 
X7 
X7 
X7 
Microbiological tests-NP swabs  
   
   
   
   
   
Viral variant and metagenomics 
sequencing   
X5  
X5  
X6  
X6   
X6   
EDTA whole blood 
 
 
 
 
 
Genetic and epigenetic analysis 
X 
 
X 
X 
X 
Stool Sample (faeces or rectal swab) 
 
 
 
 
 
Metagenomic sequencing 
X 
 
X 
 
 
Questionnaires1 
 
 
 
 
 
Risk perception and behavior 
X1 
 
 
X 
X 
Socio-economic indicators 
X1 
 
 
X 
X 
Use of resources (COVID-19 
unrelated)  
X1 
 
 
X 
X 
Adverse Events dedicated 
questionnaire 
 
X 
X 
X 
X 
Immune response 
 
 
 
 
 
Quantitative Ab anti-S IgG5 
X 
X 
X 
X 
X 
Qualitative Ab anti-N IgM+IgG5 
X 
 
 
 
 
Anti-SARS-CoV-2 sero-neutralization 
X 
X 
X 
X 
X 
Cytokine / chemokines 
X 
X 
X 
X 
X 
Anti-SARS-CoV-2 cell immunity 
 
X 
 
X6 
X6 
 
Footnotes 
Modular data capture according to level of commitment (level I, level II). 
 
Level I 
Assessments in level I are core and should be prioritized 
Level II 
Customized according to the feasibility of each cohort 
 
*3, 6 and 12 months counted from the 1st dose... 
1For centers able to follow patients since the administration of the first and second vaccination doses the 
baseline information and the risk perception behavior should be collected at one of these time-points, in 
the other cases such information should be collected during the first post-vaccination follow-up.  

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
2The assessment at 2nd dose is mandatory in patients who will receive such dose within 8-12 weeks after 
fist dose (current AstraZeneca vaccination schedule).   
3If COVID-19 diagnosis, assessments reported in Table1 are required. 
4Relevant new medical events or worsening of previous conditions, including new malignancies (type of 
cancer, overall stage, Kaposi skin-visceral, Lymphoma localization-type, Leukemia lymphoid-myeloid 
acute-chronic), deep venous thrombosis, pulmonary embolism, infections (including a new SARS-CoV-2-
infection during follow-up) 
5At least one time-point at 1st and 2nd dose is required. 
6 At least one of the three timepoints (month 3, month 6, month 12) is required. 
7 At least one of the four timepoints (2nd dose, month 3, month 6, month 12) is required. 
 
  
 
 
 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
4. LIST OF ABBREVIATIONS 
 
AOU – Azienda Ospedaliera-Universitaria 
AP-HP - Public Assistance Hospital of Paris 
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 
CINECA - Interuniversity Consortium 
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 
EPPICC - European Pregnancy and Paediatric Infections Cohort Collaboration 
FEV - Forced Expiratory Volume 
FRC – Functional Residual Capacity 
FVC - Forced Vital Capcity 
GCP - Good Clinical Practice 
GMBH German Research Center for Health and Environment 
GPAQ - Global Physical Activity, Questionnaire 
HADS - Anxiety and Depression Scale 
HIV - Human Immunodeficiency Virus 
HMGU - Helmholtz Zentrum Muenchen 
ICF - Informal Consent Form 
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 

 
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INSP - National Institute of Public Health 
IQR - Interquartile Range 
IRB – Institutional Review Board 
IRB/IEC – Institutional review board/inidipendent ethics 
IRCCS – Italian scientific health institutions 
ISGLOBAL-Barcelona Private Foundation Global Health Institut 
K10 - Kessler Psychological Distress Scale 
LCTF – Long term care facilities 
LIH - Luxembourg Institute of Health 
LMU MUENCHEN - Ludwig-Maximilians-University Munich 
LTCF – Long Term Care Facilities 
mMRC - Modified Medical Research Council 
MOS SF - Medical Outcome Study Short Form (MOS SF)-36 Score 
MRI - Magnetic Resonance Imaging 
PCFS - Post-COVID-19 Functional Status Scale 
PCR- polymerase chain reaction 
PD – Parkinson Disease 
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; 
UCD - University College Dublin, Ireland 
UHC - Clinic of the University of Cologne 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
UKHD - Universitatsklinikum Heidelberg, Gemany 
UMCG - University Medical Center Groningen 
UNIBO - University of Bologna 
UNIVR– University of Verona 
WGS – Whole genome sequencing 
WES – Whole exome sequencing 
WP – Work Package 
 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
5. BACKGROUND 
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 scientific evidence for the prevention and treatment of the infections caused by SARS-CoV-2 
assessing 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 individuals and thereby enable a prospective 
follow-up and an analysis of vaccination response. The cohort will involve four different populations: 
general population, COVID-19 patients, fragile individuals (children, elderly, transplanted, 
oncological, HIV infected, and those with Parkinson disease), and health-care workers. Each of 
these “perpetual” cohorts can answer different research questions and vaccine strategies.  
Within the ORCHESTRA project, the Work Package 4 (WP4) will focus on the cohort of fragile 
patients including children, pregnant women and new-borns, elderly (≥ 65 year age), transplant 
recipients, cancer patients, patients with HIV infection, and patients with Parkinson Disease. 
 
6. STUDY RATIONALE 
Since the beginning of COVID-19 pandemic, several special settings have been identified regarding 
the susceptibility to SARS-CoV-2 infection, the associated clinical spectrum and outcome. They 
include pregnant women, pediatric patients, elderly in particular those whole live in long-term care 
facilities (LTCFs), and immunocompromised hosts including solid organ transplant recipients (SOT), 
hematopoietic stem cell transplant (HSCT) recipients and patients with cancer.  
Among pregnant women and children, asymptomatic or mild diseases have been frequently 
reported, prompting controversial concerns about their role in the infection transmission in 
community and hospital settings (1, 2). On the other hand, a high impact of COVID-19 on morbidity 
and mortality has been described in elderly and immunocompromised hosts (3, 4). Thus, 
optimization of prevention strategies, screening practices and therapeutic management is strongly 
advocated in fragile patients (5, 6). Indeed, these settings have been established as priority groups 
for vaccines. However, safety and efficacy of vaccination in these populations should be careful 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
assessed. Thus, preliminary epidemiological data are strongly needed to design further intervention 
trials and health policies.  
 
Besides, an increasing body of evidence suggests that the gut microbiota plays a role in determining 
the severity of COVID-19, possibly through the modulation of immune responses (8, 9). Furthermore, 
dysbiosis of the gut microbiota could contribute to the persistence of symptoms, even after the 
resolution of the disease (10). For the same reasons, the microbiota could be involved in the onset 
of adverse reactions induced by vaccination, especially in fragile populations, as recently discussed 
(11). Defining the impact of the microbiota on immunity to vaccination and therefore on its 
effectiveness is currently considered a priority in various clinical settings (12).  
 
Moreover, early observations show that vaccines do not induce an immune response conferring 
protection to many fragile patients, resulting in severe Covid-19 cases. It is important to understand 
what cellular networks and molecular pathways are switched on/off by the administration of vaccines, 
and to identify the biological patterns that characterize responders and non-responders. DNA 
methylation and gene expression analyses may inform on the genomic patterns involved in response 
to vaccines and in the differences between responders and non-responders. Indeed, DNA 
sequencing may reveal that the perturbation detected at the regulatory levels may be influenced by 
alterations in the genome of the divergent subjects.  
   
With this premise, we deem that a WP dedicated to fragile patients in the ORCHESTRA project is 
necessary to inform about the peculiarities of the fragile cohort as a whole, and of each subgroup as 
well, providing clinical and biological information useful to design targeted prevention and therapeutic 
strategies.  
 
7.  OBJECTIVES 
The primary objective of the ORCHESTRA fragile cohorts is to provide an extensive and harmonized 
collection of epidemiological and clinical data, and biological samples, to assess several features of 
COVID-19 in each cohort of fragile patients. These include: 
● To assess the prevalence of SARS-CoV2 infection and COVID-19 disease in fragile 
patients; 
● To describe the clinical spectrum of COVID-19 disease in different types of fragile patients; 
● To analyze epidemiological, clinical and biological predictors of SARS-CoV-2 infection and 
prognosis in several types of fragile patients; 
● To investigate the therapeutic management of COVID-19 and /or of the underlying 
condition and its impact on the outcome in fragile patients; 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
● To describe safety and efficacy of COVID-19 vaccination in selected cohorts of fragile 
patients; 
● To describe the relationship between risk perception and adherence to preventive measures 
over time, including vaccine acceptance; 
● To analyze the impact of COVID-19 pandemic on the use of health care services; 
● To analyze the socio-economic determinants of infection and disease severity, and the 
socio-economic impact of the pandemic on cohort specific socio-economic indicators; 
● To investigate the psychological consequences of lockdown in fragile patients. 
● Identify human genetic markers and epigenetic characteristics in frail patients during post-
vaccination monitoring and in the onset of breakthrough infections as well as to analyse the 
gut microbiome in such patients 
 
8. STUDY DESIGN  
This is an observational retrospective and prospective longitudinal cohort study of several types of 
fragile patients (see details below). All ages and comorbidities will be included. All the cohorts will 
be mixed, including patients with and without past or new diagnosis of SARS-CoV2 infection 
(COVID-19 positive and COVID-19 negative groups) during the study period.  
Study will start soon after the approval by the Ethics Committee and will end in June 2023. For the 
baseline characteristics and past COVID-19 diagnosis, data will be recorded from February 2020. 
For the prospective part, patients will be recruited until June 2022. Minimum follow-up duration will 
be of: i) 12 months from the first vaccination dose for fragile patients who will receive anti-COVID-
19 vaccination; ii) 18 months from diagnosis of SARS-CoV-2 infection for COVID-19 positive group, 
allowing a partial follow-up for participants whose SARS-COV-2 infection occur after January 2022; 
and iii) up to the end of recruitment for the other fragile patients (see Figure 1).  
 
9. PATIENT COHORTS 
The ORCHESTRA fragile population consists of 29 cohorts of 10 different fragile populations 
including pregnant women/new-born, children, patients with HIV infection, patients with autoimmune 
disease, solid organ transplant recipients, patients with oncological and hematological diseases, 
patients with cystic fibrosis, patients with Parkinson Disease and rheumatological diseases from from 
14 countries (5 European and 9 non-European countries), with approximately 19784 subjects. 
Among these, 10.300 individuals are already registered in local databases and on active follow-up 
in the respective centers. A description of the cohorts is presented in the following sections. 
 
9.1. University of Bologna (UNIBO) – Italy 
UNIBO will participate in the recruitment of the following COVID-19 and non-COVID-19 
fragile populations: HIV positive, solid organ transplant recipients (liver, kidney, heart, lung), 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
hematopoietic stem cell transplant recipients, patients with cancer and autoimmune disease 
patients. 
 
9.2. Azienda-Ospedaliera Universitaria di Parma (AOU di Parma) – Italy 
AOU Parma will participate in the recruitment of COVID19 and non-COVID-19 solid organ 
transplant (kidney).  
 
9.3. Servicio Andaluz de Salud (SAS) – Spain 
SAS will participate in the recruitment of the following COVID-19 and non-COVID-19 fragile 
populations: HIV positive subjects, solid organ transplant recipients, oncological and hemato- 
oncological patients, hemodialysis patients and rheumatological patients. 
 
9.4. University of Verona (UNIVR) – Italy 
UNIVR will participate in the recruitment of the following COVID-19 and non-COVID-19 fragile 
populations: HIV positive, solid organ transplant recipients, patients with cystic fibrosis, 
oncological and hematological patients. 
 
9.5. Regione Veneto (REG VEN)– Italy 
The Transplant Centers of Treviso, Vicenza and Padova will participate in the recruitment of 
COVID-19 and non-COVID-19 solid organ transplant recipients (kidney, liver, heart, lung).  
  
9.6. Catholics Bishops Conference of India, Society for Medical Education (CBCI) – India 
CBCI will participate in the recruitment of the following COVID-19 and non-COVID-19 fragile 
populations: HIV positive subjects and patients with auto-immune and rheumatological 
disorders. 
 
9.7. Universidad de Buenos Aires (UBA) – Argentina 
UBA will participate in the recruitment of COVID-19 and non-COVID-19 HIV positive subjects. 
The cohort, of approximately 100 patients, will be coordinated by the School of Medicine, 
involving sub-investigators that are already following HIV patients (some of them who already 
acquired COVID-19) since the beginning of the pandemic in March. Patients will be 
prospectively enrolled once the study protocol is approved by the Ethics Committee. 
 
 
 
 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
9.8. PENTA Foundation ONLUS – Italy 
 
9.8.1 Italian cohorts 
PENTA will participate with the following Italian children cohorts: a large cohort of children 
followed by primary care paediatricians involved in the “Pedianet” network (COVID-19 and 
non-COVID-19 subjects) and children attending the clinic for follow-up visits after intra-family 
COVID-19 infection. Pedianet is an organised network of more than 400 family paediatricians 
in Italy aimed to collect anonymous data from the electronic system they use in their daily 
activities to be used for clinical and epidemiological research. 
 
9.8.2 EPPICC cohort 
European Pregnancy and Paediatric Infections Cohort Collaboration (EPPICC) is a multi-
country cohort of children and adolescents living with HIV across Europe and attending 
routine HIV care.  The initial purpose of the cohort was to address key research questions 
related to long term outcomes of HIV infection and treatment in the paediatric population. The 
sample size is ~10,000 children/adolescents EVER in follow up included in the database, of 
which approximately 3000 were in active follow up during 2020. 
 
Pregnant women cohort coordinated by Instituto Gonçalo Moniz- Fiocruz- Bahia, hospital 
partner: Maternidade de Referência Prof José Maria de Magalhães Neto (Maternidade RPJ). 
In April 2020, a pregnancy surveillance study that involves pregnant women admitted to 
Maternidade RPJ with suspected COVID-19 was started, with the aim to increase 
understanding of the risks and impact of COVID-19 during pregnancy and birth outcomes. 
The study population will be composed of women with confirmed COVID-19 as well as those 
with COVID-19-compatible symptoms who test negative for SARS-CoV-2 (control group). At 
present, Maternidade RPJ has recorded over 300 pregnancies with confirmed or suspected 
COVID-19 since March 2020 
 
9.8.3 EPICO cohort 
EPICO is a Spanish cohort of currently 1035 children attended in hospitals with COVID-19 
with respiratory symptoms or suspected COVID-19 with infection by SARS-CoV-2 confirmed 
by PCR or serology, from March 2020. 
 
