key: cord-310096-a242g5kg
authors: Yokota, I.; Shane, P. Y.; Okada, K.; Unoki, Y.; Yang, Y.; Inao, T.; Sakamaki, K.; Iwasaki, S.; Hayasaka, K.; Sugita, J.; Nishida, M.; Fujisawa, S.; Teshima, T.
title: Mass screening of asymptomatic persons for SARS-CoV-2 using saliva
date: 2020-08-14
journal: nan
DOI: 10.1101/2020.08.13.20174078
sha: 
doc_id: 310096
cord_uid: a242g5kg

Background COVID-19 has rapidly evolved to become a global pandemic due largely to the transmission of its causative virus through asymptomatic carriers. Detection of SARS-CoV-2 in asymptomatic people is an urgent priority for the prevention and containment of disease outbreaks in communities. However, few data are available in asymptomatic persons regarding the accuracy of PCR testing. Additionally, although self-collected saliva has significant logistical advantages in mass screening, its utility as an alternative specimen in asymptomatic persons is yet to be determined. Methods We conducted a mass-screening study to compare the utility of nucleic acid amplification, such as reverse transcriptase polymerase chain reaction (RT-PCR) testing, using NPS and saliva samples from each individual in two cohorts of asymptomatic persons: the contact tracing cohort and the airport quarantine cohort. Results In this mass-screening study including 1,924 individuals, the sensitivity of nucleic acid amplification testing with nasopharyngeal and saliva specimens were 86% (90%CI:77-93%) and 92% (90%CI:83-97%), respectively, with specificities greater than 99.9%. The true concordance probability between the nasopharyngeal and saliva tests was estimated at 0.998 (90%CI:0.996-0.999) on the estimated airport prevalence, 0.3%. In positive individuals, viral load was highly correlated between NPS and saliva. Conclusion Both nasopharyngeal and saliva specimens had high sensitivity and specificity. Self-collected saliva is a valuable specimen to detect SARS-CoV-2 in mass screening of asymptomatic persons.

Background COVID-19 has rapidly evolved to become a global pandemic due largely to the transmission of its causative virus through asymptomatic carriers. Detection of SARS-CoV-2 in asymptomatic people is an urgent priority for the prevention and containment of disease outbreaks in communities. However, few data are available in asymptomatic persons regarding the accuracy of PCR testing.

Additionally, although self-collected saliva has significant logistical advantages in mass screening, its utility as an alternative specimen in asymptomatic persons is yet to be determined.

We conducted a mass-screening study to compare the utility of nucleic acid amplification, such as reverse transcriptase polymerase chain reaction (RT-PCR) testing, using NPS and saliva samples from each individual in two cohorts of asymptomatic persons: the contact tracing cohort and the airport quarantine cohort.

Since its discovery in Wuhan, China in late 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly created a global pandemic of coronavirus disease 2019 . The fast evolution of this pandemic has been attributed to the majority of transmissions occurring through people who are presymptomatic or asymptomatic [1] [2] [3] . Accordingly, detection of the virus in asymptomatic people is a problem that requires urgent attention for the prevention and containment of the outbreak of COVID-19 in communities [4] .

Currently, the diagnosis of COVID-19 is made by the detection of the nucleic acids of SARS-CoV-2 typically by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) testing of specimens collected by nasopharyngeal swabs (NPS) [5, 6] . However, few data are available regarding the accuracy of qRT-PCR testing in asymptomatic persons upon which the implications of the current testing strategy depend. The sensitivity and specificity of PCR testing need to be elucidated in order to save unnecessary quarantine and contact-tracing, while minimizing new infections from presymptomatic persons.

Recently, specimen collection by NPS has been under scrutiny, as this method requires specialized health care workers and the use of personal protective equipment (PPE) to mitigate the risk of viral exposure. Consequently, self-collected saliva has been reported to have several advantages over NPS.

As the name implies, self-collection of saliva eliminates the close contact in sampling, obviating the need for PPE. Additionally, providing saliva is painless and minimizes discomfort for the test subject. However, although we and others have shown the value of saliva as a diagnostic specimen in symptomatic patients [7] [8] [9] [10] [11] [12] , the utility of saliva in detecting the virus in asymptomatic persons remains to be elucidated.

We conducted a mass-screening study to determine and compare the sensitivity and specificity of nucleic acid amplification using paired samples (self-collected saliva and NPS) for the detection of SARS-CoV-2 in two cohorts of asymptomatic individuals.

