Longitudinal monitoring of SARS-CoV-2 viral load in self-collected saliva from health care workers during breakthrough infections to spare working days
Authors: Caneparo Valeria , Rinaldi Carmela , Ferrante Daniela , Ravanini Paolo , Lo Cigno Irene , Cavalieri Silvia , Gariglio Marisa , Borgogna Cinzia 
Published: November 2023

In this prospective study, we used droplet digital (dd)PCR to determine the SARS-CoV-2 viral load determination in saliva samples collected from two cohorts of Italian health care workers (HCWs) who had tested positive for SARS-CoV-2 breakthrough infections in nasopharyngeal swabs (NPS). The study encompasses two distinct periods: one from February to May 2021 (
n
= 16), characterized by Alpha variant predominance, and another from January to March 2022 (
n
= 23), marked by the prevalence of the Omicron variant. To assess the temporal pattern of virus shedding, we performed dd-PCR analysis of self-collected longitudinal saliva harvested the day after NPS testing and every 3 days until SARS-CoV-2 negativization, determined by real-time PCR (RT-PCR). Using saliva harvested within 24 h from the initial SARS-CoV-2-positive NPS tested by real-time quantitative PCR (RT-qPCR), we found that 11/16 [68.7%, confidence interval (CI) 95% 44.0%–93.4%] HCWs in the “Alpha wave” and 6/23 (26.1%, CI 95% 7.1%–45.1%) in the “Omicron wave” were negative for the presence of SARS-CoV-2 by ddPCR. The viral loads obtained with these samples were negatively correlated with the cycle threshold (Ct) values measured in the NPS specimens (
P
< 0.0001). The median Ct values of the double-positive (RT-qPCR and ddPCR) subjects were significantly higher when compared to the single-positive (RT-qPCR only) ones. In addition, we found that using ddPCR in self-collected saliva, 80% of the HCWs in the “Alpha wave” and 53% in the “Omicron wave” would have been freed from isolation within four working days from the initial positive NPS, sparing 176 working days among the HCWs enrolled in the study. Overall, this study highlights the effectiveness of using ddPCR in self-collected saliva as part of a surveillance program. It ultimately aims to identify individuals who still test positive by RT-PCR in NPS but retains a residual, non-active infection. This diagnostic approach is likely to not only prevent unnecessary isolation but also spare working days.

IMPORTANCE

Real-time quantitative PCR (RT-qPCR) on nasopharyngeal swabs (NPS) has been used as the standard method for detecting and monitoring SARS-CoV-2 infection during the pandemic. However, NPS collection often causes discomfort and poses a higher risk of transmission to health care workers (HCW). Furthermore, RT-qPCR only provides relative quantification and does not allow distinguishing those samples with residual, no longer active infection, whereas droplet digital PCR (ddPCR) allows for precise quantification of viral load, offering greater sensitivity and reproducibility. This study highlights the effectiveness of using self-collected saliva as a convenient and reliable sampling method. By utilizing ddPCR to measure the SARS-CoV-2 viral load in saliva samples, individuals with low or undetectable viral loads can be quickly identified. This approach is particularly advantageous for surveillance programs targeting HCW, as it enables the early identification and release of uninfected personnel, minimizing lost workdays. Additionally, analyzing viral load in saliva samples by ddPCR is valuable in determining virus shedding duration across different SARS-CoV-2 variants, informing transmission and disease control. Finally, testing saliva could overcome the detection of historic cases due to prolonged RNA swabbing past-infection and the unnecessary exclusion of those individuals from the workplace.