Data collection tool and variables.

During the second phase of the study, data will be collected through questionnaires and analytical procedures. This integrated approach will yield a comprehensive dataset, enhancing the study’s clinical and environmental assessment as a whole.

1. Questionnaires. The questionnaires have undergone a meticulous design process, incorporating both newly formulated sections and standardized or adapted questions from previously employed surveys in published studies [24–28]. To ensure their effectiveness, these questionnaires have been subjected to multiple revisions by experts across various departments within the field. This comprehensive review aimed to guarantee that the questions align with the constructs intended for assessment. Upon the initial phase of data collection, the robustness of these questionnaires will be further evaluated. Internal consistency will be assessed using the Cronbach’s alpha test, ensuring that the questions within each questionnaire measure the intended constructs reliably. This rigorous evaluation process is integral to maintaining the quality and precision of the data we aim to collect.

The following information will be collected through the questionnaires:

• Environmental characteristics of the settings (including surfaces, volumes, ventilation, and common areas), daily activities performed, and hygiene practices within the facilities
• Individual participant characteristics including general attributes, existing medical conditions such as respiratory infections and allergies, vaccination history, exposure to risk factors, socio-economic factors, any acute illnesses experienced during the study period, contact history with other individuals, and their consent for potential nasopharyngeal swab testing.

1.1 Environmental questionnaire: The purpose of this questionnaire is to understand the current hygiene policies and practices within the facilities and to identify factors that might influence the risk of ARIs.

The environmental questionnaire (EQ) is specifically designed for the managers of the various settings. It evaluates the environmental structure and conditions, as well as individual and group behaviour and practices within these settings.

The questionnaire will cover several aspects, including personal and environmental hygiene practices, cleaning and disinfection of common areas, ventilation, air quality and daily activities. It will consist of a series of multiple-choice and open-ended questions to cover different environmental factors in order to obtain specific information on participants behaviour, their movements and contacts within the facility. In addition, the questionnaire will assess the efficacy of existing policies on preventing the spread of respiratory viruses, as well as the facility’s ability to adopt any policy changes in a timely and effective manner. The EQ will be administered once at the beginning of the survey to the facility managers and will be administered again later if there are substantial changes in the facility. The questionnaire will take approximately 8 minutes to complete.

1.2 Baseline clinical questionnaire: The objective of the baseline clinical assessment questionnaire (BCQ) is to provide a complete and accurate picture of the participant’s clinical status, identifying potential risk factors and individuals susceptible to developing complications from ARIs. The questions have been crafted by combining established question formats, including inquiries about pre-existing respiratory symptoms, allergic history, and risk factors [24–26], with the integration of newly devised questions designed to align with the objectives of our project.

The BCQ is intended for all participants and consists of a series of multiple-choice and open-ended questions covering different aspects of the participant’s medical history, such as the presence of previous illnesses, risk factors for respiratory infections, previous vaccinations, respiratory and allergic symptoms, socio-economic factors, and exposure to risk factors such as smoking and alcohol consumption. The data collected by the BCQ will allow a baseline assessment of the subject’s condition and evaluate a possible correlation with respiratory virus infections. The BCQ will only be administered once at the beginning of the survey, or when a new participant joins the study (e.g. transfer to the facility). The questionnaire will take approximately 12 minutes to complete.

1.3 Acute clinical assessment questionnaire: The objective of the Acute Clinical Assessment Questionnaire (ACAQ) is to collect information that will allow us to monitor the evolution of the subject’s condition over time, to identify possible clusters of respiratory infections within the community environment, and to take the necessary prevention and control measures to prevent the spread of infection. The questions have been formulated by merging established question formats pertaining to acute symptoms and manifestations [27, 28] with newly integrated inquiries that specifically align with the objectives of our project.

The ACAQ is an essential tool for the early identification of any symptoms associated with ARIs and for monitoring the evolution of the subject’s condition over time. It is designed to identify all participants presenting with respiratory symptoms, such as cough, sore throat, difficulty breathing or fever, and is intended to provide a detailed and timely clinical assessment of participants. The questionnaire consists of a series of specific questions regarding the subject’s current symptoms, the duration of symptoms, any current therapies and contact with other individuals who have experienced similar symptoms. The completion of each questionnaire will take approximately 6 minutes.

1.4 Methods of questionnaire administration: The questionnaires will be administered with different methods and supports based on the target. The method of administration, support and frequency are summarised in Table 1 for each setting and target population.

