In December 2019, China reported the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which rapidly evolved into the ongoing coronavirus disease 2019 (COVID-19) pandemic. However, there are signs of a gradual return to normalcy from the pandemic following vaccine development, mass vaccinations, and the identification of effective treatment strategies. With the easing of restrictions, the number of individuals with SARS-CoV-2 infection continues to increase. Consequently, the key to preventing infection spread and alleviating pressure on medical facilities is the early detection of infected people using simple, safe, and accurate diagnostic kits. Currently, the reverse transcription-polymerase chain reaction (RT-PCR) assay for detecting SARS-CoV-2 RNA using nasopharyngeal swab (NPS) samples is the gold standard for COVID-19 diagnosis. However, due to concerns about the burden on patients, the risk of infection to healthcare workers, and the paucity of personal protective equipment, the use of self-collected upper respiratory specimens for RT-PCR has been increasingly considered (Wehrhahn et al., 2020, To et al., 2020, Tsujimoto et al., 2021). In addition, the collection of saliva specimens is less burdensome for patients due to the ease of self-collection of samples, and these specimens are preferred in settings with a shortage of medical personnel (e.g., remote areas, airport quarantine, quarantine before entering a remote island, etc.) or when a wide range of screening tests are performed on asymptomatic patients (Terada-Hirashima et al., 2022). In particular, it is recommended that saliva specimens from individuals with confirmed or suspected COVID-19 should be transported as UN3373, “Biological Substance, Category B” (World Health Organization, 2021). Therefore, to reduce the risk of viral transmission to individuals handling specimens, specimen inactivation is necessary before they are transported to the laboratory. Currently, several saliva test kits containing such inactivators are commercially available, and these inactivators are diverse. Notably, guanidine-based inactivators, which have been established as the standard method for viral inactivation and nucleic acid extraction, have been reported to potentially inhibit RT-PCR by interfering with both DNA and Taq polymerase (Thompson et al., 2014). Additionally, guanidine-free inactivators have recently been shown to reduce SARS-CoV-2 titers for virus-infected Vero E6 cells; however, the effect of their addition to saliva samples on the accuracy and efficacy of RT-PCR in COVID-19 diagnosis remains unknown (Katherine et al., 2021).

We hypothesized that the use of guanidine-based and guanidine-free inactivator for saliva specimens affect the accuracy and efficacy of RT-PCR in the diagnosis of COVID-19. Therefore, we conducted this prospective observational study to assess the accuracy of RT-PCR for detecting SARS-CoV-2 RNA in hospitalized patients using saliva samples treated with inactivators, both with and without guanidine.