9.8.4 Bahia cohort 
The Instituto Gonçalo Moniz- Fiocruz- Bahia, partnered with the Maternidade de Referência 
Prof José Maria de Magalhães Neto (Maternidade RPJMMN), will also participate in the 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
ORCHESTRA project with a pregnancy surveillance study that involves pregnant women 
admitted to Maternidade RPJMMN with suspected COVID-19. The study population will be 
composed of women with confirmed COVID-19 as well as those with COVID-19-compatible 
symptoms who test negative for SARS-CoV-2 (control group).  
At present, Maternidade RPJMMN has recorded over 300 pregnancies with confirmed or 
suspected COVID-19 since March 2020. 
 
9.9. University College Dublin (UCD) – Ireland 
The All Ireland Infectious Diseases (AIID) Cohort study was initially established at a single 
hospital site (Mater Misericordiae University Hospital) in 2013. It has since expanded to five 
hospital sites across Ireland. The aim of the cohort is to create a data-rich prospective dataset 
derived from patients who present with suspected Infectious Diseases focusing on HIV and 
Hepatitis and now includes COVID-19. UCD will participate in this protocol recruiting HIV 
positive subjects including COVID-19 and non-COVID-19 patients. 
 
9.10. 
Luxembourg Institute of Health (LIH) – Luxembourg 
LIH will participate in the recruitment of COVID-19 and non-COVID-19 subjects with 
Parkinson disease (PD). The Luxembourg Parkinson’s Study is a nation-wide and 
comprehensive clinical, molecular and device-based cohort comprising more than 1,600 
participants from Luxembourg and the Greater Region. The cohort includes patients with 
typical PD and atypical parkinsonism, irrespective of their disease stage, age, comorbidities, 
or linguistic background (followed-up yearly) and matching control subjects (followed-up 
every 4 years). 
 
9.11. 
Translational Health Science and Technology Institute (THSTI) – India 
THSTI will participate in the recruitment of a large cohort of COVID-19 and non-COVID-19 
pregnant women participating in the GARBH-Ini (interdisciplinary Group for Advanced 
Research on BirtH outcomes - DBT India Initiative) program. GARBH-Ini Cohort was initiated 
in May 2015 at the Civil hospital in Gurugram (GCH), Haryana, India. Women are enrolled 
within 20 weeks of gestation and are followed until delivery and once at postpartum. The 
enrolled women are followed up at 4-5 time-points across 3 trimesters of pregnancy to 
document extensive clinical & epidemiological information, varied maternal and neonatal 
biospecimens and for serial ultrasonographic examination. Accounting for a cohort design to 
detect epidemiological risk factors & nested case-control design for identifying biomarkers, 
an apriori sample size of 8000 was estimated.  More than 8534 pregnant mothers have been 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
enrolled till February 10, 2020 since May 2015 with documentation of 7260 pregnancy 
outcomes. 
 
9.12. 
ZIKAlliance (ORCHESTRA partners: UMCG & UKHD) 
ZIKAlliance is a multi-country network coordinated by the ORCHESTRA Partners 
Academisch Ziekenhuis Groningen (UMCG), Netherlands, and Universitatsklinikum 
Heidelberg (UKHD), Germany. The initial purpose of this cohort was the evaluation of risks 
of congenital malformations and other adverse pregnancy outcomes after Zika virus infection 
in pregnant women and their children. Further, ZIKAlliance has integrated the investigation 
of SARS-CoV-2 infection and COVID-19 disease into the on-going Zika pregnant women 
cohort study. ZIKAlliance will participate in this Project with a SARS-CoV-2 substudy 
including the cohorts of pregnant women and their newborns from Venezuela, Colombia, 
Ecuador and Peru. Mother and children are followed from birth up to 2 years of age. 
 
9.13. 
Centre de Recherches Medicales de Lambaréné (CERMEL) – Gabon 
CERMEL will participate in the recruitment of children including COVID-19 and non-COVID-
19 subjects. 
 
 
 
 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
 
10. INCLUSION AND EXCLUSION CRITERIA 
Inclusion criteria are: 
● 
Any age 
● 
Person (or attorney or deputy who has been authorized to make the decision for patients who 
lack capacity) consent to participate, or appropriate local waiver of consent. 
 
The only exclusion criteria will be the lack of consent to participate to the study. 
 
11. STUDY PROCEDURES  
 
In Figure 1, a study work flow is shown. 
 
Figure 1: study work flow 
 
 
All included patients will be evaluated during the scheduled visits for the underlying condition. During 
such visits, the following actions are required: 
- 
Distribution of a questionnaire to capture the impact of COVID-19 pandemic on the socio-
economic indicators, on the access to health care services, and to investigate risk perception 
and behavior towards preventing infection and COVID-19 disease, including vaccine 
acceptance. This questionnaire should be administered at two different time-points with an 
interval of at least 6 months to capture changes in behavior over the study period (Annex 1a 
or Annex 1b in case of children cohorts).  

 
30 
ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
- 
Distribution of psychological scale questionnaires to investigate the impact of lockdown 
measures on mental health (Annex 2).  
- 
Screening for past and/or current COVID-19 disease by medical interview, serology 
determination and nasopharyngeal swab or saliva specimens’ collection. 
- 
Collection, processing and storage of plasma for cytokine and chemokine analysis. 
- 
Collection, processing and storage of serum for the subsequent sero-neutralization and type 
I IFNs autoantibodies detection. 
- 
Collection, processing and storage of PBMCs for the subsequent anti-COVID-19 cellular 
immunity analysis. 
- 
Collection, processing and storage of whole blood for genetic and epigenetic analysis. 
- 
Collection, processing and storage of nasopharyngeal samples for SARS-CoV-2 detection 
and variant identification if clinically relevant. 
- 
Collection, processing and storage of stool samples for metagenomic sequencing.  
- 
In immunocompromised fragile cohorts, laboratory tests to assess the baseline “net state of 
immunosuppression” 
including 
whole 
blood 
count, 
lymphocyte 
differentiation, 
immunoglobulin levels, and complement (C3) levels should be done at the baseline (first 
clinical evaluation).  
 
For patients with a past and/or new diagnosis of SARS-CoV2 infection/COVID-19 disease a pre-
established long-term follow-up including multiple diagnostic levels in order to adapt to all the 
participating cohorts is requested. A detailed overview of the schedule of the study visits and the 
clinical parameters to be recorded is shown in Table 1. Patients who have already had COVID, and 
new patients being diagnosed with COVID, who can be followed up to at least 12 months, and for 
whom data can be collected according to Table 1 should be included. In specific fragile cohorts, 
such as children cohorts, virtual assessments are allowed.  
 
For patients who have been or will be vaccinated against COVID-19 during the study period, a pre-
established monitoring of safety and efficacy in terms of immunological response will be performed.  
A detailed overview of the schedule of the clinical and laboratory parameters to be recorded is shown 
in Table 2. Patients who have already been vaccinated, or will be vaccinated, who can be followed 
for 12 months, and for whom data can be collected according to Table 2 should be included. The 
monitoring should start from the days of first and second dose administration when possible (i.e. 
vaccination administered in the same center where the patient is followed for the fragile condition), 
alternatively patients will be monitored at 3±1, 6±2 and 12±3 months after the first vaccination dose.  
- 
Vaccination safety will be assessed by a dedicated questionnaire (Annex 3) distributed to 
patients at baseline or during the first monitoring visit.  

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
- 
Vaccination efficacy will be established analyzing the immune response at the predefined 
time-points. The humoral immune response to vaccination will be assessed by a serological 
assay able to detect anti-Spike and anti-Nucleocapsid antibodies as well as sero-
neutralization assays in sera samples. The latter will be useful to exclude possible natural 
infection at the baseline (1st/2nd dose, or first post-vaccination monitoring visit). The cytokine 
and cell-mediated immune response to vaccination will be assessed in a subgroup of patients 
by centralizing samples to a referral laboratory, according to local ethic rules and budget. 
The number of SARS-CoV-2 paucisymptomatic or symptomatic infection despite vaccination 
will be also assessed by SARS-CoV-2 detection in respiratory/saliva sample. 
 
During the post-vaccination monitoring and in the onset of a breakthrough infection after one or two 
doses of the anti-SARS-CoV-2 vaccine, several analyses (genomic, transcriptomic, cytokine, viral, 
genetic and epigenetic) will be performed in a local or centralised laboratory according to the type of 
testing realized (see Table 3):  
- 
PBMCs for characterisation of T-cell immune response will be sent to University of Antwerpen 
- 
Plasma for cytokinome analysis will be sent to University of Antwerpen 
- 
Serum for autoantibodies against type I IFNs detection will be sent to INSERM or University 
of Antwerpen 
- 
Whole blood samples for epigenetic and genetic analysis will be sent to UNIBO, INSERM 
and HMGU. In-depth human genetic analysis will be conducted using WGS or whole exome 
sequencing (WES) followed by functional analyses of the most promising variants.  
- 
Nasal swabs for characterization of viral markers and respiratory microbiome dynamics wiill 
be sent to University of Antwerpen or INSERM.  
- 
Stool samples for intestinal microbiome profiling will be sent to University of Bologna.  
 
 
Detailed instructions on sample collection, processing, storage, shipment and destination sites are 
provided in the WP6 protocol (WP6, D 6.1) attached as Annex 4. 
 
 
 
 
 
 
 
 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
Table 3 – Overview of samples collection, processing, storage and destination sites. 
Sample 
Aliquot Sample type 
Volume 
Storage 
solution 
Storage 
temp. (°C) 
Shipping 
temp. 
(°C) 
Task 
Comment 
Partner 
PBMC 
1 or 2 
samples 
PBMC 
 
0.5 ml 
FBS/DMSO 
20% 
-70°C or 
below or 
liquid 
nitrogen 
Dry ice 
Characterisat
ion of T-cell 
immune 
response 
 
UANTW
ERPEN 
Blood 
1 
EDTA 
plasma, but 
heparin 
plasma or 
serum can 
also be used, 
if EDTA 
plasma is 
absolutely 
unavailable 
350 µL 
EDTA plasma 
has to be 
processed 
according to the 
protocol 
provided before 
freezing 
(preferably at     
-80°C directly) 
Short term at 
-20 °C, long 
term at        
-70°C or 
below 
Dry ice 
Cytokinome 
analysis 
Please 
process 
and freeze 
within 2 
hours. 
UANTWERP
EN 
2 
Serum/plasm
a 
200 µL 
NA 
-20°C, long 
term at -
70°C or 
below 
Dry ice 
Auto-
antibodies 
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; 
UANTWERP
EN 
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 
extracted 
locally or 
at HMGU. 
INSERM/UNI
BO 
2 
DNA or 
whole blood 
750 ng in 
45 µL if 
extracted 
DNA; 
otherwise 
1 aliquot 
TE buffer or 
water if 
extracted DNA 
-20°C 
Dry ice 
Illumina 
EPIC DNA 
methylation 
DNA could 
be 
extracted 
locally or 
at HMGU. 
HMGU 
NP 
swab 
1 
NP swab 
400 µL 
 
TRIzol; RNA 
later; 
DNA/RNA 
shield 
 
-70°C or 
below 
Dry ice 
Characterizat
ion of viral 
markers 
 
Respiratory 
microbiome 
dynamics 
 
UANTWERP
EN- INSERM 
Stool 
sample 
or 
rectal 
swab 
1 
Stool (faecal 
swab if stool 
is 
unavailable) 
1-2 g 
RNA later if 
possible, 
otherwise 
frozen. 
+4°C 
(up to 24 h) 
 
long term at 
-70°C or 
below 
Dry ice 
Intestinal 
microbiome 
profiling 
 
UNIBO 
 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
 
12. STUDY VARIABLES 
Endpoint variables: 
- 
Infection with SARS-CoV2 detected by RT-PCR on respiratory/saliva specimens; 
- 
Reinfection and/or clinical relapse defined according to recent proposed definitions (7);  
- 
Clinical spectrum of COVID-19 according to WHO mild, moderate, severe, and critical criteria 
(https://www.who.int/publications/i/item/WHO-2019-nCoV-clinical-2021-1); 
- 
Duration of viable viral shedding in fragile patients diagnosed with COVID-19; 
- 
Immune response in patients diagnosed with and/or vaccinated against COVID-19 assessed 
according to study protocol procedures (see above); 
- 
All-cause mortality during the follow-up period; 
- 
Change in basal clinical condition assessed by questionnaire on functional status and/or 
according to specific parameters of underlying fragile condition (i.e. graft function, cancer 
stage etc). 
Baseline variables will include:  
- 
Demographic data (sex, date of birth, ethnicity, blood group); 
- 
Underlying conditions; 
- 
Vaccination status against respiratory infectious diseases (i.e. Influenza, pneumococcus, 
BCG) if available. 
 
13. STATISTICAL ANALYSIS 
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 epidemiological features, laboratory results, therapeutic and 
preventive (including vaccination) strategies will be analysed using chi-square tests, t-tests or Mann-
Whitney tests, depending on the data. To evaluate potential 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 factors associated with each 
endpoint (including death). In addition, we will use cumulative incidence functions, such as the Fine-
Gray sub-distribution hazard regression model, to account for competing events (i.e., death in 
evaluating graft function etc.). For missing values, a different strategy to understand the causes and 
the significance for the analysis will be developed and a graduated procedure for dealing with 
censorship and imputations via linked regressions will be developed. The significance level is defined 
with a p-value <0.05. Risk perception and socio-economic factors will be analysed using quasi-

 
34 
ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
experimental techniques (eg: propensity score matching, difference-in-differences) by exploiting 
several sources of variation over time and across individuals.  All statistical analyses will be carried 
out with STATA, Python and/or R statistics software by trained staff (epidemiologists, statisticians) 
using the latest analysis 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 e protocol, protocol amendments, ICF, and other relevant documents must be 
submitted to an IRB/IEC by the investigator and reviewed and approved by the IRB/IEC before the 
study is initiated. 
 