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Table 1. Types and methods used for the questionnaires in the various settings of the Stell-ARI study.

https://doi.org/10.1371/journal.pone.0309111.t001

For individuals who are unable to fill out the questionnaire due to health or social reasons (such as residents in LTCFs and inmates), the questionnaires will be administered during a face-to-face interview. Specifically trained interviewers will ask the questions and record the answers on paper forms or tablets, depending on the setting. For other participants (such as parents of students and staff), the questionnaires will be self-administered in paper form, on tablet, or through the web portal Influweb (for primary school). These questionnaires are designed to be simple, easy to understand and fill, and are accompanied by short and clear instructions. They will be distributed along with a brief explanation of the survey’s objectives, to motivate the participation and cooperation of participants. Completed paper questionnaires will be collected in a dedicated closed box to guarantee the anonymity of the participant and will be transported to the Department of Biology Department of the University of Pisa. For tablet or web portal administration (https://influweb.org/welcome), study participants will receive a letter of introduction and detailed instructions.

For the school setting, questionnaire data is collected through the platform of Influweb, which is an Italian specialised digital platform designed for the efficient administration of surveys and questionnaires related to data on Influenza-Like Illness (ILI). It is a project supported by Fondazione ISI, Fondazione CRT and from the European projects Epipose and PANDEM-2, part of the EU Horizon 2020 program. It provides a user-friendly interface for data collection and analysis, making it particularly valuable in research and public health initiatives. This technology enhances our ability to effectively detect and respond to ILI outbreaks, ultimately contributing to the prevention and control of these illnesses [29].

2. Analytical procedures. Analytical procedures will involve:

• Nasopharyngeal swabs in symptomatic subjects: detection of the etiological agents for confirmation of the infection
• Air analysis: assessment of microclimatic conditions, chemical indicators, microbiological indicators, and the presence of key pathogens in the air
• Surface Analysis: assessment of microbiological indicators and the presence of key pathogens on various surfaces
• Wastewater analysis: quantification of genomic copies of target viruses in wastewater samples.
• Proximity sensors (in school setting): detection of close encounters among students to analyse the risk of transmission within the school environment.

2.1 Clinical sampling: Biological material, such as surface cells, mucus, and secretions from the respiratory tract, will be collected using nasopharyngeal swabs. These diagnostic tests are designed to detect pathogenic microorganisms that cause upper respiratory tract infections. The swabs are minimally invasive, easy to perform, and painless. The sampling procedure involves using a thin, sterile, single-use wadded stick. This procedure will be carried out by the facility staff (nursing staff in the LTCFs and prison settings) and by parents and staff themselves in school setting, with the consent of the study subject or their guardian. To ensure proper execution and prevent sample contamination, appropriate personal protective equipment (PPE) will be used, as recommended by the Centers for Disease Control and Prevention (CDC) [30]. After collection, the swab will be placed in a sterile tube containing a transport medium. Following hand hygiene protocols, the collector will label the tube with a pseudonymised code and arrange for its transportation to the laboratory via a dedicated service.

2.2 Environmental microbiological sampling: To identify highly contaminated areas, an extensive distribution of "exposed plates" containing a non-selective medium will be deployed. After a 6-hour exposure period, these plates will be incubated, and colony-forming units will be counted [31].

For air sampling, automatic samplers will be positioned in common areas to detect bacterial and viral contamination on a monthly basis. Active samplers will be employed, which draw in air and either impact it on a plate (e.g., SAS—Surface Air System sampler) or place it into a liquid (e.g., impinger sampler). In particular:

• Bacterial counts will be obtained by using an impactor sampler, with Rodac plates containing Plate Count Agar (PCA) or selective medium. Subsequently, the plates will be incubated.
• Viral analysis will be performed by using Impinger Sampler, consisting of an air inlet structure and a collection vessel filled with a liquid medium, Viral Transport Medium (VTM) [32].

Surface sampling will also be conducted in these areas on a monthly basis. Sterilized swabs will be swiped across 36 cm² areas [33]. For viral analysis, virus will be eluted from the swabs with 1 mL of 3% beef extract solution at pH 9, whereas for bacteriological analysis elution will be performed with 0.9% NaCl solution [34].

In wastewater analysis, the primary focus will be on target viruses. Trained personnel will perform weekly sampling at the exit point of each facility via a sewer drain outside the building. Necessary safety measures, including the use of PPE, will be strictly applied. A passive sampling method will be employed, as suggested for small communities with irregular sewage flow [35]: this involves immersing passive materials (electronegative filter membranes or gauze swabs) into the wastewater flow and leaving them for an extended duration [36]. Viruses will subsequently be eluted from these passive materials with 1 aliquot of sterile phosphate buffer solution. The solution will be centrifuged at 4500 rcf for 10 minutes to remove debris and then analysed using a commercial kit.

