Exploring the potential of a saliva-based, RNA-extraction-free PCR test for the multiplexed detection of key respiratory pathogens

Abstract

ABSTRACTIntroductionEfforts 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.MethodsWe 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.ResultsThe 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.ConclusionOur 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.