key: cord-0719914-hr3r17mz
authors: Yokota, Isao; Shane, Peter Y; Okada, Kazufumi; Unoki, Yoko; Yang, Yichi; Inao, Tasuku; Sakamaki, Kentaro; Iwasaki, Sumio; Hayasaka, Kasumi; Sugita, Junichi; Nishida, Mutsumi; Fujisawa, Shinichi; Teshima, Takanori
title: Mass screening of asymptomatic persons for SARS-CoV-2 using saliva
date: 2020-09-25
journal: Clin Infect Dis
DOI: 10.1093/cid/ciaa1388
sha: 724115b39532dce88ab90bf688bcd8be3e89ff1a
doc_id: 719914
cord_uid: hr3r17mz

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 nasopharyngeal swabs (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 at 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.

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.

A c c e p t e d M a n u s c r i p t 6

We conducted a mass-screening study to determine and compare the sensitivity and specificity of nucleic acid amplification using paired samples (NPS and self-collected saliva) 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. Close contact was defined as a person who was within approximately 2 meters of an infected person. Contact-tracing was implemented by tracing the links of each infected person identified by two public health centers between June 12 and July 7, 2020. A separate cohort enrolled asymptomatic travellers arriving at Tokyo and Kansai international airports (airport quarantine [AQ] cohort) between June 12 to June 23, 2020. In both cohorts, the subjects were requested to provide saliva in addition to mandatory NPS sampling by the medical officers. Saliva samples were self-collected in a sterilized 15mL polystyrene sputum collection tube (Toyo Kizai, Warabi, Japan) at partitioned booth. Multiple partitioned booths enabled parallel sample collection with expedious flow of test subjects, with high feasibility of saliva testing especially in the context of mass-screening. All specimens were transported at 4°C and analyzed within 48 hours at the central laboratory (SRL, Tokyo, Japan).

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 

The method of collection for both saliva and NPS was the same across all participants at all sites. All neat saliva specimens were self-collected in a sterilized 15mL polystyrene sputum collection tube (Toyo Kizai, Warabi, Japan) and transported at 4°C without transport media. while RT-LAMP assay for saliva was performed at a central laboratory SRL by using the same system and methods. Regardless of the test site, all qRT-PCR tests for both NPS and saliva were performed using the same methods, according to the manual by National Institute of Infectious Diseases (NIID, https://www.niid.go.jp/niid/images/epi/corona/2019-nCoVmanual20200217-en.pdf). Briefly, 5uL of the extracted RNA was used as a template.

One step qRT-PCR was performed using THUNDERBIRD® Probe One-step qRT-PCR Kit (TOYOBO, Osaka, Japan) and 7500 Real-time PCR Systems (Thermo Fisher Scientific, Waltham, USA). The cycle threshold (Ct)-values were obtained using N2 primers (NIID_2019-nCOV_N_F2, NIID_2019-nCOV_N_R2) and a probe (NIID_2019-nCOV_N_P2). 

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 the infected population and specificity was negative probability in the 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.

Although qRT-PCR using NPS may be the best performing test available, it is not a "gold standard" without known clinical outcomes. Therefore, the Se NPS , Se saliva , Sp NPS , Sp saliva and p were jointly estimated using a Bayesian latent class model [15] [16] [17] 

Of the 2,558 persons screened, consent was obtained from 1,940 persons (75.8%) and

1,924 persons were included for analysis ( Figure 1 ). The most common reason for exclusion was the presence of symptoms (n=95; 33%) and declined consent (n=493; 22%) in the CT and AQ cohorts, respectively. Only 16 persons (0.82%) out of who agreed to participate were excluded due to insufficient saliva volume, confirming the feasibility of self-collection. (Table 2a) . 114 persons were negative in both tests, which resulted in 152 of 161 matches. In the AQ cohort, viral RNA was detected in NPS and saliva in five and four samples, respectively, out of 1763 individuals (Table 2b ).

The sensitivity of NPS and saliva were 86% (90% CI: 77-93%) and 92% (90% CI: 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. 

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 A c c e p t e d M a n u s c r i p t 12 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. These significant advantages became immediately apparent during our sample collection at the airport quarantine, where queue of international arrivals filtered smoothly through multiple collection booths. Self-collection of saliva enables parallel sample collection, which is simply more conducive to simultaneous mass screening of large number of individuals, in settings such as social and sporting events.

Previous studies comparing the viral load between NPS and saliva samples report 

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We thank Tokyo and Kansai airport quarantine stations for their cooperation; we thank 

A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t 24 Figure 4