At-Home Saliva Sampling in Healthy Adults Using CandyCollect, a Lollipop-Inspired Device.

Respiratory infections are common in children, and there is a need for user-friendly collection methods. Here, we performed the first human subjects study using the CandyCollect device, a lollipop-inspired saliva collection device .We showed that the CandyCollect device can be used to collect salivary bacteria from healthy adults using Streptococcus mutans and Staphylococcus aureus as proof-of-concept commensal bacteria. We enrolled healthy adults in a nationwide (USA) remote study in which participants were sent study packages containing CandyCollect devices and traditional commercially available oral swabs and spit tubes. Participants sampled themselves at home, completed usability and user preference surveys, and mailed the samples back to our laboratory for analysis by qPCR. Our results showed that for participants in which a given bacterium (S. mutans or S. aureus) was detected in one or both of the commercially available methods (oral swab and/or spit tubes), CandyCollect devices had a 100% concordance with the positive result (n = 14 participants). Furthermore, the CandyCollect device was ranked the highest preference sampling method among the three sampling methods by 26 participants surveyed (combining survey results across two enrollment groups). We also showed that the CandyCollect device has a shelf life of up to 1 year at room temperature, a storage period that is convenient for clinics or patients to keep the CandyCollect device and use it any time. Taken together, we have demonstrated that the CandyCollect is a user-friendly saliva collection tool that has the potential to be incorporated into diagnostic assays in clinic visits and telemedicine.

homeRNA self-blood collection enables high-frequency temporal profiling of pre-symptomatic host immune kinetics to respiratory viral infection: a prospective cohort study.

Background: Early host immunity to acute respiratory infections (ARIs) is heterogenous, dynamic, and critical to an individual's infection outcome. Due to limitations in sampling frequency/timepoints, kinetics of early immune dynamics in natural human infections remain poorly understood. In this nationwide prospective cohort study, we leveraged a self-blood collection tool (homeRNA) to profile detailed kinetics of the pre-symptomatic to convalescence host immunity to contemporaneous respiratory pathogens. Methods: We enrolled non-symptomatic adults with recent exposure to ARIs who subsequently tested negative (exposed-uninfected) or positive for respiratory pathogens. Participants self-collected blood and nasal swabs daily for seven consecutive days followed by weekly blood collection for up to seven additional weeks. Symptom burden was assessed during each collection. Nasal swabs were tested for SARS-CoV-2 and common respiratory pathogens. 92 longitudinal blood samples spanning the pre-shedding to post-acute phase of eight SARS-CoV-2-infected participants and 40 interval-matched samples from four exposed-uninfected participants were subjected to high-frequency longitudinal profiling of 773 host immune genes. Findings: Between June 2021 - April 2022, 68 participants across 26 U.S. states completed the study and self-collected a total of 691 and 466 longitudinal blood and nasal swab samples along with 688 symptom surveys. SARS-CoV-2 was detected in 17 out of 22 individuals with study-confirmed respiratory infection. With rapid dissemination of home self-collection kits, two and four COVID-19+ participants started collection prior to viral shedding and symptom onset, respectively, enabling us to profile detailed expression kinetics of the earliest blood transcriptional response to contemporaneous variants of concern. In pre-shedding samples, we observed transient but robust expression of T-cell response signatures, transcription factor complexes, prostaglandin biosynthesis genes, pyrogenic cytokines, and cytotoxic granule genes. This is followed by a rapid induction of many interferon-stimulated genes (ISGs), concurrent to onset of viral shedding and increase in nasal viral load. Finally, we observed increased expression of host defense peptides (HDPs) in exposed-uninfected individuals over the 4-week observational window. Interpretation: We demonstrated that unsupervised self-collection and stabilization of capillary blood can be applied to natural infection studies to characterize detailed early host immune kinetics at a temporal resolution comparable to that of human challenge studies. The remote (decentralized) study framework enables conduct of large-scale population-wide longitudinal mechanistic studies. Expression of cytotoxic/T-cell signatures in pre-shedding samples preceding expansion of innate ISGs suggests a potential role for T-cell mediated pathogen control during early infection. Elevated expression of HDPs in exposed-uninfected individuals warrants further validation studies to assess their potential role in protective immunity during pathogen exposure. Funding: This study was funded by R35GM128648 to ABT for in-lab developments of homeRNA, Packard Fellowship from the David and Lucile Packard Foundation to ABT, and R01AI153087 to AW.

At-home saliva sampling in healthy adults using CandyCollect, a lollipop-inspired device.

Respiratory infections are common in children, and there is a need for user-friendly collection methods. Here, we performed the first human subjects study using the CandyCollect device, a lollipop inspired saliva collection device. 1 We showed the CandyCollect device can be used to collect salivary bacteria from healthy adults using Streptococcus mutans and Staphylococcus aureus as proof-of-concept commensal bacteria. We enrolled healthy adults in a nationwide (USA) remote study in which participants were sent study packages containing CandyCollect devices and traditional commercially available oral swabs and spit tubes. Participants sampled themselves at home, completed usability and user preference surveys, and mailed the samples back to our laboratory for analysis by qPCR. Our results showed that for participants in which a given bacterium ( S. mutans or S. aureus ) was detected in one or both of the commercially available methods (oral swab and/or spit tubes), CandyCollect devices had a 100% concordance with the positive result (n=14 participants). Furthermore, the CandyCollect device was ranked the highest preference sampling method among the three sampling methods by 26 participants surveyed (combining survey results across two enrollment groups). We also showed that the CandyCollect device has a shelf life of up to 1 year at room temperature, a storage period that is convenient for clinics or patients to keep the CandyCollect device and use it any time. Taken together, we have demonstrated that the CandyCollect is a user-friendly saliva collection tool that has the potential to be incorporated into diagnostic assays in clinic visits and telemedicine.

