Change in Saliva RT-PCR Sensitivity Over the Course of SARS-CoV-2 Infection

While real-time reverse transcriptase–polymerase chain reaction (RT-PCR) on nasopharyngeal swabs is the current standard for SARS-CoV-2 detection, saliva is an attractive alternative for diagnosis and screening due to ease of collection and minimal supply requirements.^1,2 Studies on the sensitivity of saliva-based SARS-CoV-2 molecular testing have shown considerable variability.³ We conducted a prospective, longitudinal study to investigate the testing time frame that optimizes saliva sensitivity for SARS-CoV-2 detection.

Between June 17, 2020, and February 15, 2021, a convenience sample of individuals exposed to a household member with RT-PCR–confirmed SARS-CoV-2 within 2 weeks were recruited from Children’s Hospital Los Angeles and nearby community testing sites into the Household Exposure and Respiratory Virus Transmission and Immunity Study (HEARTS). Paired nasopharyngeal and saliva samples were collected every 3 to 7 days for up to 4 weeks or until 2 negative nasopharyngeal test results. RT-PCR for SARS-CoV-2 N1 and N2 genes was performed; cycle threshold less than 40 defined a positive result. A nasopharyngeal N1 cycle threshold of 34 or less was defined as high viral load.⁴ Detailed specimen collection and RT-PCR methods are reported in the eMethods in the Supplement.

Saliva sensitivity was calculated using nasopharyngeal-positive RT-PCR as the reference standard. COVID-19 onset was defined as the earlier date between first symptom (collected by questionnaire daily) or first RT-PCR positivity. Pre- and postsymptomatic were defined as asymptomatic time points before and after a symptomatic interval, respectively. Saliva sensitivity by week of collection and between symptomatic and asymptomatic individuals were compared using the χ² test or the Fisher exact test. Generalized estimating equations were used to determine clinical characteristics (Table) associated with saliva sensitivity in nasopharyngeal-positive pairs while accounting for repeated samples from the same individuals. Analyses were performed using SPSS version 27.0 (IBM Corp) with a 2-sided P < .05 considered significant. Written informed consent was obtained from participants. The study was approved by the institutional review board of Children’s Hospital Los Angeles.

We tested 889 paired nasopharyngeal swab-saliva samples from 404 participants, of which SARS-CoV-2 was detected in 524 nasopharyngeal (58.9%) and 318 saliva (35.7%) specimens. SARS-CoV-2 was detected in both specimens in 258 pairs (29.0%). Of the 256 nasopharyngeal SARS-CoV-2–positive participants (63.4%), the mean age was 28.2 years (range, 3.0-84.5 years); 108 (42.2%) were male. Participants returned for a median of 3 visits (interquartile range, 2-4). Ninety-three participants (36.3%) were asymptomatic throughout their infection; 126 (77.3%) of 163 symptomatic individuals reported mild severity.

Saliva sensitivity was highest in samples collected during the first week of infection at 71.2% (95% CI, 62.6%-78.8%) but decreased each subsequent week (Figure, A). Participants who presented with COVID-19–associated symptoms on the specimen collection day during week 1 of infection had significantly higher saliva sensitivity compared with asymptomatic participants (88.2% [95% CI, 77.6%-95.1%] vs 58.2% [95% CI, 46.3%-69.5%]; P < .001). Saliva sensitivity remained significantly higher in symptomatic participants in week 2 (83.0% [95% CI, 70.6%-91.8%] vs 52.6% [95% CI, 42.6%-62.5%]; P < .001). No difference was observed more than 2 weeks after COVID-19 onset (Figure, B). Sensitivities did not significantly differ for never-symptomatic (34.7% [95% CI, 27.3%-42.7%]), presymptomatic (57.1% [95% CI, 31.7%-80.2%]), and postsymptomatic (42.9% [95% CI, 36.8%-49.1%]) time points (P = .26).

For each day after COVID-19 onset, the odds ratio for saliva detection was 0.94 (95% CI, 0.91-0.96) compared with the previous day (P < .001) (Table). Participants presenting with COVID-19–associated symptoms at the time of specimen collection or with high nasopharyngeal viral loads had 2.8 (95% CI, 1.6-5.1; P < .001) and 5.2 (95% CI, 2.9-9.3; P < .001) higher odds of having a saliva-positive RT-PCR result compared with those with asymptomatic presentation or low nasopharyngeal viral loads, respectively.

Saliva was sensitive for detecting SARS-CoV-2 in symptomatic individuals during initial weeks of infection, but sensitivity in asymptomatic SARS-CoV-2 carriers was less than 60% at all time points. As COVID-19 testing strategies in workplaces, schools, and other shared spaces are optimized, low saliva sensitivity in asymptomatic infections must be considered.⁵ This study suggests saliva-based RT-PCR should not be used for asymptomatic COVID-19 screening.

This study has limitations. Samples were collected following household exposure; therefore, pretest probability was high. Nasopharyngeal swab testing was the reference standard, but this is not a perfect test for SARS-CoV-2 infection, and a positive RT-PCR result from any sample past 10 days of infection may not be predictive of viral replication or infectivity.⁶

Accepted for Publication: August 1, 2021.

Published Online: August 13, 2021. doi:10.1001/jama.2021.13967

Corresponding Author: Pia S. Pannaraj, MD, MPH, Pediatrics and Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Division of Infectious Diseases, Children’s Hospital Los Angeles, 4650 Sunset Blvd, MS #51, Los Angeles, CA 90027 (ppannaraj@chla.usc.edu).

Author Contributions: Mr Congrave-Wilson and Dr Pannaraj had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Dr Pannaraj is accountable for all aspects including accuracy and integrity of the work.

Concept and design: Congrave-Wilson, Lee, Pannaraj.

Acquisition, analysis, or interpretation of data: All authors.

Drafting of the manuscript: Congrave-Wilson, Lee, Jumarang, Perez.

Critical revision of the manuscript for important intellectual content: Congrave-Wilson, Bender, Dien Bard, Pannaraj.

Statistical analysis: Congrave-Wilson, Lee, Pannaraj.

Obtained funding: Pannaraj.

Administrative, technical, or material support: Jumarang, Perez, Dien Bard.

Supervision: Lee, Pannaraj.

Conflict of Interest Disclosures: Dr Pannaraj reported receiving consultant fees from Sanofi-Pasteur and Seqirus, research funding from Pfizer and AstraZeneca for unrelated studies, and grants from MedImmune. No other disclosures were reported.

Funding/Support: This study was funded by grant U01AI144616-02S1 from the National Institutes of Health/National Institute of Allergy and Infectious Diseases. The funds were used for salary support for staff and materials to conduct the study.

Role of the Funder/Sponsor: The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Additional Contributions: We thank Ariana Peralta, AS, Melissa Lucero Tanaka, MD, Adrianna Sainz, BS, Carolyn Jennifer Marentes Ruiz, MD, and Lauren Turner, MS, who helped to collect specimens; and Wesley Cheng, BS, for help with REDCap. All received salary support from the study and are staff at Children’s Hospital Los Angeles.