Serum Prevalence of Dars-Cov-2 Antibodies in Pre-pandemic Blood Samples (SPARE)
Serum Prevalence of Dars-Cov-2 Antibodies in Pre-pandemic Blood Samples (SPARE)
The first reports of infections caused by SARS-CoV-2 were released from Wuhan, China in
 December 2019. From there, the infection quickly spread into a pandemic form. The clinical
 manifestation of the infection varies enormously, from totally asymptomatic or mildly
 symptomatic forms, with nonspecific and flu-like manifestations, to an acute respiratory
 distress syndrome which, in patients requiring hospitalization in the ICU and mechanical
 ventilation invasive, can lead to death, especially in elderly subjects and carriers of
 co-morbidities. Recently, the association of blood groups ABO as possible biological markers
 of susceptibility to COVID-19, has been evaluated, linking blood type O with a lower chance
 of infection, blood type A with the highest risk, and blood group B with the greatest
 complications.

 In Italy, the first non "imported" case dates back to February 2020, although new evidence on
 subjects tested positive for the antibody assay on serum samples suggests that the virus
 started to circulate before the official date.

 Few are the data relating to asymptomatic infections or with mild non-specific and nuanced
 symptoms that have been quantified in about 85% of the total number of infected. Moreover,
 thanks to the availability of serological tests that identify the presence of anti-SARS-CoV-2
 antibodies, it emerged that a proportion of the population was infected by the virus and
 developed an antibody response and that almost 30% of the people with antibodies were
 asymptomatic. In order to evaluate the seroprevalence of COVID-19 infection among
 asymptomatic subjects we will conduct specific serological tests (total antibodies) to
 identify the prevalence of SARS-2-CoV antibodies among healthy blood donors who went to
 transfusion facilities of the DIMT in Venice during the pandemic (about 2500 periodic donors
 who referred to the Transfusion Center in the period between May and October 2019). Patients
 afferent to the Interinstitutional Multidisciplinary Biobank (BioBIM) of the Research Center
 of the IRCCS San Raffaele Pisana in Rome, with or without concomitant co-morbidities (about
 1000 subject) will be also tested. All samples will be analyzed for any association with sex,
 age group and blood group.
1. Title Serum prevalence of Sars-Cov-2 antibodies in pre-pandemic blood samples

 2. Background The first reports of infections caused by SARS-CoV-2, a Beta Corona virus,
 single-stranded RNA, were released from Wuhan, China in December 2019 [1,2]. From there,
 the infection quickly spread into a pandemic form.

 The clinical manifestation of the infection varies enormously, from totally asymptomatic
 or mildly symptomatic forms, with nonspecific and flu-like manifestations, to an acute
 respiratory distress syndrome which, in patients requiring hospitalization in the ICU
 and mechanical ventilation invasive, can lead to death [3]. The greater severity of the
 course of the infection depends greatly on the general conditions of the patients, being
 maximum in elderly subjects and carriers of co-morbidities [4]. Recent studies have also
 evaluated the association of blood groups ABO as possible biological markers of
 susceptibility to COVID-19, suggesting that people with blood type O have a lower chance
 of infection while individuals with blood type A are most at risk, and people with blood
 group B have the greatest complications in the event of infection [5,6].

 In Italy, the first non "imported" case dates back to February 2020 in a person residing
 in Lombardy, in the province of Lodi [7]. However, new evidence on subjects tested
 positive for the antibody assay on serum samples suggests that the virus started to
 circulate before the official date.

 Initially, due to the sudden explosion of cases, epidemiological surveillance focused on
 patients related to health facilities because they were symptomatic, suspicious and
 high-risk contacts. Few are the data relating to asymptomatic infections or with mild
 non-specific and nuanced symptoms that, data from China, would quantify in about 85% of
 the total number of infected. Precisely on the role of person-to-person transmission in
 the course of asymptomatic infection and in the subclinical phases of the disease,
 epidemiological data are lacking despite they could prove to be very important. In fact,
 thanks to the availability of serological tests that identify the presence of
 anti-SARS-CoV-2 antibodies, it emerged that a proportion of the population was infected
 by the virus and developed an antibody response [8] and that almost 30% of the people
 with antibodies were asymptomatic. [9]. Therefore, the understanding of the prevalence
 of the disease and the prevalence of the antibody response appear to be of considerable
 interest, as it seems to be established that asymptomatic subjects can transmit the
 SARS-CoV-2 infection for a period that in some cases has proved greater than 14 days
 [10].

 In order to evaluate the seroprevalence of COVID-19 infection among asymptomatic
 subjects, thus obtaining a real estimate of the beginning of the circulation of the
 virus in Italy, in our study we intend to conduct specific serological tests (total
 antibodies) to identify the prevalence of SARS-2-CoV antibodies among healthy blood
 donors who went to transfusion facilities of the DIMT in Venice during the pandemic
 (about 2500 periodic donors who referred to the Transfusion Center in the period between
 May and October 2019).