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 (ICF) must be obtained and documented in accordance with 
local regulations, ICH-GCP requirements, and the ethical principles that have their origin in the 
principles of the 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 Investigator (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 

 
35 
ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
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 his/her consent to participate in the study at any time. Consent may 
be waived with documented approval as per local guidelines, for example in the case where only 
routinely collected data from medical records are collected for this study. 
 
14.4 Data protection 
The participant must be informed that his/her personal study-related data will be used by the local 
Principal Investigator in accordance with local data protection law. The level of disclosure must also 
be explained to the participant 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 
of Bologna/CINECA. 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 should be performed also retrospectively after a patient case has been completed 
(treatment or follow-up is finished or patient's death). This process will be compliant with all 
applicable European and German federal data protection regulations, including EU directive 
2016/679 and the German DS-GVO. 
 
14.5 Description of the data collected 
Only data that is strictly necessary and relevant to meet the objectives of the research are collected. 
The data collected via the medical file as part of the research falls into the categories following: 
Demographics data: age, sex, race, education, cigarette smoking, usual residence. 
Detailed clinical data, including about possible previous and concomitant illnesses, medical findings 
and therapies in the context of SARS-CoV-2 infection. 
Data collected by self-administered questionnaires: questionnaires to address the functional status, 
respiratory impairment, mental health, and socio-economic issues with related behavior.  
 
Data will be collected by a standardized electronic case report form (eCRF) and managed using 
REDCap capture tool. Collected data will be periodically checked for accuracy by an investigator of 
the coordinating unit (UNIBO). Queries for incongruous or missing data will be submitted to 

 
36 
ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
investigators. There will be 12 mailing cycles a year in which baseline and post-COVID-19 follow-up 
and/or post vaccination monitoring information will be requested. This policy will ensure that the 
cohort statistics will be based on a data set that is current, complete and as accurate as possible. 
 
14.6 Data quality assurance 
All participant data relating to the study will be recorded on the electronic-CRF. The Investigator is 
responsible for verifying that data entries are accurate and correct by physically or electronically 
signing the eCRF or paper CRF. The local investigator must maintain accurate documentation 
(source data) that supports the information entered in the eCRF or paper CRF. The local investigator 
must permit study-related monitoring, audits, IRB/IEC review, and regulatory agency inspections and 
provide direct access to source data documents. The promotor is responsible for the data 
management of this study including quality checking of the data as described above in study 
procedures’ section. 
 
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 will start after the approval by the ethics committee approximately on April 2021 and it will 
end 12 months after the last patient enrollment. The overall duration of the study is 26 months. 
 
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 

 
37 
ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
does not justify authorship. Publications will be planned by the PI and the scientific and statistical 
committees. Publication of partial or local data must be approved by the PI. 
 
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. ADD-ON SUBSTUDIES 
 
15.1 Pregnant Women (PW) Cohort for evaluation of risks of congenital malformations and 
other adverse pregnancy outcomes after Zika virus infection (part of ZIKAlliance) - SARS-
CoV-2 sub study. 
15.2 Children (CH) cohort for the evaluation of developmental and neurological abnormalities 
in infants born to mothers residing in areas with Zika virus transmission during pregnancy 
(part of ZIKALLIANCE) - SARS-CoV-2 sub study. 
15.3 Epidemiologic study of respiratory infections by novel coronavirus (SARS-CoV-2) in the 
paediatric population 
 
15.4 UNIVR: Rete Oncologica Veneta (ROV) 
15.5 UNIBO: CONTRAST study 
 
 

 
38 
ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
16. REFERENCES 
1. 
Castagnoli R, Votto M, Licari A, Brambilla I, Bruno R, Perlini S, et al. Severe Acute 
Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection in Children and Adolescents: A 
Systematic Review. JAMA pediatrics. 2020. Epub 2020/04/23. 
2. 
Dashraath P, Jing Lin Jeslyn W, Mei Xian Karen L, Li Min L, Sarah L, Biswas A, et al. 
Coronavirus Disease 2019 (COVID-19) Pandemic and Pregnancy. American journal of obstetrics 
and gynecology. 2020. Epub 2020/03/29. 
3. 
McMichael TM, Currie DW, Clark S, Pogosjans S, Kay M, Schwartz NG, et al. Epidemiology 
of Covid-19 in a Long-Term Care Facility in King County, Washington. The New England journal of 
medicine. 2020. Epub 2020/03/29. 
4. 
Pereira MR, Mohan S, Cohen DJ, Husain SA, Dube GK, Ratner LE, et al. COVID-19 in solid 
organ transplant recipients: Initial report from the US epicenter. American journal of transplantation 
: official journal of the American Society of Transplantation and the American Society of Transplant 
Surgeons. 2020. Epub 2020/04/25. 
5. 
Lloyd-Sherlock PG, Kalache A, McKee M, Derbyshire J, Geffen L, Casas FG, et al. WHO 
must prioritise the needs of older people in its response to the covid-19 pandemic. BMJ (Clinical 
research ed). 2020;368:m1164. Epub 2020/03/25. 
6. 
Fishman JA, Grossi PA. Novel Coronavirus-19 (COVID-19) in the immunocompromised 
transplant recipient: #Flatteningthecurve. American journal of transplantation: official journal of the 
American Society of Transplantation and the American Society of Transplant Surgeons. 2020. Epub 
2020/04/02. 
7. 
Yahav D, Yelin D, Eckerle I, Eberhardt CS, Wang J, Cao B, et al. Definitions for coronavirus 
disease 2019 reinfection, relapse and PCR re-positivity. Clinical microbiology and infection: the 
official publication of the European Society of Clinical Microbiology and Infectious Diseases. 
2021;27(3):315-8. Epub 2020/12/08. 
 
8.     Gaibani P, D'Amico F, Bartoletti M, Lombardo D, Rampelli S, Fornaro G, Coladonato S, 
Siniscalchi A., Re M C, Viale P, Brigidi P, Turroni S, Giannella M. The Gut Microbiota of Critically Ill 
Patients 
With 
COVID-19. 
Front 
Cell 
Infect 
Microbiology 2021;11:670424. 
doi: 
10.3389/fcimb.2021.670424. eCollection 2021. 

 
39 
ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
9.        Yeoh YK, Zuo T, Lui GC, Zhang F, Liu Q, Li AY, Chung AC, Cheung CP, Tso EY, Fung KS, 
Chan V, Ling L, Joynt G, Hui DS, Chow KM, Ng SSS, Li TC, Ng RW, Yip TC, Wong GL, Chan FK, 
Wong CK, Chan PK, Ng SC. Gut microbiota composition reflects disease severity and dysfunctional 
immune responses in patients with COVID-19. Gut 2021; 70(4):698-706. doi: 10.1136/gutjnl-2020-
323020. Epub 2021 Jan 11. 
 
10.      Chen Y, Gu S, Chen Y, Lu H, Shi D, Guo J, Wu WR, Yang Y, Li Y, Xu KJ , Ding C, Luo R, 
Huang C, Yu L, Xu M, Yi P, Liu J, Tao JJ, Zhang H, Lv L, Wang B, Sheng J, Li L. Six-month follow-
up of gut microbiota richness in patients with COVID-19. Gut 2021. doi: 10.1136/gutjnl-2021-324090. 
 
11.     Torjesen et al. Covid-19: Norway investigates 23 deaths in frail elderly patients after 
vaccination. British Medical Journal 2021;372:n149. doi: 10.1136/bmj.n149. 
 
12.       Lynn DJ, Benson SC, Lynn MA, Pulendran B. Modulation of immune responses to vaccination 
by the microbiota: implications and potential mechanisms. Nature Reviews Immunology 20211-
14. doi: 10.1038/s41577-021-00554-7. 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
ANNEX 1a and ANNEX 1b – Time points for the socio-economic questionnaires 
 
 
Day 0 of this questionnaire refers to the first contact with the patient. Day 0 may be allocated in different time points 
depending on the cohort and study. For WP2 patients, Day 0 is the first prospective follow-up point, which could be 
month 3, month 6, month 12 or month 18, depending on the time of infection diagnosis. For WP4, Day 0 corresponds to 
either the first contact for an underlying condition visit or to vaccine administration. If Day 0 cannot correspond to the 
time of vaccine administration then Day 0 will correspond to the first post-vaccination follow-up.  
Information will be collected at least at two time points for each patient, unless Day 0 corresponds to month 18 in WP2 
(in this case it is unfeasible to interview the patient again). The two time points will be ≈12 months apart unless not 
feasible, that is when Day 0 is at month 12 or 18 for WP2. 
The second time point will be: 
- 
For WP2, month 18. For example, if the first prospective data collection point corresponded to the follow-up 
visit at month 12, then the second and last contact with such patient would take place at month 18. 
- 
For WP4, month 12, which corresponds to either the 12 month vaccination follow-up or the visit of end of 
study.  
In addition to the two time points, information could also be collected ≈6 months after Day 0 whenever possible 
(although not mandatory). 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
 
ANNEX 1a – Socio-economic questionnaire 
 
To be asked on Day 0 ONLY: 
Day 0 refers to the first contact with the patient, independently of whether they have been 
vaccinated, whether they are positive or negative, or of the time of infection, i.e. all cohort patients 
should be interviewed. It is crucial to record the date of the contact/interview.  
 
1. (This is the ID that allows to identify the patient as part of ORCHESTRA and/or the original 
cohort):  Patient ID ____________   / Date of interview ________________ 
 
Demographic information: 
 
2. What is the highest level of education that you have achieved to date? 
a. No formal school 
b. Primary school 
c. Secondary school 
d. Some university 
e. Undergraduate degree 
f. Post-graduate degree 
 
3. What is your current marital status? 
a. Married 
b. Cohabitation 
c. Divorced/separated 
d. Widowed 
e. Single  
 
Defining household as the family unit, 
 
4. what is the total number of household members? 
 
5. what is the number of household members below age 14? 
Use of health care resources (COVID-19 UNRELATED/RELATED TO YOUR UNDERLYING CONDITIONS): 
Since the beginning of the pandemic (February/March 2020), have you experienced: 
6. any disruption in your routine visits (these are related to underlying conditions) and/or 
periodic check-ups (these may be, for example, regular gynecological visits for preventative 
reasons)?   
- 
 Cancellation                              Yes/No/NA 
- 
Postponement                           Yes/No/NA 
7. any interruption to the treatment you regularly follow?  

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
- 
Problems accessing medication                          Yes/No/NA 
- 
Postponement of your treatment session        Yes/No/NA 
- 
Cancellation of your treatment session            Yes/No/NA 
 
Socio-economic indicators 
8. Which one of the following status best describes your current labour situation? 
a. Paid Employee (in a firm/office/other) 
b. Formal and remunerated self-employed 
c. Informal/occasional work (with no contract) 
d. Volunteer or other non-remunerated work  
e. Housekeeping/look after family members 
f. Permanent inability to work (with benefits) 
g. Permanent inability to work (without benefits) 
h. Temporary inability to work (with benefits): e.g., maternity leave 
i. Unemployed without benefits 
j. Unemployed with benefits 
k. Retired  
l. Student 
m. Other  
Question 9 is only for those replying a, b, c or d to question 8: 
 
 
9.  How many total hours a week do you work on average? 
 
10. Thinking about your income and the income of everyone who lives in your household and 
contributes to the household budget, what is your current total monthly household net 
income (after taxes)?  
 
a. Less than 100€ 
b. 100€ to 299€ 
c. 300€ to 499€ 
d. 500€ to 999€ 
e. 1,000€ to 1,999€ 
f. 2,000€ to 2,999€ 
g. 3,000€ to 3,999€ 
h. 4,000€ to 4,999€ 
i. 5,000€ to 5,999€ 
j. More than 6,000€ 
 
Since the beginning of the pandemic (February/March 2020), have you: 
 
11. had financial or liquidity problems (e.g. problems with paying rent, school fees, loan, utility 
bills, etc.)?  Yes/No 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
 
12. received any financial or material aid (e.g. public assistance, cash-transfers, food banks, etc)?        
Yes/No 
 
Knowledge, Risk perception, Behaviour 
13. How often have you recently looked for information on COVID19 (eg: efficacy and safety of 
vaccines, development of vaccination plans in your country, number of new cases) in the 
newspapers, television, radio, social networks, etc? 
a. Several times each day 
b. Once a day  
c. 2-3 times a week 
d. Once a week 
e. Less than once a week 
 
14. On a theoretical scale from 0 to 10 (where 0 means no risk at all and 10 means maximum 
risk), what do you think is your current risk of infection/ re-infection?    
 
15. On a theoretical scale from 0 to 10 (where 0 means no risk at all and 10 means maximum 
risk), what do you think is your current risk that, in case of infection/re-infection, there will 
be severe complications or even death?    
 
16. What do you do to protect yourself against infection or re-infection, and how often? (provide 
an answer on frequency for all measures specified) 
 
Never 
Rarely 
Often  Very 
often 
Wash my hands with hand-soap / hydro-
alcoholic solutions  
□ 
□ 
□ 
□ 
Keep a distance of at least 2 meters from 
others  
□ 
□ 
□ 
□ 
Stay at home / Avoid gathering with 
friends and relatives not belonging to 
my bubble 
□ 
□ 
□ 
□ 
Use mask 
□ 
□ 
□ 
□ 
 
17. Have you been offered a vaccine by the public health system? Yes/No/NA 
18. Have you accepted or will you accept to be vaccinated once you get offered a vaccine? 
Yes/No/NA 
19. IF NOT (for those who have not accepted or would not accept to get vaccinated): Why 
did/would you not accept the vaccine? (multiple replies allowed) 
a. Lack of trust in its efficacy. 
b. Fear of adverse effects. 
c. Having passed COVID-19 already, I am afraid that adverse effects will be stronger 
than the disease itself. 
d. Not helpful in my case as I adopt many other measures (eg: I always wear a mask). 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
e. I already had COVID-19 and I am sure I will not get it again. 
f. I prefer someone else gets it before me. 
g. I believe there are other (better) ways to prevent COVID-19 than with a vaccine. 
h. Other 
 
To be asked at the next contacts (6, 12, 18 months, whatever applies): 
Follow-up interviews after Day 0. 
 
