Clinical Evaluation of Self-Collected Saliva by Quantitative Reverse Transcription-PCR (RT-qPCR), Direct RT-qPCR, Reverse Transcription-Loop-Mediated Isothermal Amplification, and a Rapid Antigen Test To Diagnose COVID-19

Mayu Nagura-Ikeda, Kazuo Imai, Sakiko Tabata, Kazuyasu Miyoshi, Nami Murahara, Tsukasa Mizuno, Midori Horiuchi, Kento Kato, Yoshitaka Imoto, Maki Iwata, Satoshi Mimura, Toshimitsu Ito, Kaku Tamura, Yasuyuki Kato, Mayu Nagura-Ikeda, Kazuo Imai, Sakiko Tabata, Kazuyasu Miyoshi, Nami Murahara, Tsukasa Mizuno, Midori Horiuchi, Kento Kato, Yoshitaka Imoto, Maki Iwata, Satoshi Mimura, Toshimitsu Ito, Kaku Tamura, Yasuyuki Kato

Abstract

The clinical performances of six molecular diagnostic tests and a rapid antigen test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were clinically evaluated for the diagnosis of coronavirus disease 2019 (COVID-19) in self-collected saliva. Saliva samples from 103 patients with laboratory-confirmed COVID-19 (15 asymptomatic and 88 symptomatic) were collected on the day of hospital admission. SARS-CoV-2 RNA in saliva was detected using a quantitative reverse transcription-PCR (RT-qPCR) laboratory-developed test (LDT), a cobas SARS-CoV-2 high-throughput system, three direct RT-qPCR kits, and reverse transcription-loop-mediated isothermal amplification (RT-LAMP). The viral antigen was detected by a rapid antigen immunochromatographic assay. Of the 103 samples, viral RNA was detected in 50.5 to 81.6% of the specimens by molecular diagnostic tests, and an antigen was detected in 11.7% of the specimens by the rapid antigen test. Viral RNA was detected at significantly higher percentages (65.6 to 93.4%) in specimens collected within 9 days of symptom onset than in specimens collected after at least 10 days of symptoms (22.2 to 66.7%) and in specimens collected from asymptomatic patients (40.0 to 66.7%). Self-collected saliva is an alternative specimen option for diagnosing COVID-19. The RT-qPCR LDT, a cobas SARS-CoV-2 high-throughput system, direct RT-qPCR kits (except for one commercial kit), and RT-LAMP showed sufficient sensitivities in clinical use to be selectively used in clinical settings and facilities. The rapid antigen test alone is not recommended for an initial COVID-19 diagnosis because of its low sensitivity.

Keywords: RT-LAMP; RT-qPCR; SARS-CoV-2; antigen test; saliva.

Copyright © 2020 Nagura-Ikeda et al.

Figures

FIG 1
FIG 1
Cycle threshold (CT) values and detection times for each molecular diagnostic test of saliva specimens. CT value for each RT-qPCR primer set and detection time by reverse transcription–loop-mediated isothermal amplification (RT-LAMP). Horizontal lines indicate the mean CT value or detection time.
FIG 2
FIG 2
Relation of RT-qPCR, RT-LAMP, and the rapid antigen test (RAT) results for saliva specimens. (A) Relation between the detection time of reverse transcription–loop-mediated isothermal amplification (RT-LAMP) and the CT value of target 2 (SARS-CoV-2 envelope gene) in the cobas SARS-CoV-2 test. The blue slope line represents the fitted regression curve. The gray shadow indicates the 95% confidence interval around the regression curve. (B) Distribution of the CT values of target 2 for the cobas SARS-CoV-2 test of saliva with positive and negative results. Horizontal lines indicate the mean CT value. The P value was calculated using Student’s t test.

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