**Background:** Real-time RT-PCR is the standard diagnostic method for COVID-19, but detection of viral genome does not equate to presence of infectious virus. Previous work by the authors showed that while SARS-CoV-2 could be efficiently isolated from nasal/nasopharyngeal swabs, many genome-positive saliva specimens failed to yield infectious virus in culture. This study systematically compared viral isolation rates between specimen types, across SARS-CoV-2 variants, and investigated which salivary components might be responsible for reduced infectivity.
**Methods:** A total of 327 nasal/nasopharyngeal swab and 268 saliva specimens from rRT-PCR-positive COVID-19 patients (August 2020–March 2022) were used. Specimens were stored at −80°C, then 20 μL was inoculated onto VeroE6/TMPRSS2 cells and cultured for 5 days; cytopathic effect (CPE) was assessed microscopically. Variants were identified by RT-PCR with specific primers (N501Y for Alpha, L452R for Delta, G339D for Omicron) or next-generation sequencing. For simulated specimens, the Wuhan strain was diluted to five genome copy levels (5.0×10², 5.0×10¹, 5.0×10⁰, 5.0×10⁻¹, 5.0×10⁻² copies/μL) in swab or saliva from healthy donors (n=20 swab donors, n=10 saliva donors) who were SARS-CoV-2-negative and unvaccinated. For inhibitory assays, VeroE6 cells were pre-treated with lactoferrin, amylase, cathelicidin, or mucin at indicated concentrations for 1 hour, then infected with SARS-CoV-2 pseudotyped virus (SARS-CoV-2pv) or VSV pseudotyped virus (VSVpv). Luciferase activity was measured after 1 day. Cell viability was assessed using CellTiter-Glo 2.0. Statistical significance was determined by χ² test (Fisher's exact test when expected frequency <5) or Dunnett's test, with P<0.05 considered significant.
**Key Results:** Overall viral isolation was 45.6% (149/327) from nasal/nasopharyngeal swabs vs. 23.1% (62/268) from saliva (P<0.05). By Ct value group, isolation was significantly lower from saliva in the Ct 20–25 group (NS: 81.3% vs. S: 56.3%) and Ct 25–30 group (NS: 43.1% vs. S: 18.8%), but not in the Ct ≥30 group. By variant, isolation from saliva was significantly lower for the Wuhan strain (NS: 53.1% vs. S: 21.8%), Delta variant (NS: 46.7% vs. S: 22.7%), and Omicron variant (NS: 33.3% vs. S: 8.5%), but not for the Alpha variant (NS: 52.0% vs. S: 38.2%). Among variants within the same specimen type, the Omicron variant had significantly lower isolation rates than other variants in both swab and saliva specimens. In simulated specimens, isolation from saliva was significantly lower at 1.0×10² copies (2.8×10⁻¹ PFU); at ≥1.0×10³ copies (2.8×10⁰ PFU) all specimens yielded isolation, and below 1.0×10¹ copies (2.8×10⁻² PFU) none did. In inhibitory assays, lactoferrin inhibited SARS-CoV-2pv infection in a dose-dependent manner without cytotoxicity. Amylase significantly inhibited both SARS-CoV-2pv and VSVpv infection at higher concentrations (≥100 U/mL). Cathelicidin showed no inhibition up to 100 ng/mL and enhanced infection at 1000 ng/mL. Mucin showed no effect on either pseudovirus. No cytotoxicity was observed for any substance at tested concentrations.
**Clinical Implications:** This study demonstrates that saliva specimens yield significantly lower rates of infectious SARS-CoV-2 isolation compared to nasal/nasopharyngeal swabs, even when viral genome copies are equivalent by RT-PCR. The inhibitory effect is attributable at least in part to salivary lactoferrin and amylase. These findings suggest that the oral cavity may have a natural defensive mechanism against SARS-CoV-2 infection, and that positive RT-PCR results from saliva may overestimate the presence of infectious virus. This has implications for specimen selection in diagnostic testing, infection control policies, and understanding transmission risk. The authors note that physiological concentrations of lactoferrin (~10 µg/mL) are lower than those used experimentally, but may still contribute to anti-SARS-CoV-2 activity, potentially in synergy with amylase. Limitations include insufficient low-Ct-value saliva specimens for some variants and the need for further study of additional salivary components.