1. (This is the ID that allows to identify the patient as part of ORCHESTRA and/or the original 
cohort):  Patient ID ________________ / Date of interview ________________ 
Demographic information: 
 
Defining household as the family unit, 
2. what is the total number of household members? 
3. what is the number of household members below age 14? 
Use of health care resources (COVID-19 UNRELATED/RELATED TO YOUR UNDERLYING CONDITIONS): 
Over the last 6 or 12 months, have you experienced: 
4. any disruption in your routine visits (these are related to underlying conditions) and/or 
periodic check-ups (these may be, for example, regular gynecological visits for preventative 
reasons)?   
 
- 
Cancellation                              Yes/No/NA 
- 
Postponement                           Yes/No/NA 
5. any interruption to the treatment you regularly follow?  
- 
problems accessing medication                          Yes/No/NA 
- 
Postponement of your treatment session        Yes/No/NA 
- 
Cancellation of your treatment session             Yes/No/NA 
 
Socio-economic indicators 
6. Which one of the following status best describes your current labour situation? 
a. Paid Employee (in a firm/office/other) 
b. Formal and remunerated self-employed 
c. Informal/occasional work (with no contract) 
d. Volunteer or other non-remunerated work  
e. Housekeeping/look after family members 
f. Permanent inability to work (with benefits) 
g. Permanent inability to work (without benefits) 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
h. Temporary inability to work (with benefits): e.g., maternity leave 
i. Unemployed without benefits 
j. Unemployed with benefits 
k. Retired  
l. Student 
m. Other  
 
Questions 7 is only for those replying a, b, c or d to question 6: 
 
7.  How many total hours a week do you work on average? 
 
8. Thinking about your income and the income of everyone who lives in your household and 
contributes to the household budget, what is your current total monthly household net 
income (after taxes)? 
 
a. Less than 100€ 
b. 100€ to 299€ 
c. 300€ to 499€ 
d. 500€ to 999€ 
e. 1,000€ to 1,999€ 
f. 2,000€ to 2,999€ 
g. 3,000€ to 3,999€ 
h. 4,000€ to 4,999€ 
i. 5,000€ to 5,999€ 
j. More than 6,000€ 
 
Over the last 6 or 12 months, have you: 
9. had financial or liquidity problems (e.g. problems with paying rent, school fees, loan, utility 
bills, etc.)?  Yes/No 
 
10. received any financial or material aid (e.g. public assistance, cash-transfers, food banks, etc)?        
Yes/No 
 
 
Knowledge, Risk perception, Behaviour 
11. How often have you recently looked for information on COVID19 (eg: efficacy and safety of 
vaccines, development of vaccination plans in your country, number of new cases) in the 
newspapers, television, radio, social networks, etc? 
a. Several times each day 
b. Once a day  
c. 2-3 times a week 
d. Once a week 
e. Less than once a week 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
 
12. On a theoretical scale from 0 to 10 (where 0 means no risk at all and 10 means maximum 
risk), what do you think is your current risk of infection/ re-infection?    
 
 
13. On a theoretical scale from 0 to 10 (where 0 means no risk at all and 10 means maximum 
risk), what do you think is your current risk that, in case of infection/re-infection, there will 
be severe complications or even death?    
 
14. What do you do to protect yourself against infection or re-infection, and how often? (provide 
an answer on frequency for all measures specified) 
 
Never 
Rarely 
Often  Very 
often 
Wash my hands with hand-soap / hydro-
alcoholic solutions  
□ 
□ 
□ 
□ 
Keep a distance of at least 2 meters from 
others  
□ 
□ 
□ 
□ 
Stay at home / Avoid gathering with 
friends and relatives not belonging to 
my bubble 
□ 
□ 
□ 
□ 
Use mask 
□ 
□ 
□ 
□ 
 
15. Have you been offered a vaccine by the public health system? Yes/No/NA 
16. Have you accepted or will you accept to be vaccinated once you get offered a vaccine? 
Yes/No/NA 
17. IF NOT (for those who have not accepted or would not accept to get vaccinated): Why 
did/would you not accept the vaccine? (multiple replies allowed) 
a. Lack of trust in its efficacy 
b. Fear of adverse effects 
c. Having passed COVID-19 already, I am afraid that adverse effects will be stronger 
than the disease itself 
d. Not helpful in my case as I adopt many other measures (eg: I always wear a mask) 
e. I already had COVID-19 and I am sure I will not get it again 
f. I prefer someone else gets it before me 
g. I believe there are other (better) ways to prevent COVID-19 than with a vaccine. 
h. Other 
 
 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
ANNEX 1b – Socio-economic questionnaire for CHILDREN cohorts 
 
SCHOOL AGE CHILDREN COHORTS WP4 - Socio-economic questions for the prospective protocol 
(from WP8) 
In the case of children, questions will be asked to the caretaker. 
 
To be asked on Day 0: 
Day 0 refers to the first contact with the patient, independently of whether they have been 
vaccinated, whether they are positive or negative, or of the time of infection, i.e. all cohort patients 
should be interviewed. It is crucial to record the date of the contact/interview.  
 
1. (This is the ID that allows to identify the patient as part of ORCHESTRA and/or the original 
cohort):  Patient ID ____________ / Date of interview ________________ 
 
Demographic information: 
 
2. What is the school year you are attending? 
 
Use of health care resources (COVID-19 UNRELATED/RELATED TO YOUR UNDERLYING 
CONDITIONS): 
Since the beginning of the pandemic (February/March 2020), have you experienced: 
3. any disruption in your routine visits (for eventual underlying conditions) and/or periodic 
check-ups (such as pediatric visits, routine vaccination schedules)?   
- 
 Cancellation                              Yes/No/NA 
- 
Postponement                           Yes/No/NA 
4. any interruption to the treatment you regularly follow?  
- 
problems accessing medication                          Yes/No/NA 
- 
Postponement of your treatment session        Yes/No/NA 
- 
Cancellation of your treatment session            Yes/No/NA 
 
Since the beginning of the pandemic (February/March 2020),  
 
5. Were classes suspended at your school?  Yes/No 
 
If yes: 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
 
6. For how long were classes suspended? 
 
7. Were you able to follow some forms of distance learning?        Yes/No 
 
If not: 
 
8. Why could you not follow distance learning? 
a. distance learning was not organised at my school 
b. I was not interested/I was bored 
c. I could not rely on good technical means (e.g. computer or wifi) 
d. Other  
 
9. In total, how many weeks of school can you estimate you have lost?  
 
 To be asked at month 6 or/and 12: 
Follow-up interviews after Day 0. 
 
1. (This is the ID that allows to identify the patient as part of ORCHESTRA and/or the original 
cohort):  Patient ID ____________ / Date of interview ________________ 
Use of health care resources (COVID-19 UNRELATED/RELATED TO YOUR UNDERLYING 
CONDITIONS): 
Over the last 6/12 months, have you experienced: 
2. any disruption in your routine visits and/or periodic check-ups?   
- 
 Cancellation                              Yes/No/NA 
- 
Postponement                           Yes/No/NA 
3. any interruption to the treatment you regularly follow?  
- 
Problems accessing medication                          Yes/No/NA 
- 
Postponement of your treatment session        Yes/No/NA 
- 
Cancellation of your treatment session            Yes/No/NA 
Over the last 6/12 months: 
4. Were classes suspended at your school?  Yes/No 
 
If yes: 
 
5. For how long were classes suspended? 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
 
6. Were you able to follow some forms of distance learning?        Yes/No 
 
If not: 
 
7. Why could you not follow distance learning? 
a. distance learning was not organised at my school 
b. I was not interested/I was bored 
c. I could not rely on good technical means (e.g. computer or wifi) 
d. Other  
 
8. In total, how many weeks of school can you estimate you have lost?  
 
 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
 
ANNEX 2 – Psychological scale Questionnaires 
 
0. Time log for synchronizing analyses across cohorts and countries 
1. Generalized Anxiety Disorder 7-item (GAD-7) scale 
2. Perceived Stress Scale - 4 Item version 
3. UCLA Loneliness Scale-short version 
4. Brief Resilience Scale (BRS) 
5. Depression Scale (CES-D Scale)  
 
0. Time log for simplifying analyses across cohorts and countries [BASELINE][FOLLOW-UP] 
 
Rational for this part: In order to compare effects of pandemic measures across different 
countries, we need to synchronize and include these questions about the prevention measures 
in place at the time assessment. 
 
0.1 During the last two weeks have you been in a lockdown? 
1 
Yes 
2 
No 
0.2 Du ring the last two weeks have the schools been closed? 
1 
2 
3 
Yes 
No 
Not relevant for me, as I do not have children or I’m not working in a school 
0.3 Du ring the last two weeks did you need to do home office (either completely or only on several days/week)? 
1 
2 
Yes 
No 
0.4 Du 
cinema
ring the last two weeks were you obliged to wear masks in closed public areas (e.g. theaters, restaurants, 
,…)? 
1 
2 
Yes 
No 
0.5 Du ring the last two weeks were you obliged to wear masks in open public areas (e.g. parks, playgrounds,)? 
1 
2 
Yes 
No 
0.6 Du ring the last two weeks were you obliged to follow the ‘vaccinated-tested-recovered’ rule to: 
 
1 
2 
3 
4 
5 
6 
7 
go to work 
go to a restaurant/bar 
go to a shopping center 
go to the cinema 
go to a museum 
go to sports events 
go to any other place/event not mentioned before 
0.7 If ‘ 
0.8  
7 - go to any other place/event not mentioned before’ in 0.6, please specify – open field. 
During the last two weeks did you have to keep a minimum distance of 1.5 or 2 meters when contacting people 
outside  your household? 
1 
2 
Yes 
No 

 
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ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
 
 
1. Generalized Anxiety Disorder 7-item (GAD-7) scale [BASELINE][FOLLOW-UP] 
 Over the last 2 weeks, how often have you been bothered by the following problems? 
Not at all 
Several days 
More than half the 
days 
Nearly every day 
Feeling nervous, anxious, or on edge 
 
 
 
 
Not being able to stop or control worrying 
 
 
 
 
Worrying too much about different things 
 
 
 
 
Trouble relaxing 
 
 
 
 
Being so restless that it is hard to sit still 
 
 
 
 
Becoming easily annoyed or irritable 
 
 
 
 
Feeling afraid as if something awful might 
happen 
 
 
 
 
 
2. Perceived Stress Scale - 4 Item version [BASELINE][FOLLOW-UP] 
 The questions in this scale ask you about your feelings and thoughts during the last month. In 
each case, please indicate with a check how often you felt or thought a certain way. 
Never 
Almost 
never 
Sometimes 
Fairly often 
Very often 
In the last month, how often have you felt 
that you were unable to control the important 
things in your life? 
 
 
 
 
 
In the last month, how often have you felt 
confident about your ability to handle your 
personal problems? 
 
 
 
 
 
In the last month, how often have you felt 
that things were going your way? 
 
 
 
 
 
 
 
3. UCLA Loneliness Scale-short version [BASELINE][FOLLOW-UP] 
 The next questions are about how you feel about different aspects of your life. For each one, 
tell how often you feel that way. 
Hardly ever or never Some of the time 
Often 
How often do you feel that you lack companionship? 
 
 
 
How often do you feel left out? 
 
 
 
How often do you feel isolated from others? 
 
 
 
 
 
4. Brief Resilience Scale (BRS) [BASELINE][FOLLOW-UP] 
 Please indicate the extent to which you agree with each of the following statements by using 
the following scale: 
Strongly 
disagree 
Disagree 
Neutral 
Agree 
Strongly 
agree 
I tend to bounce back quickly after hard times 
 
 
 
 
 
I have a hard time making it through stressful 
events 
 
 
 
 
 
It does not take me long to recover from a 
stressful event 
 
 
 
 
 
It is hard for me to snap back when something 
bad happens 
 
 
 
 
 
I usually come through difficult times with 
little trouble 
 
 
 
 
 
I tend to take a long time to get over set- 
backs in my life 
 
 
 
 
 
 
 

 
52 
ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
 
5. Center for Epidemiologic Studies Depression Scale (CES-D Scale) [BASELINE][FOLLOW-UP] 
 Below is a list of the ways you might have felt or behaved. Please tell me how often you have 
felt this way during the past week. 
During the last week … 
Rarely or 
None of the 
time (less 
than 1 day) 
Some or a 
Little of the 
time (1 - 2 
days) 
Occasionally or a 
moderate amount 
of time (3 - 4 days) 
Most or All of 
the time (5 - 7 
days) 
I was bothered by things that usually don’t bother 
me 
 
 
 
 
I did not feel like eating; my appetite was poor 
 
 
 
 
I felt that I could not shake off the blues even with 
help from my family or friends 
 
 
 
 
I felt that I was just as good as other people 
 
 
 
 
I had trouble keeping my mind on what I was 
doing 
 
 
 
 
I felt depressed 
 
 
 
 
I felt that everything I did was an effort 
 
 
 
 
I felt hopeful about the future 
 
 
 
 
I thought my life had been a failure 
 
 
 
 
I felt fearful 
 
 
 
 
My sleep was restless 
 
 
 
 
I was happy 
 
 
 
 
I talked less than usual 
 
 
 
 
I felt lonely 
 
 
 
 
People were unfriendly 
 
 
 
 
I enjoyed life 
 
 
 
 
I had crying spells 
 
 
 
 
I felt sad 
 
 
 
 
I felt that people dislike me 
 
 
 
 
I could not get “going” 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
53 
ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
 
 
ANNEX 3 – Questionnaire for monitoring of adverse events to COVID-19 vaccination 
 
A) Demographics – Anamnesis 
Age: __________ 
Sex:           □  M      □  F 
Special population:    □ HIV      □  Cancer    □  Solid organ transplant   □  Haematological stem cell 
transplant      
If solid organ transplant, please specify:        □   Liver    □   Kidney     □   Heart     □  Lung 
Drug/food allergies:    □ Y   □  N      If yes, please specify:_____________________ 
Previous serious adverse events to vaccination:    □  Y      □  N 
Underlying diseases:     
□  Renal  □ Cardiovascular  □  Asthma  □  Diabetes mellitus □ Anaemia/Blood disorders  □ 
Seizure/Epilepsy 
Other nervous system disease:    □ Y   □  N      If yes, please specify:__________________ 
Concomitant medications:___________________________________________________ 
Blood transfusion in the last year:  □ Y   □  N 
Treatment with immunoglobulin or antivirals in the last year: □ Y   □  N 
Other relevant conditions: __________________________________________ 
 
