key: cord-0811557-ovv37leg authors: Bognanni, Antonio; Schiaffino, Armando; Pimpinelli, Fulvia; Donzelli, Sara; Celesti, Ilaria; Strano, Sabrina; Solari, Elena; Schiaffino, Giorgia; Solari, Gabriele; Solari, Domenico; Delbue, Serena; Rigoni, Marta; Nollo, Giandomenico; Muti, Greta E.; Muti Schünemann, Giovanna E.U.; Schünemann, Holger J; Blandino, Giovanni; Morrone, Aldo; Muti, Paola title: The Conundrum of Giglio Island: unraveling the dynamics of an apparent resistance to COVID-19 – A descriptive study date: 2021-03-02 journal: Comput Struct Biotechnol J DOI: 10.1016/j.csbj.2021.02.008 sha: 0c1bd4e9679d609124677f6db1ec674de0944f1c doc_id: 811557 cord_uid: ovv37leg OBJECTIVES: Despite an extensive risk of exposure to COVID-19, the residents of Giglio Island, Italy, did not develop any symptom of SARS-CoV-2. The present study aims to characterize the nature of exposure and to describe the local population dynamics underlying its apparent resistance to COVID-19. METHODS: Descriptive study of an islander partially-segregated population cohort based on a seroprevalence screening conducted from Aprile 29 to May 3, 2020 and including SARS-CoV-2 seroprevalence and viral prevalence in samples of saliva assessed through RT-qPCR. Serologic testing was performed using a COVID-19 IgG/IgM rapid test while molecular analyses were carried out by Allplex 2019-nCoV Assay (Seegene). RESULTS: A total of 634 residents out of 748 (84.8%) present at the time, and 89 non-residents underwent serological testing. 364 males and 359 females with a median age of 58.5 years. The serological screening identified one positive, asymptomatic subject. The Nucleic Acid Amplification Tests (NAAT) did not yield any positive result. CONCLUSION: Despite extensive exposure to SARS-CoV-2, possibly only one new asymptomatic infection occurred in this population, as documented by IgM positivity not confirmed by RT-qPCR. This may be due to unknown protective factors or chance. On the basis of this baseline study, using its population as a reference model, further investigations will be conducted to test the advanced hypotheses, focusing on the evaluation of a possible cross-reactivity to SARS-CoV-2 from exposure to endemic viruses. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease . (1) It is a beta-coronavirus with a close structural and phylogenetic relationship to other pathogens of the "Coronaviridae" viral family, such as SARS-CoV-1(2) and MERS-CoV. (3) While both the international and Italian scenarios were dominated by uncertainties, born out of the fragile balance between the need for drastic interventions and the inevitable economic repercussions, the virus found its way to "Isola del Giglio", the same island off the coast of Tuscany, where, in 2012, the Italian cruise ship Costa Concordia ran aground and overturned after striking an underwater rock. It remains one of the few places in the world where despite the arrival of the SARS-CoV-2, no case of symptomatic COVID19 has been diagnosed among the inhabitants. This observation has caught a broad international media coverage (4) and is the subject of this article. The aim of the study was to assess whether the observed "resistance" to SARS-CoV-2 had a serological foundation or to identify asymptomatic cases, seeking to detect SARS-CoV-2 antibodies through the screening process, simultaneously laying the foundation to future investigations focused on unraveling the causal dynamics of the apparent resistance. The imported cases will be thoroughly described later in the article. All of these, except for one, reached the island prior to implementation of distancing measures, while being asymptomatic and later presented symptoms, exposing the population to significant infectious risk, as both phases have been associated with virus transmission.(5-9) Despite the prolonged exposure in a densely populated scenario, the infectious potential of the affected subjects (5) (6) (7) (8) and the lack of distancing measures, the resident population did not develop COVID-19 syndromic features in the following months raising profound interest in the local medical community. This study received ethical approval on April 25, 2020 by the Ethical Committee of the "Istituto Nazionale Malattie Infettive -Lazzaro Spallanzani". Geographically, Giglio Island has a mountainous terrain, with ninety percent of its surface being inhabitable or inaccessible due to natural barriers. The last census dates back to May 2019, counting 1,423 inhabitants, in three main settlements. The island covers a surface of 24.1 km 2 with a population density of 59 inhabitants/km. 2 To estimate the island population density at the time of the serological screening, our study adjusted the surface area as illustrated in Table 1 , excluding extra-urban lots, using the official structural plan and maps, provided as a guidance by the municipality and publicly available online.(10) Legend: areas A -B -B* (high-density areas), C -C** (low-density areas). Public areas for communal activities (F1-3) have been considered as well to account for potential physical interactions. Given the adjusted surface of 0.44771 km 2 , the resulting population density is 1670.7 inhabitants/km 2 . By omitting "Giglio Campese" to avoid a negatively skewed distribution, the resulting estimate in population density is 2511.62 pp/Km 2 , which would rank the local municipality as the 122 th most densely populated in Italy (Top 1.54 percentile out of 7903 Municipalities). This is an important information as population density is a likely factor correlated with spread of COVID-19.