To determine the frequency of respiratory viral co-infections in a post-pandemic cohort, and the impact of SARS-CoV-2, influenza, and RSV.
This is a longitudinal, prospective, observational cohort study evaluating patterns of AVRIs in children and their qualitative effects on the immune system by age and virus. Figure 1 illustrates the sequence of study visits. A total of 200 participants will be followed for two years with in-person visits and bio-specimen collection. Enrollment will be staggered over a four-year period, with study activities continuing for five years to allow for longitudinal follow-up. We plan to enroll 30 participants in Year 1, 60 in Year 2, 60 in Year 3, and 50 in Year 4. This phased approach ensures that the number of active participants at any given time remains manageable, allowing adequate capacity for timely sample collection and data capture during respiratory viral illnesses across all enrolled age groups. Accrual will be on a rolling basis, and dropouts will be replaced.
The study is being conducted at the Uniformed Services University (USU) through the Translational Medicine Unit (TMU), located on the Naval Support Activity (NSA) Bethesda military installation in Bethesda, Maryland. The TMU is equipped with state-of-the-art facilities for conducting clinical research and is dedicated to advancing translational science, particularly in the context of military health. The location facilitates the integration of military-specific considerations and enables recruitment of military-connected children, while also allowing for the enrollment of civilian children, providing a diverse study population. The NSA Bethesda campus is frequented by both children of military and non-military families who work on the installation. The campus also houses two daycare centers and two military medical facilities, including the David E. Cabrera University Family Health Center at USU and the Walter Reed National Military Medical Center, both of which routinely provide pediatric care.
We will recruit up to 220 participants, stratified by age group, to account for an anticipated 10% attrition rate, ensuring a final sample size of 200 participants. The power analysis was based on 3D relationship between power, sample size, and the Cohen's omega effect size for a Chi-square test with 3 degrees of freedom. This analysis showed that a total sample size of N = 200 would be needed to detect Cohen's omega effect sizes as small as w = 0.23 with 80% power. This target may be adjusted if infection rates among study participants differ significantly from expected estimates. This sample size is designed to provide robust statistical power to examine age-based immune responses to SARS-CoV-2, influenza, and RSV, as well as the impact of co-infection on immune regulation. Participants will be divided into age groups as follows:
To ensure valid interpretation of immune responses, the study enrolls healthy participants with unaltered immunity. Therefore, individuals with known immunodeficiencies or those receiving immunomodulatory therapies are excluded. A study physician reviews all medical conditions and medications to assess eligibility, and any uncertainty regarding potential immunosuppressive effects is referred to the Principal Investigator for final determination. Additionally, because sample collection involves nasopharyngeal (NP) swabs and peripheral blood draws, individuals with conditions that could interfere with or contraindicate these procedures are excluded.
Participants are currently recruited through multiple strategies. Approximately twice weekly, study recruiters conduct tabling sessions at the Walter Reed National Military Medical Center Pediatric Immunization Clinic and the Pediatric waiting area, where they engage with parents and families either waiting for appointments or recently completing clinical visits. Recruiters also participate in health fairs and special events held on the NSA Bethesda campus by setting up informational tables and engaging with passersby. Flyers containing study details, contact information, and a link to the study website for self-referral are posted in and around the Walter Reed Pediatrics Clinic, University Family Health Center, outpatient pharmacies, USU, and NSA Bethesda daycare centers. Additional flyers are distributed at other military installations within a 50-mile radius of the research site, as well as community locations -- such as public libraries and civilian daycare centers -- within a 25-mile radius. Online recruitment methods include paid social media advertisements and listings through the clinical research promotion platform ResearchMatch®.
The informed consent process is completed electronically or via paper consent forms. The Informed Consent Document (ICD) and Assent Form were reviewed and approved by the USU Institutional Review Board (IRB) prior to study initiation. The ICD outlines the potential benefits and risks of participation, and states that participation is voluntary, withdrawal is allowed at any time, and refusal involves no penalty. Participants and/or their parent/guardian are provided the opportunity to ask questions to ensure understanding of the protocol prior to signing the ICD. For participants aged 7-17, an Assent Form (with parental/guardian permission) is signed before any study-related procedures. Parental/guardian permission is documented before obtaining assent from minors. For children aged 6 and under, a Waiver of Documentation of Assent is used, confirming the study is explained in age-appropriate terms and that the child either expresses verbal assent or lack of the capacity to do so.