B) Adverse event report 
First dose (dd/mm/yy): __________ 
Second dose (dd/mm/yy): __________ 
 
Clinical events after the first dose:  □ Y   □  N 
Clinical events after the first dose:  □ Y   □  N 
Headache: □ Y   □  N      
Asthenia: □ Y   □  N 
Fever (≥38°C): □ Y   □  N 
Nausea:  □ Y   □  N 
Vomiting:  □ Y   □  N 
Lymphadenopathy:  □ Y   □  N 
Tachycardia:  □ Y   □  N 
Flushing:  □ Y   □  N 
Syncope:  □ Y   □  N 
Pruritus, erythema or pain in injection site:  □ Y   □  N 

 
54 
ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
Generalised urticaria:  □ Y   □  N 
Serious allergic events (anaphylaxis, angioedema): □ Y   □  N 
COVID infection after vaccination:  □ Y   □  N 
Use of paracetamol or non-steroidal anti-inflammatory drugs:  □ Y   □  N 
Requirement for use of other drugs:  □ Y   □  N   If Yes, please specify:  _________________ 
Demyelinating disorders:   □ Y   □  N 
Encephalitis/Encephalopathy:  □ Y   □  N 
Peripheral neuropathy:  □ Y   □  N 
Muscular weakness and/or paralysis: □ Y   □  N 
Visual impairment (including optic neuritis):  □ Y   □  N 
Walking disturbance:  □ Y   □  N 
Blood disorders (anaemia, thrombocytopenia, leukopenia):  □ Y   □  N 
Seizures/Epilepsy:  □ Y   □  N 
Weakness and/or paralysis of facial muscle:  □ Y   □  N 
Vasculitis:  □ Y   □  N 
Renal disorders:  □ Y   □  N 
Liver function disorders:  □ Y   □  N 
Cardiac disorders: □ Y   □  N 
Other adverse events:     □ Y   □  N  If Yes, please specify:  _________________________ 
 
If yes (complete for each reported event):   day of onset (dd/mm/yy):________   
hospitalization/emergency department admission/general practitioner visit:    □ Y   □  N 
Description of the event (laboratory data/administered drugs/other relevant information): 
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
___________________________ 
Recovery:  □ Y   □  N 
 
 
 
 
 
 
 
 
 
 

 
55 
ORCHESTRA_WP4 prospective protocol_ Version 1.1, September 28th, 2021 
 
 
 
ANNEX 4 – WP6 Deliverable 6.1_Mapping of retrospective samples and development of 
standardized protocols for prospective sampling 
 
 
 
 

 
 
 
 
1 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
 
 
 
 
 
 
 
WP6 Deliverable 6.1  
Mapping of retrospective samples and development of 
standardised protocols for prospective sampling 
UANTWERPEN 
 
 
 
 
 
 
 
Connecting European Cohorts 
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 Effective 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  
  
WP No:  
WP6 
Deliverable No:  
D6.1 
Title:  
Standardised protocol and manual on collection and 
storage of samples 
Lead Beneficiary:  
UANTWERPEN 
Other Involved  
Beneficiaries: 
UNIBO, INSERM, HMGU 
Nature:  
Report, 
Dissemination Level:  
Confidential 
Due Delivery Date:  
M6 / 31 May, 2021 
Submission Date:  
31 May, 2021 
Justification of delay: 
N/A 
Status:  
Final 
Version: 
1.0 
Author(s): 
Surbhi Malhotra-Kumar, Matilda Berkell 
 
 
History of Changes  
  
Version   
Date  
Created/Modified by  
1  
Surbhi Malhotra-Kumar, Matilda Berkell 
  
 
 

 
 
 
 
3 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
Table of contents 
 
Executive summary .................................................................................................................... 3 
WP Context ............................................................................................................................. 3 
Purpose of the document ....................................................................................................... 3 
Content of the document ........................................................................................................ 3 
Target of the document .......................................................................................................... 4 
Dissemination level ................................................................................................................. 4 
Core content ............................................................................................................................... 4 
References ................................................................................................................................. 4 
Acknowledgments ...................................................................................................................... 4 
Appendix 1 .................................................................................................................................. 5 
 
Executive summary 
WP Context 
The objective of WP6 in the ORCHESTRA project is to study human (epi)genetic, 
immunological, microbial, and viral features to identify markers of disease severity and to study 
the long-term impact of SARS-CoV-2 infection as well as the effect of immunization and 
characteristics underlying breakthrough infections post vaccination. Samples will be included 
retrospectively as well as prospectively within the study to study severe COVID-19 infection in 
hospitalized and outpatients during the acute disease stage as well as study long-term effects 
of infection and immunization on the host immune response. 
Purpose of the document 
The purpose of the WP6 protocols is to provide an overview of the biosamples to be collected 
within the prospective ORCHESTRA study, at which timepoints, and which analyses these 
samples are to be subjected to. 
Content of the document 
The WP6 protocol outlines the objectives of the biological studies foreseen within the project, 
the methodology to be utilized to meet these goals, which patient populations that are to be 
analyzed, and how these samples are to be handled and stored. 
 
 

 
 
 
 
4 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
Target of the document 
The target audience of the document are the clinical WPs 2-5 within the ORCHESTRA study 
in which prospective sample collection is conducted. 
Dissemination level 
Confidential, only for members of the consortium (including the Commission Services). 
Core content 
The WP6 prospective protocol document is available in Appendix 1. 
References 
N/A 
Acknowledgments 
N/A 
 
 

 
 
 
 
5 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
Appendix 1 
 
 
 
 
 
 
PROSPECTIVE SAMPLE COLLECTION AND MANAGEMENT DOCUMENT 
WP6 – Biobanking, genomics, and viral-host interactions 
 
University of Antwerp (UANTWERPEN) in collaboration with all WP6 partners 
 
Version dated 11-05-2021 
 
 
 
 
 
Connecting European Cohorts to Incre
ase Common 
and Effective Response to SARS- CoV-
2 Pandemic 

 
 
 
 
6 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
Thank you for your participation in the ORCHESTRA study. The purpose of this document is to provide 
you with additional information and instructions on the collection and processing of samples for WP6 
within the prospective tier of ORCHESTRA. This document is a guideline for research staff including 
(sub-) investigators, research nurses, and laboratory staff. The clinical protocol takes precedence over 
this document. If there is a discrepancy between the sample collection and management document and 
the clinical protocol, the ORCHESTRA clinical protocol should be followed. 
This document outlines how samples should be handled after patient inclusion. Specifically, it describes 
the time points desired for biosampling. 
 
 

 
 
 
 
7 
 
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 Effective 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:   
PROSPECTIVE SAMPLE COLLECTION AND 
MANAGEMENT DOCUMENT 
Lead Beneficiary:   
UANTWERPEN  
Other Involved   
Beneficiaries:  
INSERM, APHP, UNIBO, HMGU 
PIs, Participating centres 
Surbhi-Malhotra-Kumar, UANTWERPEN 
Samir Kumar-Singh, UANTWERPEN 
Patrizia Brigidi, UNIBO 
Valerio Carelli, UNIBO 
Benoit Visseaux, INSERM/APHP 
Laurent Abel, INSERM 
Anette Peters, HMGU 
Authors: 
Surbhi Malhotra-Kumar, Samir Kumar-Singh, Matilda Berkell, 
Patrizia Brigidi, Benoit Visseaux 
 
History of Changes   
Version    
Date   
Modifications  
1.0  
 
Current version. 
2.0  
 
 
   
 
 

 
 
 
 
8 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
TABLE OF CONTENTS 
List of Abbreviations .............................................................................................................................. 10 
List of Tables .......................................................................................................................................... 11 
List of Figures ........................................................................................................................................ 11 
1. 
Introduction to ORCHESTRA ....................................................................................................... 12 
2. 
Objectives and brief description of substudies within WP6 .......................................................... 12 
2.1 
Characterisation of SARS-CoV-2 viral variants (Task 6.2) ................................................... 12 
2.2 
Characterisation of serological markers of SARS-CoV-2 infection (Task 6.3) ..................... 14 
2.3 
Characterisation of cellular immunity for SARS-CoV-2 infection (Task 6.4) ....................... 14 
2.4 
Cytokinome analysis (Task 6.5) ............................................................................................. 14 
2.5 
Next generation sequencing (NGS) of COVID-19 cohorts (Task 6.6) .................................. 14 
2.6 
Epigenome-wide analyses (Task 6.7) ..................................................................................... 14 
2.7 
Intestinal Microbiome Profiling (Task 6.8) ............................................................................ 14 
2.8 
Respiratory microbiome dynamics (Task 6.9) ........................................................................ 16 
3. 
Sample Timepoints ......................................................................................................................... 16 
3.1 
Sampling timepoints for COVID-19 patients (Including Reinfections and breakthrough 
infections in vaccinated individuals) .................................................................................................. 17 
3.1.1 
Sample collection and management .................................................................................. 17 
3.2 
Sampling timepoints for vaccinated individuals ..................................................................... 18 
3.2.1 
Sample collection and management .................................................................................. 18 
4. 
Detailed sample collection and storage instructions ...................................................................... 19 
4.1 
NP swab collection ................................................................................................................. 19 
4.1.1 
Recommended materials.................................................................................................... 19 
4.1.2 
Sample collection .............................................................................................................. 19 
4.1.3 
Storage conditions ............................................................................................................. 20 
4.2 
Serum sample collection ......................................................................................................... 21 
4.2.1 
Recommended materials.................................................................................................... 21 
4.2.2 
Sample collection .............................................................................................................. 21 
4.2.3 
Sample processing ............................................................................................................. 21 
4.2.4 
Storage conditions ............................................................................................................. 21 
4.3 
EDTA plasma isolation ........................................................................................................... 22 
4.3.1 
Recommended materials.................................................................................................... 22 
4.3.2 
Sample collection .............................................................................................................. 22 
4.3.3 
Sample processing ............................................................................................................. 22 
4.3.4 
Storage conditions ............................................................................................................. 23 

 
 
 
 
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ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
4.4 
Heparin Plasma collection and PBMC isolation .................................................................... 24 
4.4.1 
Recommended materials.................................................................................................... 26 
4.4.2 
Sample collection .............................................................................................................. 26 
4.4.3 
Sample processing ............................................................................................................. 28 
4.4.4 
Storage conditions ............................................................................................................. 33 
4.5 
Whole blood collection ........................................................................................................... 34 
4.5.1 
Recommended materials.................................................................................................... 34 
4.5.2 
Sample collection .............................................................................................................. 34 
4.5.3 
Sample processing ............................................................................................................. 34 
4.5.4 
Storage conditions ............................................................................................................. 34 
4.6 
Stool sample collection ........................................................................................................... 35 
4.6.1 
Recommended materials.................................................................................................... 35 
4.6.2 
Sample collection .............................................................................................................. 35 
4.6.3 
Sample processing ............................................................................................................. 35 
4.6.4 
Storage conditions ............................................................................................................. 35 
4.7 
Rectal swab sample collection ................................................................................................ 36 
4.7.1 
Recommended materials.................................................................................................... 36 
4.7.2 
Sample collection .............................................................................................................. 36 
4.7.3 
Sample processing ............................................................................................................. 36 
4.7.4 
Storage conditions ............................................................................................................. 36 
5. 
Storage instructions and destination sites ...................................................................................... 38 
6. 
Contact details ................................................................................................................................ 38 
6.1 
Questions concerning protocols for biological sampling ....................................................... 38 
NP swabs ........................................................................................................................................ 38 
Serum – Plasma .............................................................................................................................. 38 
PBMC ............................................................................................................................................. 38 
Whole blood ................................................................................................................................... 38 
Stool – Rectal swabs ...................................................................................................................... 39 
6.2 
Coordinating Laboratory for sample shipments ..................................................................... 39 
 
 
 

 
 
 
 
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ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
LIST OF ABBREVIATIONS 
APHP 
Assistance Publique – Hôpitaux de Paris 
COVID-19 
Coronavirus Disease 19 
DMSO 
Dimethyl Sulfoxide 
eCRF 
Electronic Clinical Research Form 
EDTA  
Ethylenediaminetetraacetic Acid 
ELISA  
Enzyme-linked Immunosorbent Assay  
FBS 
Fetal Bovine Serum 
HMGU 
Helmholtz Zentrum München 
IFN 
Interferon 
INSERM 
Institut National de la Santé et de la Recherche Médicale 
LMM 
Laboratory of Medical Microbiology 
MSD 
MesoScale Discovery 
NGS 
Next Generation Sequencing  
NP  
Nasopharyngeal 
PBMC 
Peripheral Blood Mononuclear Cell 
PBS 
Phosphate Buffered Saline 
PCR 
Polymerase Chain Reaction  
RBC 
Red Blood Cells 
RPMI-1640 
Roswell Park Memorial Institute 1640 medium 
SARS-CoV-2 
Severe Acute Respiratory Syndrome Coronavirus 2 
UANTWERPEN University of Antwerp 
UNIBO 
University of Bologna 
UTM 
Universal Transport Medium 
VOC 
Variant of Concern  
WES 
Whole Exome Sequencing  
WGS 
Whole Genome Sequencing  
 
 

 
 
 
 
11 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
LIST OF TABLES  
Table 1. Overview of required biosamples associated analysis within WP6. Task numbers correspond 
to tasks described in the Description of Work, Amendment 1, Annex 1. .............................................. 16 
Table 2. Overview sampling and data collection in ORCHESTRA cohorts for long-term sequelae in 
COVID-19 patients. ............................................................................................................................... 17 
Table 3. Overview sampling and data collection in ORCHESTRA cohorts for vaccinated individuals.
 ................................................................................................................................................................ 18 
Table 4. Common PBMC isolation procedures. .................................................................................... 24 
Table 5. Cost of different centrifuge tubes and separation media.......................................................... 25 
Table 6. Overview of samples to be shipped per time point per patient and their shipping conditions. 38 
LIST OF FIGURES 
Figure 1. PBMC isolation procedure using SepMate tubes. .................................................................. 28 
Figure 2. Liquid phases at different PBMC isolation stages. ................................................................. 32 
 