The contact-tracing (CT) cohort included asymptomatic persons that have been in close contact with clinically confirmed COVID-19 patients with a positive qRT-PCR by NPS. Subjects in the CT cohort participated between June 12 and July 7, 2020 at several centres in Japan. Asymptomatic travelers arriving at Tokyo and Kansai international airports were enrolled from June 12 to June 23, 2020 as a separate cohort (airport quarantine (AQ) cohort). In both cohorts, all subjects were requested to provide NPS and saliva samples. All NPS samples in the CT cohort were tested by qRT-PCR. The NPS samples in the AQ cohort was tested by either qRT-PCR or reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) [13, 14] at the discretion of the airport quarantine. All 

Test value of qRT-PCR and RT-LAMP methods were illustrated by scatter plots and Kendall's coefficient of concordance W as nonparametric intraclass correlation coefficient taken non-linearity and censored value into consideration.

The performance of diagnostic tests was evaluated by sensitivity Se NPS (NPS)/ Se saliva (saliva) and specificity Sp NPS (NPS)/ Sp saliva (saliva). Sensitivity was positive probability in infected population and specificity was negative probability in non-infected population. To evaluate the concordance between NPS and saliva test, true concordance probability was defined by

, that p was the prevalence of SARS-CoV-2.

The Se NPS , Se saliva , Sp NPS , Sp saliva and p were jointly estimated using a Bayesian latent class model [15] [16] [17] The point estimate and 90% credible interval were used for the median and 5th to 95th percentile, respectively. All statistical analyses were conducted by SAS® Ver 9.4(Cary, NC).

Of the 2,558 persons screened, consent was obtained from 2,035 persons (80%) and 1,924 persons were included for analysis ( Figure 1 ). Figure 3 , when the prevalence was varied from 0% to 30%, the point estimate for the true concordance probability ranged from 0.934 to 0.999 and the lower limit of the 90% CI was never below 0.9. True concordance probability with varying estimation constraints of sensitivity is shown to be very high (supplement 1), and therefore the qRT-PCR results from saliva and NPS appeared to be sufficiently consistent. 

To confirm the equivalence of the qRT-PCR and RT-LAMP methods, a scatter 

This study examined the accuracy of detecting SARS-CoV-2 by qRT-PCR using NPS and saliva in a significant number (n=1,924) of asymptomatic individuals.

Our results showed that qRT-PCR in both specimens had specificity greater than 99.9% and sensitivity approximately 90%, validating the current practice of detecting infection by nucleic acid amplification.

We report for the first time the accuracy of viral detection using natural clinical specimens of asymptomatic persons [18] , that the sensitivity is higher than the 52% to 71% reported in symptomatic patients [5, [19] [20] [21] [22] . COVID-19

literature to date have been consistent in identifying the peak viral load at symptom onset with subsequent decline [7, 19, [23] [24] [25] [26] , suggesting the possibility of higher presymptomatic viral load. More recent studies have also shown that infectiousness peaks on or before symptom onset [27] , and that live virus can be isolated from asymptomatic individuals [28] . Concomitantly, there have been reports of discrepancy between viral load as detected by qRT-PCR and contagiousness [28] [29] [30] , which may be of utmost importance in controlling outbreaks, as the potential to infect close contacts lends credibility to the current strategy of self-quarantine. Although the relationship of contagiousness and viral load is a subject in need of further investigation, abrogation of early infectiousness may also be an effective drug development target.

The current study further extends that saliva may be a beneficial alternative to nasopharyngeal fluid in detecting SARS-CoV-2 in asymptomatic carriers. The comparison between paired samples have shown equivalent utility with similar sensitivity and specificity. However, self-collected saliva has significant advantages over NPS sampling especially in the setting of mass screening. For example, saliva collection is non-invasive and does not require specialized personnel nor the use of PPE, which saves time and cost.

Additionally, providing saliva is painless and minimizes discomfort for the patient. labelling one third of all positive tests. As PPV is dependent on the prevalence of disease, mass testing using a highly specific test will remain effective as long as test positivity remains relatively high.

RT-LAMP is an isothermal nucleic acid amplification technique that allows results to be obtained in approximately 30-60 minutes and a recent study showed the equivalent efficacy of RT-PCR and RT-LAMP in symptomatic patients [12] . In this study, we confirmed this in a large population of asymptomatic persons using saliva samples; there were no samples that were 

We declare no competing interests.

This study was funded by Health, Labour and Welfare Policy Research Grants 20HA2002.

and Hana Wakasa for assistance in collecting saliva samples. Kendall's W =0.87 Kendall's W =0.98

Investigation of a COVID-19 outbreak in Germany resulting from a single travel-associated primary case: a case series

Presumed asymptomatic carrier transmission of COVID-19

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Saliva is a reliable tool to detect SARS-CoV-2

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Saliva is more sensitive for SARS-CoV-2 detectionin COVID-19 patients than nasopharyngeal swabs

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Sensitivity of nasopharyngeal swabs and saliva for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

Saliva is less sensitive than nasopharyngeal swabs for COVID-19 detection in the community setting

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We thank Tokyo international airport quarantine and Kansai international airport