(11) During lockdown, the connection to the mainland was through a ferry route with two daily trips covering 11 marine miles. Despite the maximum capacity of 600 passengers, the ferries carried a daily average of ten individuals, rendering the island practically isolated. From February to April 2020, 5 people were tested positive for SARS-CoV-2 while on Giglio Island. Four of these individuals (three males and one female) reached the island before the Italian government imposed social distancing and preventive measures on March 9, 2020. The following is a description of the cases that reached the island, as summarized in Table 2 . After arriving, the man resided in his family home, together with his parents and sister. On March 21, 2020 the patient developed low-grade fever and cough and was ordered to isolate. On March 24, 2020 the man tested positive for COVID-19 and was asked to quarantine in Giglio-Porto. The man's condition remained stable and did not require advanced medical care. On April 9, 2020 he was tested negative by RT-PCR. The population screening by serological rapid test on SARS-CoV-2 began on April 29 and ended on May 3, 2020. We initiated population screening for SARS-CoV-2 by inviting all adult residents who were present on the island. Eighty-five people, including healthcare professionals and volunteers, after testing negative at the serological antibody and salivary molecular tests, assisted in conducting the screening. The participation of island inhabitants was assessed and monitored through cross-checking the updated documents provided by the municipality. The testing was performed in pre-determined locations. Subjects signed an informed consent. A health professional administered a questionnaire asking for background information (age,gender) permanence on the island and smoking status. All data were recorded in digital form. Each participant underwent rapid serological testing and provided a self-collected sample of saliva. The island population has been tested through the use of COVID The IgG/IgM out of 723 participants. The asymptomatic subject found IgM-positive was tested twice more to corroborate the finding. The man had reached the island on the same ferry as Case 4. Case 1 could not be tested as deceased prior to study beginning; Cases 2 and 5 refused to be tested or provide a biological specimen. A total of 723 samples of saliva were collected from the population. Of these, 66 could not be analyzed due to collection of insufficient material. The NAAT on patients' saliva did not identify any subject positive for SARS-CoV-2 RNA. Despite of the introduction of SARS-CoV-2 on Giglio Island by five cases, known to have had physical interaction with local population, our study found one asymptomatic subject, positive to IgM but negative to RT-qPCR and confirmed the absence of either current or past viral infection in 633 residents of this highly populated area through qualitative assessment of anti-SARS-CoV-2 antibodies and RNA in saliva. We monitored the island population for two additional weeks after the study termination, so to detect possible false negatives to rapid ki, but no inhabitants developed any COVID19 related signs or sypmtoms. There are several strengths to our study. The fact that the island population remained isolated throughout the lockdown period creates a closed cohort model with few confounding factors and a reasonably well characterized exposure. Also, the data and sample collection of our study has been properly standardized on the basis of predetermined criteria, as to increase reproducibility and limit bias. The high participation rate also reduces concerns of selection bias. There are also some limitations. Despite extensive exposure to SARS-CoV-2, only one new asymptomatic infection occurred in this population, as documented by IgM but not by RT-qPCR. This may be due to unknown protective factors or chance. On the basis of this first descriptive study, we will carry out further investigations to test the advanced hypotheses using its population as a reference model. In particular, our immediate research activities will focus on examining crossreactivity to SARS-CoV-2, investigating a possible humoral contribution, both systemic (IgG/IgM mediated) and localized (IgA mediated), accounting also for potential interaction with the residing microbiota. We will also conduct a second serological screening following the population's exposure during a busy summer season that brought thousands of tourists to the island. We will study whether and how the island inhabitants were potentially infected with SARS-Co V-2 during that period. 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Science (80-) SARS-CoV-2-reactive T cells in healthy donors and patients with COVID-19 Author Statement Conceptualization methodology, Investigation, Data Evaluation, Writing Armando Schiaffino: Conceptualization Methodology, Investigation Fulvia Pimpinelli: Validation-Investigation Sara Donzelli: Investigation Ilaria Celesti : Investigation Sabrina Strano: Writing -Review & Editing Elena Solari: Investigation Giorgia Schiaffino: Investigation Gabriele Solari: Investigation Domenico Solari: Investigation Serena Delbue: Investigation Marta Rigoni: Conceptualization methodology, Writing-Review & Editing Giandomenico Nollo:C onceptualization methodology, Writing-Review & Editing Greta E. Muti: Investigation Giovanna E.U. Muti Schünemann: Investigation Holger J Schünemann: Conceptualization methodology -Writing -Review & Editing Giovanni Blandino: Resources, Writing -Review & Editing Aldo Morrone: Resources -Writing -Review & Editing Conceptualization Methodology -Resources -Writing -Review & Editing, Supervision We would like to express our graditude to the population of Giglio Island who generously participated in the study, the island volunteers who directly worked on the study planning and implementation, including the "Confraternita Misericordia" in Giglio Island. We also thank the Municipality of Giglio Island, the Regional Health System local Unit "Toscana Sud-Est" Director and managers, the pharmacy in Giglio Island, the Association "Together in Pink" from Castiglione della Pescaia, the Grosseto General Hospital, the Carabinieri of Giglio island local station and Municipality Police for their important and efficient support to the study. Our graditude is also directed to Elisa Milano, Giulia Orlandi e Alina Catalina Palcau from the Oncogenomic and Epigenetic Unit at IRCCS Regina Elena National Cancer Institute, who all assisted in preparing the biologic sample for genetic testing. Furthermore, we thank the "Istituto Nazionale Malattie Infettive -Lazzaro Spallanzani", and in particular both the Scientific Director Giuseppe Ippolito and the Institutional Review Board for their important inputs about the study implementation. Finally, we thank the General Director of the Tuscany Region for the strong support of the study and the availability to provide kits for serological assessment of the island population.