 

 
 
 
 
12 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
1. INTRODUCTION TO ORCHESTRA 
ORCHESTRA is a three-year international research project aimed at tackling the coronavirus pandemic, 
led by the University of Verona and involving 26 partners (extending to a wider network of 37 partners) 
from 15 countries: Argentina, Belgium, Brazil, Congo, France, Gabon, Germany, India, Italy, 
Luxemburg, Netherlands, Romania, Slovakia, Spain, Venezuela. The project is divided into a 
retrospective and a prospective tier in which clinical data and biosamples are analysed in order to 
• 
develop evidence-based recommendations for effective prevention, protection and optimized 
treatment of COVID-19 patients (including long-term sequelae) with a special focus on ‘at risk’ 
populations, including healthcare workers and fragile individuals 
• 
assess impact of environmental factors, socio-economic determinants, lifestyle and confinement 
measures on the spread of COVID-19 
• 
provide knowledge on the efficacy of vaccines against SARS-CoV-2 
• 
provide a model for responsiveness for future pandemic outbreaks. 
2. OBJECTIVES AND BRIEF DESCRIPTION OF SUBSTUDIES WITHIN WP6 
Several different sample types will be analysed within the ORCHESTRA study for the purposes of 
identifying human and viral genetic markers indicative of disease severity as well as to study immune 
responses over time as a result of infection and immunization. Specifically, samples will be collected 
from patients developing COVID-19 (including breakthrough and reinfection) in order to study both 
short- and long-term effects of infection on host immunity, respiratory and intestinal microbiome 
dynamics as well as host and viral genetic determinants underlying infection. Additionally, samples will 
be collected from vaccinated fragile populations as well as vaccinated healthcare workers in order to 
study effects of vaccination on host immunity and respiratory and intestinal microbiome dynamics. 
Samples collected within the framework of the ORCHESTRA study will in many cases be subjected to 
more than one type of analysis. To provide an overview of research questions of interest within the 
project, a brief summary of each analysis can be found below. 
2.1 CHARACTERISATION OF SARS-COV-2 VIRAL VARIANTS (TASK 6.2) 
This task will target two main points among selected patient populations: [i] to characterise the viral 
variants and to identify variants of concern (VOCs) by whole genome sequencing (WGS) in COVID-
19 patients and in vaccinated individuals with breakthrough infections, and [ii] to study potential 

 
 
 
 
13 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
mutation selection in populations presenting with long viral replication or receiving immunoglobulin 
therapies, and [iii] to study the viral replication (viral load and excretion duration). 
 
 

 
 
 
 
14 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
2.2 CHARACTERISATION OF SEROLOGICAL MARKERS OF SARS-COV-2 INFECTION 
(TASK 6.3) 
This task will characterize the host antibody responses with quantitative serology (anti-S and anti-N) on 
Abbott, Roche, MesoScale Discovery (MSD), or similar platforms as well as (pseudo-) 
seroneutralisation assays. These assays will be performed on populations with various degrees of 
COVID-19 severity and vaccinated individuals, also with breakthrough infections. 
2.3 CHARACTERISATION OF CELLULAR IMMUNITY FOR SARS-COV-2 INFECTION (TASK 
6.4) 
Vaccinated and non-vaccinated COVID-19 patients with varying degrees of disease severity and SARS-
CoV-2-positive non-symptomatic individuals will be studied for the balance and the phenotypes of T 
and B cells as a function of disease course and severity as well as response to vaccination. This task will 
chiefly employ flow cytometric analyses with CD45, CD3, CD4, CD19, FOXP3, Ki-67, CD38 markers, 
viability and IFNγ release assays. 
2.4 CYTOKINOME ANALYSIS (TASK 6.5) 
Vaccinated and non-vaccinated COVID-19 patients with varying degrees of disease severity, SARS-
CoV-2-positive non-symptomatic individuals and vaccinated individuals will be studied on MSD 
panels, Luminex panels and on select ELISAs. As an outcome, panels of cytokine markers predicting 
disease severity, mortality, breakthrough infections, and long term sequalae will be generated. 
2.5 NEXT GENERATION SEQUENCING (NGS) OF COVID-19 COHORTS (TASK 6.6) 
In-depth human genetic analysis will be conducted using WGS or whole exome sequencing (WES) 
followed by functional analyses of the most promising variants. Additionally, serum samples will be 
utilized for detection of auto-antibodies against type I interferons (IFNs). 
2.6 EPIGENOME-WIDE ANALYSES (TASK 6.7) 
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. 
2.7 INTESTINAL MICROBIOME PROFILING (TASK 6.8) 
This task will profile compositional and functional structures of the microbiome from faecal samples by 
NGS approaches, in order to elucidate the role of the intestinal microbiome in the susceptibility, 
progression and severity of COVID-19 infection. 

 
 
 
 
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ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
 
 

 
 
 
 
16 
 
ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
2.8 RESPIRATORY MICROBIOME DYNAMICS (TASK 6.9) 
We will investigate differences in the respiratory microbiome composition by combining meta-
transcriptomic and metagenomic sequencing to analyse both RNA and DNA viruses and the bacterial 
and fungal fractions. This will firstly elucidate the role of commensal flora and of co-infecting 
respiratory pathogens in influencing COVID-19 disease severity. Secondly, long-term carriage and 
impact of SARS-CoV-2 on the respiratory microbiome will be assessed on longitudinally collected 
prospective samples (6-12 months post-recovery and new infections). 
3. SAMPLE TIMEPOINTS 
An overview of sample types to be collected at each time point can be found in Table 1 below. No 
collection material will be provided within this study, but recommended materials to be used for each 
collection can be found in the section entitled “Detailed sample collection and storage instructions”. 
Table 1. Overview of required biosamples associated analysis within WP6. Task numbers correspond to 
tasks described in the Description of Work, Amendment 1, Annex 1. 
Sample type 
Task 
Analysis 
NP swab 
Task 6.2 
Viral variant sequencing 
Task 6.9 
Respiratory microbiome analysis 
Serum 
Task 6.3 
Serology 
Task 6.6 
Assessment of auto-antibodies against type I IFNs 
EDTA plasma 
Task 6.5 
Cytokinome analysis 
Heparin blood (PBMCs) 
Task 6.4 
Cellular immunity characterization 
EDTA whole blood 
Task 6.6 
Human WGS or WES 
Task 6.7 
Epigenomics 
Stool sample (faeces or rectal swab) 
Task 6.8 
Intestinal microbiome analysis 
 
 
 

 
 
 
 
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ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
3.1 SAMPLING TIMEPOINTS FOR COVID-19 PATIENTS (INCLUDING REINFECTIONS AND 
BREAKTHROUGH INFECTIONS IN VACCINATED INDIVIDUALS) 
3.1.1 Sample collection and management 
Sampling is required to be performed on the day of diagnosis and at following timepoints as shown in 
Table 2. Patient inclusion will primarily be based on availability of informed consent for the outlined 
tasks as well as availability of multiple samples per patient. Informed consent forms should clearly 
request permission to perform human genetic and epigenetic  analyses, without which these analyses 
cannot be undertaken. 
Table 2. Overview sampling and data collection in ORCHESTRA cohorts in COVID-19 patients including 
long-term sequelae. 
D01 
3 months1 
±1 month 
6 months1 
±1 month 
12 months1 
±1 month 
18 months1 
±2 month 
 Objective 
NP swab 
x 
x2,3 
x2,3 
x2,3 
x2 
Viral variant and 
metagenomic sequencing  
2 x 2 mL serum tube 
(serum) 
x 
x 
x 
x 
x 
Immune - serology and 
type I IFNs autoantbodies 
4 mL EDTA blood tube 
(plasma) 
x 
x4 
x4 
x4 
x2 
Immune - cytokine and 
chemokine 
1 (if possible 2) 9 mL 
heparin tube (PBMCs) 
x 
x4 
x4 
x4 
x2 
Immune - cellular 
2 x 2 mL EDTA tube 
(whole blood)  
x 
x 
x 
x 
x 
Genetic and epigenetic 
analyses 
Stool sample 
(faeces or rectal swab) 
x 
 
x 
 
x 
Metagenomic sequencing  
 
1. Day 0: first positive SARS-CoV-2 PCR test. Follow-up of 3, 6, 12, and 18 months start from Day 0. 
2. Reassessed only if outside the normal ranges at the previous assessment or if clinically indicated. 
3. At least one of the three timepoints (month 3, month 6, month 12) is required to perform metagenomic 
analyses. 
4. At least one of the three timepoints (month 3, month 6, month 12) is required. 
Level I 
Assessments of Level I are mandatory 
Level II 
Customized according to the feasibility of each cohort 
 
 
 

 
 
 
 
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ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
3.2 SAMPLING TIMEPOINTS FOR VACCINATED INDIVIDUALS 
3.2.1 Sample collection and management 
Vaccinated individuals in WP4 and WP5 will be sampled according to the time points outlined in Table 
3. In case of breakthrough infections post vaccination, samples will be collected as outlined in Table 2. 
Vaccination is (mostly) performed in two doses. Collection is to be performed prior to administration of 
dose 1 and dose 2, and 3, 6, and 12 months after the first dose (Table 3). Patient inclusion will primarily 
be based on availability of informed consent for the outlined tasks as well as availability of multiple 
samples per patient. Informed consent forms should clearly request permission to perform human 
genetic and epigenetic  analyses, without which these analyses cannot be undertaken. 
Table 3. Overview sampling and data collection in ORCHESTRA cohorts for vaccinated individuals. 
  
1st dose 
2nd dose1 
3 months2  
(± 1 month) 
6 months2 
(± 2 months) 
12 months2  
(± 3 months) 
 Objective 
NP swab 
x3 
x3 
x4 
x4 
x4 
Viral variant and 
metagenomic sequencing  
2 x 2 mL serum tube 
(serum) 
x 
x 
x 
x 
x 
Immune - serology and  
type I IFNs autoantbodies 
4 mL EDTA blood tube 
(plasma) 
x 
x5 
x5 
x5 
x5 
Immune - cytokine and 
chemokine 
1 (if possible 2) 9 mL 
heparin tube (PBMCs) 
x 
x5 
x5 
x5 
x5 
Immune - cellular 
2 x 2 mL EDTA tube  
(whole blood)  
x 
 
x 
x 
x 
Genetic and epigenetic 
analyses 
Stool sample 
(faeces or rectal swab) 
x 
 
x 
 
 
Metagenomic sequencing  
 
1. The assessment at 2nd dose is mandatory in patients who will receive such dose within 8-12 weeks 
after first dose (current AstraZeneca vaccination schedule). 
2. 3, 6, and 12 months counted from 1st dose. 
3. At least one timepoint at 1st or 2nd dose is required. 
4. At least one of the three timepoints (month 3, month 6, month 12) is required. 
5. At least one of the four timepoints (2nd dose, month 3, month 6, month 12) is required. 
Level I 
Assessments of Level I are mandatory 
Level II 
Customized according to the feasibility of each cohort 
 
 

 
 
 
 
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ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
4. DETAILED SAMPLE COLLECTION AND STORAGE INSTRUCTIONS 
4.1 NP SWAB COLLECTION 
4.1.1 Recommended materials 
Swabs 
• 
NP FLOQSwabs – Regular flocked swab (Copan Italia, Cat. No. 503CS01) 
Storage media (in order of preference) – use 1mL storage media per swab 
• 
DNA/RNAShield (Zymo Research, Cat. No. R1100-50 / R1100-250)  
• 
TRIzol (Invitrogen, Thermofisher Scientific, Cat. No. 15596026) 
• 
UTM Tubes – 12x80 mm tube size prefilled with 1 mL UTM (COPAN Italia, Cat. No. 
350C)  
• 
RNALater (Thermofisher Scientific, Cat. No. AM7021)  
Swab-medium combinations (in order of preference) 
• 
DNA/RNA Shield collection tube with swab – 12x80 mm screwcap vial pre-filled 
with 1 mL DNA/RNA Shield (Zymo Research, Cat. No. R1107) 
• 
NP UTM flocked Swabs – Regular NP FLOQSwab (Sterile) with tube (12x80 mm) 
prefilled with 1 mL UTM (COPAN Italia, Cat. No. 360C) 
• 
Sigma Virocult – Liquid viral transport media (1 to 2 mL) and regular flocked swab (Cat. 
MW951S or MW951S2ML) 
Due to the large number of available materials, we cannot provide an exhaustive list of acceptable swab-
medium combinations. We have presented some of the most common and well accepted solutions. If 
another is used for collection, it should contain a universal viral transport medium and not only be 
adapted to a single PCR technology (such as those provided by several RT-PCR manufacturers) with a 
transport medium volume ranging from 1 to 2 mL, and using a flocked swab. 
4.1.2 Sample collection 
1. Label the tube containing the storage medium as instructed in the section entitled “Labelling 
instructions”. 
2. Register the sample collection in the Requisition Form displayed in Appendix X. 
3. Collect the sample according to your routine practice protocol. 
4. Immediately after collection, place the swab into the tube containing storage medium. Ensure 
that the entire swab is immersed in medium. 
5. Break the swab at the scored line as instructed by the manufacturer. 

 
 
 
 
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ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
6. Using the aid of the cap, push that swab in the tube and close the cap tightly 
7. Transfer the sample to the Local Laboratory. 
4.1.3 Storage conditions 
• 
At arrival in the Local Laboratory, store the tube containing storage medium and the swab in 
designated boxes in the freezer at -70°C or below until shipment. 
• 
In case you do not have immediate access to a -70°C freezer, store them at -20°C and transfer 
them to a -70°C as soon as possible and within 2 days. Keep them at -70°C until shipment is 
arranged. 
 