 Samples belonging to healthy volunteers (about 500) who have agreed to donate their
 blood will also be evaluated to establish a pool of control subjects belonging to the
 Interinstitutional Multidisciplinary Biobank (BioBIM) of the Research Center of the
 IRCCS San Raffaele Pisana in Rome. Finally, in order to evaluate the impact of
 seroprevalence in subjects with chronic diseases (about 1000), such as cardiovascular,
 oncological, respiratory and neurodegenerative ones, which represent settings of
 COVID-19 positive patients with a poorer prognosis, patient samples with the listed
 comorbidities, already present in BioBIM, will be analyzed. It has recently emerged that
 seroprevalence increases with increasing age and that younger groups have a lower
 susceptibility to infection [11]. Therefore, all samples will be analyzed for any
 association with sex, age group and blood group.

 3. Promoter Prof.ssa Fiorella Guadagni

 4. Sponsor/Promotor IRCCS San Raffaele Pisana

 5. Site IRCCS San Raffaele Pisana

 6. Study type Multicentric, retrospective, observational, non pharmacological, on
 biological samples, conducted at the IRCCS San Raffaele Pisana, Rome, aimed at verifying
 the seroprevalence of SARS-COV-2 antibodies in the population.

 7. Objectives 7.1 PRIMARY Objective: The aim of the study is to determine the proportion of
 people in the general population who developed an antibody response to antigenic
 determinants of SARS-CoV-2 in the period preceding the spread of the virus in Italy.

 7.2 SECONDARY Objective: Secondary objectives are represented by the possibility to
 characterize the differences in prevalence between genders, age groups and blood groups,
 also in relation to more unfavourable courses in subjects with other chronic diseases.

 8. Participants

 The study protocol will involve a total of 3500 subjects (3000 healthy subjects and 1000
 patients with chronic diseases) aged> 18 years, of both sexes. The two groups will be
 formed as follows:

 A- Healthy subjects among periodic blood donors of the Transfusion Center and volunteer
 donors of BioBIM B- Patients with chronic diseases including cardiovascular,
 respiratory, oncological and neurodegenerative diseases related to BioBIM

 9. Inclusion/exclusion Criteria 9.1 Inclusion criteria - Both sexes

 -  Age >18 years

 -  Signed informed consent for the donation / sample storage in the Biological Bank
 9.2 Exclusion criteria

 -  for healthy subjects, all those who have shown any symptoms, albeit mild, in the 28
 days prior to the donation.

 -  patients who have not signed the informed consent for the conservation of their
 samples in the Biological Bank.

 9.3 Drop out Not applicable.

 10. Study design

 The study will last 9 months, and includes 3 phases as follows:

 Phase 1: identification of samples Phase 2: sample analysis Phase 3: statistical
 analysis

 11. Methods of contact with possible participants in the study Not applicable. The study
 will be carried out only on samples stored in the Biobank.

 12. Methods of acquisition, statistical analysis of data and storage of raw data, compliance
 with privacy regulations Data collection will be carried out on a dedicated and
 anonymized database by the Principal Investigator. Data will be stored on a dedicated
 and protected server.

 13. Statistical Methodology 13.1 Statistical analysis Statistical analysis of all parameters
 will be conducted under the responsibility of the Biomarker Discovery and Advanced
 Technologies (BioDAT) Unit of IRCCS San Raffaele Pisana.

 Data analysis will be carried out using frequency and percentage for the categorical
 variables, and mean and standard deviation for the quantitative variables. The t-test will be
 used to compare means and a p-value less than 0.05 will be considered statistically
 significant. All analysis will be performed on the SPSS Statistical Package for Social
 Sciences.

 13.2 Sample size The SPARE study is a pilot study, designed to verify the adequacy and
 feasibility of the project, and to obtain preliminary data that allow to determine the size
 of the sample of the final study. The sample size is therefore represented by a consecutive
 number of subjects who donated blood in the period May-October 2019 (about 2500) and all
 subjects (healthy and with chronic diseases) who referred to BioBIM in 2019 (about 1500 ).

 14) Requirements of originality and scientific soundness of the study The SPARE study aims to
 evaluate the seroprevalence of COVID-19 infection among asymptomatic subjects and thus obtain
 a real estimate of the beginning of the circulation of the virus in Italy. The evidence
 currently present in the literature refers mainly to the period of the actual pandemic, but
 there are no data relating to the months prior to autumn 2019. Same for the official data
 released by the Ministry of Health in collaboration with the National Institute of Statistics
 (ISTAT) [11]. This study will allow us to define the spread of the SARS-2-CoV virus in a
 period long before the date to which the pandemic began in Italy.

