Exploring the Potential of a Saliva-Based, RNA-extraction-free PCR Test for the Multiplexed Detection of Key Respiratory Pathogens

Introduction Efforts to diagnose and monitor transmissible respiratory infections can be impaired by invasive or resource-intensive sample collection. Having extensively demonstrated the feasibility of saliva for SARS-CoV-2 detection, we sought to validate its potential for other common upper respiratory tract pathogens. Methods We modified our RNA-extraction-free SARS-CoV-2 PCR test for multiplexed detection of influenza A/B (IAV/IBV), respiratory syncytial virus (RSV) and human metapneumovirus (hMPV). Stability of virus detection in saliva from virus-positive patients was tested after storage at +4°C, room temperature (∼19°C), 30°C and 40°C for up to 7 days and through simulated shipping conditions. De-identified saliva samples were collected from individuals (≥18 years) with respiratory symptoms who were undergoing nasal-swab-based testing for SARS-CoV-2 (New Haven, CT). Saliva samples from SARS-CoV-2-negative individuals were tested with the multiplexed assay, with and without RNA extraction. Results The limit of assay detection ranged from 3-6 copies/μl, virus target depending. Detection remained stable after prolonged sample storage at elevated temperatures and through shipping conditions. From the symptomatic testing sites, 1,095 clinical specimens tested SARS-CoV-2-negative. Upon multiplexed testing of their paired saliva, 41 (3.7%) tested positive (IAV, n=20; RSV, n=5; hMPV, n=7). Additionally, upon screening samples in singleplex for pneumococcus, 29 (3%) samples tested positive. Conclusion Our findings emphasize the adaptability of a low-cost, open-source saliva-based PCR test for common respiratory pathogens, beyond SARS-CoV-2. We demonstrated its utility in symptomatic individuals, identifying viral infection missed when testing focused solely on a singular target, such as SARS-CoV-2. ### Competing Interest Statement ALW has received consulting and/or advisory board fees from Pfizer, Diasorin, PPS Health, Co-Diagnostics, and Global Diagnostic Systems for work unrelated to this project, and and is Principal Investigator on research grants from Pfizer, Merck and Flambeau Diagnostics to Yale University. All other co-authors declare no potential conflict of interest. ### Funding Statement This work was supported by a sponsored research agreement from Flambeau Diagnostics to ALW. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Institutional Review Board of the Yale Human Research Protection Program gave ethical approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.