 

 
 
 
 
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ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
4.2 SERUM SAMPLE COLLECTION 
4.2.1 Recommended materials 
• 
2 mL BD Vacutainer Serum tube (e.g., BD #368492) 
• 
2 mL Cryovial (e.g., Simport # T309-2A) 
• 
Disposable plastic pipettes (2.5, 5 mL size) 
4.2.2 Sample collection 
1. Label the Serum tubes as instructed in the section entitled “Labelling instructions”. 
2. Register the sample collection in the Requisition Form displayed in Appendix X.  
3. Draw the patient’s blood into two Serum tubes (2 mL).  
4. Slowly and gently invert the tubes 180° and back 5-6 times. 
5. Transfer as soon as possible (within one hour) to the Local Laboratory. 
4.2.3 Sample processing 
6. Before centrifugation, allow blood to clot thoroughly for 60 minutes.  
7. Label the tubes as instructed in the section entitled “Labelling instructions”. 
8. Centrifuge the sample at 1300 g for 10 min at 20°C WITH THE BRAKE ON.  
9. Transfer approx. 1 mL supernatant from each tube into separate cryovials using sterile 
disposable pipette taking care to not disturb the buffy coat. 
4.2.4 Storage conditions 
• 
After processing, store the cryovials as soon as possible in your freezer at -70°C or below until 
shipment. 
• 
In case you do not have immediate access to a -70°C freezer, store them at -20°C and transfer 
them to a -70°C as soon as possible and within 2 days. Keep them at -70°C until shipment is 
arranged. 
 
 

 
 
 
 
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ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
4.3 EDTA PLASMA ISOLATION 
In this section, two protocols for EDTA plasma isolation have been described. Depending on the 
laboratory protocol utilized, material required for isolation may vary. 
4.3.1 Recommended materials 
Common materials 
• 
4 mL BD Vacutainer K2E (EDTA) (e.g., BD Cat. No. 368861) 
• 
3 mL vial (e.g., Simport Cat. No. T309-3A) 
• 
Disposable plastic pipettes (2.5, 5 mL size) 
EDTA Plasma Protocol 1: Single-spin EDTA plasma isolation 
• 
No additional material is required. 
EDTA Plasma Protocol 2: Double-spin EDTA plasma isolation 
• 
3 mL BD Vacutainer EST Tubes (e.g., BD Cat. No. 362725) 
4.3.2 Sample collection 
1. Label the K2E (EDTA) tube as instructed in the section entitled “Labelling instructions”. 
2. Register the sample collection in the Requisition Form displayed in Appendix X. 
3. Draw the patient’s blood into the EDTA tube (4 mL).  
4. Slowly and gently invert the tube 180° and back 8-10 times. 
5. Transfer as soon as possible (within one hour) to the Local Laboratory. 
4.3.3 Sample processing 
EDTA Plasma Protocol 1: Single-spin EDTA plasma isolation 
6. The samples should be processed within 120 minutes. 
7. Label the 3 mL vial as instructed in the section entitled “Labelling instructions”. 
8. Centrifuge the sample at 1300 g for 10 min at 20°C WITH THE BRAKE ON. This will give 
three layers: (from top to bottom) plasma, leucocytes (buffy coat), and erythrocytes. 
9. Transfer approx. 2 mL of plasma into the 3 mL vial using sterile disposable pipette taking care 
to not disturb the buffy coat. 
EDTA Plasma Protocol 2: Double-spin EDTA plasma isolation 
6. The samples should be processed within 120 minutes. 
7. Label the 3 mL vial as instructed in the section entitled “Labelling instructions”. 
8. Register the sample collection in the Requisition Form displayed in Appendix X. 

 
 
 
 
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ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
9. Centrifugation I: Centrifuge the sample at 1500 g for 15 min at 20°C WITH THE BRAKE ON. 
This will give three layers: (from top to bottom) plasma, leucocytes (buffy coat), and 
erythrocytes. 
10. Collection of supernatant I: Transfer the plasma in a 3 mL centrifugation tube (e.g. 3 mL BD 
Vacutainer EST Tube) using sterile disposable pipette taking care to not disturb the buffy coat. 
11. Centrifugation II: Centrifugation at 2000 g for 15 min at 20°C WITH THE BRAKE ON to 
remove all potentially remaining cells. 
12. Collection of supernatant II: Transfer approx. 2 mL of plasma into the 3 mL vial using sterile 
disposable pipette taking care to not disturb the buffy coat. 
4.3.4 Storage conditions 
• 
After processing, store the cryovial as soon as possible in your freezer at -70°C or below until 
shipment. 
• 
In case you do not have immediate access to a -70°C freezer, store it at -20°C and transfer it to 
a -70°C as soon as possible and within 2 days. Keep them at -70°C until shipment is arranged. 
 
 

 
 
 
 
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ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
4.4 HEPARIN PLASMA COLLECTION AND PBMC ISOLATION 
In this section, three protocols for PBMC isolation have been described (Table 4). Depending on the 
laboratory protocol utilized, material required for isolation may vary. PBMC Protocol 1 and PBMC 
Protocol 2 are slightly more expensive but are time saving and easier to perform. These protocols employ 
specialized tubes that provide a clear separation, especially PBMC Protocol 1. PBMC Protocol 3 is a 
conventional protocol of PBMC isolation based on Ficoll-Paque as a separating medium, no specialized 
tubes are needed here. 
Table 4 also provides an approximate cost per sample by each of these procedures. These prices include 
the cost of tubes and separation media. Please note that these calculations are based on prices offered to 
UANTWERPEN in Belgium (Table 5). Sites are advised to get the quotations by the local distributors. 
Table 4. Common PBMC isolation procedures. 
PBMC isolation  
procedures 
  
Cost per sample 
  
 
  
Low  
volume 
Bulk 
Comment 
1 
SepMate tube /  
Ficoll-Paque PLUS 
Protocol 1 
8.83 € 
5.07 € 
Sepmate utilizes 15 mL separation medium  
(can process 4 mL to 17 mL blood) 
2 
LeucoSep tube /  
Lymphoprep medium 
Protocol 2 
7.94 € 
4.59 € 
LeucoSep utilizes 15 mL separation medium  
(can process 4 mL to 17 mL blood) 
3 
Falcon 50 mL tube /  
Ficoll-Paque PLUS 
Protocol 3 
5.93 € 
3.84 € 
Ficoll-Paque Plus separation utilizes 15 mL 
separation medium (can process 4 mL to 17 mL 
blood) 
4 
LeucoSep tube with  
pre-filled Lymphoprep 
optional 
7.20 € 
7.20 € 
LeucoSep tubes are pre-filled with Lymphoprep  
5 
BD CPT (tube with  
pre-filled Ficoll-Paque) 
optional 
15.11 € 
15.11 € 
CPT tubes are pre-filled with Ficoll-Paque  
 
 

 
 
 
 
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ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
Table 5. Cost of different centrifuge tubes and separation media. 
Product name 
Supplier 
Catalogue number 
Cost 
Unit price 
(EUR)  
per mL/tube 
Media 
Ficoll-Paque PLUS 
Cytiva 
17144003 
(6x100mL) 
6 x 100 mL – 225EUR 
0.38 
Ficoll-Paque PLUS 
Cytiva 
17144002 
(6x500mL) 
6 x 500 mL - 706EUR 
0.24 
Lymphoprep 
StemCell 
07801 (250mL) 
250 mL - 100 EUR 
0.40 
Lymphoprep 
StemCell 
07851 (500mL) 
500 mL - 156 EUR 
0.31 
Lymphoprep 
StemCell 
07811 (4x250mL) 
4x250 mL - 227 EUR 
0.23 
Lymphoprep 
StemCell 
07861 (6x500mL) 
6x500 mL - 531 EUR 
0.18 
Centrifuge tubes 
Falcon 50 mL tubes 
Greiner 
227261 
500 tubes - 51,40 EUR 
0.10 
LeucoSep 
Greiner 
227289 (non-sterile) 
300 tubes - 520,25EUR 
1.73 
LeucoSep 
Greiner 
227290 (sterile) 
300 tubes - 430,00EUR 
1.43 
SepMate 
StemCell 
85450 (100 tubes) 
100 tubes - 310EUR 
3.10 
SepMate 
StemCell 
85460 (500 tubes) 
500 tubes - 882EUR 
1.76 
Centrifuge tubes prefilled with separation medium 
BD CPT (pre-filled Ficoll-Paque) 
BD 
362753 
60 tubes - 894EUR 
14.90 
LeucoSep (pre-filled 
Lymphoprep) 
Greiner 
227288 (prefilled) 
25 tubes - 174,96EUR 
7.00 
 
 
 

 
 
 
 
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ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
4.4.1 Recommended materials 
Common materials 
• 
9 mL Lithium Heparin Tube (e.g., Greiner #455084) 
• 
Phosphate Buffered Saline (PBS) (e.g., Lonza #17-516F) 
• 
Fetal Bovine Serum (FBS), heat inactivated (e.g., Sigma 
#F7524) 
• 
Falcon tubes, 50 mL (e.g., Greiner #227261) 
• 
Dimethyl Sulfoxide (DMSO) (e.g., Sigma #D2650) 
• 
Cryo freezing tubes (e.g., Greiner #126263) 
• 
Cryo freezing container (e.g., Nalgene #5100-0001) 
• 
Pipette tips, 5-1000 µL 
• 
Disposable plastic pipettes (5, 10, 25, and 50 mL size) 
PBMC Protocol 1: PBMC isolation with SepMate tube/ Ficoll-
Paque Plus or Lymphoprep 
• 
Ficoll-Paque 
Plus 
(e.g., 
Cytiva 
#GE17-1440-02). 
Alternatively, Lymphoprep (StemCell #1114547) can also be 
used 
• 
SepMate tubes (e.g., StemCell #15440) 
PBMC Protocol 2: PBMC isolation with Leucosep / Lymphoprep 
• 
RPMI-1640 medium with L-glutamine and Hepes 25 mM 
(e.g., Lonza #12-115F) 
• 
L-glutamine (e.g., Lonza #BE17-605E) 
• 
Gentamycin (e.g., Sigma #G1397) 
• 
Penicillin-Streptomycin (e.g., Sigma #DE17-602E) 
• 
Lymphoprep (e.g., Lymphoprep #1114547) 
• 
Leucosep tubes (e.g., Greiner #227290) 
• 
Trypan blue stain (0,4%) (e.g., Gibco #15250-061) 
PBMC Protocol 3: Conventional Ficoll-Paque PBMC isolation protocol 
• 
Ficoll-Paque Plus (e.g., Cytiva #GE17-1440-02) 
4.4.2 Sample collection 
1. Label the Heparin tube as instructed in the section entitled “Labelling instructions”. 
2. Register the sample collection in the Requisition Form displayed in Appendix X. 

 
 
 
 
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ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
3. Draw the patient’s blood into one or two Heparin tubes (9 ml). 
4. Gently invert the tube 180° and back 5-6 times. 
5. Transfer as soon as possible (within one hour) to the Local Laboratory. 
 
 

 
 
 
 
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ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
4.4.3 Sample processing 
PBMC Protocol 1: PBMC isolation with SepMate tube/ Ficoll-Paque Plus or Lymphoprep 
 
Figure 1. PBMC isolation procedure using SepMate tubes. 
Buffer and other reagents preparation 
1. Warm up PBS + 2% FBS at 37°C. 
2. Ficoll-Paque or Lymphoprep (stored at ambient temperature of 18-20°C). 
3. Heat inactivated (56°C – 30 min) FBS and heat inactivated FBS-DMSO 20% at 4°C. 
4. Keep cryo freezing container (with isopropanol 100%) at 4°C. Replace isopropanol after 5 
freezings. 
PBMC Isolation (day of scheduled study visit) 
5. Label each tube as instructed in the section entitled “Labelling instructions”. 
6. Add 15 mL density gradient medium to the SepMate tube by carefully pipetting it through the 
central hole of the SepMate insert. The top of the density gradient medium will be above the 
insert. 
7. Spin tubes at 200 x g for 1 minute at room temperature. 
8. Dilute 8-9 mL of blood sample with an equal volume of PBS + 2% FBS. Mix gently (up to 16 
mL of blood with equal volume of PBS + 2% FBS can be loaded on one tube) 
9. Keeping the SepMate tube vertical, add the diluted sample by pipetting it down the side of the 
tube. The sample will mix with the density gradient medium above the insert.  
NOTE: The sample can be poured down the side of the tube. Take care not to pour the diluted 
sample directly through the central hole.  
10. Centrifuge at 1200 x g for 10 minutes at room temperature, WITH BRAKE ON.  

 
 
 
 
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ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
11. Pour off the top layer, which contains the enriched PBMCs, into a new tube. Do not hold the 
SepMate tube in the inverted position for longer than 2 seconds.  
NOTE: Some red blood cells (RBCs) may be present on the surface of the SepMate insert after 
centrifugation. These RBCs will not affect performance. 
12. Wash enriched PBMCs by adding PBS + 2% FBS to top off the 50 mL tubes. Cap the tubes and 
mix by gently inverting tubes several times. 
13. Spin tubes at 300 x g for 8 minutes at room temperature WITH BRAKE ON. 
14. Dump supernatant into a waste in one smooth motion being careful not to disturb the cell pellet. 
15. Repeat the wash of enriched PBMCs by adding PBS + 2% FBS to top off the 50 mL tubes. Cap 
the tubes and mix by gently inverting tubes several times. 
16. Spin tubes at 300 x g for 8 minutes at room temperature WITH BRAKE ON. 
17. Dump supernatant into a waste in one smooth motion being careful not to disturb the cell pellet. 
To speed up the procedure, PBMCs are not counted before freezing. Instead, one cryotube of 1 mL is 
prepared per 8-9 mL of blood. 
(ONE TO TWO 9 ML TUBES ARE RECOMMENDED FOR EACH SAMPLE) 
18. Resuspend the pellet in multiples of 0.5ml FBS (previously kept at 4°C) for every 8-9 mL of 
processed blood volume. 
19. Label cryo-tubes as instructed in the section entitled “Labelling instructions”. 
20. Transfer 0.5 mL of the resuspended PBMCs to the labelled cryo-tubes containing 0.5 ml 
FBS/DMSO 20% (previously kept at 4°C). The final storage solution is therefore FBS/DMSO 
10%. 
21. Close and invert cryo-tubes and transfer to cryo-freezing container (previously kept at 4°C). 
 
