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

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.

FIG 1

Cycle threshold (C_T) values and detection times for each molecular diagnostic test of saliva specimens. C_T value for each RT-qPCR primer set and detection time by reverse transcription–loop-mediated isothermal amplification (RT-LAMP). Horizontal lines indicate the mean C_T value or detection time.

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 C_T 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 C_T values of target 2 for the cobas SARS-CoV-2 test of saliva with positive and negative results. Horizontal lines indicate the mean C_T value. The P value was calculated using Student’s t test.