Following enrollment, presentation for an Acute Visit is participant-driven. Reminders to contact the study team upon development of respiratory symptoms will include study-specific refrigerator magnets placed in the home and quarterly electronic newsletters summarizing study findings for participants and their parents/guardians. An attrition rate of 10% over the two-year study period has been projected. This estimate is informed by retention data from comparable pediatric longitudinal clinical trial cohorts, including those requiring peripheral blood collection, where attrition has ranged from 2 to 17% (median 8%) under active retention management. A proportion of our study population will be military-connected, and we recognize that frequent relocations among military families, in addition to general study burden, may increase the risk of attrition. To mitigate these risks, we have incorporated proactive retention strategies. Study staff will conduct routine participant contact (via phone, text, or email) to reinforce engagement, provide anticipatory guidance regarding upcoming visits, and address logistical or clinical concerns in real time. Frequent contact will also allow early identification of families planning to relocate, enabling timely replacement of participants who withdraw. Visit scheduling will remain highly flexible, with early morning and late afternoon availability, and study procedures will be coordinated with routine clinical encounters whenever feasible. These strategies are designed to strengthen rapport with families, reduce barriers to participation, and minimize the impact of anticipated moves, thereby increasing the likelihood of achieving the projected 10% attrition rate and preserving statistical power for both primary and subgroup analyses.
An overview of biospecimen procurement by visit type is depicted in Fig. 1. Participants have a minimum of two study visits: an Enrollment Visit and, for those who do not present with an acute illness within the first year of enrollment, a Second Baseline Visit. The number of Acute Visits depends on how many AVRIs participants develop each year, but Acute Visits do not occur more frequently than every 2 months, resulting in a maximum of six Acute Visits per year in addition to enrollment. Acute Visits specifically associated with SARS-CoV-2, influenza, or RSV infection require an additional Post-Acute visit. At every visit, participants are asked for an update to their health history, immunizations, and the date of their last symptoms of respiratory infection. Participants receive compensation for study-related sample collection, including NP swabs and peripheral blood draws.
Participants are enrolled when they are perceived to be in good health and do not meet the study-defined criteria for an acute respiratory viral infection for at least 14 days prior to the enrollment date. After informed consent is obtained, a study physician performs a history and physical exam to confirm eligibility. Two NP swabs are collected for pathogen identification via research-grade multiplex polymerase chain reaction (PCR) and downstream immunologic analysis. Peripheral blood is collected for baseline humoral immunity measures, following minimum risk guidelines.
Participants are eligible for a Second Baseline Visit if they either report no ARVIs during the 12-month period following enrollment or were unable to present for an acute visit within seven days of respiratory viral symptom onset. This visit includes an updated medical history, vital signs, and the collection of two NP swabs and peripheral blood, following minimum risk guidelines.
Participants present for an Acute Visit within seven days of meeting study-defined criteria for an acute respiratory viral infection as defined in Fig. 2. The visit includes an updated medical history, vital signs, and two NP swabs. One swab is pathogen detection via PCR, and the other is used for innate immune analysis and microbiome analysis. Participants receive a paper log to record viral symptoms for 10 days, as well as a follow-up email prompting electronic completion of the symptom questionnaire. If a swab is positive for SARS-CoV-2, influenza, or RSV, then a Post-Acute Visit is scheduled. If negative for those 3 pathogens (even if positive for a different virus), participants continue monitoring for subsequent AVRIs and return for additional Acute Visits, which occur no more than every 2 months. Of note, participants who test positive for pathogens other than SARS-CoV-2, influenza, or RSV, as well as those who test negative for all three, will not have a scheduled Post-Acute Visit.
Participants with a positive SARS-CoV-2, influenza, or RSV swab from an Acute Visit return for a Post-Acute Visit 10-21 days after symptom onset. This visit includes an updated medical history, vital signs, and the collection of two NP swabs and peripheral blood, following minimum risk guidelines.
Following an Acute Visit, participants or their caregivers complete a symptom questionnaire to assess type and frequency of symptoms and assess illness impact (see Supplementary Material S1). Participants or their caregivers rate the severity of 25 symptoms using a Likert scale (0-3, where 3 is severe) daily for 10 days. Additionally, the questionnaire assesses if medication was used for symptomatic relief, when the participant returned to baseline health, if medical attention was sought (and type if applicable), the number of days the participant missed school or daycare, number of days their caregiver missed work, and the number of people in the household who developed symptoms following the participant. Participants receive compensation for completing the symptom questionnaire.
Two NP swabs are collected per visit. The first NP swab is placed in 3 mL of universal transport media (UTM), the second into 1 mL of UTM. After collection, NP swabs are immediately transported to the lab. Swabs are stored at 4 °C and processed within 1 h of collection. Multiplex PCR testing typically occurs within 2 h of sample collection; however, PCR testing is validated for up to seven days when stored at 4 °C.
Peripheral blood samples are collected in acid citrate dextrose (ACD) tubes. Immediately after drawing the sample, the tube is gently inverted 8-10 times and subsequently stored at room temperature (20 °C) prior to processing.
Specimens collected in this study, including both NP swabs and peripheral blood, undergo comprehensive analyses to evaluate both respiratory viral infections and immune responses. From the NP swab supernatant, 800 ml of universal transport medium is extracted and used for a multiplex PCR assay designed to detect a broad spectrum of upper respiratory tract pathogens, targeting 18 viruses, including SARS-CoV-2, influenza, and RSV, and four bacteria with high sensitivity and specificity overall and for each individual pathogen (see Supplementary Table S2). Any residual supernatant is stored for potential future testing with expanded pathogen panels, allowing for surveillance of emerging respiratory infections as new pathogens are identified.
