Accurate reporting of COVID-19 infections to monitor populations and control outbreaks has been a primary goal worldwide from the start of the pandemic. Among the various methods currently in use, tests using saliva have the advantage of easy sample acquisition, which could allow collection without a health-care practitioner. However, our study has indicated that neat saliva poses a problem for conventional RT-PCR assays due to complications surrounding its physical attributes (viscosity, heterogeneity) and unidentified chemical constituents.
A key observation was that dispersal of saliva particulates via sample mixing prior to the RT-PCR assay increased the number of observed false negative results and increased Ct values generally, even in positive samples (Figure 1a). Allowing the prepared sample to settle for at least 5 minutes after processing greatly improved RT-PCR detectability. In our hands, brief centrifugation worked as well as settling (Supplementary Figure S3), although Ranoa et al.3 did not observe the same benefit. Based on the visual assessment of several hundred assessment center negative saliva samples including and beyond those used for this study, there was no obvious correlation between any physical attribute and an erroneous RT-PCR result. Furthermore, the rate of false negative results did not seem to depend on the sample group (NPS positive, NPS negative, Healthy volunteer) (Figure 1b; Supplementary Figure S2).
Pre-heating of seven saliva samples that consistently returned false negative results after spiking with heat-inactivated virus showed marked improvement—four samples returned a positive result (Figure 2a, black triangles)—suggesting that some inhibitory components in the saliva could be disrupted or degraded. However, the remaining three samples showed resistance to the treatment, and it was unclear why the long heating step already included in the covidSHIELD method was not sufficient for inactivation of the interferants. Sample dilution was unable to produce correct results for false negative saliva samples (NPS positive, saliva RT-PCR negative) (Figure 2b, grey circle and square), though this method did remedy false negative results from spiked healthy samples.
While the utility of saliva as a diagnostic specimen cannot be disputed13, and even considering the documented reduction in detection sensitivity relative to NPS samples14, our observations provide evidence for a potentially significant issue with a high false negative rate when using unprocessed saliva samples for COVID-19 RT-PCR testing. Indeed, the original manuscript describing the covidSHIELD method3 reported 11% false negative results upon initial testing, but 100% sensitivity upon retesting; Kandel et al.15 reported 38 of 432 samples (8.8%) as invalid upon initial testing, improving to three invalid samples after retesting; and Sahajpal et al.5 reported 5% invalid results (though no false negatives).
Our study indicated that valid samples showing expected Ct values for the control RNaseP sequence could repeatedly result in a false negative SARS-CoV-2 result, suggesting an inherent issue with interferants in some saliva samples. We note that several other factors could lead to variability in saliva samples, such as the timing of sample collection with respect to disease progression, symptoms, and time of day; and the volume of saliva provided. We also recognize that disease prevalence in the population at the time of sample collection and our limited number of samples may have contributed to our observations. However, general agreement in the rate of false negative SARS-CoV-2 RT-PCR results across 137 samples from assessment centre attendees and healthy volunteers presented here, and seen in other studies, points to unknown factors that produce significant false negative results with a widely used RT-PCR method. These results indicate that while direct use of unprocessed saliva is desirable to increase the simplicity and reduce the cost for COVID-19 testing, the relatively high rate of false negative results may require additional sample processing, such as RNA extraction, to reduce the possibility of COVID-19 positive citizens unknowingly transmitting the virus within the community.