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Mass screening of asymptomatic persons for SARS-CoV-2 using saliva
Isao Yokota, PhD, MPH1*; Peter Y Shane, MD2*; Kazufumi Okada, MPH1; Yoko
Unoki, BSN1; Yichi Yang, MPH1; Tasuku Inao, BS1; Kentaro Sakamaki, PhD,
MPH3; Sumio Iwasaki, BS4; Kasumi Hayasaka4; Junichi Sugita, MD, PhD4;
Mutsumi Nishida, PhD4; Shinichi Fujisawa, BS4; Takanori Teshima, MD, PhD2,4,5.
* Co-first author
Author Affiliations
1 Department of Biostatistics, Hokkaido University Graduate School of Medicine,
Sapporo, Japan.
2 International Medical Department, Hokkaido University Hospital, Sapporo,
Japan.
3 Center for Data Science, Yokohama City University, Yokohama, Japan.
4 Division of Laboratory and Transfusion Medicine, Hokkaido University Hospital,
Sapporo, Japan.
5 Department of Hematology, Hokkaido University Graduate School of Medicine,
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Sapporo, Japan.
Correspondence to:
Prof Takanori Teshima, Department of Hematology, Hokkaido University
Graduate School of Medicine, N15, W7, Kita-ku, Sapporo, 060-8638, Japan,
teshima@med.hokudai.ac.jp
and
Dr Isao Yokota, Department of Biostatistics, Hokkaido University Graduate
School of Medicine, N15, W7, Kita-ku, Sapporo, 060-8638, Japan,
yokotai@pop.med.hokudai.ac.jp
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Abstract
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.
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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.
Keywords
SARS-CoV-2, COVID-19, saliva, PCR, LAMP
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Introduction
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 (COVID-19). The fast evolution of this pandemic has
been attributed to the majority of transmissions occurring through people who
are presymptomatic or asymptomatic[1-3]. 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
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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[7-12], the utility of saliva in detecting the virus in asymptomatic persons
remains to be elucidated.
Methods
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.
Design and Population
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
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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
saliva samples in both cohorts were subjected to both qRT-PCR and RT-LAMP
testing. This study was approved by the Institutional Ethics Board (Hokkaido
University Hospital Division of Clinical Research Administration Number:
020-0116) and informed consent was obtained from all individuals.
Diagnostic test
Saliva was diluted 4-fold with phosphate buffered saline (PBS) and centrifuged
at 2000 × g for 5 min to remove cells and debris. RNA was extracted from 200 µL
of the supernatant or nasopharyngeal swab samples using QIAsymphony DSP
Virus/Pathogen kit and QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany).
Nucleic acids of SARS-CoV-2 were detected by qRT-PCR or RT-LAMP.
qRT-PCR tests were performed, according to the manual by National Institute of
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Infectious Diseases (NIID,
https://www.niid.go.jp/niid/images/epi/corona/2019-nCoVmanual20200217-en.p
df). 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). RT-LAMP was carried out to detect SARS-CoV-2
2019-SARS-CoV-2 Detection Reagent Kit (Eiken
Chemical, Tokyo, Japan). The final reaction volume containing 10µl of viral RNA
extract and 15µl of Primer Mix containing SARS-CoV-2 specific primers was
dispensed into a reaction tube with dried amplification reagents including Bst
DNA polymerase and AMV reverse transcriptase. This tube was incubated at
62.5oC with turbidity readings (optical density at 650 nm) and monitored for 35
minutes using the Loopamp Real-time Turbidimeter (Eiken Chemical Co., Ltd.,).
Statistical analysis
Test value of qRT-PCR and RT-LAMP methods were illustrated by scatter plots
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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 SeNPS (NPS)/
Sesaliva (saliva) and specificity SpNPS (NPS)/ Spsaliva (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 SeNPS, Sesaliva, SpNPS, Spsaliva and p were jointly estimated using a
Bayesian latent class model[15-17] since this method accounts for change of
plans, rare positive cases. The prior distribution of specificity SpNPS, Spsaliva were
Beta(201,1), reflecting the results of the in-hospital screening, all negative in
more than 200 consecutive individuals with none subsequently developing
COVID-19 (data not shown). The prior distribution of SeNPS, Sesaliva and p were
Beta(1,1). The corresponding true concordance probability was estimated under
varying prevalence values. For a sensitivity analysis, we estimated the true
concordance probability when we imposed the constraint that the sensitivity of
saliva test was equal to and 10% less than the sensitivity of NPS test.