 15) Expected Results

 The main expected results are:

 1. Identification of seroprevalence of anti-SARS-2-CoV antibodies in the population prior
 to the date on which the beginning of the infection in Italy is officially traced;

 2. Description of any associations with sex, age, blood group, outcomes of patients with
 chronic diseases.

 16) Progress report The proposers will submit a report on the progress of the study to the
 Ethics Committee at the end of each year and at the end of the study.

 17) Feasibility

 - n. patient samples that may be included is consistent with the study design

 -  presence of the necessary equipment

 -  adequacy of personnel 18) Concomitant treatments Not applicable. The SPARE study is a
 study on biological samples only 19) Cost/benefit ratio The study is supported by the
 IRCCS San Raffaele Pisana. The inclusion of samples belonging to healthy donors and
 patients in this clinical study may provide benefits in terms of more accurate
 management and acquisition of indications on the spread of the Sars-COV-2 virus in the
 asymptomatic population. There are no risks.

 20) Ethical relevance The study is conducted on patient samples stored in the Biological
 Banks, therefore there are no problems of an ethical nature to its execution.

 21) Insurance The procedures provided for the study in this centre do not directly
 involve patients, but the analysis of samples taken in the centres they enrol. The
 researcher assigned to the analysis of the samples is covered by an insurance policy
 included in the contract stipulated with the IRCCS San Raffaele Pisana.

 22) Result publication In compliance with the provisions in force regarding the
 confidentiality of sensitive data and patent protection, the results of the study will
 be submitted for publication within 6 months of after the closure of the database.

 23) Informed Consent The submission of specific informed consent for this study on
 anonymized samples from biological banks or large collections does not apply.

 24) Ethical aspects This protocol will be conducted in accordance with the ethical
 principles that draw their origin from the Declaration of Helsinki, respects the GCP and
 applicable regulatory provisions.

 25) References

 1. Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, et al. China Novel Coronavirus
 Investigating and Research Team A Novel Coronavirus from Patients with Pneumonia in
 China, 2019. N Engl J Med. 2020;382(8):727-33. 10.1056/NEJMoa2001017

 2. World Health Organization (WHO). Novel coronavirus - China. Geneva: WHO; 12 Jan
 2020. [Accessed 09 Oct 2020]. Available from:
 https://www.who.int/csr/don/12-january-2020-novel-coronavirus-china/en/

 3. Santos-Sánchez NF, Salas-Coronado R. Medwave. Origin, structural characteristics,
 prevention measures, diagnosis and potential drugs to prevent and COVID-19. 2020
 Sep 25;20(8):e8037. doi: 10.5867/medwave.2020.08.8037.

 4. Luo L, Fu M, Li Y, Hu S, Luo J, Chen Z, Yu J, Li W, Dong R, Yang Y, Tu L, Xu X. The
 potential association between common comorbidities and severity and mortality of
 coronavirus disease 2019: A pooled analysis. Clin Cardiol. 2020 Oct 7. doi:
 10.1002/clc.23465.

 5. Zaho J, Yang Y, Huang H, Li D, Gu D., Lu X, et al. Relationship between the ABO
 Blood Group and the COVID-19 susceptibility. 10.1101/2020.03.11.20031096

 6. Zalba Marcos S, Luisa Antelo M, Galbete A, Etayo M, Ongay E, García-Erce JA.
 Infection and thrombosis associated with COVID-19: Possible role of the ABO blood
 group. Med Clin (Engl Ed). 2020 Sep 26. doi: 10.1016/j.medcle.2020.06.013.

 7. Livingston E, Bucher K. Coronavirus Disease 2019 (COVID-19) in Italy. JAMA.
 2020;323(14):1335. 10.1001/jama.2020.4344

 8. Mizumoto K, Kagaya K, Zarebski A, Chowell G. Estimating the asymptomatic proportion
 of coronavirus disease 2019 (COVID-19) cases on board the Diamond Princess cruise
 ship, Yokohama, Japan, 2020. Euro Surveill 2020;25(10):2000180.

 9. ISTAT. Primi risultati dell'indagine di sieroprevalenza sul SARS-CoV-2. Accessible
 presso https://www.istat.it/it/archivio/246156

 10. Oran DP, et al. Prevalence of Asymptomatic SARS-CoV-2 Infection - A Narrative
 Review. Ann Int Med 2020; 173:362-376. doi.org/10.7326/M20-3012.

 11. Pagani G, et al. Seroprevalence of SARS-CoV-2 significantly varies with age:
 Preliminary results from a mass population screening. J Infect. 2020 Sep
 19;S0163-4453(20)30629-0. doi: 10.1016/j.jinf.2020.09.021.
Inclusion Criteria:

 -  Both sexes

 -  Age >18 years

 -  Signed informed consent for the donation / sample storage in the Biological Bank

 Exclusion Criteria:

 -  for healthy subjects: all those who have shown any symptoms, albeit mild, in the 28
 days prior to the donation.

 -  for patients: who have not signed the informed consent for the conservation of their
 samples in the Biological Bank.