2.3 Microbiological analyses: Air and surface samples will be analysed for indicators of general/human microbial contamination (total bacterial load at both 22°C and 37°C, as well as Staphylococcus aureus and Escherichia coli) using conventional cultural methods. Specific respiratory viruses: Human Adenovirus (HAdV), Influenza Virus A and B (IVA and IVB), SARS-CoV-2, Respiratory Syncytial Virus Type A and Type B (RSVA and RSVB) will be detected using PCR techniques. The same RT-PCR protocols, summarised in Table 2, will be applied to both clinical and environmental samples. For wastewater samples, the amount of genome copies obtained will be adjusted on the basis of the sewage flow to estimate the daily viral emissions from the community.

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Table 2. PCR protocols used for the detection of viral targets.

https://doi.org/10.1371/journal.pone.0309111.t002

2.4 Environmental chemical samples: To identify the most contaminated sampling points within the three structures being studied, a preliminary chemical air characterization will be performed using diffusive samplers coupled with gas chromatography and mass spectrometry (TD-GC-MS). This protocol is able to monitor a wide range (C3-C30) of volatile organic compounds (VOCs) and semi-volatile organic compounds (SVOCs) [40]. The passive sampler will be placed at various sampling points for 6 hours allowing VOCs and SVOCs to be retained by the sorbent material of the sampler. After sampling, the diffusive bodies of the samplers will be hermetically sealed with Swagelock caps avoiding any external contamination. Routine chemical analysis will be carried out monthly by transferring an aliquot (1 L) of air samples with a suction pump into stainless steel absorbent systems packed with 250 mg of Tenax Gr 60/80 mesh. Prior to sampling, each absorbent tube will be spiked with 30 ng of labelled 8D-toluene to ensure an accurate quantification of the analytes. The result of the chemical analysis will contain information on the qualitative chemical composition of the air samples as well as the concentration levels of each contaminant found. An Arduino-based platform will be installed at the different sites to continuously monitor temperature, humidity, CO2 levels, PM2.5, and total VOCs.

2.5 Proximity sensors (students only): Proximity sensors gather the time-resolved proximity relationships between each sensor and the others within the system, along with information on the sensor’s state of motion obtained through an on-board accelerometer in the device. This technology offers a promising approach for monitoring and managing social interactions, providing both the frequency and duration of contact. The sensors collect data, which will be stored internally in the devices (tags) and extracted at the end of data collection, via a physical connection between the sensors and the operators’ computers [23]. All students who have given their consent will be required to wear the proximity devices every day in class for a period of 10 to 15 days.

The data collected will include patterns of close person-to person encounters, interaction dynamics between different groups, and pathways of environmental exposure, using diverse indicators. The high spatial and temporal resolution infrastructure will enable monitoring the number of individual contacts, encounter durations, cumulative interaction times, and encounter frequencies between any two individuals. For each pair, we will collect the following data: occurrence and frequency of each contact, time spent in each encounter, and cumulative contact duration per subject. In relation to the collected data, the following metrics will be obtained: number of distinct contacts, total number of contacts and cumulative contact time. In addition to representing a minimally invasive method of data collection, the tags require only minimal effort from participants. Proximity sensors will enable accurate, automated and pseudonymised detection of close encounters among students within the school environment.

Statistical analysis.

Subject characteristics will be described by means of medians and interquartile ranges for numerical variables and absolute frequencies and percentages for categorical variables. The time course of the environmental variables will be presented through graphical displays.

Associations between infections detected via ACAQ and confirmed by clinical swabs (when necessary) and exposure factors detected by the QCB and the environmental air and surface monitoring will be assessed employing logistic regression models taking potential confounders into account. Statistical methods and analysis algorithms will be used to examine the collected data by proximity sensors and identify contact patterns, interaction frequencies and other relevant metrics.

WBE data will be normalised and adjusted to reduce variations arising from excretion and sewage flow. Environmental data will be treated as continuous variables, with regression models featuring splines used to estimate the time course of the measured parameters. The relationship between ARIs incidence and environmental data will be explored via time series regression models. In the event of missing data, imputation models will be employed, and sensitivity analysis will evaluate the impact of outliers on results. All analyses will encompass the entire dataset or stratified by setting. All statistical tests will be two-tailed with a significance level set at α = 0.05. Data analysis will be carried out using the R statistical software [41].

QMRA.