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Sample size in the CT cohort was calculated as 250 based on the
prevalence of 0.1 and 25 positive samples were needed in order to keep the
width of the 90% credible interval of sensitivity within 0.3 under the sensitivity at
0.7. Sample size in the AQ cohort was calculated 1,818 based on the probability
that 90% credible interval of specificity over 99.0% would be 0.8 (likes statistical
power) under the expected specificity being 99.5%.
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).
Results
Demographics
Of the 2,558 persons screened, consent was obtained from 2,035 persons
(80%) 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 (0.78%) were excluded due to insufficient saliva volume, confirming the
feasibility of self-collection. Background characteristics of the 161 and 1,763
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persons in the CT and AQ cohorts, respectively, are shown in Table 1. In the CT
cohort, age and gender data were not made available from many subjects due to
procedural reasons. This population mainly consisted of relatively young people
between 20 and 50 years old. In the AQ cohort, the number of participants by the
last point of embarkation was 467 (26%) from Europe (Amsterdam, Frankfurt,
and London), 583 (33%) from Asia and Oceania (Bangkok, Jakarta, Manila,
Seoul, Shanghai, Sydney, and Taipei), and 713 (40%) from North America
(Chicago, Los Angeles, Seattle, and Vancouver). Because of the reduced
number of international flights during this period, passengers from Central and
South Americas, Africa, and the Middle East may have arrived via transit through
any of the aforementioned regions.
Sensitivity, Specificity and True concordance
In the CT cohort, SARS-CoV-2 was detected in 41 NPS samples and in 44 saliva
samples, of which 38 individuals had both samples test positive (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).
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The sensitivity of NPS and saliva were 86% (90% CI: 77-93%) and 92%
(90% CI: 83-97%), respectively (Figure 2a), and the specificity of NPS and saliva
were 99.93% (90% CI: 99.77-99.99%) and 99.96% (90%CI: 99.85-100.00%),
respectively (Figure 2b). The estimated prevalence at the CT and AQ cohort was
29.6% (90%CI: 23.8-35.8%) and 0.3% (90%CI: 0.1-0.6%), respectively. The true
concordance probability was estimated at 0.998 (90% CI: 0.996-0.999) in the AQ
cohort. As shown in 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.
Comparison of the viral load between NPS and saliva samples
Scatter plot of the Ct values of qRT-PCR from the 45 positive specimens (either
NPS or saliva) is depicted in Figure 4a. All three samples that were negative by
saliva and positive by NPS had Ct values of 40 on NPS qRT-PCR test. On the
other hand, six samples that were negative by NPS and positive by saliva had Ct
values between 33.7 and 37.2 by saliva qRT-PCR. Kendall's coefficient of
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concordance was 0.87, indicating that the viral load was equivalent between
NPS and saliva samples.
Test values of RT-PCR and RT-LAMP methods
To confirm the equivalence of the qRT-PCR and RT-LAMP methods, a scatter
plot of time for detecting positive results (Tp) with RT-LAMP against Ct values of
qRT-PCR test using 44 saliva samples is shown in Figure 4b. Four samples that
were negative by RT-LAMP and positive by qRT-PCR had Ct values ranging
from 36.0 to 37.3, indicating very low viral loads (Kendall's coefficient of
concordance = 0.98). Excluding these four samples, concordance between
qRT-PCR and RT-LAMP was demonstrated in saliva specimens in 87 samples
(36 positive and 51 negative) in the CT cohort. In the AQ cohort, all 1763
samples (4 positive and 1759 negative) were concordant.
Discussion
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
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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, 19-22]. COVID-19
literature to date have been consistent in identifying the peak viral load at
symptom onset with subsequent decline[7, 19, 23-26], 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[28-30], 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
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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. Obtaining saliva 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 conflicting results. Wyllie et al. showed that the viral load was
five-times higher in saliva than NPS[23], while others have reported results to
the contrary[9, 26]. Our results clearly show the viral loads to be equivalent
between NPS and saliva in asymptomatic individuals and both specimens may
be useful in detecting viral RNA.