At-home blood collection and stabilization in high temperature climates using homeRNA.

Expanding whole blood sample collection for transcriptome analysis beyond traditional phlebotomy clinics will open new frontiers for remote immune research and telemedicine. Determining the stability of RNA in blood samples exposed to high ambient temperatures (>30°C) is necessary for deploying home-sampling in settings with elevated temperatures (e.g., studying physiological response to natural disasters that occur in warm locations or in the summer). Recently, we have developed homeRNA, a technology that allows for self-blood sampling and RNA stabilization remotely. homeRNA consists of a lancet-based blood collection device, the Tasso-SST™ which collects up to 0.5 ml of blood from the upper arm, and a custom-built stabilization transfer tube containing RNAlater™. In this study, we investigated the robustness of our homeRNA kit in high temperature settings via two small pilot studies in Doha, Qatar (no. participants = 8), and the Western and South Central USA during the summer of 2021, which included a heatwave of unusually high temperatures in some locations (no. participants = 11). Samples collected from participants in Doha were subjected to rapid external temperature fluctuations from being moved to and from air-conditioned areas and extreme heat environments (up to 41°C external temperature during brief temperature spikes). In the USA pilot study, regions varied in outdoor temperature highs (between 25°C and 43.4°C). All samples that returned a RNA integrity number (RIN) value from the Doha, Qatar group had a RIN ≥7.0, a typical integrity threshold for downstream transcriptomics analysis. RIN values for the Western and South Central USA samples (n = 12 samples) ranged from 6.9-8.7 with 9 out of 12 samples reporting RINs ≥7.0. Overall, our pilot data suggest that homeRNA can be used in some regions that experience elevated temperatures, opening up new geographical frontiers in disseminated transcriptome analysis for applications critical to telemedicine, global health, and expanded clinical research. Further studies, including our ongoing work in Qatar, USA, and Thailand, will continue to test the robustness of homeRNA.

An open microfluidic coculture model of fibroblasts and eosinophils to investigate mechanisms of airway inflammation.

Interactions between fibroblasts and immune cells play an important role in tissue inflammation. Previous studies have found that eosinophils activated with interleukin-3 (IL-3) degranulate on aggregated immunoglobulin G (IgG) and release mediators that activate fibroblasts in the lung. However, these studies were done with eosinophil-conditioned media that have the capacity to investigate only one-way signaling from eosinophils to fibroblasts. Here, we demonstrate a coculture model of primary normal human lung fibroblasts (HLFs) and human blood eosinophils from patients with allergy and asthma using an open microfluidic coculture device. In our device, the two types of cells can communicate via two-way soluble factor signaling in the shared media while being physically separated by a half wall. Initially, we assessed the level of eosinophil degranulation by their release of eosinophil-derived neurotoxin (EDN). Next, we analyzed the inflammation-associated genes and soluble factors using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and multiplex immunoassays, respectively. Our results suggest an induction of a proinflammatory fibroblast phenotype of HLFs following the coculture with degranulating eosinophils, validating our previous findings. Additionally, we present a new result that indicate potential impacts of activated HLFs back on eosinophils. This open microfluidic coculture platform provides unique opportunities to investigate the intercellular signaling between the two cell types and their roles in airway inflammation and remodeling.

CandyCollect: at-home saliva sampling for capture of respiratory pathogens.

Streptococcus pyogenes is a major human-specific bacterial pathogen and a common cause of a wide range of symptoms from mild infection such as pharyngitis (commonly called strep throat) to life-threatening invasive infection and post-infectious sequelae. Traditional methods for diagnosis include collecting a sample using a pharyngeal swab, which can cause discomfort and even discourage adults and children from seeking proper testing and treatment in the clinic. Saliva samples are an alternative to pharyngeal swabs. To improve the testing experience for strep throat, we developed a novel lollipop-inspired sampling platform (called CandyCollect) to capture bacteria in saliva. The device can be used in clinics or in the home and shipped back to a lab for analysis, integrating with telemedicine. CandyCollect is designed to capture bacteria on an oxygen plasma treated polystyrene surface embedded with flavoring substances to enhance the experience for children and inform the required time to complete the sampling process. In addition, the open channel structure prevents the tongue from scraping and removing the captured bacteria. The flavoring substances did not affect bacterial capture and the device has a shelf life of at least 2 months (with experiments ongoing to extend the shelf life). We performed a usability study with 17 participants who provided feedback on the device design and the dissolving time of the candy. This technology and advanced processing techniques, including polymerase chain reaction (PCR), will enable user-friendly and effective diagnosis of streptococcal pharyngitis.