 

 
 
 
 
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ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
PBMC Protocol 2: PBMC isolation with Leucosep / Lymphoprep 
Buffer and other reagents preparation 
1. RPMI complete media (supplemented with 10% fetal bovine serum, 1% L-glutamine, 0.1% 
gentamicin and 1% Pen/Strep). 
2. Warm up RPMI and RPMI complete media at 37°C. 
3. Lymphoprep (stored at ambient temperature of 18-20°C). 
4. Heat inactivated (56°C – 30 min) FBS and heat inactivated FBS-DMSO 20% at 4°C. 
5. Keep cryo freezing container (with isopropanol 100%) at 4°C. Replace isopropanol after 5 
freezings. 
PBMC Isolation (day of scheduled study visit) 
6. For each sample, pipet 15 mL of lymphoprep into a 50 mL leucosep tube. Prepare appropriate 
number of tubes per sample (max 35 ml of diluted blood per leucosep tube). 
7. Label each tube as instructed in the section entitled “Labelling instructions”. 
8. Spin tubes at 200 x g for 1 minute at room temperature. 
9. Add blood into a leucosep tube and fill till 50 mL with warm RPMI 1640 complete media. Mix 
gently. 
10. Spin tubes at 880 x g, for 20 minutes at room temperature WITHOUT BRAKE. 
11. After centrifugation, the tubes will contain 4 layers (from top to bottom): 1) a plasma layer, 2) 
a cloudy interface layer of PBMCs containing white blood cells: lymphocytes + monocytes, 
3) lymphoprep layer, 4) RBC layer with the granulocyte layer on top. 
12. Collect the ring (PBMC layer) and place cloudy interface layer into a new 50 mL centrifuge 
tube, combining cells from the various tubes of each individual participant sample. 
13. Add RPMI to top off the 50 mL tubes containing the PBMCs. Cap the tubes and mix by gently 
inverting tubes several times. 
14. Spin tubes at 690 x g, for 10 minutes at room temperature WITH BRAKE ON. 
15. Dump supernatant into a waste in one smooth motion being careful not to disturb the cell pellet. 
16. Add 1 mL RPMI to tube and resuspend the cell pellet by gently swirling the tubes. 
17. Add RPMI to top off the 50 mL tubes containing the PBMCs. Cap the tubes and mix by gently 
inverting tubes several times. 
18. Spin tubes at 480 x g, for 10 minutes at room temperature WITH BRAKE ON. 
19. Dump supernatant into a waste in one smooth motion being careful not to disturb the cell pellet. 
20. Add 1 mL RPMI to tube and resuspend the cell pellet by gently swirling the tubes. 

 
 
 
 
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ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
21. Add RPMI to top off the 50 mL tubes containing the PBMCs. Cap the tubes and mix by gently 
inverting tubes several times. 
22. Spin tubes at 260 x g, for 10 minutes at room temperature WITH BRAKE ON. 
23. Dump supernatant into a waste in one smooth motion being careful not to disturb the cell pellet. 
To speed up the procedure, PBMCs are not counted before freezing. Instead, one cryotube of 1 mL is 
prepared per 8-9 mL of blood. 
(ONE TO TWO 9 ML TUBES ARE RECOMMENDED FOR EACH SAMPLE) 
24. Resuspend the pellet in multiples of 0.5 ml FBS (previously kept at 4°C) for every 8-9 mL of 
processed blood volume. 
25. Label cryo-tubes as instructed in the section entitled “Labelling instructions”. 
26. Transfer 0.5 mL of the resuspended PBMCs to the labelled cryo-tubes containing 0.5 ml 
FBS/DMSO 20% (previously kept at 4°C). The final storage solution is therefore FBS/DMSO 
10%. 
27. Close and invert cryo-tubes and transfer to cryo-freezing container (previously kept at 4°C). 
 
 

 
 
 
 
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ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
PBMC Protocol 3: Conventional Ficoll-Paque PBMC isolation protocol 
 
Figure 2. Liquid phases at different PBMC isolation stages.  
Buffer and other reagents preparation 
1. Warm up PBS at 37°C. 
2. Ficoll-Paque Plus (stored at ambient temperature of 18-20°C). 
3. Heat inactivated (56°C – 30 min) FBS and heat inactivated FBS-DMSO 20% at 4°C. 
4. Keep cryo freezing container (with isopropanol 100%) at 4°C. Replace isopropanol after 5 
freezings. 
PBMC Isolation (day of scheduled study visit) 
5. Dilute the blood 1:1 in PBS.  
6. Mix the blood and PBS by inverting the tube several times or by drawing the mixture in and out 
of a pipette.  
7. Invert the bottle of Ficoll-Paque Plus several times to ensure proper mixing.  
8. Add Ficoll-Paque Plus (15 mL) to a new 50mL centrifuge tube. 
9. Carefully layer the diluted blood sample onto the Ficoll-Paque Plus.  
IMPORTANT: When layering the sample do not mix the Ficoll-Paque Plus and the blood 
sample. 
10. Centrifuge at 400 g for 30-40 min at 18-20°C with WITHOUT BRAKE.  
11. Remove the upper layer containing plasma and platelets using a sterile Pasteur pipette, leaving 
the mononuclear cell layer undisturbed at the interface. The upper layer, which contains the 
plasma, may be discarded or saved for later use.  
12. Using a clean Pasteur pipette transfer the lymphocyte layer to a clean 50 mL centrifuge tube. It 
is critical to remove all the material at the interface but in a minimum volume. 
13. Estimate the volume of the transferred mononuclear cells. Add at least 3 volumes (~ 6 mL) of 
PBS to the PBMCs in the centrifuge tube.  

 
 
 
 
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ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
14. Suspended the cells by gently drawing them in and out of a Pasteur pipette. 
15. Centrifuge at 400-500 g for 10-15 min at 18-20°C WITH BRAKE ON. 
16. Discard the supernatant.  
17. Suspend the lymphocytes in 6-8 mL of PBS by gently drawing them in and out of a Pasteur 
pipette.  
18. Centrifuge at 400-500 g for 10-15 min at 18-20°C WITH BRAKE ON  
19. Discard the supernatant. 
To speed up the procedure, PBMCs are not counted before freezing. Instead, one cryotube of 1 mL is 
prepared per 8-9 mL of blood. 
(ONE TO TWO 9 ML TUBES ARE RECOMMENDED FOR EACH SAMPLE) 
20. Resuspend the pellet in multiples of 0.5mL FBS (previously kept at 4°C) for every 8-9 mL of 
processed blood volume. 
21. Label cryo-tubes. 
22. Transfer 0.5 mL of the resuspended PBMCs to the labelled cryo-tubes containing 0.5 mL 
FBS/DMSO 20% (previously kept at 4°C). The final storage solution is therefore FBS/DMSO 
10%. 
23. Close and invert cryo-tubes and transfer to cryo-freezing container (previously kept at 4°C). 
4.4.4 Storage conditions 
• 
Transfer the cryo-freezing container to -70°C or below. 
• 
After 1-3 days, transfer cryo-tubes to liquid N2 or store at -70°C until shipment is arranged. 
 
 

 
 
 
 
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ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
4.5 WHOLE BLOOD COLLECTION 
4.5.1 Recommended materials 
• 
 2 mL BD Vacutainer K2E (EDTA) (e.g., BD Cat. No. 368841) 
4.5.2 Sample collection 
1. Label two EDTA tubes as instructed in the section entitled “Labelling 
instructions”. 
2. Register the sample collection in the Requisition Form displayed in Appendix X. 
3. Draw the patient’s blood into two EDTA tubes (2 mL).  
4. Slowly and gently invert the tubes 180° and back 8-10 times. 
4.5.3 Sample processing 
5. Snap freeze the EDTA tubes containing whole blood samples in liquid nitrogen or freeze them 
at -20 °C or -80 °C. 
4.5.4 Storage conditions 
• 
Store the cryovial as soon as possible in your freezer at -70°C or below until shipment. 
• 
In case you do not have immediate access to a -70°C freezer, store them at -20°C and transfer 
them to a -70°C as soon as possible and within 2 days. Keep them at -70°C until shipment is 
arranged.  
 
 

 
 
 
 
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ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
4.6 STOOL SAMPLE COLLECTION 
4.6.1 Recommended materials 
• 
Faecal sampling TUBE (e.g APTACA #2688) 
• 
RNALater solution (e.g, Thermo Fisher Scientific #AM7021 / 
#AM7024 (500 and 100mL respectively) or Sigma-Aldrich 
#R0901) 
• 
Freezer for storage (ideally -80°C, also -20°C can be temporarily used) 
4.6.2 Sample collection 
1. Label the collection tube as instructed in the section entitled “Labelling instructions”. 
2. Collect of faecal matter according to your routine practice protocol. 
3. Transfer the sample to the laboratory 
NOTE: In case the sample cannot be transferred to the laboratory immediately, the sample can 
be stored at +4°C up to 24 h in a proper container (RNase- DNase- free, sterile tube e.g.,Thermo 
Fisher Scientific #AM12501) or directly in the faecal sampling tube. 
4.6.3 Sample processing 
4. Label transfer tube as described in Section 5. 
5. Transfer 1-2 g faecal matter to the faecal sampling tube. 
6. Add 5-10mL of RNALater solution (all the specimen must be covered) and gently mix by 
inverting the tube few times. 
NOTE: In case RNALater is unavailable, direct freezing at -70°C without adding any carrier 
fluid is also acceptable. 
4.6.4 Storage conditions 
• 
At arrival in the lab, store the tube containing faecal matter (and storage medium where 
applicable) in the freezer at -70°C or below until shipment. 
• 
In case you do not have access to a -70°C freezer, samples can be stored temporarily at -20°C 
for 2-3 weeks. 
• 
If needed, samples can be stored +4°C for up to 24 h immediately after collection before transfer 
to the Local Laboratory. 
 

 
 
 
 
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ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
4.7 RECTAL SWAB SAMPLE COLLECTION 
4.7.1 Recommended materials 
• 
Fecal SWAB (e.g. APTACA CliniSwab #2160/SG or #2170/SG). 
NOTE: if you choose other brands pay attention the swab comes 
without any carrier fluid or surface treatment (dry swab) 
• 
RNALater solution (e.g, Thermo Fisher Scientific #AM7021 / 
#AM7024 (500 and 100mL respectively) or Sigma-Aldrich #R0901) 
• 
Freezer for storage (ideally -80°C, also -20°C can be temporarily 
used) 
4.7.2 Sample collection 
1. Label transfer tube as instructed in the section entitled “Labelling instructions”. 
2. Remove the swab from the tube (do not touch the swab tip, always hold the shaft applicator 
above the marked breakpoint) 
3. Insert the flocked swab through the rectal sphincter 1-1.5 inches (2.5-4cm) and gently rotate it 
between your fingers 
4. Remove the swab and examine to make sure there is faecal material visible on the tip of the 
swab 
5. Transfer the swab into the tube. Holding the end of the swab shaft, place the marked breaking 
point against the rim of the tube and bend it to break at the marked breakpoint. Discard the 
broken upper part and tighten the screw cap. 
6. Transfer the sample to the laboratory 
NOTE: In case the sample cannot be transferred to the laboratory immediately, the sample can 
be stored at +4°C up to 24 h 
4.7.3 Sample processing 
7. Add 2-10mL of RNALater solution, the sufficient volume to fully submerge the swab tip, which 
may vary according to the chosen brand/model of the swab.  
NOTE: In case RNALater is unavailable, direct freezing at -70°C without adding any carrier 
fluid is also acceptable. 
4.7.4 Storage conditions 
• 
At arrival in the lab, store the tube containing the swab (and storage medium where applicable) 
in the freezer at -70°C or below until shipment. 
• 
In case you do not have access to a -70°C freezer, samples can be stored temporarily at -20°C 
for 2-3 weeks. 

 
 
 
 
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ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
• 
If needed, samples can be stored +4°C for up to 24 h immediately after collection before transfer 
to the Local Laboratory. 
 
 

 
 
 
 
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ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
5. STORAGE INSTRUCTIONS AND DESTINATION SITES 
Table 6. Overview of samples to be shipped per time point per patient and their shipping conditions. 
Sample type 
Number of 
tubes/samples 
Storage temp. (°C) 
Shipping 
temp. (°C) 
Destination site 
NP swab 
1 
-70°C or below 
Dry ice 
INSERM 
UANTWERPE
N 
Serum 
2 
Short-term at -20°C, 
long-term at -70°C 
or below 
Dry ice 
INSERM 
UANTWERPE
N 
EDTA plasma 
1 
Short-term at -20°C, 
long-term at -70°C 
or below 
Dry ice 
UANTWERPE
N 
PBMC 
1 or 2 
-70°C or below, or 
liquid nitrogen 
Dry ice 
UANTWERPE
N 
2 mL EDTA whole 
blood 
2 
-20°C or -70°C or 
below 
Dry ice 
INSERM 
UNIBO 
HMGU 
Stool or rectal swab 
1 
+4°C (up to 24 h), 
long-term at -70°C 
or below 
Dry ice 
UNIBO 
 
6. CONTACT DETAILS 
6.1 QUESTIONS CONCERNING PROTOCOLS FOR BIOLOGICAL SAMPLING 
NP swabs 
Benoit Visseaux (INSERM/APHP); email: benoit.visseaux@aphp.fr  
Surbhi Malhotra-Kumar (UANTWERPEN); email: surbhi.malhotra@uantwerpen.be  
Serum – Plasma 
Samir Kumar-Singh (UANTWERPEN); email: samir.kumarsingh@uantwerpen.be  
Benoit Visseaux (INSERM/APHP); email: benoit.visseaux@aphp.fr 
PBMC 
Samir Kumar-Singh (UANTWERPEN); email: samir.kumarsingh@uantwerpen.be  
Whole blood 
Laurent Abel (INSERM); email: laurent.abel@inserm.fr  
Valerio Carelli (UNIBO); email: valerio.carelli@unibo.it  

 
 
 
 
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ORCHESTRA has received funding from the European Union’s Horizon 2020 research and innovation programme under grant 
agreement No 101016167 
 
Stool – Rectal swabs 
Patrizia Brigidi (UNIBO); email: patrizia.brigidi@unibo.it  
 
6.2 COORDINATING LABORATORY FOR SAMPLE SHIPMENTS 
University of Antwerp, Laboratory of Medical Microbiology, 
Universiteitsplein 1, S 620 
2610 Wilrijk, BELGIUM 
Email:  christine.lammens@uantwerpen.be 
 
matilda.berkell@uantwerpen.be 
Tel.: +32 3 265 93 32 
Working hours: Weekdays from 08:30 to 17:30 (GMT +1)