Among the limitations of any diagnostic modality is the possibility of
obtaining false results with serious consequences. While persons infected with
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SARS-CoV-2 with falsely negative test may be left in society without the
necessary precautions to keep him/her from transmitting the virus, false positive
non-infected persons may undergo unnecessary quarantine and
labour-intensive contact tracing measures. Although the high specificity of
qRT-PCR reported herein may be reassuring in individual cases, the implications
of mass testing depends on the prevalence of disease in the subject population.
However, point prevalence is unknowable a priori and extremely difficult to
assess in rapidly evolving outbreaks from carriers with relatively long
presymptomatic periods. Rather, insights on mass testing may be gained
through carefully monitoring test positivity in relation to the total number of tests
performed. For example, with greater than 99.9% specificity, a positive result in
five percent of all tests would indicate that more than 4.9% (out of the 5%) are
true positives, with a positive predictive value (PPV) of at least 98%. On the
other hand, if only 0.3% of all tests return positive (e.g. in isolated localities with
very few disease), the PPV would be (0.3%-0.1%)/0.3% = 0.67, erroneously
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.
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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
negative by NPS RT-LAMP and positive by saliva. It is unlikely that the sensitivity
of the RT-LAMP method is significantly less than that of qRT-PCR, and the
RT-LAMP testing has little impact on our conclusions. Our study suggests that
RT-LAMP is a useful alternative to RT-PCR for the diagnosis of SARS-CoV-2.
The current study lacks longitudinal data and clinical confirmation of
positive cases. Nonetheless, this is the first study in asymptomatic individuals
comparing paired samples of NPS and saliva. Rapid detection of asymptomatic
infected patients is critical for the prevention of outbreaks of COVID-19 in
communities and hospitals. Mass screening of the virus using self-collected
saliva can be performed easily, non-invasively, and with minimal risk of viral
transmission to health care workers.
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Contributors
IY, KS, JS, MN and TT determined the study design. IY, PS, YU, SI, KH, MN, SF
and TT collected the data. IY, KO, YU, YY, TI, KS did statistical analysis. IY, PS,
TT drafted the manuscript and all authors reviewed critically and approved the
final manuscript.
Declaration of interests
We declare no competing interests.
Funding
This study was funded by Health, Labour and Welfare Policy Research Grants
20HA2002.
Acknowledgement
We thank Tokyo airport quarantine station and Kansai airport quarantine station
for cooperation; Megumi Aoki, Miwa Aoki, Nana Arai, Satomi Araki, Cao Cuicui,
Kazumi Hasegawa, Masato Horiuchi, Dr. Nao Kurita, Dr. Aki Nakamura, Chiho
Okabe, Mana Okamura, Yusuke Sakai, Dr. Akahito Sako, Natsumi Satake, Maki
Shimatani, Kaki Tanaka, Maina Toguri, Sachiko Tominaga and Hana Wakasa for
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assistance in collecting saliva samples.
Reference
1. Bohmer MM, Buchholz U, Corman VM, et al. Investigation of a COVID-19
outbreak in Germany resulting from a single travel-associated primary case: a
case series. Lancet Infect Dis 2020.
2. Bai Y, Yao L, Wei T, et al. Presumed asymptomatic carrier transmission of
COVID-19. JAMA 2020.
3. Moghadas SM, Fitzpatrick MC, Sah P, et al. The implications of silent
transmission for the control of COVID-19 outbreaks. Proc Natl Acad Sci U S A
2020.
4. Abbasi J. The promise and peril of antibody testing for COVID-19. JAMA 2020.
5. Wang W, Xu Y, Gao R, et al. Detection of SARS-CoV-2 in different types of
clinical specimens. JAMA 2020.
6. Wang X, Tan L, Wang X, et al. Comparison of nasopharyngeal and
oropharyngeal swabs for SARS-CoV-2 detection in 353 patients received tests
with both specimens simultaneously. Int J Infect Dis 2020; 94: 107-9.