Cells are isolated from NP swabs through gentle mechanical action and centrifugation. After centrifugation supernatant is removed and stored at -80 °C. Cells from both NP swabs are combined, and an aliquot of cells is taken for analysis of cell surface markers by flow cytometry. The remainder of the cells are cryopreserved in 10% DMSO for single-cell RNA sequencing.
Peripheral blood samples are analyzed for systemic circulating immune responses. Peripheral blood mononuclear cells (PBMCs) and plasma are isolated and cryopreserved for downstream analysis to include measurement of antibody responses and T cell phenotype, and function.
Data variables collected in this study are detailed in Table 1. Data obtained at Enrollment, Acute, Post-Acute, and Second Baseline Visits are directly entered or transcribed from paper source documents into electronic case report forms (CRFs) at the study site via Research Electronic Data Capture (REDCap®). Data entered on CRFs, as well as corrections made, are only performed by delegated study staff.
All data transmitted to and from the application is done using a secure, encrypted transmission
(SSL/HTTPS). For increased security, the application additionally verifies the SSL certificate of the REDCap® server in order to validate the server's identity. The REDCap® mobile application employs encryption-at-rest on the mobile device's hard drive; therefore, data and information stored on the device are protected from unauthorized users. REDCap® has built-in safeguards such as username, personal identification number, and remote lockout. Access to study files (e.g., CRFs) is restricted to research team members with task-specific access.
Data collected through this protocol is coded and does not contain personal identifiers. Much of the subsequent research using the coded materials collected under the auspices of this study will be accomplishable through approved exempt or non-human use sub-studies. All research using the materials collected under this protocol is conducted in accordance with an IRB-reviewed protocol, and future analysis will require a new or modified IRB-reviewed protocol. Data and specimens released for research use are labeled by a unique identifier only; data or specimen are shared without any identifiers or links to individuals.
This study was approved by the USU Institutional Review Board under protocol number USUHS.2023-121.
The primary objectives are to assess the frequency of viral co-infections in an outpatient pediatric population and to evaluate differences in innate immune responses across infection types, stratified by age group and respiratory pathogen. In the analysis, we will cross-tabulate the frequency of infection/non-infection events into a (2 × 2 × 2 × 4) contingency table having dimensions of SARS-CoV-2, influenza (Yes, No), RSV (Yes, No), and age category (4 groups). The null hypothesis of interest is that the age-category differences in log-odds ratios equal zero for all pairwise differences in age category. We will also perform a Breslow-Day analysis for the homogeneity of for the odds ratios of one infection type across age groups by another infection type. For example, are the odds of influenza infection profile by age the same for RSV-positive and RSV-negative participants?
A secondary analysis will analyze the seasonality of co-infections as a time series. To analyze this data, we will cross-tabulate the frequency of (SARS-CoV-2, influenza, RSV) infection by week. Once this is accomplished, we will perform a seasonal decomposition of the (SARS-CoV-2, influenza, RSV) time-series, which decomposes each time-series into (trend, seasonal, residual) parts. Once each seasonal decomposition is complete, we will then analyze the correlations between the seasonal and trend components of each timeseries. All analyses will be adjusted for age and other relevant covariates, with significance set at p < 0.05.
Another analysis will include an omics approach to assess whether differences in means for various inflammatory cytokines or RNA transcriptomic biomarkers vary between infection types (yes/no). To perform this analysis, we will fit a Linear Mixed Effects (LME) model, which uses the collection of inflammatory or RNA transcriptomic biomarkers as the outcome variable. The fixed effect of the model will be a binary indicator of infection for each of the three infections (SARS-CoV-2, influenza, RSV). The random effects of the LME model will be subject-specific intercepts. The null hypotheses are that the (yes-no) difference in mean equals zero. To account for the vast number of hypotheses, the Benjamini-Hochberg procedure will be utilized to maintain the False Discovery Rate (FDR) at 10%.
To analyze whether the mean cytokine levels differ by age group and infection type, we will fit a 4-way ANOVA-type linear mixed effects (LME) model to the data. The response variable in this analysis will be the cytokine level. The fixed effects of this LME model will include age group, SARS-CoV-2 (Yes, No), influenza (Yes, No), and RSV (Yes, No), as well as the two-way interactions of age group with each infection type (Age-SARS-CoV-2, Age-influenza, Age-RSV). The random effects will consist of subject-specific intercepts. The null hypotheses are that the pairwise differences in mean cytokine levels by age group are equal to zero for SARS-CoV-2, influenza, and RSV positive groups. Tukey's Honest Significant Difference (Tukey's HSD) method will be used to control the Type I error rate due to multiple hypothesis testing.