QMRA is a formal risk assessment process (based on a modified chemical risk approach) that includes the classical steps of hazards identification and characterization, exposure assessment, and risk characterization. This process involves formulating and applying models using fields and literature data to represent a scenario while addressing uncertainties. QMRA models will be designed and applied to the four selected target viruses and for the different settings in order to assess the risk of ARI based on environmental contamination measured by the microbiological air and surface monitoring. The contamination values, along with the estimated frequency and duration of exposure through inhalation and contact obtained from questionnaires, will be used to calculate the dose, which is the amount of pathogen assumed in a unit of time. Probabilities of infection and disease will then be estimated by applying virus-specific dose-response relationships. These probabilities will be expressed as functions that take into account variability and uncertainties, using Monte Carlo Analysis applied through Vensim software. A sensitivity analysis will also be performed to identify the most influential variables [42].

Ethical considerations and privacy.

The study has been approved by the Bioethics Committee of the University of Pisa, on October 11, 2023, under protocol number 0136245/2023 Review No. 43/2023. The investigators ensure that the study will be conducted in full compliance with the European legislation on clinical studies [Regulation (EU) No 536/2014] and its national transposition [DM 15 July 1997; D.Lvo 211/2003; D.L.vo 200/2007 and D.L.vo. 52/2019] and the principles of the Declaration of Helsinki in order to ensure maximum protection of the subjects involved. The principal investigator agrees that the study will be conducted in accordance with this protocol and Good Clinical Practice (GCP). The principal investigator has provided to the relevant Ethics Committee and Competent Authorities the study protocol and any related documents provided to the patient (Information Notice and Informed Consent Form). If changes to the protocol become necessary during the course of the study, the sponsor will submit an appropriate protocol amendment request to the relevant Ethics Committee, the approval process will align with the Committee’s established procedures.

The project with the planned interventions and the administration of questionnaires at various times during the course of the study, will not have an impact on the health status of the participants. Respect for persons and human dignity, fair distribution of the benefits and burdens of research will be ensured, while protecting the values, rights and interests of study participants. The project will also consider the social, cultural and historical experiences of the patients involved. The research process will not perpetrate or endorse, directly or indirectly, any aspect of discrimination, stigma or exclusion. During the activities, the project will uphold the Avoiding Harm Principle: risks will be assessed with respect to their physical, social and psychological implications for participants.

In addition, participants are invited to take part in the project following the procedure outlined in the ‘Method of administration of questionnaires’ section. Obtaining informed consent from the participants involves informational sessions where explanation of the study’s objectives and methods were carried out. Leaflets has been distributed in multiple languages to accommodate the diverse backgrounds of our participants. Actual participation starts only after individuals have signed the informed consent form, indicating their willingness to join. It is the responsibility of the investigators, or their representatives, to obtain informed consent from surveyed subjects (or their parents, for students) after they have been adequately informed about the aims, methods, expected benefits and foreseeable risks of the study. The investigators or designees also inform the participants that non-participation or discontinuation will not result in any harm or negative consequences.

Participation is entirely voluntary, and the signed informed consent forms will be kept by the respective facilities (LTCFs, Prison and Primary school) for the duration of the study.

Communication and dissemination.

The project will be illustrated in the enrolled facilities, intermediate and final results will be published in peer reviewed journals, scientific reports, presentations at conferences, seminars and symposia and will be used to inform public agencies and authorities about evidence-based strategies to link wastewater, air and surface matrices with respiratory health in closed communities, the data will be presented in an aggregated and anonymous manner and no information that could identify participants will be reported in any way. Intermediate and final results will be shared through scientific publications and academic conferences. Information material concerning the use of the results will be produced for relevant stakeholders, including the project’s sponsor (Ministry of University and Research with reference to mission 4—Education and Research—of the National Recovery and Resilience Plan, NRRP).

However, the views and opinions expressed are solely those of the authors and do not necessarily reflect those of the European Union or the European Commission. Neither the European Union nor the European Commission can be held responsible for them.

Role of the promoter and the investigators.

The role of the promoter and the investigator in the study is established in the technical annex of the Stell-ARI project, on the basis of which the financial resources corresponding to the planned commitment have been allocated. In detail, the promoter is responsible for developing the study design, the data collection tools and the analysis plan. The investigators are involved in the definition of the study design and are responsible for enrolment. Data collection will be performed by the investigators, commissioned facilities and, limited to schools, by the ISI Foundation via the Influweb platform. The promoter, with input from all investigators, is responsible for writing the scientific and dissemination reports. All investigators will be included in the authorship of the products resulting from this study.

Ownership of data processing.

The University of Pisa, with headquarter at Lungarno Pacinotti 43 in Pisa, Italy represented by the acting Rector, and, exclusively for the school setting, in collaboration with the ISI Foundation, headquartered at Via Chisola 5 in Turin, Italy represented by the President.