7. To KK, Tsang OT, Chik-Yan Yip C, et al. Consistent detection of 2019 novel
coronavirus in saliva. Clin Infect Dis 2020.
8. Azzi L, Carcano G, Gianfagna F, et al. Saliva is a reliable tool to detect
SARS-CoV-2. J Infect 2020.
9. Williams E, Bond K, Zhang B, Putland M, Williamson DA. Saliva as a
Noninvasive Specimen for Detection of SARS-CoV-2. J Clin Microbiol 2020;
58(8).
10. Tu YP, Jennings R, Hart B, et al. Swabs collected by patients or health care
workers for SARS-CoV-2 testing. N Engl J Med 2020.
11. Iwasaki S, Fujisawa S, Nakakubo S, et al. Comparison of SARS-CoV-2
detection in nasopharyngeal swab and saliva. J Infect 2020.
12. Nagura-Ikeda M, Imai K, Tabata S, et al. Clinical evaluation of self-collected
saliva by RT-qPCR, direct RT-qPCR, RT-LAMP, and a rapid antigen test to
diagnose COVID-19. J Clin Microbiol 2020.
13. Notomi T, Okayama H, Masubuchi H, et al. Loop-mediated isothermal
amplification of DNA. Nucleic Acids Res 2000; 28(12): E63.
All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprintthis version posted August 15, 2020. .https://doi.org/10.1101/2020.08.13.20174078doi: medRxiv preprint
20
14. Park GS, Ku K, Baek SH, et al. Development of reverse transcription
loop-mediated isothermal amplification assays targeting severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2). J Mol Diagn 2020; 22(6): 729-35.
15. Joseph L, Gyorkos TW, Coupal L. Bayesian estimation of disease prevalence
and the parameters of diagnostic tests in the absence of a gold standard. Am J
Epidemiol 1995; 141(3): 263-72.
16. Johnson WO, Gastwirth JL, Pearson LM. Screening without a "gold standard":
the Hui-Walter paradigm revisited. Am J Epidemiol 2001; 153(9): 921-4.
17. Wang C, Hanson TE. Estimation of sensitivity and specificity of multiple
repeated binary tests without a gold standard. Stat Med 2019; 38(13): 2381-90.
18. Woloshin S, Patel N, Kesselheim AS. False negative tests for SARS-CoV-2
infection - challenges and implications. N Engl J Med 2020.
19. Zou L, Ruan F, Huang M, et al. SARS-CoV-2 viral load in upper respiratory
specimens of infected patients. N Engl J Med 2020; 382(12): 1177-9.
20. Fang Y, Zhang H, Xie J, et al. Sensitivity of chest CT for COVID-19:
comparison to RT-PCR. Radiology 2020: 200432.
21. Ai T, Yang Z, Hou H, et al. Correlation of chest CT and RT-PCR testing in
coronavirus disease 2019 (COVID-19) in China: a report of 1014 cases.
Radiology 2020: 200642.
22. Guo L, Ren L, Yang S, et al. Profiling early humoral response to diagnose novel
coronavirus disease (COVID-19). Clin Infect Dis 2020.
23. Wyllie AL, Fournier J, Casanovas-Massana A, et al. Saliva is more sensitive for
SARS-CoV-2 detectionin COVID-19 patients than nasopharyngeal swabs.
medRxiv 2020.
24. To KK, Tsang OT, Leung WS, et al. Temporal profiles of viral load in posterior
oropharyngeal saliva samples and serum antibody responses during infection by
SARS-CoV-2: an observational cohort study. Lancet Infect Dis 2020; 20(5):
565-74.
25. Jamal MA, Mohammad M, Coomes E, et al. Sensitivity of nasopharyngeal
swabs and saliva for the detection of severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2). medRxiv 2020.
26. Becker D, Sandoval E, Amin A, al. e. Saliva is less sensitive than
nasopharyngeal swabs for COVID-19 detection in the community setting.
medRxiv 2020.
27. He X, Lau EHY, Wu P, et al. Temporal dynamics in viral shedding and
transmissibility of COVID-19. Nat Med 2020; 26(5): 672-5.
All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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21
28. Arons MM, Hatfield KM, Reddy SC, et al. Presymptomatic SARS-CoV-2
infections and transmission in a skilled nursing facility. N Engl J Med 2020;
382(22): 2081-90.
