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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. Studies on the sensitivity of saliva-based SARS-CoV-2 molecular testing have shown considerable variability.We conducted a prospective, longitudinal study to investigate the testing time frame that optimizes saliva sensitivity for SARS-CoV-2 detection.Methods

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. 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.Results

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 . 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 . 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) . 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. 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.


Our study is concordant with multiple published works supporting saliva as an alternative sample for COVID-19 screening and diagnosis, and one of a minority where saliva was shown to be more sensitive than the corresponding NP swab8,9,13, although the results by Leung et al. (53.7% saliva vs. 47.4% NP swab, 95 subjects) were not statistically different8. Several reasons may account for this difference in the studies, including enrichment from nasal and oropharyngeal secretions, where the viral load is potentially higher8,9, or a higher volume of samples collection, where approximately 10 mL of saliva was collected for testing13. Steps were taken to minimize biases and errors—NP swabs performed by trained healthcare staff, environmental testing of CAP-accredited laboratory (no evidence of contamination), conduction of tests for most of the samples in the same laboratory, and pre-processing of saliva samples with dithiothreitol before RNA extraction to resolve the issues of saliva specimen viscosity, which can lead to false negatives.


Interestingly but perhaps unsurprisingly, the use of different RT-PCR kits in the present study resulted in different test-positive rates in saliva, suggesting that this can potentially be an important consideration for clinical laboratories, where more sensitive laboratory protocols should be deployed for clinical diagnosis as opposed to mass screening for low-prevalence populations. More validation would be required to confirm this finding.

SN swabs, however, appeared less sensitive compared to both saliva and NP swabs for the diagnosis of COVID-19. Although it was convenient, less time-consuming to perform relative to saliva collection, and caused less discomfort compared to NP swabs, the markedly lower sensitivity should preclude its use where other sample types can be collected.

In our study, NGS provided efficient whole-genome profiling of SARS-CoV-2 for phylogenetic analysis directly from the clinical samples without culture. NGS detection sensitivity was excellent with a threshold of 1.7% genome coverage or 5 amplicons targets, confirming all CDC-LDT positives tested. Other groups have reported highly sensitive performance for NGS with limits of detection ranging between a threshold of 5% genome coverage or 84 genome-equivalents per mL21, or at least 5 SARS-CoV-2 targets for detection22. The phylogeny results were consistent with the virus belonging to a viral type (Clade O, lineage B.6) known to be circulating in the geographical regions of Singapore and India.

There are several limitations to our work. Firstly, the study population was confined to young and middle-aged men who were either asymptomatic or had mild disease. The results cannot be extrapolated to other populations (e.g., paediatric), where there is a clear need for alternate sample types to NP swabs. Secondly, we did not extend the follow-up testing sufficiently to determine when saliva viral shedding stopped for the majority of subjects, although this has been explored in other studies. Thirdly, we did not test for the difference, if any, between saliva obtained from naso-oropharyngeal or the mouth alone, although it is biologically plausible that the latter would result in lower sensitivity for COVID-19 diagnosis.

In conclusion, our study adds to the body of evidence supporting saliva as a sensitive and less intrusive sample for COVID-19 diagnosis and further defines the role of naso-oropharyngeal secretions and the impact of different RT-PCR kits in increasing the sensitivity of testing. In our study, SN swabs were inferior to both saliva and NP swabs. Our study also provides evidence to support NGS in challenging samples for sensitive COVID-19 molecular diagnosis. Such an NGS workflow can also provide direct-from-sample phylogenetic analysis for public health decision-making, such as contact tracing.

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