29. La Scola B, Le Bideau M, Andreani J, et al. Viral RNA load as determined by
cell culture as a management tool for discharge of SARS-CoV-2 patients from
infectious disease wards. Eur J Clin Microbiol Infect Dis 2020; 39(6): 1059-61.
30. Bullard J, Dust K, Funk D, et al. Predicting infectious SARS-CoV-2 from
diagnostic samples. Clin Infect Dis 2020.
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Table 1. Background characteristics
contact-tracing cohort airport cohort
N (%) N (%)
Sex
Female 26 (16.1) 832 (47.2)
Male 44 (27.3) 927 (52.6)
unknown 91 (56.5) 4 (0.2)
Age
Median [IQR] 44.9 [29.8, 66.4] 33.5 [22.6, 47.4]
-19 2 (1.2) 299 (17.0)
20-29 16 (9.9) 433 (24.6)
30-39 13 (8.1) 344 (19.5)
40-49 9 (5.6) 324 (18.4)
50-59 8 (5.0) 230 (13.0)
60-69 9 (5.6) 97 (5.5)
70- 13 (8.1) 34 (1.9)
unknown 91 (56.5) 2 (0.1)
Last point of embarkation
North America - 713 (40.4)
Asia and Oceania - 583 (33.1)
Europe - 467 (26.5)
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Table 2. Diagnostic results of nasopharyngeal swab (NPS) and saliva test
(a) Contact-tracing cohort (n=161)
saliva
NPS positive negative
positive 38 3
negative 6 114
(b) Airport Quarantine cohort (n=1,763)
saliva
NPS positive negative
positive 4 1
negative 0 1758
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Figure 1. Flow diagram of participants
Figure 2. The sensitivity and specificity of nasopharyngeal swab and saliva
Histograms of posterior distribution of (a) sensitivity and (b) specificity. Point
estimates and 90% credible interval (90%CI) defined by 5th to 95th percentile
are shown.
Figure 3. True concordance probability with varying rates of prevalence.
The true concordance probability of diagnosis between nasopharyngeal swab
and saliva test in populations with various prevalence. Solid line indicates point
estimates and dashed lines indicate 90% credible interval.
Figure 4. Comparison of the viral load between NPS and saliva
(a) Ct values determined with the qRT-PCR test of nasopharyngeal swab and
saliva are plotted. (b) Times to detecting positive results (Tp) determined by the
RT-LAMP test of saliva are plotted against Ct values determined by the
qRT-PCR test of saliva. W indicates Kendall’s coefficient of concordance. Data
were plotted with one of the tests being positive and the values being measured.
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Supplement 1. True concordance probability under several scenarios.
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Contact-Tracing (CT) cohort Airport Quarantine (AQ) cohort
2,270 persons screened288 persons screened
161 persons were analyzed
Declined to participate (n=30)
Symptomatic persons (n=95)
Insufficient saliva volume (n=2)
Declined to participate (n=493)
Insufficient saliva volume (n=14)
1,763 persons were analyzed
Figure 1
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50 60 70 80 90 100
0246 810
50 60 70 80 90 100
0246 810
99.0 99.2 99.4 99.6 99.8 100.0
0 600 1200 1800
99.0 99.2 99.4 99.6 99.8 100.0
0 600 1200 1800
NPS saliva
(a) sensitivity
(b) specificity
86% (90%CI: 77-93%) 92% (90%CI: 83-97%)
99.93%
(90%CI: 99.77-99.99%)
99.96%
(90%CI: 99.85-100.00%)
%%
%%
NPS saliva
Figure 2
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0102030
0.0 0.2 0.4 0.6 0.8 1.0
True concordance probability
%
Prevalence
Figure 3
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(a) qRT-PCR between NPS and saliva (n=45) (b) qRT-PCR and RT-LAMP in saliva (n=44)
Figure 4
010203040
0 10203040
unde termined
unde termined
C
t
value in nasopharyngeal swabs
C
t
value in saliva
010203040
0 10203040
unde termined
C
t
value
Tp [min]
Kendall’s W =0.87 Kendall’s W =0.98
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